SKIN TISSUE ENGINEERING: From the biomaterials development … · 2020. 2. 4. · Totipotency All...
Transcript of SKIN TISSUE ENGINEERING: From the biomaterials development … · 2020. 2. 4. · Totipotency All...
SKIN TISSUE ENGINEERING:
From the biomaterials
development to in vivo tests
SKIN IS A COMPLEX TISSUE…AN ORGAN
http://trekity.com/skin-care-routine/
D. Markeson, et al., J Tissue Eng Regen Med, 2013
MAIN NORMAL SKIN COMPONENTS
Adapted from Kern et al., 2011
ADULT NORMAL WOUND HEALING EVENTS
Adapted from M. T. Cerqueira, R. P. Pirraco, R. L. Reis, A. P. Marques, Advances in Wound Care, 2016, 5(4): 164-175. doi:10.1089/wound.2014.0607
Gurtner, et. al, Wound Repair and Regeneration, Nature 453, 314-321
Repair
Regeneration
Scar
Myofibroblast presence - Contribution to wound contraction
Non-functioning mass of fibrotic tissue
Adaptation of normal skin to tissue damage
No Scar
Restoration of normal structure and function of the injured tissue
Functional tissue
Regenerative potential in Adult mammals is limited
Wound Healing
REGENERATION VS REPAIR
COMPARISON OF FETAL SCARLESS HEALING AND
HYPERTROPHIC SCAR FEATURES
S. Sanon et al., Skin Tissue Engineering and Regenerative Medicine, 2016, Pages 19–47
Worldwide Wound Management
MAIN PROBLEMATIC IN SKIN HOMEOSTASIS
Source: MedMarket Diligence, LLC, Report #S190
R. Harries et al., Skin Tissue Engineering and Regenerative Medicine, 2016, Pages 125–143
PRODUCT
CLINICALLY
AVAILABLE
SINCE
DESCRIPTION INDICATION
Integra Integra Life Sciences 1984 Polysiloxane membrane and complex
matrix (collagen and glycosaminoglycans) Burns
Transcyte Advanced Tissue
Sciences 1997
Silicone membrane and ECM from neonatal human fibroblast cells cultured on
a nylon mesh DFU
Alloderm Life Cell 2001 Decellularized human cadaver dermis Soft Tissue
Repair
PRODUCT
CLINICALLY
AVAILABLE
SINCE
DESCRIPTION INDICATION
Epicel Genzyme Biosurgery 1987 Autologous keratinocytes and xenogenic proliferation arrested
mouse fibroblasts in petroleum gauze dressing Full thickness burns taking >30%
of body area
Epidex Modex Therapeutics 1999 Cultured epidermal skin equivalent derived from keratinocyte
precursors of human hair follicles Chronic Leg ulcers
Dermagraft Advanced Biohealing Inc 1999 Allogeneic neonatal fibroblasts seeded in a polyglycolic acid (Dexon)
or polyglactin-9-10Vicryl scaffold. Full thickness DFU; Epidermolysis
Bullosa
Transcyte Advanced Biohealing Inc 1999 Human allogeneic fibroblasts cultured on a nylon mesh pre-coated
with collagen Burns; Transparent dressing
Laserskin and Hyalograft Fidia Farmaceutical 2000 Autologous fibroblasts and keratinocytes cultured on a hyaluronic
acid laser perforated membrane DFU and Chronic
Wounds
Orcel Ortec International 2001 Type 1 Bovine collagen seeded with allogeneic neonatal fibroblasts
and keratinocytes Donor sites for autografting, DFU;
Epidermolysis Bullosa
Apligraft /Graftskin Organogenesis 2001 Allogeneic neonatal foreskin keratinocytes and fibroblasts seeded in
a type 1 bovine collagen
DFU; Venous leg ulcers; Partial thickness burns; Epidermolysis
Bullosa
Bioseed-S BioTissue Technologies 2001 Autologous keratinocytes resuspended in a fibrin sealant Venous leg ulcers
Myskin Altrika 2008 Autologous keratinocytes grown on a silicone layer with irradiated
murine fibroblasts Non-healing wounds; DFU, Burns,
Pressure ulcers
Permaderm Regenicin
Pre-market approval
Autologous keratinocytes and fibroblasts seeded on collagen biomaterial Burns; Chronic Wounds
AC
EL
LU
LA
R
CE
LL
UL
AR
SKIN SUBSTITUTES AND THE CLINICAL APPLICABILITY
SKIN REGENERATION: THE GOAL TO ATTAIN
M. T. Cerqueira, et al., unpublished data
Totipotency All somatic cell types,
germ cells, Placenta
Fertilized Egg
Pluripotency All somatic Cell Types,
Germ Cells
Blastocyst – Inner Cell Mass
Multipotency Lineage-restricted cell
types
Unipotency
Neural Stem Cell
Mesenchymal Stem Cells
Epidermal Stem Cells
Single Cell Type
Eckfeldt et al, Nat Rev Molecular Cell Biology, doi:10.1038/nrm1713
TYPES OF STEM CELLS
PROMISE OF STEM CELLS AS THERAPY FOR SKIN REGENERATION
M. T. Cerqueira, R. P. Pirraco, R. L. Reis, A. P. Marques, Advances in Wound Care, 2016, 5(4): 164-175. doi:10.1089/wound.2014.0607
M. T. Cerqueira, A. P. Marques, R. L. Reis, Using Stem Cells in Skin Regeneration: Possibilities and Reality. Stem Cells and Development, 21 (2012) 1201-1214, doi: 10.1089/scd.2011.0539
BUILDING BLOCKS : DIFFERENTIATION OF STEM CELLS IN
EPIDERMAL LINEAGE
STEM CELLS AS SECRETOME UNITS DURING HEALING
M. T. Cerqueira, R. P. Pirraco, R. L. Reis, A. P. Marques, Advances in Wound Care, 2016, 5(4): 164-175. doi:10.1089/wound.2014.0607
POSSIBLE STRATEGIES TO MAXIMIZE STEM CELLS EFFECT
M. T. Cerqueira, R. P. Pirraco, R. L. Reis, A. P. Marques, Advances in Wound Care, 2016, 5(4): 164-175. doi:10.1089/wound.2014.0607
SKIN EXTRACELLULAR-MATRIX (ECM)
M. Climov et al., Skin Tissue Engineering and Regenerative Medicine, 2016, Pages 145–161
M. T. Cerqueira, R. L. Reis, A. P. Marques,, , Current Tissue Engineering, 2 (2013) 145
SKIN EXTRACELLULAR MATRIX (ECM)
Glycosaminoglycans
Proteoglycans
Collagens
Non-collagenous glycoproteins (Elastin,
Laminin, Fibronectin)
THE IMPORTANCE OF CELL-ECM INTERACTIONS
DIFFERENT TYPES OF ECM-MIMICKING
M. Climov et al., Skin Tissue Engineering and
Regenerative Medicine, 2016, Pages 145–161
Natural Vs Artificial ECM – Example at 3B’s Research Group
hASCs CS, after 5 days in culture
Phalloidin/DAPI
Trigger local responses towards
the regenerative pathway
Deposited ECM
Intact
- Cell-Cell
- Cell-Matrix
• High water content
• Enhanced
microstructural and
mechanical
performance
• Cell adhesive
features
• Facilitated diffusion
Freezing Freeze-
-drying
Re-
-hydration
Frozen
Hydrogel Hydrogel Sublimated
Hydrogel
Dried
Polymeric Networks Spongy-like
Hydrogel
da Silva LP, … , Reis RL, Marques AP Acta Biomater, 2014
da Silva LP, …, Reis RL, WO/2014/167513, 2014
HYDROPHILIC
DEADHESION
HYDROPHOBIC
CELL ADHESION
Above 32ºC
Below 32ºC
Adapted from http://www.twmu.ac.jp/ABMES/ja/cellsheet
Natural ECM Cell Sheet Engineering
Artificial ECM Spongy-like Hydrogels
Human Adipose-derived Stem Cells
Human Adipose-derived
Microvascular Endothelial Cells
Human Microvascular
Endothelial Cells
Total cells from the
epidermal fraction
Total cells from the
dermal fraction
ONGOING: Human
dermal fibroblasts, ...
Human keratinocytes
Melanin
Hyaluronic acid
SPONGY-LIKE HYDROGELS
CELL SHEETS Human
Microvascular
Endothelial Cells
Human keratinocytes
Human dermal
fibroblasts
SKIN LINEAGES
Human Adipose-derived
Stem Cells
STEM CELLS
SPONGY-LIKE HYDROGELS AND CELL SHEETS VERSATILITY
CELL SHEET TECHNOLOGY FOR SKIN WOUND HEALING
H &E
Collagen I
Rete Ridges -like Structures
Hair-Folicle Formation
Early Neovascularization
hD
Fb
+ h
DM
EC
s +
hK
Cs
hD
Fb
+ h
KC
s
hD
Fb
+ h
DM
EC
s
Enhanced Re-epithelialization
HUMAN ADIPOSE STEM CELLS
PROMOTED EPIDERMAL
REGENERATION
SKIN CELLS CELL SHEETS IMPACTED
WOUND HEALING, DEPENDING ON THEIR
CELLULAR COMPOSITION
Cell sheets
Cerqueira MT…Marques AP, Acta Biomaterialia, 2014 Cerqueira MT ….Marques AP, Biomacromolecules 2013
Stable Stem Cell-based
Cell Sheet Constructs
GELLAN GUM-BASED SPONGY-LIKE HYDROGELS
90°C
37°C
CaCl2
Ca2+
PBS
Na+
K+
Medium
Na+
K+
Ca2+
Temperature
decrease
Linear repeating tetrasaccharide unit composed of glucose, rhamose
and glucuronic acid.
Oliveira, J. T. et al. Gellan Gum Based Hydrogels For Regenerative Medicine And Tissue Engineering Applications, Its System, And Processing Devices,
WO/2009/101518 ; PCT/IB2009/000258
Da Silva L. P.. et Al. Gellan Gum Spongy-like Hydrogel, Its Prepararion And Biomedical Applications Thereof, WO 2014/167513 ; PCT/IB2014/060563
Hydrogel
Spongy-like Hydrogel
TAILORING SPONGY-LIKE HYDROGELS PROPERTIES
Hydrogel Spongy-like Hydrogel
GG
0.7
5 %
G
G 1
.25
%
0
10
20
30
40
50
GG 0.75 %
GG 1.25 %
Hydrogel Spongy-like
hydrogel
***
Co
mp
res
siv
e M
od
ulu
s (
KP
a)
0
2000
4000
6000
GG 0.75 %
GG 1.25 %
Hydrogel Spongy-like
hydrogel
**
**
Wa
ter
co
nte
nt
(%)
L. P. da Silva,…A.P. Marques, Acta Biomaterialia 10 (2014) 4787-97
Da Silva L. P.. et Al. Gellan Gum Spongy-like Hydrogel, Its Prepararion And Biomedical Applications Thereof, WO 2014/167513 ; PCT/IB2014/060563
SPONGY-LIKE HYDROGELS PROCESSING VERSATILITY
L. P. da Silva,…A.P. Marques, Acta Biomaterialia 10 (2014) 4787-97 Da Silva L. P.. et Al. Gellan Gum Spongy-like Hydrogel, Its Prepararion And Biomedical Applications Thereof, WO 2014/167513 ; PCT/IB2014/060563
CELL ADHESION TO GELLAN GUM SPONGY-LIKE HYDROGELS
Hyd
rog
el
Sp
on
gy-l
ike
Hyd
rog
el
Phalloidin/DAPI
Phalloidin/DAPI
100 µm
100 µm
hASCs hDMECs hKCs0
20
40
60
80
100 ******
% o
f Entrapped C
ells
hDMECs
Pre
-so
akin
g in
fib
ron
ectin
100 µm
Phalloidin/DAPI
hKCs
100 µm
K14/Phalloidin/DAPI
Cell-type dependent
Sp
on
gy-l
ike
Hyd
rog
el
100 µm
Calcein/PI
100 µm
hASCs
Ki-67/DAPI
100 µm
hASCs
100 µm
hASCs
Phalloidin/DAPI
Absence of
serum
Sp
on
gy-l
ike H
yd
rog
el
L. P. da Silva,…A.P. Marques, Acta Biomaterialia 10 (2014) 4787-97 M. T. Cerqueira, …, A.P. Marques, Materials Today 18 (8), 468-469
SPONGY-LIKE HYDROGELS FOR SKIN REGENERATION
Early neovascularization in the presence of
endothelial cells
Vessel density (microvessels/mm2)
CD31 α-SMA
Ctrl 8.5 ± 3.5 7.9 ± 3.5
GG-HA 1% 17.7 ± 8.7 (p<0.001) 11.1 ± 3.8
GG-HA 2% 10.9 ± 3.3 10.0 ± 4.7
Innervation in the condition with stem cells
Neovascularization tailored by hyaluronic acid
degradation products
Day 7 Day 14 8 Weeks post injury
Con
trol
GG-H
A
GG-H
A +
hAS
Cs
hASCs
A
GG-H
A +
con
d
0
10
20
30
40
50
604 3 4 4
IEN
F
(ne
rve
s/m
m)
Ctrl
GG-HA + hASCs 20μm
20μm
Control Heterotypic spongy-like
hydrogel 20μm 20μm
GG-HA 2 % 100μm GG-HA 1 %
14 days 21 days
Improved Re-epithelialization
Neoepidermis Maturation
3 days 7 days
Early Neoepidermis Keratinocytes Migration
da Silva LP, … Marques AP ACS Appl. Mater. Interfaces, 2016 Cerqueira MT, …,Marques AP, Tissue Eng Part A, 2014
Cerqueira
MT, …,
Marques AP,
ACS Appl
Mater
Interfaces,
2014 da Silva LP, … Marques AP, J Invest Dermatol, 2017
SKIN REGENERATION: PROBLEMATIC OF VASCULARIZATION
Common methods of TE skin Pre-vascularization using different cell sources
J. Boulard et al., Skin Tissue Models, 2018, Pages 177–200
M. T. Cerqueira, A. P. Marques, R. L. Reis, Using Stem Cells in Skin Regeneration: Possibilities and Reality. Stem Cells and Development, 21 (2012) 1201-1214, doi: 10.1089/scd.2011.0539
ALTERNATIVE TO ENDOTHELIAL CELLS: DIFFERENTIATION
OF STEM CELLS
VASCULARIZED DERMAL SUBSTITUTES
J. Boulard et al., Skin Tissue Models, 2018, Pages 177–200
0 10 20 30 40 500.0
0.2
0.4
0.6
0.8
1.0CtrlGGHA 1%GGHA 2%
Time (days)
Perf
usio
n R
atio o
f is
ch
em
ic
limb
/non
-isch
em
ic lim
b
(%)
GELLAN GUM-BASED STRUCTURES WITH ANGIOGENIC POTENTIAL
GG
-HA
1%
2 weeks 4 weeks
Ctr
l G
G-H
A 2
%
Low
High
GG-HA 1%
*
m m
a
m
*
GG-HA 2%
a
Control
* - material, a – adipose tissue; m – muscle
Amount of HA fragments existing in the solutions collected from degradation (HA-F) after 7 (HA-Fa,c)
and 21 (HA-Fb,d) days of hyaluronidase (50U) degradation
HA1 HA2 HA4 HA>20
GGHA 1% HA-Fa 55,2 ± 13,0 % 7,0 ± 1,5 % 0,2 ± 0,2 % 1,5 ± 0,2 %
HA-Fb 55,8 ± 0,3 % 6,2 ± 3,2 % 0,0 ± 0,0 % 0,0 ± 0,0 %
GGHA 2%
HA-Fc 47,2 ± 2,3 % 5,5 ± 4,8 % 0,5 ± 0,1 % 8,4 ± 0,2 %
HA-Fd 64,3 ± 4,0 % 7,5 ± 7,2 % 0,1 ± 0,1 % 1,5 ± 1,4 %
Vessel density (microvessels/mm2)
CD31 α-SMA α-SMA/CD31
Ctrl 8.5 ± 3.5 7.9 ± 3.5 1.1 ± 0.5
GGHA 1% 17.7 ± 8.7 (p<0.0001) 11.1 ± 3.8 0.8 ± 0.3
GGHA 2% 10.9 ± 3.3 10.0 ± 4.7 1.3 ± 0.9
L. P. da Silva… A.P. Marques, ACS Applied Materials & Interfaces 8 (2016) 33464
IN VIVO MODELS FOR SKIN WOUND HEALING
I. Pastar et al., Skin
Tissue Models,
2018, Pages
223–253
SIMILARITIES AND DIFFERENCES BETWEEN ANIMALS/HUMANS
G. Ignacio, Skin Tissue Engineering and Regenerative Medicine, 2016, Pages 387–400
Need of human skin models that are representative of human skin mechanisms in healing and in skin diseases/disorders
DIFFERENT IN VITRO SKIN MODELS
C. Pellevoisin et al., Skin Tissue Models, 2018, Pages 3–37
Overview of different types of reconstructed skin models, depending on the cells incorporated, as well as the matrix/organization employed
SKIN MODELS FOR DRUG PERMEATION, METABOLISM AND EFFICACY
H. Ruffner, Skin Tissue Engineering and Regenerative
Medicine, 2016, Pages 357–386
Drug Delivery and Metabolism
Efficacy Studies
EPIDERMAL MODELS FOR SAFETY CHEMICAL EVALUATION
C. Pellevoisin et al., Skin Tissue Models, 2018, Pages 3–37
SKIN MODELS TO STUDY SKIN DISEASES - Genodermatoses
E. Chacón-Solano et al., Skin Tissue Models, 2018, Pages 77–102
SKIN MODELS TO STUDY SKIN DISEASES - Genodermatoses
E. Chacón-Solano et al., Skin Tissue Models, 2018, Pages 77–102
SKIN MODELS TO STUDY SKIN DISEASES – Squamous Carcinoma
Normal 3D Organotypic Skin Models
3D Skin Cancer invasion Model
Characterization of the invasion process
Demonstration of tumor-cell-specific invasion
M. Berning et al., Skin Tissue Models, 2018, Pages 151–173
SKIN MODELS FOR WOUND-HEALING STUDIES
I. Pastar et al., Skin Tissue Models, 2018, Pages 223–253