Tissue engineering and periodontal regeneration
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P.Prathahini, IIIrd MDS Tissue engineering and Periodontal Regeneration
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Transcript of Tissue engineering and periodontal regeneration
PPrathahini
IIIrd MDS
Tissue engineering and Periodontal Regeneration
Introduction
bull See the hole fill it with anything - Perio 2000 vol 50
Principle Objective hellip
ldquoTo re-create functional healthy tissues and organs
in order to replace diseased dying or dead tissuesrdquo
Periodontal Regeneration
bull Appropriate cell types amp Signals
bull Local environment
ndash Recruitment of the right cells and
preventing the wrong cell
(Local Environment includes
Cementum matrix amp CEJ)
- Affects cell migration adhesion
proliferation amp differentiation
Epithelial cells ndashjunctionalepithelium
Fibroblasts ndashgingival ampperiodontal ligament fibres
Blastic cells -Osteoblasts for Alveoalr bone cementoblastsfor cementum
Cells Growth factors ndash FGF-1 amp 2 IGF-1amp2 BMP EGFPdgf
Adhesion molecules ndashfibronectin osteopontin laminin bsp collagens CAP
Structural proteins -Types IIIIV XII and XIV collagens Proteoglycans Hyaluran tenascin non-collagenous proteins osteonectin dentinenamel proteins
Molecules
To say periodontal regeneration has occurred 4
criteria should be fulfilled
bull Functional epithelial seal
bull New connective tissue attachment
bull Acellular extrinsic fiber cementum
bull Alveolar bone height restored
Reasons for Failures in Periodontal Regeneration technique
bull Formation of a long junctional epithelium
bull Inadequate seal
bull Wound closure
bull Restriction of regeneration
bull Precise definition
bull Sufficient discrimination
bull Infection
- Tissue engineering is defined as the reconstruction of living
tissues to be used for the replacement of damaged or lost
tissueorgans of living organisms and is founded on the principles
of cell biology developmental biology and biomaterials science
(Narem R et al1995 Rosso F et al 2004
- ldquoan interdisciplinary field that applies the principles of
engineering and life sciences towards the development of biological
substitutes that restore maintain or improve tissue function or a
whole organ
Langer and Vacanti
Tissue engineering
bull Sixteenth century Tagliacozzi of Bologna Italy
ldquoDe Custorum Chirurigia per Insitionemrdquo
bull 1970 ndash WT Green (Orthopaedic surgeon) ndash
created cartilage
bull 1986 ndash Karl Meyer - created a skin
substitute by using a collagen matrix
bull DrLangler - designed appropriate scaffolds
Key components
Scaffold
Progenitor cells
Biosignals
Diagrammatic representation of arteriovenous
shunt loop model in the rat ndash Mian R et al 2000
Artery
Venous graft
Hole of insertionof vessel loop
Vein
Transparent chamber
Femoral vessels Leg
Two main criteria for successful tissue engineering
Biomechanical properties
bull Scaffold
bull Architectural geometry
bull Space-maintaining properties
Biological functions
bull Cell recruitment proliferation survival in culture and at the site of implantation
bull Neovascularization
bull Delivery of morphogenetic- regulatory- and growth factors
Stem Cells
Primal undifferentiated cells
that retain the ability to
produce an identical copy of
themselves when they divide
(clone) and differentiate into
other cell types
Potency
The potency specifies the differentiation potential of the
stem cell
bull Totipotent stem cells
bull Pluripotent stem cells
bull Multipotent stem cells
bull Unipotent stem cells
Key events in stem cell researchbull 1960s - Joseph Altman and Gopal Das presented
evidence of adult neurogenesis ongoing stem cell activity in the brain
their reports contradict Cajals no new neurons dogma are largely
ignored
bull 1963 - McCulloch and Till illustrate the presence of self-renewing stem
cells in mouse bone marrow
bull 1968 - bone marrow transplant between two siblings successfully treats
SCID
bull 1978 - haematopoietic stem cells are discovered in human cord blood
bull 1981 - mouse embryonic stem cells are derived from the inner cell mass
bull 1992 - neural stem cells are cultured in vitro as neurospheres
bull 1995 - President Bill Clinton signs into law the Dickey Amendment which
makes it illegal for Federal money to be used for research where stem cells
are derived from the destruction of the embryo
Key events in stem cell research
bull 1997 - Leukemia was shown to originate from a
haematopoietic stem cell the first direct evidence for cancer stem cells
bull 1998 - James Thomson and coworkers derive the first human embryonic
stem cell line at the University of Wisconsin-Madison
bull 2000s - several reports of adult stem cell plasticity are published
bull 2003 - Dr Songtao Shi of NIH discovers new source of adult stem cells in
childrens primary teeth
bull 2004-2005 - Hwang Woo-Suk claims to have created several human
embryonic stem cell lines from unfertilised human oocytes The lines are
later shown to be fabricated
bull July 19 2006 - President George W Bush votes a bill which would have
allowed Federal money to be used for research where stem cells are
derived from the destruction of the embryo
Types of Stem cells
Adult stem
cellshellip
Embryonic stem cells
in vitro
ADVANTAGES
The ability to examine the material as it is formed and to
perform specific measurements prior
to implantation
DISADVANTAGES
- The absence of a physiologic amp mechanical environment during the
formation of tissue in vitro
- Union of the implanted tissues with the host organ
requires remodeling ndashdegradation and new tissue formation at the interface of implant with the host
tissue
in vivoADVANTAGES
Tissue formation takes place under the influence of physiologic mechanical
environment
Incorporation of the tissues being formed with
the surrounding structures
DISADVANTAGES
Regenerating tissues may be dislodged or degraded by the mechanical forces
normally acting at the site before the regenerating
tissue is fully formed and incorporated
I) Hematopoietic stem cells
2) Bone marrow -
stem cells
-Friedenstein et al 1976-All cell lineages - Mesenchymal fibroblasts cells isolated- Specific surface markers
Locally derived uncommitted cells
bull Periodontal ligament stem cells
bull Dental pulp stem cells
- Cell surface markers ( Gronthos amp co workers 2005)
The dental pulp stem cells represent a clonogenic and highly proliferative cell
population
- Densely calcified nodules = in vitro
- Dentin like and dentin sialo-phospho- protein rich mineralisation =
Invivo
Periodontal ligament stem cellsbull Highly fibrous and vascularhellip High turn over
rates
- Cementum like mineralized structure formed in vitrohellip
Progenitor cells hellipidentified in vivo cell kinetic studieshellip
bull Enriched locations like around the blood vessels
bull Exhibit stem cell features
ndash Small size
ndash Responsiveness to stimulating factor
ndash Slow cycle time
Most compelling evidence McCulloch and coworkers
1985198719951996
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Introduction
bull See the hole fill it with anything - Perio 2000 vol 50
Principle Objective hellip
ldquoTo re-create functional healthy tissues and organs
in order to replace diseased dying or dead tissuesrdquo
Periodontal Regeneration
bull Appropriate cell types amp Signals
bull Local environment
ndash Recruitment of the right cells and
preventing the wrong cell
(Local Environment includes
Cementum matrix amp CEJ)
- Affects cell migration adhesion
proliferation amp differentiation
Epithelial cells ndashjunctionalepithelium
Fibroblasts ndashgingival ampperiodontal ligament fibres
Blastic cells -Osteoblasts for Alveoalr bone cementoblastsfor cementum
Cells Growth factors ndash FGF-1 amp 2 IGF-1amp2 BMP EGFPdgf
Adhesion molecules ndashfibronectin osteopontin laminin bsp collagens CAP
Structural proteins -Types IIIIV XII and XIV collagens Proteoglycans Hyaluran tenascin non-collagenous proteins osteonectin dentinenamel proteins
Molecules
To say periodontal regeneration has occurred 4
criteria should be fulfilled
bull Functional epithelial seal
bull New connective tissue attachment
bull Acellular extrinsic fiber cementum
bull Alveolar bone height restored
Reasons for Failures in Periodontal Regeneration technique
bull Formation of a long junctional epithelium
bull Inadequate seal
bull Wound closure
bull Restriction of regeneration
bull Precise definition
bull Sufficient discrimination
bull Infection
- Tissue engineering is defined as the reconstruction of living
tissues to be used for the replacement of damaged or lost
tissueorgans of living organisms and is founded on the principles
of cell biology developmental biology and biomaterials science
(Narem R et al1995 Rosso F et al 2004
- ldquoan interdisciplinary field that applies the principles of
engineering and life sciences towards the development of biological
substitutes that restore maintain or improve tissue function or a
whole organ
Langer and Vacanti
Tissue engineering
bull Sixteenth century Tagliacozzi of Bologna Italy
ldquoDe Custorum Chirurigia per Insitionemrdquo
bull 1970 ndash WT Green (Orthopaedic surgeon) ndash
created cartilage
bull 1986 ndash Karl Meyer - created a skin
substitute by using a collagen matrix
bull DrLangler - designed appropriate scaffolds
Key components
Scaffold
Progenitor cells
Biosignals
Diagrammatic representation of arteriovenous
shunt loop model in the rat ndash Mian R et al 2000
Artery
Venous graft
Hole of insertionof vessel loop
Vein
Transparent chamber
Femoral vessels Leg
Two main criteria for successful tissue engineering
Biomechanical properties
bull Scaffold
bull Architectural geometry
bull Space-maintaining properties
Biological functions
bull Cell recruitment proliferation survival in culture and at the site of implantation
bull Neovascularization
bull Delivery of morphogenetic- regulatory- and growth factors
Stem Cells
Primal undifferentiated cells
that retain the ability to
produce an identical copy of
themselves when they divide
(clone) and differentiate into
other cell types
Potency
The potency specifies the differentiation potential of the
stem cell
bull Totipotent stem cells
bull Pluripotent stem cells
bull Multipotent stem cells
bull Unipotent stem cells
Key events in stem cell researchbull 1960s - Joseph Altman and Gopal Das presented
evidence of adult neurogenesis ongoing stem cell activity in the brain
their reports contradict Cajals no new neurons dogma are largely
ignored
bull 1963 - McCulloch and Till illustrate the presence of self-renewing stem
cells in mouse bone marrow
bull 1968 - bone marrow transplant between two siblings successfully treats
SCID
bull 1978 - haematopoietic stem cells are discovered in human cord blood
bull 1981 - mouse embryonic stem cells are derived from the inner cell mass
bull 1992 - neural stem cells are cultured in vitro as neurospheres
bull 1995 - President Bill Clinton signs into law the Dickey Amendment which
makes it illegal for Federal money to be used for research where stem cells
are derived from the destruction of the embryo
Key events in stem cell research
bull 1997 - Leukemia was shown to originate from a
haematopoietic stem cell the first direct evidence for cancer stem cells
bull 1998 - James Thomson and coworkers derive the first human embryonic
stem cell line at the University of Wisconsin-Madison
bull 2000s - several reports of adult stem cell plasticity are published
bull 2003 - Dr Songtao Shi of NIH discovers new source of adult stem cells in
childrens primary teeth
bull 2004-2005 - Hwang Woo-Suk claims to have created several human
embryonic stem cell lines from unfertilised human oocytes The lines are
later shown to be fabricated
bull July 19 2006 - President George W Bush votes a bill which would have
allowed Federal money to be used for research where stem cells are
derived from the destruction of the embryo
Types of Stem cells
Adult stem
cellshellip
Embryonic stem cells
in vitro
ADVANTAGES
The ability to examine the material as it is formed and to
perform specific measurements prior
to implantation
DISADVANTAGES
- The absence of a physiologic amp mechanical environment during the
formation of tissue in vitro
- Union of the implanted tissues with the host organ
requires remodeling ndashdegradation and new tissue formation at the interface of implant with the host
tissue
in vivoADVANTAGES
Tissue formation takes place under the influence of physiologic mechanical
environment
Incorporation of the tissues being formed with
the surrounding structures
DISADVANTAGES
Regenerating tissues may be dislodged or degraded by the mechanical forces
normally acting at the site before the regenerating
tissue is fully formed and incorporated
I) Hematopoietic stem cells
2) Bone marrow -
stem cells
-Friedenstein et al 1976-All cell lineages - Mesenchymal fibroblasts cells isolated- Specific surface markers
Locally derived uncommitted cells
bull Periodontal ligament stem cells
bull Dental pulp stem cells
- Cell surface markers ( Gronthos amp co workers 2005)
The dental pulp stem cells represent a clonogenic and highly proliferative cell
population
- Densely calcified nodules = in vitro
- Dentin like and dentin sialo-phospho- protein rich mineralisation =
Invivo
Periodontal ligament stem cellsbull Highly fibrous and vascularhellip High turn over
rates
- Cementum like mineralized structure formed in vitrohellip
Progenitor cells hellipidentified in vivo cell kinetic studieshellip
bull Enriched locations like around the blood vessels
bull Exhibit stem cell features
ndash Small size
ndash Responsiveness to stimulating factor
ndash Slow cycle time
Most compelling evidence McCulloch and coworkers
1985198719951996
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Periodontal Regeneration
bull Appropriate cell types amp Signals
bull Local environment
ndash Recruitment of the right cells and
preventing the wrong cell
(Local Environment includes
Cementum matrix amp CEJ)
- Affects cell migration adhesion
proliferation amp differentiation
Epithelial cells ndashjunctionalepithelium
Fibroblasts ndashgingival ampperiodontal ligament fibres
Blastic cells -Osteoblasts for Alveoalr bone cementoblastsfor cementum
Cells Growth factors ndash FGF-1 amp 2 IGF-1amp2 BMP EGFPdgf
Adhesion molecules ndashfibronectin osteopontin laminin bsp collagens CAP
Structural proteins -Types IIIIV XII and XIV collagens Proteoglycans Hyaluran tenascin non-collagenous proteins osteonectin dentinenamel proteins
Molecules
To say periodontal regeneration has occurred 4
criteria should be fulfilled
bull Functional epithelial seal
bull New connective tissue attachment
bull Acellular extrinsic fiber cementum
bull Alveolar bone height restored
Reasons for Failures in Periodontal Regeneration technique
bull Formation of a long junctional epithelium
bull Inadequate seal
bull Wound closure
bull Restriction of regeneration
bull Precise definition
bull Sufficient discrimination
bull Infection
- Tissue engineering is defined as the reconstruction of living
tissues to be used for the replacement of damaged or lost
tissueorgans of living organisms and is founded on the principles
of cell biology developmental biology and biomaterials science
(Narem R et al1995 Rosso F et al 2004
- ldquoan interdisciplinary field that applies the principles of
engineering and life sciences towards the development of biological
substitutes that restore maintain or improve tissue function or a
whole organ
Langer and Vacanti
Tissue engineering
bull Sixteenth century Tagliacozzi of Bologna Italy
ldquoDe Custorum Chirurigia per Insitionemrdquo
bull 1970 ndash WT Green (Orthopaedic surgeon) ndash
created cartilage
bull 1986 ndash Karl Meyer - created a skin
substitute by using a collagen matrix
bull DrLangler - designed appropriate scaffolds
Key components
Scaffold
Progenitor cells
Biosignals
Diagrammatic representation of arteriovenous
shunt loop model in the rat ndash Mian R et al 2000
Artery
Venous graft
Hole of insertionof vessel loop
Vein
Transparent chamber
Femoral vessels Leg
Two main criteria for successful tissue engineering
Biomechanical properties
bull Scaffold
bull Architectural geometry
bull Space-maintaining properties
Biological functions
bull Cell recruitment proliferation survival in culture and at the site of implantation
bull Neovascularization
bull Delivery of morphogenetic- regulatory- and growth factors
Stem Cells
Primal undifferentiated cells
that retain the ability to
produce an identical copy of
themselves when they divide
(clone) and differentiate into
other cell types
Potency
The potency specifies the differentiation potential of the
stem cell
bull Totipotent stem cells
bull Pluripotent stem cells
bull Multipotent stem cells
bull Unipotent stem cells
Key events in stem cell researchbull 1960s - Joseph Altman and Gopal Das presented
evidence of adult neurogenesis ongoing stem cell activity in the brain
their reports contradict Cajals no new neurons dogma are largely
ignored
bull 1963 - McCulloch and Till illustrate the presence of self-renewing stem
cells in mouse bone marrow
bull 1968 - bone marrow transplant between two siblings successfully treats
SCID
bull 1978 - haematopoietic stem cells are discovered in human cord blood
bull 1981 - mouse embryonic stem cells are derived from the inner cell mass
bull 1992 - neural stem cells are cultured in vitro as neurospheres
bull 1995 - President Bill Clinton signs into law the Dickey Amendment which
makes it illegal for Federal money to be used for research where stem cells
are derived from the destruction of the embryo
Key events in stem cell research
bull 1997 - Leukemia was shown to originate from a
haematopoietic stem cell the first direct evidence for cancer stem cells
bull 1998 - James Thomson and coworkers derive the first human embryonic
stem cell line at the University of Wisconsin-Madison
bull 2000s - several reports of adult stem cell plasticity are published
bull 2003 - Dr Songtao Shi of NIH discovers new source of adult stem cells in
childrens primary teeth
bull 2004-2005 - Hwang Woo-Suk claims to have created several human
embryonic stem cell lines from unfertilised human oocytes The lines are
later shown to be fabricated
bull July 19 2006 - President George W Bush votes a bill which would have
allowed Federal money to be used for research where stem cells are
derived from the destruction of the embryo
Types of Stem cells
Adult stem
cellshellip
Embryonic stem cells
in vitro
ADVANTAGES
The ability to examine the material as it is formed and to
perform specific measurements prior
to implantation
DISADVANTAGES
- The absence of a physiologic amp mechanical environment during the
formation of tissue in vitro
- Union of the implanted tissues with the host organ
requires remodeling ndashdegradation and new tissue formation at the interface of implant with the host
tissue
in vivoADVANTAGES
Tissue formation takes place under the influence of physiologic mechanical
environment
Incorporation of the tissues being formed with
the surrounding structures
DISADVANTAGES
Regenerating tissues may be dislodged or degraded by the mechanical forces
normally acting at the site before the regenerating
tissue is fully formed and incorporated
I) Hematopoietic stem cells
2) Bone marrow -
stem cells
-Friedenstein et al 1976-All cell lineages - Mesenchymal fibroblasts cells isolated- Specific surface markers
Locally derived uncommitted cells
bull Periodontal ligament stem cells
bull Dental pulp stem cells
- Cell surface markers ( Gronthos amp co workers 2005)
The dental pulp stem cells represent a clonogenic and highly proliferative cell
population
- Densely calcified nodules = in vitro
- Dentin like and dentin sialo-phospho- protein rich mineralisation =
Invivo
Periodontal ligament stem cellsbull Highly fibrous and vascularhellip High turn over
rates
- Cementum like mineralized structure formed in vitrohellip
Progenitor cells hellipidentified in vivo cell kinetic studieshellip
bull Enriched locations like around the blood vessels
bull Exhibit stem cell features
ndash Small size
ndash Responsiveness to stimulating factor
ndash Slow cycle time
Most compelling evidence McCulloch and coworkers
1985198719951996
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Epithelial cells ndashjunctionalepithelium
Fibroblasts ndashgingival ampperiodontal ligament fibres
Blastic cells -Osteoblasts for Alveoalr bone cementoblastsfor cementum
Cells Growth factors ndash FGF-1 amp 2 IGF-1amp2 BMP EGFPdgf
Adhesion molecules ndashfibronectin osteopontin laminin bsp collagens CAP
Structural proteins -Types IIIIV XII and XIV collagens Proteoglycans Hyaluran tenascin non-collagenous proteins osteonectin dentinenamel proteins
Molecules
To say periodontal regeneration has occurred 4
criteria should be fulfilled
bull Functional epithelial seal
bull New connective tissue attachment
bull Acellular extrinsic fiber cementum
bull Alveolar bone height restored
Reasons for Failures in Periodontal Regeneration technique
bull Formation of a long junctional epithelium
bull Inadequate seal
bull Wound closure
bull Restriction of regeneration
bull Precise definition
bull Sufficient discrimination
bull Infection
- Tissue engineering is defined as the reconstruction of living
tissues to be used for the replacement of damaged or lost
tissueorgans of living organisms and is founded on the principles
of cell biology developmental biology and biomaterials science
(Narem R et al1995 Rosso F et al 2004
- ldquoan interdisciplinary field that applies the principles of
engineering and life sciences towards the development of biological
substitutes that restore maintain or improve tissue function or a
whole organ
Langer and Vacanti
Tissue engineering
bull Sixteenth century Tagliacozzi of Bologna Italy
ldquoDe Custorum Chirurigia per Insitionemrdquo
bull 1970 ndash WT Green (Orthopaedic surgeon) ndash
created cartilage
bull 1986 ndash Karl Meyer - created a skin
substitute by using a collagen matrix
bull DrLangler - designed appropriate scaffolds
Key components
Scaffold
Progenitor cells
Biosignals
Diagrammatic representation of arteriovenous
shunt loop model in the rat ndash Mian R et al 2000
Artery
Venous graft
Hole of insertionof vessel loop
Vein
Transparent chamber
Femoral vessels Leg
Two main criteria for successful tissue engineering
Biomechanical properties
bull Scaffold
bull Architectural geometry
bull Space-maintaining properties
Biological functions
bull Cell recruitment proliferation survival in culture and at the site of implantation
bull Neovascularization
bull Delivery of morphogenetic- regulatory- and growth factors
Stem Cells
Primal undifferentiated cells
that retain the ability to
produce an identical copy of
themselves when they divide
(clone) and differentiate into
other cell types
Potency
The potency specifies the differentiation potential of the
stem cell
bull Totipotent stem cells
bull Pluripotent stem cells
bull Multipotent stem cells
bull Unipotent stem cells
Key events in stem cell researchbull 1960s - Joseph Altman and Gopal Das presented
evidence of adult neurogenesis ongoing stem cell activity in the brain
their reports contradict Cajals no new neurons dogma are largely
ignored
bull 1963 - McCulloch and Till illustrate the presence of self-renewing stem
cells in mouse bone marrow
bull 1968 - bone marrow transplant between two siblings successfully treats
SCID
bull 1978 - haematopoietic stem cells are discovered in human cord blood
bull 1981 - mouse embryonic stem cells are derived from the inner cell mass
bull 1992 - neural stem cells are cultured in vitro as neurospheres
bull 1995 - President Bill Clinton signs into law the Dickey Amendment which
makes it illegal for Federal money to be used for research where stem cells
are derived from the destruction of the embryo
Key events in stem cell research
bull 1997 - Leukemia was shown to originate from a
haematopoietic stem cell the first direct evidence for cancer stem cells
bull 1998 - James Thomson and coworkers derive the first human embryonic
stem cell line at the University of Wisconsin-Madison
bull 2000s - several reports of adult stem cell plasticity are published
bull 2003 - Dr Songtao Shi of NIH discovers new source of adult stem cells in
childrens primary teeth
bull 2004-2005 - Hwang Woo-Suk claims to have created several human
embryonic stem cell lines from unfertilised human oocytes The lines are
later shown to be fabricated
bull July 19 2006 - President George W Bush votes a bill which would have
allowed Federal money to be used for research where stem cells are
derived from the destruction of the embryo
Types of Stem cells
Adult stem
cellshellip
Embryonic stem cells
in vitro
ADVANTAGES
The ability to examine the material as it is formed and to
perform specific measurements prior
to implantation
DISADVANTAGES
- The absence of a physiologic amp mechanical environment during the
formation of tissue in vitro
- Union of the implanted tissues with the host organ
requires remodeling ndashdegradation and new tissue formation at the interface of implant with the host
tissue
in vivoADVANTAGES
Tissue formation takes place under the influence of physiologic mechanical
environment
Incorporation of the tissues being formed with
the surrounding structures
DISADVANTAGES
Regenerating tissues may be dislodged or degraded by the mechanical forces
normally acting at the site before the regenerating
tissue is fully formed and incorporated
I) Hematopoietic stem cells
2) Bone marrow -
stem cells
-Friedenstein et al 1976-All cell lineages - Mesenchymal fibroblasts cells isolated- Specific surface markers
Locally derived uncommitted cells
bull Periodontal ligament stem cells
bull Dental pulp stem cells
- Cell surface markers ( Gronthos amp co workers 2005)
The dental pulp stem cells represent a clonogenic and highly proliferative cell
population
- Densely calcified nodules = in vitro
- Dentin like and dentin sialo-phospho- protein rich mineralisation =
Invivo
Periodontal ligament stem cellsbull Highly fibrous and vascularhellip High turn over
rates
- Cementum like mineralized structure formed in vitrohellip
Progenitor cells hellipidentified in vivo cell kinetic studieshellip
bull Enriched locations like around the blood vessels
bull Exhibit stem cell features
ndash Small size
ndash Responsiveness to stimulating factor
ndash Slow cycle time
Most compelling evidence McCulloch and coworkers
1985198719951996
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
To say periodontal regeneration has occurred 4
criteria should be fulfilled
bull Functional epithelial seal
bull New connective tissue attachment
bull Acellular extrinsic fiber cementum
bull Alveolar bone height restored
Reasons for Failures in Periodontal Regeneration technique
bull Formation of a long junctional epithelium
bull Inadequate seal
bull Wound closure
bull Restriction of regeneration
bull Precise definition
bull Sufficient discrimination
bull Infection
- Tissue engineering is defined as the reconstruction of living
tissues to be used for the replacement of damaged or lost
tissueorgans of living organisms and is founded on the principles
of cell biology developmental biology and biomaterials science
(Narem R et al1995 Rosso F et al 2004
- ldquoan interdisciplinary field that applies the principles of
engineering and life sciences towards the development of biological
substitutes that restore maintain or improve tissue function or a
whole organ
Langer and Vacanti
Tissue engineering
bull Sixteenth century Tagliacozzi of Bologna Italy
ldquoDe Custorum Chirurigia per Insitionemrdquo
bull 1970 ndash WT Green (Orthopaedic surgeon) ndash
created cartilage
bull 1986 ndash Karl Meyer - created a skin
substitute by using a collagen matrix
bull DrLangler - designed appropriate scaffolds
Key components
Scaffold
Progenitor cells
Biosignals
Diagrammatic representation of arteriovenous
shunt loop model in the rat ndash Mian R et al 2000
Artery
Venous graft
Hole of insertionof vessel loop
Vein
Transparent chamber
Femoral vessels Leg
Two main criteria for successful tissue engineering
Biomechanical properties
bull Scaffold
bull Architectural geometry
bull Space-maintaining properties
Biological functions
bull Cell recruitment proliferation survival in culture and at the site of implantation
bull Neovascularization
bull Delivery of morphogenetic- regulatory- and growth factors
Stem Cells
Primal undifferentiated cells
that retain the ability to
produce an identical copy of
themselves when they divide
(clone) and differentiate into
other cell types
Potency
The potency specifies the differentiation potential of the
stem cell
bull Totipotent stem cells
bull Pluripotent stem cells
bull Multipotent stem cells
bull Unipotent stem cells
Key events in stem cell researchbull 1960s - Joseph Altman and Gopal Das presented
evidence of adult neurogenesis ongoing stem cell activity in the brain
their reports contradict Cajals no new neurons dogma are largely
ignored
bull 1963 - McCulloch and Till illustrate the presence of self-renewing stem
cells in mouse bone marrow
bull 1968 - bone marrow transplant between two siblings successfully treats
SCID
bull 1978 - haematopoietic stem cells are discovered in human cord blood
bull 1981 - mouse embryonic stem cells are derived from the inner cell mass
bull 1992 - neural stem cells are cultured in vitro as neurospheres
bull 1995 - President Bill Clinton signs into law the Dickey Amendment which
makes it illegal for Federal money to be used for research where stem cells
are derived from the destruction of the embryo
Key events in stem cell research
bull 1997 - Leukemia was shown to originate from a
haematopoietic stem cell the first direct evidence for cancer stem cells
bull 1998 - James Thomson and coworkers derive the first human embryonic
stem cell line at the University of Wisconsin-Madison
bull 2000s - several reports of adult stem cell plasticity are published
bull 2003 - Dr Songtao Shi of NIH discovers new source of adult stem cells in
childrens primary teeth
bull 2004-2005 - Hwang Woo-Suk claims to have created several human
embryonic stem cell lines from unfertilised human oocytes The lines are
later shown to be fabricated
bull July 19 2006 - President George W Bush votes a bill which would have
allowed Federal money to be used for research where stem cells are
derived from the destruction of the embryo
Types of Stem cells
Adult stem
cellshellip
Embryonic stem cells
in vitro
ADVANTAGES
The ability to examine the material as it is formed and to
perform specific measurements prior
to implantation
DISADVANTAGES
- The absence of a physiologic amp mechanical environment during the
formation of tissue in vitro
- Union of the implanted tissues with the host organ
requires remodeling ndashdegradation and new tissue formation at the interface of implant with the host
tissue
in vivoADVANTAGES
Tissue formation takes place under the influence of physiologic mechanical
environment
Incorporation of the tissues being formed with
the surrounding structures
DISADVANTAGES
Regenerating tissues may be dislodged or degraded by the mechanical forces
normally acting at the site before the regenerating
tissue is fully formed and incorporated
I) Hematopoietic stem cells
2) Bone marrow -
stem cells
-Friedenstein et al 1976-All cell lineages - Mesenchymal fibroblasts cells isolated- Specific surface markers
Locally derived uncommitted cells
bull Periodontal ligament stem cells
bull Dental pulp stem cells
- Cell surface markers ( Gronthos amp co workers 2005)
The dental pulp stem cells represent a clonogenic and highly proliferative cell
population
- Densely calcified nodules = in vitro
- Dentin like and dentin sialo-phospho- protein rich mineralisation =
Invivo
Periodontal ligament stem cellsbull Highly fibrous and vascularhellip High turn over
rates
- Cementum like mineralized structure formed in vitrohellip
Progenitor cells hellipidentified in vivo cell kinetic studieshellip
bull Enriched locations like around the blood vessels
bull Exhibit stem cell features
ndash Small size
ndash Responsiveness to stimulating factor
ndash Slow cycle time
Most compelling evidence McCulloch and coworkers
1985198719951996
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Reasons for Failures in Periodontal Regeneration technique
bull Formation of a long junctional epithelium
bull Inadequate seal
bull Wound closure
bull Restriction of regeneration
bull Precise definition
bull Sufficient discrimination
bull Infection
- Tissue engineering is defined as the reconstruction of living
tissues to be used for the replacement of damaged or lost
tissueorgans of living organisms and is founded on the principles
of cell biology developmental biology and biomaterials science
(Narem R et al1995 Rosso F et al 2004
- ldquoan interdisciplinary field that applies the principles of
engineering and life sciences towards the development of biological
substitutes that restore maintain or improve tissue function or a
whole organ
Langer and Vacanti
Tissue engineering
bull Sixteenth century Tagliacozzi of Bologna Italy
ldquoDe Custorum Chirurigia per Insitionemrdquo
bull 1970 ndash WT Green (Orthopaedic surgeon) ndash
created cartilage
bull 1986 ndash Karl Meyer - created a skin
substitute by using a collagen matrix
bull DrLangler - designed appropriate scaffolds
Key components
Scaffold
Progenitor cells
Biosignals
Diagrammatic representation of arteriovenous
shunt loop model in the rat ndash Mian R et al 2000
Artery
Venous graft
Hole of insertionof vessel loop
Vein
Transparent chamber
Femoral vessels Leg
Two main criteria for successful tissue engineering
Biomechanical properties
bull Scaffold
bull Architectural geometry
bull Space-maintaining properties
Biological functions
bull Cell recruitment proliferation survival in culture and at the site of implantation
bull Neovascularization
bull Delivery of morphogenetic- regulatory- and growth factors
Stem Cells
Primal undifferentiated cells
that retain the ability to
produce an identical copy of
themselves when they divide
(clone) and differentiate into
other cell types
Potency
The potency specifies the differentiation potential of the
stem cell
bull Totipotent stem cells
bull Pluripotent stem cells
bull Multipotent stem cells
bull Unipotent stem cells
Key events in stem cell researchbull 1960s - Joseph Altman and Gopal Das presented
evidence of adult neurogenesis ongoing stem cell activity in the brain
their reports contradict Cajals no new neurons dogma are largely
ignored
bull 1963 - McCulloch and Till illustrate the presence of self-renewing stem
cells in mouse bone marrow
bull 1968 - bone marrow transplant between two siblings successfully treats
SCID
bull 1978 - haematopoietic stem cells are discovered in human cord blood
bull 1981 - mouse embryonic stem cells are derived from the inner cell mass
bull 1992 - neural stem cells are cultured in vitro as neurospheres
bull 1995 - President Bill Clinton signs into law the Dickey Amendment which
makes it illegal for Federal money to be used for research where stem cells
are derived from the destruction of the embryo
Key events in stem cell research
bull 1997 - Leukemia was shown to originate from a
haematopoietic stem cell the first direct evidence for cancer stem cells
bull 1998 - James Thomson and coworkers derive the first human embryonic
stem cell line at the University of Wisconsin-Madison
bull 2000s - several reports of adult stem cell plasticity are published
bull 2003 - Dr Songtao Shi of NIH discovers new source of adult stem cells in
childrens primary teeth
bull 2004-2005 - Hwang Woo-Suk claims to have created several human
embryonic stem cell lines from unfertilised human oocytes The lines are
later shown to be fabricated
bull July 19 2006 - President George W Bush votes a bill which would have
allowed Federal money to be used for research where stem cells are
derived from the destruction of the embryo
Types of Stem cells
Adult stem
cellshellip
Embryonic stem cells
in vitro
ADVANTAGES
The ability to examine the material as it is formed and to
perform specific measurements prior
to implantation
DISADVANTAGES
- The absence of a physiologic amp mechanical environment during the
formation of tissue in vitro
- Union of the implanted tissues with the host organ
requires remodeling ndashdegradation and new tissue formation at the interface of implant with the host
tissue
in vivoADVANTAGES
Tissue formation takes place under the influence of physiologic mechanical
environment
Incorporation of the tissues being formed with
the surrounding structures
DISADVANTAGES
Regenerating tissues may be dislodged or degraded by the mechanical forces
normally acting at the site before the regenerating
tissue is fully formed and incorporated
I) Hematopoietic stem cells
2) Bone marrow -
stem cells
-Friedenstein et al 1976-All cell lineages - Mesenchymal fibroblasts cells isolated- Specific surface markers
Locally derived uncommitted cells
bull Periodontal ligament stem cells
bull Dental pulp stem cells
- Cell surface markers ( Gronthos amp co workers 2005)
The dental pulp stem cells represent a clonogenic and highly proliferative cell
population
- Densely calcified nodules = in vitro
- Dentin like and dentin sialo-phospho- protein rich mineralisation =
Invivo
Periodontal ligament stem cellsbull Highly fibrous and vascularhellip High turn over
rates
- Cementum like mineralized structure formed in vitrohellip
Progenitor cells hellipidentified in vivo cell kinetic studieshellip
bull Enriched locations like around the blood vessels
bull Exhibit stem cell features
ndash Small size
ndash Responsiveness to stimulating factor
ndash Slow cycle time
Most compelling evidence McCulloch and coworkers
1985198719951996
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
- Tissue engineering is defined as the reconstruction of living
tissues to be used for the replacement of damaged or lost
tissueorgans of living organisms and is founded on the principles
of cell biology developmental biology and biomaterials science
(Narem R et al1995 Rosso F et al 2004
- ldquoan interdisciplinary field that applies the principles of
engineering and life sciences towards the development of biological
substitutes that restore maintain or improve tissue function or a
whole organ
Langer and Vacanti
Tissue engineering
bull Sixteenth century Tagliacozzi of Bologna Italy
ldquoDe Custorum Chirurigia per Insitionemrdquo
bull 1970 ndash WT Green (Orthopaedic surgeon) ndash
created cartilage
bull 1986 ndash Karl Meyer - created a skin
substitute by using a collagen matrix
bull DrLangler - designed appropriate scaffolds
Key components
Scaffold
Progenitor cells
Biosignals
Diagrammatic representation of arteriovenous
shunt loop model in the rat ndash Mian R et al 2000
Artery
Venous graft
Hole of insertionof vessel loop
Vein
Transparent chamber
Femoral vessels Leg
Two main criteria for successful tissue engineering
Biomechanical properties
bull Scaffold
bull Architectural geometry
bull Space-maintaining properties
Biological functions
bull Cell recruitment proliferation survival in culture and at the site of implantation
bull Neovascularization
bull Delivery of morphogenetic- regulatory- and growth factors
Stem Cells
Primal undifferentiated cells
that retain the ability to
produce an identical copy of
themselves when they divide
(clone) and differentiate into
other cell types
Potency
The potency specifies the differentiation potential of the
stem cell
bull Totipotent stem cells
bull Pluripotent stem cells
bull Multipotent stem cells
bull Unipotent stem cells
Key events in stem cell researchbull 1960s - Joseph Altman and Gopal Das presented
evidence of adult neurogenesis ongoing stem cell activity in the brain
their reports contradict Cajals no new neurons dogma are largely
ignored
bull 1963 - McCulloch and Till illustrate the presence of self-renewing stem
cells in mouse bone marrow
bull 1968 - bone marrow transplant between two siblings successfully treats
SCID
bull 1978 - haematopoietic stem cells are discovered in human cord blood
bull 1981 - mouse embryonic stem cells are derived from the inner cell mass
bull 1992 - neural stem cells are cultured in vitro as neurospheres
bull 1995 - President Bill Clinton signs into law the Dickey Amendment which
makes it illegal for Federal money to be used for research where stem cells
are derived from the destruction of the embryo
Key events in stem cell research
bull 1997 - Leukemia was shown to originate from a
haematopoietic stem cell the first direct evidence for cancer stem cells
bull 1998 - James Thomson and coworkers derive the first human embryonic
stem cell line at the University of Wisconsin-Madison
bull 2000s - several reports of adult stem cell plasticity are published
bull 2003 - Dr Songtao Shi of NIH discovers new source of adult stem cells in
childrens primary teeth
bull 2004-2005 - Hwang Woo-Suk claims to have created several human
embryonic stem cell lines from unfertilised human oocytes The lines are
later shown to be fabricated
bull July 19 2006 - President George W Bush votes a bill which would have
allowed Federal money to be used for research where stem cells are
derived from the destruction of the embryo
Types of Stem cells
Adult stem
cellshellip
Embryonic stem cells
in vitro
ADVANTAGES
The ability to examine the material as it is formed and to
perform specific measurements prior
to implantation
DISADVANTAGES
- The absence of a physiologic amp mechanical environment during the
formation of tissue in vitro
- Union of the implanted tissues with the host organ
requires remodeling ndashdegradation and new tissue formation at the interface of implant with the host
tissue
in vivoADVANTAGES
Tissue formation takes place under the influence of physiologic mechanical
environment
Incorporation of the tissues being formed with
the surrounding structures
DISADVANTAGES
Regenerating tissues may be dislodged or degraded by the mechanical forces
normally acting at the site before the regenerating
tissue is fully formed and incorporated
I) Hematopoietic stem cells
2) Bone marrow -
stem cells
-Friedenstein et al 1976-All cell lineages - Mesenchymal fibroblasts cells isolated- Specific surface markers
Locally derived uncommitted cells
bull Periodontal ligament stem cells
bull Dental pulp stem cells
- Cell surface markers ( Gronthos amp co workers 2005)
The dental pulp stem cells represent a clonogenic and highly proliferative cell
population
- Densely calcified nodules = in vitro
- Dentin like and dentin sialo-phospho- protein rich mineralisation =
Invivo
Periodontal ligament stem cellsbull Highly fibrous and vascularhellip High turn over
rates
- Cementum like mineralized structure formed in vitrohellip
Progenitor cells hellipidentified in vivo cell kinetic studieshellip
bull Enriched locations like around the blood vessels
bull Exhibit stem cell features
ndash Small size
ndash Responsiveness to stimulating factor
ndash Slow cycle time
Most compelling evidence McCulloch and coworkers
1985198719951996
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
bull Sixteenth century Tagliacozzi of Bologna Italy
ldquoDe Custorum Chirurigia per Insitionemrdquo
bull 1970 ndash WT Green (Orthopaedic surgeon) ndash
created cartilage
bull 1986 ndash Karl Meyer - created a skin
substitute by using a collagen matrix
bull DrLangler - designed appropriate scaffolds
Key components
Scaffold
Progenitor cells
Biosignals
Diagrammatic representation of arteriovenous
shunt loop model in the rat ndash Mian R et al 2000
Artery
Venous graft
Hole of insertionof vessel loop
Vein
Transparent chamber
Femoral vessels Leg
Two main criteria for successful tissue engineering
Biomechanical properties
bull Scaffold
bull Architectural geometry
bull Space-maintaining properties
Biological functions
bull Cell recruitment proliferation survival in culture and at the site of implantation
bull Neovascularization
bull Delivery of morphogenetic- regulatory- and growth factors
Stem Cells
Primal undifferentiated cells
that retain the ability to
produce an identical copy of
themselves when they divide
(clone) and differentiate into
other cell types
Potency
The potency specifies the differentiation potential of the
stem cell
bull Totipotent stem cells
bull Pluripotent stem cells
bull Multipotent stem cells
bull Unipotent stem cells
Key events in stem cell researchbull 1960s - Joseph Altman and Gopal Das presented
evidence of adult neurogenesis ongoing stem cell activity in the brain
their reports contradict Cajals no new neurons dogma are largely
ignored
bull 1963 - McCulloch and Till illustrate the presence of self-renewing stem
cells in mouse bone marrow
bull 1968 - bone marrow transplant between two siblings successfully treats
SCID
bull 1978 - haematopoietic stem cells are discovered in human cord blood
bull 1981 - mouse embryonic stem cells are derived from the inner cell mass
bull 1992 - neural stem cells are cultured in vitro as neurospheres
bull 1995 - President Bill Clinton signs into law the Dickey Amendment which
makes it illegal for Federal money to be used for research where stem cells
are derived from the destruction of the embryo
Key events in stem cell research
bull 1997 - Leukemia was shown to originate from a
haematopoietic stem cell the first direct evidence for cancer stem cells
bull 1998 - James Thomson and coworkers derive the first human embryonic
stem cell line at the University of Wisconsin-Madison
bull 2000s - several reports of adult stem cell plasticity are published
bull 2003 - Dr Songtao Shi of NIH discovers new source of adult stem cells in
childrens primary teeth
bull 2004-2005 - Hwang Woo-Suk claims to have created several human
embryonic stem cell lines from unfertilised human oocytes The lines are
later shown to be fabricated
bull July 19 2006 - President George W Bush votes a bill which would have
allowed Federal money to be used for research where stem cells are
derived from the destruction of the embryo
Types of Stem cells
Adult stem
cellshellip
Embryonic stem cells
in vitro
ADVANTAGES
The ability to examine the material as it is formed and to
perform specific measurements prior
to implantation
DISADVANTAGES
- The absence of a physiologic amp mechanical environment during the
formation of tissue in vitro
- Union of the implanted tissues with the host organ
requires remodeling ndashdegradation and new tissue formation at the interface of implant with the host
tissue
in vivoADVANTAGES
Tissue formation takes place under the influence of physiologic mechanical
environment
Incorporation of the tissues being formed with
the surrounding structures
DISADVANTAGES
Regenerating tissues may be dislodged or degraded by the mechanical forces
normally acting at the site before the regenerating
tissue is fully formed and incorporated
I) Hematopoietic stem cells
2) Bone marrow -
stem cells
-Friedenstein et al 1976-All cell lineages - Mesenchymal fibroblasts cells isolated- Specific surface markers
Locally derived uncommitted cells
bull Periodontal ligament stem cells
bull Dental pulp stem cells
- Cell surface markers ( Gronthos amp co workers 2005)
The dental pulp stem cells represent a clonogenic and highly proliferative cell
population
- Densely calcified nodules = in vitro
- Dentin like and dentin sialo-phospho- protein rich mineralisation =
Invivo
Periodontal ligament stem cellsbull Highly fibrous and vascularhellip High turn over
rates
- Cementum like mineralized structure formed in vitrohellip
Progenitor cells hellipidentified in vivo cell kinetic studieshellip
bull Enriched locations like around the blood vessels
bull Exhibit stem cell features
ndash Small size
ndash Responsiveness to stimulating factor
ndash Slow cycle time
Most compelling evidence McCulloch and coworkers
1985198719951996
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Key components
Scaffold
Progenitor cells
Biosignals
Diagrammatic representation of arteriovenous
shunt loop model in the rat ndash Mian R et al 2000
Artery
Venous graft
Hole of insertionof vessel loop
Vein
Transparent chamber
Femoral vessels Leg
Two main criteria for successful tissue engineering
Biomechanical properties
bull Scaffold
bull Architectural geometry
bull Space-maintaining properties
Biological functions
bull Cell recruitment proliferation survival in culture and at the site of implantation
bull Neovascularization
bull Delivery of morphogenetic- regulatory- and growth factors
Stem Cells
Primal undifferentiated cells
that retain the ability to
produce an identical copy of
themselves when they divide
(clone) and differentiate into
other cell types
Potency
The potency specifies the differentiation potential of the
stem cell
bull Totipotent stem cells
bull Pluripotent stem cells
bull Multipotent stem cells
bull Unipotent stem cells
Key events in stem cell researchbull 1960s - Joseph Altman and Gopal Das presented
evidence of adult neurogenesis ongoing stem cell activity in the brain
their reports contradict Cajals no new neurons dogma are largely
ignored
bull 1963 - McCulloch and Till illustrate the presence of self-renewing stem
cells in mouse bone marrow
bull 1968 - bone marrow transplant between two siblings successfully treats
SCID
bull 1978 - haematopoietic stem cells are discovered in human cord blood
bull 1981 - mouse embryonic stem cells are derived from the inner cell mass
bull 1992 - neural stem cells are cultured in vitro as neurospheres
bull 1995 - President Bill Clinton signs into law the Dickey Amendment which
makes it illegal for Federal money to be used for research where stem cells
are derived from the destruction of the embryo
Key events in stem cell research
bull 1997 - Leukemia was shown to originate from a
haematopoietic stem cell the first direct evidence for cancer stem cells
bull 1998 - James Thomson and coworkers derive the first human embryonic
stem cell line at the University of Wisconsin-Madison
bull 2000s - several reports of adult stem cell plasticity are published
bull 2003 - Dr Songtao Shi of NIH discovers new source of adult stem cells in
childrens primary teeth
bull 2004-2005 - Hwang Woo-Suk claims to have created several human
embryonic stem cell lines from unfertilised human oocytes The lines are
later shown to be fabricated
bull July 19 2006 - President George W Bush votes a bill which would have
allowed Federal money to be used for research where stem cells are
derived from the destruction of the embryo
Types of Stem cells
Adult stem
cellshellip
Embryonic stem cells
in vitro
ADVANTAGES
The ability to examine the material as it is formed and to
perform specific measurements prior
to implantation
DISADVANTAGES
- The absence of a physiologic amp mechanical environment during the
formation of tissue in vitro
- Union of the implanted tissues with the host organ
requires remodeling ndashdegradation and new tissue formation at the interface of implant with the host
tissue
in vivoADVANTAGES
Tissue formation takes place under the influence of physiologic mechanical
environment
Incorporation of the tissues being formed with
the surrounding structures
DISADVANTAGES
Regenerating tissues may be dislodged or degraded by the mechanical forces
normally acting at the site before the regenerating
tissue is fully formed and incorporated
I) Hematopoietic stem cells
2) Bone marrow -
stem cells
-Friedenstein et al 1976-All cell lineages - Mesenchymal fibroblasts cells isolated- Specific surface markers
Locally derived uncommitted cells
bull Periodontal ligament stem cells
bull Dental pulp stem cells
- Cell surface markers ( Gronthos amp co workers 2005)
The dental pulp stem cells represent a clonogenic and highly proliferative cell
population
- Densely calcified nodules = in vitro
- Dentin like and dentin sialo-phospho- protein rich mineralisation =
Invivo
Periodontal ligament stem cellsbull Highly fibrous and vascularhellip High turn over
rates
- Cementum like mineralized structure formed in vitrohellip
Progenitor cells hellipidentified in vivo cell kinetic studieshellip
bull Enriched locations like around the blood vessels
bull Exhibit stem cell features
ndash Small size
ndash Responsiveness to stimulating factor
ndash Slow cycle time
Most compelling evidence McCulloch and coworkers
1985198719951996
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Diagrammatic representation of arteriovenous
shunt loop model in the rat ndash Mian R et al 2000
Artery
Venous graft
Hole of insertionof vessel loop
Vein
Transparent chamber
Femoral vessels Leg
Two main criteria for successful tissue engineering
Biomechanical properties
bull Scaffold
bull Architectural geometry
bull Space-maintaining properties
Biological functions
bull Cell recruitment proliferation survival in culture and at the site of implantation
bull Neovascularization
bull Delivery of morphogenetic- regulatory- and growth factors
Stem Cells
Primal undifferentiated cells
that retain the ability to
produce an identical copy of
themselves when they divide
(clone) and differentiate into
other cell types
Potency
The potency specifies the differentiation potential of the
stem cell
bull Totipotent stem cells
bull Pluripotent stem cells
bull Multipotent stem cells
bull Unipotent stem cells
Key events in stem cell researchbull 1960s - Joseph Altman and Gopal Das presented
evidence of adult neurogenesis ongoing stem cell activity in the brain
their reports contradict Cajals no new neurons dogma are largely
ignored
bull 1963 - McCulloch and Till illustrate the presence of self-renewing stem
cells in mouse bone marrow
bull 1968 - bone marrow transplant between two siblings successfully treats
SCID
bull 1978 - haematopoietic stem cells are discovered in human cord blood
bull 1981 - mouse embryonic stem cells are derived from the inner cell mass
bull 1992 - neural stem cells are cultured in vitro as neurospheres
bull 1995 - President Bill Clinton signs into law the Dickey Amendment which
makes it illegal for Federal money to be used for research where stem cells
are derived from the destruction of the embryo
Key events in stem cell research
bull 1997 - Leukemia was shown to originate from a
haematopoietic stem cell the first direct evidence for cancer stem cells
bull 1998 - James Thomson and coworkers derive the first human embryonic
stem cell line at the University of Wisconsin-Madison
bull 2000s - several reports of adult stem cell plasticity are published
bull 2003 - Dr Songtao Shi of NIH discovers new source of adult stem cells in
childrens primary teeth
bull 2004-2005 - Hwang Woo-Suk claims to have created several human
embryonic stem cell lines from unfertilised human oocytes The lines are
later shown to be fabricated
bull July 19 2006 - President George W Bush votes a bill which would have
allowed Federal money to be used for research where stem cells are
derived from the destruction of the embryo
Types of Stem cells
Adult stem
cellshellip
Embryonic stem cells
in vitro
ADVANTAGES
The ability to examine the material as it is formed and to
perform specific measurements prior
to implantation
DISADVANTAGES
- The absence of a physiologic amp mechanical environment during the
formation of tissue in vitro
- Union of the implanted tissues with the host organ
requires remodeling ndashdegradation and new tissue formation at the interface of implant with the host
tissue
in vivoADVANTAGES
Tissue formation takes place under the influence of physiologic mechanical
environment
Incorporation of the tissues being formed with
the surrounding structures
DISADVANTAGES
Regenerating tissues may be dislodged or degraded by the mechanical forces
normally acting at the site before the regenerating
tissue is fully formed and incorporated
I) Hematopoietic stem cells
2) Bone marrow -
stem cells
-Friedenstein et al 1976-All cell lineages - Mesenchymal fibroblasts cells isolated- Specific surface markers
Locally derived uncommitted cells
bull Periodontal ligament stem cells
bull Dental pulp stem cells
- Cell surface markers ( Gronthos amp co workers 2005)
The dental pulp stem cells represent a clonogenic and highly proliferative cell
population
- Densely calcified nodules = in vitro
- Dentin like and dentin sialo-phospho- protein rich mineralisation =
Invivo
Periodontal ligament stem cellsbull Highly fibrous and vascularhellip High turn over
rates
- Cementum like mineralized structure formed in vitrohellip
Progenitor cells hellipidentified in vivo cell kinetic studieshellip
bull Enriched locations like around the blood vessels
bull Exhibit stem cell features
ndash Small size
ndash Responsiveness to stimulating factor
ndash Slow cycle time
Most compelling evidence McCulloch and coworkers
1985198719951996
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Two main criteria for successful tissue engineering
Biomechanical properties
bull Scaffold
bull Architectural geometry
bull Space-maintaining properties
Biological functions
bull Cell recruitment proliferation survival in culture and at the site of implantation
bull Neovascularization
bull Delivery of morphogenetic- regulatory- and growth factors
Stem Cells
Primal undifferentiated cells
that retain the ability to
produce an identical copy of
themselves when they divide
(clone) and differentiate into
other cell types
Potency
The potency specifies the differentiation potential of the
stem cell
bull Totipotent stem cells
bull Pluripotent stem cells
bull Multipotent stem cells
bull Unipotent stem cells
Key events in stem cell researchbull 1960s - Joseph Altman and Gopal Das presented
evidence of adult neurogenesis ongoing stem cell activity in the brain
their reports contradict Cajals no new neurons dogma are largely
ignored
bull 1963 - McCulloch and Till illustrate the presence of self-renewing stem
cells in mouse bone marrow
bull 1968 - bone marrow transplant between two siblings successfully treats
SCID
bull 1978 - haematopoietic stem cells are discovered in human cord blood
bull 1981 - mouse embryonic stem cells are derived from the inner cell mass
bull 1992 - neural stem cells are cultured in vitro as neurospheres
bull 1995 - President Bill Clinton signs into law the Dickey Amendment which
makes it illegal for Federal money to be used for research where stem cells
are derived from the destruction of the embryo
Key events in stem cell research
bull 1997 - Leukemia was shown to originate from a
haematopoietic stem cell the first direct evidence for cancer stem cells
bull 1998 - James Thomson and coworkers derive the first human embryonic
stem cell line at the University of Wisconsin-Madison
bull 2000s - several reports of adult stem cell plasticity are published
bull 2003 - Dr Songtao Shi of NIH discovers new source of adult stem cells in
childrens primary teeth
bull 2004-2005 - Hwang Woo-Suk claims to have created several human
embryonic stem cell lines from unfertilised human oocytes The lines are
later shown to be fabricated
bull July 19 2006 - President George W Bush votes a bill which would have
allowed Federal money to be used for research where stem cells are
derived from the destruction of the embryo
Types of Stem cells
Adult stem
cellshellip
Embryonic stem cells
in vitro
ADVANTAGES
The ability to examine the material as it is formed and to
perform specific measurements prior
to implantation
DISADVANTAGES
- The absence of a physiologic amp mechanical environment during the
formation of tissue in vitro
- Union of the implanted tissues with the host organ
requires remodeling ndashdegradation and new tissue formation at the interface of implant with the host
tissue
in vivoADVANTAGES
Tissue formation takes place under the influence of physiologic mechanical
environment
Incorporation of the tissues being formed with
the surrounding structures
DISADVANTAGES
Regenerating tissues may be dislodged or degraded by the mechanical forces
normally acting at the site before the regenerating
tissue is fully formed and incorporated
I) Hematopoietic stem cells
2) Bone marrow -
stem cells
-Friedenstein et al 1976-All cell lineages - Mesenchymal fibroblasts cells isolated- Specific surface markers
Locally derived uncommitted cells
bull Periodontal ligament stem cells
bull Dental pulp stem cells
- Cell surface markers ( Gronthos amp co workers 2005)
The dental pulp stem cells represent a clonogenic and highly proliferative cell
population
- Densely calcified nodules = in vitro
- Dentin like and dentin sialo-phospho- protein rich mineralisation =
Invivo
Periodontal ligament stem cellsbull Highly fibrous and vascularhellip High turn over
rates
- Cementum like mineralized structure formed in vitrohellip
Progenitor cells hellipidentified in vivo cell kinetic studieshellip
bull Enriched locations like around the blood vessels
bull Exhibit stem cell features
ndash Small size
ndash Responsiveness to stimulating factor
ndash Slow cycle time
Most compelling evidence McCulloch and coworkers
1985198719951996
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Stem Cells
Primal undifferentiated cells
that retain the ability to
produce an identical copy of
themselves when they divide
(clone) and differentiate into
other cell types
Potency
The potency specifies the differentiation potential of the
stem cell
bull Totipotent stem cells
bull Pluripotent stem cells
bull Multipotent stem cells
bull Unipotent stem cells
Key events in stem cell researchbull 1960s - Joseph Altman and Gopal Das presented
evidence of adult neurogenesis ongoing stem cell activity in the brain
their reports contradict Cajals no new neurons dogma are largely
ignored
bull 1963 - McCulloch and Till illustrate the presence of self-renewing stem
cells in mouse bone marrow
bull 1968 - bone marrow transplant between two siblings successfully treats
SCID
bull 1978 - haematopoietic stem cells are discovered in human cord blood
bull 1981 - mouse embryonic stem cells are derived from the inner cell mass
bull 1992 - neural stem cells are cultured in vitro as neurospheres
bull 1995 - President Bill Clinton signs into law the Dickey Amendment which
makes it illegal for Federal money to be used for research where stem cells
are derived from the destruction of the embryo
Key events in stem cell research
bull 1997 - Leukemia was shown to originate from a
haematopoietic stem cell the first direct evidence for cancer stem cells
bull 1998 - James Thomson and coworkers derive the first human embryonic
stem cell line at the University of Wisconsin-Madison
bull 2000s - several reports of adult stem cell plasticity are published
bull 2003 - Dr Songtao Shi of NIH discovers new source of adult stem cells in
childrens primary teeth
bull 2004-2005 - Hwang Woo-Suk claims to have created several human
embryonic stem cell lines from unfertilised human oocytes The lines are
later shown to be fabricated
bull July 19 2006 - President George W Bush votes a bill which would have
allowed Federal money to be used for research where stem cells are
derived from the destruction of the embryo
Types of Stem cells
Adult stem
cellshellip
Embryonic stem cells
in vitro
ADVANTAGES
The ability to examine the material as it is formed and to
perform specific measurements prior
to implantation
DISADVANTAGES
- The absence of a physiologic amp mechanical environment during the
formation of tissue in vitro
- Union of the implanted tissues with the host organ
requires remodeling ndashdegradation and new tissue formation at the interface of implant with the host
tissue
in vivoADVANTAGES
Tissue formation takes place under the influence of physiologic mechanical
environment
Incorporation of the tissues being formed with
the surrounding structures
DISADVANTAGES
Regenerating tissues may be dislodged or degraded by the mechanical forces
normally acting at the site before the regenerating
tissue is fully formed and incorporated
I) Hematopoietic stem cells
2) Bone marrow -
stem cells
-Friedenstein et al 1976-All cell lineages - Mesenchymal fibroblasts cells isolated- Specific surface markers
Locally derived uncommitted cells
bull Periodontal ligament stem cells
bull Dental pulp stem cells
- Cell surface markers ( Gronthos amp co workers 2005)
The dental pulp stem cells represent a clonogenic and highly proliferative cell
population
- Densely calcified nodules = in vitro
- Dentin like and dentin sialo-phospho- protein rich mineralisation =
Invivo
Periodontal ligament stem cellsbull Highly fibrous and vascularhellip High turn over
rates
- Cementum like mineralized structure formed in vitrohellip
Progenitor cells hellipidentified in vivo cell kinetic studieshellip
bull Enriched locations like around the blood vessels
bull Exhibit stem cell features
ndash Small size
ndash Responsiveness to stimulating factor
ndash Slow cycle time
Most compelling evidence McCulloch and coworkers
1985198719951996
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Potency
The potency specifies the differentiation potential of the
stem cell
bull Totipotent stem cells
bull Pluripotent stem cells
bull Multipotent stem cells
bull Unipotent stem cells
Key events in stem cell researchbull 1960s - Joseph Altman and Gopal Das presented
evidence of adult neurogenesis ongoing stem cell activity in the brain
their reports contradict Cajals no new neurons dogma are largely
ignored
bull 1963 - McCulloch and Till illustrate the presence of self-renewing stem
cells in mouse bone marrow
bull 1968 - bone marrow transplant between two siblings successfully treats
SCID
bull 1978 - haematopoietic stem cells are discovered in human cord blood
bull 1981 - mouse embryonic stem cells are derived from the inner cell mass
bull 1992 - neural stem cells are cultured in vitro as neurospheres
bull 1995 - President Bill Clinton signs into law the Dickey Amendment which
makes it illegal for Federal money to be used for research where stem cells
are derived from the destruction of the embryo
Key events in stem cell research
bull 1997 - Leukemia was shown to originate from a
haematopoietic stem cell the first direct evidence for cancer stem cells
bull 1998 - James Thomson and coworkers derive the first human embryonic
stem cell line at the University of Wisconsin-Madison
bull 2000s - several reports of adult stem cell plasticity are published
bull 2003 - Dr Songtao Shi of NIH discovers new source of adult stem cells in
childrens primary teeth
bull 2004-2005 - Hwang Woo-Suk claims to have created several human
embryonic stem cell lines from unfertilised human oocytes The lines are
later shown to be fabricated
bull July 19 2006 - President George W Bush votes a bill which would have
allowed Federal money to be used for research where stem cells are
derived from the destruction of the embryo
Types of Stem cells
Adult stem
cellshellip
Embryonic stem cells
in vitro
ADVANTAGES
The ability to examine the material as it is formed and to
perform specific measurements prior
to implantation
DISADVANTAGES
- The absence of a physiologic amp mechanical environment during the
formation of tissue in vitro
- Union of the implanted tissues with the host organ
requires remodeling ndashdegradation and new tissue formation at the interface of implant with the host
tissue
in vivoADVANTAGES
Tissue formation takes place under the influence of physiologic mechanical
environment
Incorporation of the tissues being formed with
the surrounding structures
DISADVANTAGES
Regenerating tissues may be dislodged or degraded by the mechanical forces
normally acting at the site before the regenerating
tissue is fully formed and incorporated
I) Hematopoietic stem cells
2) Bone marrow -
stem cells
-Friedenstein et al 1976-All cell lineages - Mesenchymal fibroblasts cells isolated- Specific surface markers
Locally derived uncommitted cells
bull Periodontal ligament stem cells
bull Dental pulp stem cells
- Cell surface markers ( Gronthos amp co workers 2005)
The dental pulp stem cells represent a clonogenic and highly proliferative cell
population
- Densely calcified nodules = in vitro
- Dentin like and dentin sialo-phospho- protein rich mineralisation =
Invivo
Periodontal ligament stem cellsbull Highly fibrous and vascularhellip High turn over
rates
- Cementum like mineralized structure formed in vitrohellip
Progenitor cells hellipidentified in vivo cell kinetic studieshellip
bull Enriched locations like around the blood vessels
bull Exhibit stem cell features
ndash Small size
ndash Responsiveness to stimulating factor
ndash Slow cycle time
Most compelling evidence McCulloch and coworkers
1985198719951996
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Key events in stem cell researchbull 1960s - Joseph Altman and Gopal Das presented
evidence of adult neurogenesis ongoing stem cell activity in the brain
their reports contradict Cajals no new neurons dogma are largely
ignored
bull 1963 - McCulloch and Till illustrate the presence of self-renewing stem
cells in mouse bone marrow
bull 1968 - bone marrow transplant between two siblings successfully treats
SCID
bull 1978 - haematopoietic stem cells are discovered in human cord blood
bull 1981 - mouse embryonic stem cells are derived from the inner cell mass
bull 1992 - neural stem cells are cultured in vitro as neurospheres
bull 1995 - President Bill Clinton signs into law the Dickey Amendment which
makes it illegal for Federal money to be used for research where stem cells
are derived from the destruction of the embryo
Key events in stem cell research
bull 1997 - Leukemia was shown to originate from a
haematopoietic stem cell the first direct evidence for cancer stem cells
bull 1998 - James Thomson and coworkers derive the first human embryonic
stem cell line at the University of Wisconsin-Madison
bull 2000s - several reports of adult stem cell plasticity are published
bull 2003 - Dr Songtao Shi of NIH discovers new source of adult stem cells in
childrens primary teeth
bull 2004-2005 - Hwang Woo-Suk claims to have created several human
embryonic stem cell lines from unfertilised human oocytes The lines are
later shown to be fabricated
bull July 19 2006 - President George W Bush votes a bill which would have
allowed Federal money to be used for research where stem cells are
derived from the destruction of the embryo
Types of Stem cells
Adult stem
cellshellip
Embryonic stem cells
in vitro
ADVANTAGES
The ability to examine the material as it is formed and to
perform specific measurements prior
to implantation
DISADVANTAGES
- The absence of a physiologic amp mechanical environment during the
formation of tissue in vitro
- Union of the implanted tissues with the host organ
requires remodeling ndashdegradation and new tissue formation at the interface of implant with the host
tissue
in vivoADVANTAGES
Tissue formation takes place under the influence of physiologic mechanical
environment
Incorporation of the tissues being formed with
the surrounding structures
DISADVANTAGES
Regenerating tissues may be dislodged or degraded by the mechanical forces
normally acting at the site before the regenerating
tissue is fully formed and incorporated
I) Hematopoietic stem cells
2) Bone marrow -
stem cells
-Friedenstein et al 1976-All cell lineages - Mesenchymal fibroblasts cells isolated- Specific surface markers
Locally derived uncommitted cells
bull Periodontal ligament stem cells
bull Dental pulp stem cells
- Cell surface markers ( Gronthos amp co workers 2005)
The dental pulp stem cells represent a clonogenic and highly proliferative cell
population
- Densely calcified nodules = in vitro
- Dentin like and dentin sialo-phospho- protein rich mineralisation =
Invivo
Periodontal ligament stem cellsbull Highly fibrous and vascularhellip High turn over
rates
- Cementum like mineralized structure formed in vitrohellip
Progenitor cells hellipidentified in vivo cell kinetic studieshellip
bull Enriched locations like around the blood vessels
bull Exhibit stem cell features
ndash Small size
ndash Responsiveness to stimulating factor
ndash Slow cycle time
Most compelling evidence McCulloch and coworkers
1985198719951996
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Key events in stem cell research
bull 1997 - Leukemia was shown to originate from a
haematopoietic stem cell the first direct evidence for cancer stem cells
bull 1998 - James Thomson and coworkers derive the first human embryonic
stem cell line at the University of Wisconsin-Madison
bull 2000s - several reports of adult stem cell plasticity are published
bull 2003 - Dr Songtao Shi of NIH discovers new source of adult stem cells in
childrens primary teeth
bull 2004-2005 - Hwang Woo-Suk claims to have created several human
embryonic stem cell lines from unfertilised human oocytes The lines are
later shown to be fabricated
bull July 19 2006 - President George W Bush votes a bill which would have
allowed Federal money to be used for research where stem cells are
derived from the destruction of the embryo
Types of Stem cells
Adult stem
cellshellip
Embryonic stem cells
in vitro
ADVANTAGES
The ability to examine the material as it is formed and to
perform specific measurements prior
to implantation
DISADVANTAGES
- The absence of a physiologic amp mechanical environment during the
formation of tissue in vitro
- Union of the implanted tissues with the host organ
requires remodeling ndashdegradation and new tissue formation at the interface of implant with the host
tissue
in vivoADVANTAGES
Tissue formation takes place under the influence of physiologic mechanical
environment
Incorporation of the tissues being formed with
the surrounding structures
DISADVANTAGES
Regenerating tissues may be dislodged or degraded by the mechanical forces
normally acting at the site before the regenerating
tissue is fully formed and incorporated
I) Hematopoietic stem cells
2) Bone marrow -
stem cells
-Friedenstein et al 1976-All cell lineages - Mesenchymal fibroblasts cells isolated- Specific surface markers
Locally derived uncommitted cells
bull Periodontal ligament stem cells
bull Dental pulp stem cells
- Cell surface markers ( Gronthos amp co workers 2005)
The dental pulp stem cells represent a clonogenic and highly proliferative cell
population
- Densely calcified nodules = in vitro
- Dentin like and dentin sialo-phospho- protein rich mineralisation =
Invivo
Periodontal ligament stem cellsbull Highly fibrous and vascularhellip High turn over
rates
- Cementum like mineralized structure formed in vitrohellip
Progenitor cells hellipidentified in vivo cell kinetic studieshellip
bull Enriched locations like around the blood vessels
bull Exhibit stem cell features
ndash Small size
ndash Responsiveness to stimulating factor
ndash Slow cycle time
Most compelling evidence McCulloch and coworkers
1985198719951996
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Types of Stem cells
Adult stem
cellshellip
Embryonic stem cells
in vitro
ADVANTAGES
The ability to examine the material as it is formed and to
perform specific measurements prior
to implantation
DISADVANTAGES
- The absence of a physiologic amp mechanical environment during the
formation of tissue in vitro
- Union of the implanted tissues with the host organ
requires remodeling ndashdegradation and new tissue formation at the interface of implant with the host
tissue
in vivoADVANTAGES
Tissue formation takes place under the influence of physiologic mechanical
environment
Incorporation of the tissues being formed with
the surrounding structures
DISADVANTAGES
Regenerating tissues may be dislodged or degraded by the mechanical forces
normally acting at the site before the regenerating
tissue is fully formed and incorporated
I) Hematopoietic stem cells
2) Bone marrow -
stem cells
-Friedenstein et al 1976-All cell lineages - Mesenchymal fibroblasts cells isolated- Specific surface markers
Locally derived uncommitted cells
bull Periodontal ligament stem cells
bull Dental pulp stem cells
- Cell surface markers ( Gronthos amp co workers 2005)
The dental pulp stem cells represent a clonogenic and highly proliferative cell
population
- Densely calcified nodules = in vitro
- Dentin like and dentin sialo-phospho- protein rich mineralisation =
Invivo
Periodontal ligament stem cellsbull Highly fibrous and vascularhellip High turn over
rates
- Cementum like mineralized structure formed in vitrohellip
Progenitor cells hellipidentified in vivo cell kinetic studieshellip
bull Enriched locations like around the blood vessels
bull Exhibit stem cell features
ndash Small size
ndash Responsiveness to stimulating factor
ndash Slow cycle time
Most compelling evidence McCulloch and coworkers
1985198719951996
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
in vitro
ADVANTAGES
The ability to examine the material as it is formed and to
perform specific measurements prior
to implantation
DISADVANTAGES
- The absence of a physiologic amp mechanical environment during the
formation of tissue in vitro
- Union of the implanted tissues with the host organ
requires remodeling ndashdegradation and new tissue formation at the interface of implant with the host
tissue
in vivoADVANTAGES
Tissue formation takes place under the influence of physiologic mechanical
environment
Incorporation of the tissues being formed with
the surrounding structures
DISADVANTAGES
Regenerating tissues may be dislodged or degraded by the mechanical forces
normally acting at the site before the regenerating
tissue is fully formed and incorporated
I) Hematopoietic stem cells
2) Bone marrow -
stem cells
-Friedenstein et al 1976-All cell lineages - Mesenchymal fibroblasts cells isolated- Specific surface markers
Locally derived uncommitted cells
bull Periodontal ligament stem cells
bull Dental pulp stem cells
- Cell surface markers ( Gronthos amp co workers 2005)
The dental pulp stem cells represent a clonogenic and highly proliferative cell
population
- Densely calcified nodules = in vitro
- Dentin like and dentin sialo-phospho- protein rich mineralisation =
Invivo
Periodontal ligament stem cellsbull Highly fibrous and vascularhellip High turn over
rates
- Cementum like mineralized structure formed in vitrohellip
Progenitor cells hellipidentified in vivo cell kinetic studieshellip
bull Enriched locations like around the blood vessels
bull Exhibit stem cell features
ndash Small size
ndash Responsiveness to stimulating factor
ndash Slow cycle time
Most compelling evidence McCulloch and coworkers
1985198719951996
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
in vivoADVANTAGES
Tissue formation takes place under the influence of physiologic mechanical
environment
Incorporation of the tissues being formed with
the surrounding structures
DISADVANTAGES
Regenerating tissues may be dislodged or degraded by the mechanical forces
normally acting at the site before the regenerating
tissue is fully formed and incorporated
I) Hematopoietic stem cells
2) Bone marrow -
stem cells
-Friedenstein et al 1976-All cell lineages - Mesenchymal fibroblasts cells isolated- Specific surface markers
Locally derived uncommitted cells
bull Periodontal ligament stem cells
bull Dental pulp stem cells
- Cell surface markers ( Gronthos amp co workers 2005)
The dental pulp stem cells represent a clonogenic and highly proliferative cell
population
- Densely calcified nodules = in vitro
- Dentin like and dentin sialo-phospho- protein rich mineralisation =
Invivo
Periodontal ligament stem cellsbull Highly fibrous and vascularhellip High turn over
rates
- Cementum like mineralized structure formed in vitrohellip
Progenitor cells hellipidentified in vivo cell kinetic studieshellip
bull Enriched locations like around the blood vessels
bull Exhibit stem cell features
ndash Small size
ndash Responsiveness to stimulating factor
ndash Slow cycle time
Most compelling evidence McCulloch and coworkers
1985198719951996
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
I) Hematopoietic stem cells
2) Bone marrow -
stem cells
-Friedenstein et al 1976-All cell lineages - Mesenchymal fibroblasts cells isolated- Specific surface markers
Locally derived uncommitted cells
bull Periodontal ligament stem cells
bull Dental pulp stem cells
- Cell surface markers ( Gronthos amp co workers 2005)
The dental pulp stem cells represent a clonogenic and highly proliferative cell
population
- Densely calcified nodules = in vitro
- Dentin like and dentin sialo-phospho- protein rich mineralisation =
Invivo
Periodontal ligament stem cellsbull Highly fibrous and vascularhellip High turn over
rates
- Cementum like mineralized structure formed in vitrohellip
Progenitor cells hellipidentified in vivo cell kinetic studieshellip
bull Enriched locations like around the blood vessels
bull Exhibit stem cell features
ndash Small size
ndash Responsiveness to stimulating factor
ndash Slow cycle time
Most compelling evidence McCulloch and coworkers
1985198719951996
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Locally derived uncommitted cells
bull Periodontal ligament stem cells
bull Dental pulp stem cells
- Cell surface markers ( Gronthos amp co workers 2005)
The dental pulp stem cells represent a clonogenic and highly proliferative cell
population
- Densely calcified nodules = in vitro
- Dentin like and dentin sialo-phospho- protein rich mineralisation =
Invivo
Periodontal ligament stem cellsbull Highly fibrous and vascularhellip High turn over
rates
- Cementum like mineralized structure formed in vitrohellip
Progenitor cells hellipidentified in vivo cell kinetic studieshellip
bull Enriched locations like around the blood vessels
bull Exhibit stem cell features
ndash Small size
ndash Responsiveness to stimulating factor
ndash Slow cycle time
Most compelling evidence McCulloch and coworkers
1985198719951996
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Periodontal ligament stem cellsbull Highly fibrous and vascularhellip High turn over
rates
- Cementum like mineralized structure formed in vitrohellip
Progenitor cells hellipidentified in vivo cell kinetic studieshellip
bull Enriched locations like around the blood vessels
bull Exhibit stem cell features
ndash Small size
ndash Responsiveness to stimulating factor
ndash Slow cycle time
Most compelling evidence McCulloch and coworkers
1985198719951996
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
bull Fibroblastic colony forming units hellipgreater in PDL stem cell (170) as against the bone marrow stromal cell (14)
ndash Propensity hellipor hellipdifference in turn over rate
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Soe et al 2004 Isolated a population of multipotent stem cells in human PDL derived mesenchymal
stromal cells
Liu et al 2008Autologous periodontal ligament derived
mesenchymal stromal cells promoted healing of experimental periodontitis in
mini-pigs
Studies
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Growth potential of pdl stem cells
bull Differential proliferative capacityhellip- 80 Failed beyond 20 population doublings
bull Pdl Stem cells Versus the Bone marrow stemcells - 30 higher osteogenic potential ( Yu BH et al 2014)- BMSCs owned the stronger immunomodulation inlocal microenvironment via anti-inflammatoryfunctions compared to PDLSCs (Zhang J et al 2014)
bull Finite lifespan of Pdl stem cellshellipndash Postnatal stem cells (Adult stem cells)ndash Embryonic stem cellhellipvirtually immortal
bull Genetic manipulation can be done with caution
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Characterization and origin of Periodontal
stem cellsbull Differentiated from Dental Follicle during embryogenesishellip
bull Putative cell marker STRO-1 Common
ndash Isolation using immunomagnetic hellipfluorescence activatedcell selection
bull Share common expression of cell marker CD146
bull McCulloch et alhelliphellippresence of perivascular progenitor cells inPdl
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
ScaffoldsMatrices
bull The extracellular matrix consists ofproteins and glycoproteins such ascollagens laminins fibronectin andproteoglycans and also thepolysaccharide hyaluronic acid
Composition organisation amp distribution of extracellular matrix
Simple mixing
Cell isolation amp expansion
Cell on matrix
New tissue
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Provides
bull Hydration for cells
bull Elastic network
bull Cell attachment proliferation amp differentiation
bull Store amp protect Growth factors from degradation
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Interactions between ECM amp cell surafce recptors
Embryonic morphogenesis
Cell surface receptor
Feedbackto ECM
Gene expression
ECM
Receptor ligandbinding
Receptor GF binding
Presenting
GF Cytokines
Storage PoolModulation of ECM
Intracellular
Dynamic reciprocity
ECM
Cytoskeleton
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Area code hypothesisbull Hood L et al 1977
bull The presence of a recognition system that guides cell positioning
ECM proteins
bull RGD domains in fibronectin vitronectin YIGSR domain - laminin and GER domain ndashtype I collagen
Cell surface receptors
bull Integrins syndecan CD44 thrombomodulin lamininbinding protein CD36 and matrikines
Cell adhesion
MMPs ndash
breakdown
and
remodellin
g of ECM
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Ideal properties
bull Biocompatibility (no immunogenicity)
bull Space maintenance
bull Mechanical rigidity
bull Degradability-in a phased manner
bull Mechanical structure-three dimensional structure with sufficient porosity surface roughnessgreatersurface energyand availability of surface molecules
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Natural
bull Collagen hyaluronan chitosan Gelatin fibrin alginate biocover amp Matrix extracts like Matrigel
bull Biodegradable and non-toxic
bull Possess known cell binding sites
bull Disadvantages ndash Immunogenicity speed of degradation and limited ability to tailor certain specific properties
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Synthetic bull Poly(glycolic acid) poly(lactic acid) or
poly(lacticacid)poly(glycolic acid) and their copolymers poly(p-dioxanone) and copolymers trimethylene carbonate
bull Hydroxyapatite calcium sulfate and tricalcium phosphate - Osteoconductive
bull Bioactive glass ndash silicon network structure
bull Can modify the strength pore size and stability
bull Low pH hinder the cell growth
Bioactive glass
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Collagen sponge scaffold +gelatin microspheres loadedwith FGF 2 in alveolar bonedefects - Nakahara et al 2004Vascularization osteogenesisand recovery of periodontalligament function after 4 weeks
Collagen sponges + periodontalligament cells = cementum haduniformly regenerated along theroot surface of bony defects indog teeth
Sculean et al 2003 reported that theapplication of bovine derived xenograft andbioresorbable collagen in patients withperiodontal defects resulted in significantimprovement with reduction of periodontalprobing depth and clinical attachment levelsobserved 1 year post-treatment
Studies
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Tissue engineered scaffolds in periodontal therapy
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Physical forces determines longterm composition
quality volume of the tissue construct
- Mechanical forces as regulators of tissue
growth stem cell lineage commitment and
differentiation polarity motility contractility amp
apoptosis
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Functional engineering
- The aim of improving understanding of the role that mechanical
factors play in tissue regeneration
- Mechanical signals as key regulators of the cell behaviors required
for successful engineered tissue growth
- Bioreactors are already being developed that are able to
apply lineage-specific mechanical signals to populations of
stem cells
bull Rigid substrate ndash Support high level isometric tension
bull Flexible substrate ndash Cannot resist forces
- Turn off growth amp differentiation
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Competence factors (PDGF FGF)
Progression factors(IGF-I
Dexamethazone)
JE = 1-6 days
Osteoblast lineage = 20-30 daysBiosignals
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
BMPrsquos
PDGF
BMPrsquos IGF IIITGFβ
IGF IIITGFβ
Cell differentiation
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Transforming Growth factors
bull Major growth factors in bone matrix
bull Superfamily of bone morphogenic proteins
bull A peptide synthesized amp secreted in cell culture
bull Increases the pool of committed osteoblasts
bull Prevents long junctional epithelium formation
bull Increases osteoblasts chemotaxis
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Contrella M et al 1987
TGF -β
Smadsignalling pathway
Migration of Osteogenicprogenitor cells
Proliferate Increases osteoblastspool
Depends on dose amp local environment
At high concentrations of TGF -β
Inhibits DNA synthesis
Inhibits osteoblasts
Maeda et al 2004
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Miyazona K et al 2005
Cell
BMPs + BMPR1BMPR2
Increases cbfX1 gene exp
Increases MSX gene exp
Epithelial amp mesenchymalinteraction
Increases gene exp of ALP osteocalcin
Matrix formation amp mineralization
Bone morphogenic proteins
bull20 members Eg ndash BMP 247
bullBMP-2 differentiation factor
for bone amp cartilage precursor
cells during osteogenesis and
bone regeneration
bull BMP 5 through ActRIAinhibits matrix synthesis
bull Induce ectopic bone formation
bullDrive endochondral ossification
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
BMPs acts on the pluripotent cells
increases the committment amp
differentiation of osteoblasts
- Katagiri et al 1990
BMPs shown formation of bone nodules invitro
- Chen TL et al 1991
Gene expression studies
Adenovirus vector + BMP-7
Robust osteogenicresponse
Franceschi et al 2000
Limitations- No pdl fibre formation in perpendicular direction and no acellularcementum formation- No role in osteoblast induction
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Insulin like growth factors I amp II
bull Synthesized primarily in liver and locally by osteoblasts
bull Mediate effect of systemic hormones (Eg GH) cytokines
(IL-1α) amp morphogens (BMPs) in bone formation amp healing
bull Increases proliferation effects of osteoblasts
bull Local regulator of bone turnover
bull IGF -I more potent
bull IGF ndashI upregulates the osteoblast associated transcription
factor OSTERIX but not cbfx1 amp Runx2
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
IGF + PDGF increases periodontal regeneration
- Lynch SE et al 1989
IGF I + BMP 2 act synergistically on OSTERIX
- Celil AB et al 2003
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Fibroblast Growth Factors
bull Autocrine Paracrine regulators of bone formation
bull Stimulate chemotaxis proliferation amp matrix synthesis of
osteoblasts amp osteoblast precursor
bull Play role in angiogenesis amp mesenchymal Cell mitogenesis
bull Accelerates fractures healing
bull FGF-2 most potent than FGF-1
- Contrella et al 1988
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Bone regeneration
GFs IGF FGF VEGF TGF PDGF
TNF-alpha IL-1beta
Bone regeneration remodelling amp repair
BMPsCytokines GF
Mesenchymal cellsDifferentiation
Proliferation
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Role of vasculature and neovascularization
in tissue engineeringbull Cell survivalhellip
ndash O2 Supply
ndash Nourishment
ndash Disposal of waste products
bull Cells more than approximately 200 μm from a blood
supply are found to be either metabolically inactive or
necrotic
bull Tissue implantation volumes lt 2ndash3 mm3
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Vasculature = 1) vasculogenesis ndash denovo formation
2) angiogenesis ndash mature network
Major angiogenic factors ndash FGF PDGF and VEGF
Blood vesselAngiogenicSproutingVEGFPDGFAng1
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Vascular endothelial growth factor
bull 6 proteins ndash VEGF ABCDE and placental GF
bull Spliced forms like VEGF 121 165189
bull Primary target is endothelial cells
bull Also for recruitment survival and action of osteoblast amp
osteoclast
bull Osteoblasts has VEGF receptors amp also secretes VEGF
bull Mediates other GFs
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Vascular endothelial growth factor
bull Mouse model Fracture healing amp cortical defect model
- Street J et al 2002
bull Major factor coupling osteogenesis + vasculogenesis
- Gerber HP et al 2000
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Platelet derived growth factor
bull 3 isoforms ( AAAB BB) receptors a amp b
bull Chemotactic effect on osteoblast amp Ct cells
- Hughes et al 1991
Increases collagenase transcription amp increases IL-6
expression on osteoblasts
Indirect effect on Bone resorption
- Durant D et al 2000
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Platelet derived growth factor
bull PDGF +βTricalcium phosphate GEM21S
rh PDGF + freeze dried bone allograft
Excellent results in intrabony defect
- Nevins M et al 2007
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Applications
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
bullCell therapy
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Gene therapy
ldquoGene therapy is the insertion of genes into an
individuals cells and tissues to treat a disease and
hereditary diseases in particularrdquo
Typically aims to supplement a defective mutant allele
with a functional one
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
bull Transformation of bull Somatic cells
bull Germ line cells (Sperm Ova amp stem Cells)
ex vivo
(where cells are modified outside
the body and then transplanted
back in again)
in vivo
(where genes are
changed in cells still in
the body)
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
In Periodontics
A field of biomedicine
bull Not applied with success in periodontics
ndash Gene therapy Technology hellip far from Perfect
ndash Periodontal disease hellip Multifactorialhellip
ndash Genetic variations bull multiple genes
bull interactions between genes and the environment
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Gene enhanced TE
Delivery of Therapeutic protein-like growth factor
-short life (a few hours)
bull This is due to proteolytic breakdown and receptor mediated
exocytosis and solubility of the delivery vehicle
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Studies in periodontal tissue engineering
bull Sankaranarayanan S et al 2013 in a 23-year female patientwith advanced periodontitis correlated with radiographicevidence of severe horizontal bone loss extending up to theapex of mandibular incisors after debridement the defectwas implanted with Autologous Bone Marrow MononuclearCells impregnated in Thermo responsive Gelatin Polymer In6mo PPD reduced to 2mm from 6mm amp CAL 7mm from13mm At 36mo radiographic evidence in improvement ofvertical n horizontal height
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull 7 case reports (for a total of 22 patients) where MSCs had beenapplied clinically for infra-bony defects and furcation involvementafter chronic periodontitis in humans
bull The efficacy of human periodontal cell therapy by graftingautologous stromal cells from gingiva or periodontal ligamentwith a hydroxyapatite (HA) carrier has been assessed since 1992(Feng and Hou 1992 Feng et al 1995 2010 Hou et al 2003)
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Studies in periodontal tissue engineering ndash
Reviewed by Elena A Trofin et al 2013
bull Cultured mandibular periosteum-derived cell sheets withPRP have also been used in the treatment of chronicperiodontitis without (Mizuno et al 2010) or with HA inpatients suffering from advanced chronic periodontitis(Okuda et al 2009)
bull A BMSC-PRP gel has also been used in an infrabonyperiodontal defect and led to a 4 mm clinical attachmentgain (Yamada et al 2006)
These data suggest MSCs may induce efficient and
safe periodontal regeneration in humans
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Future directions in periodontics
1 Gene Therapeutics-Periodontal Vaccination
2 Genetic Approach to Biofilm Antibiotic
Resistance
3 An In vivo Gene Transfer by Electroporation
for Alveolar Remodeling
4 Tight Adherence Gene for the Control of
Periodontal Disease Progression
5 Antimicrobial Gene Therapy to Control
Disease Progression
6 Gene Therapy to Grow New Teeth
Tooth grown with urinestem cells by a team fromthe Guangzhou Institutesof Biomedicine andHealth-
Cia et al
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Cell sheet engineering ndash Okano et al 1993
bull Temperature responsive culture
dishes
bull At 37 degree celsius adhere amp
proliferate
bull Below 32 degree celsius cells detach
bull Allows non-invasive harvest of
cultured cells as an intact monolayer
cell sheet including deposited ECM
bull Enables direct transplantation
bull 3D constructs such as thick cardiac
muscle
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Cell sheet engineering
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Artificial salivary gland
Baum BJ et al
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Tissue engineering for dental implants ndash
Biomimetic materialsBiomimetic Ca-P coatings
Both the superior mechanical properties of titanium and its alloys and
excellent biocompatibility of Ca-P materials
Techniques
Radiofrequency magnetron sputtering
Pulsed-laser deposition
Ion-beam sputtering
Ion-beam-assisted deposition
Electrophoretic techniques
Biomimetic Technique
Growing a Ca-P thin layer on metals or other implant materials from a
physiologically related supersaturated calcifying solution
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Benefits Of The Biomimetic Approach Over Plasma-
sprayed Hydroxyapatitehellip
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Albumin
Plasma protein and plays a fundamental role
in the transport of other proteins and
functional molecules in the blood
Co-precipitated into the Ca-P coating
KRAGH ET AL 1990
As albumin can bind a wide diversity of
ligands reversibly with high affinity and
albumin microspheres have been used as
drug carriers
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
BMPrsquos
rhBMP-2 incorporated into Ca-P
coatings
Combination of biomimetic Ca-P
coatings and osteoinductive agents
can provide superior inductive
capability
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Bisposphonates
Incorporated into Ca ndash P coatings
bull Osteoclast inhibition
bull Reduced bone turnover
bull Increased bone mass
bull Improved mineralization
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
bull To Master the art of recreating functional viable tissues in
the laboratoryhelliptranslate the knowledge hellipto population
at large
Promise is GREAThellipbut challenges exist
bull Cost efficienthellip
bull Availability
bull Trained staff requirement
bull Ethical issues
Smile if you think science is real
Smile if you think science is real
![Enamel matrix derivative (Emdogain(R)) for periodontal ... · [Intervention Review] Enamel matrix derivative (Emdogain®) for periodontal tissue regeneration in intrabony defects](https://static.fdocuments.in/doc/165x107/5f552cf7423b6b423a7f833d/enamel-matrix-derivative-emdogainr-for-periodontal-intervention-review.jpg)
![Enamel matrix derivative [Emdogain(R)] for …...[Intervention Review] Enamel matrix derivative (Emdogain®) for periodontal tissue regeneration in intrabony defects Marco Esposito1,](https://static.fdocuments.in/doc/165x107/5e7b0259219c7e285a26632d/enamel-matrix-derivative-emdogainr-for-intervention-review-enamel-matrix.jpg)
