Stem Cells and Periodontal Regeneration

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REV I EWAustralian Dental Journal2008; 53: 108121

doi: 10.1111/j.1834-7819.2008.00019.x

Stem cells and periodontal regenerationN-H Lin,* S Gronthos,PM Bartold*

*School of Dentistry, The University of Adelaide, South Australia.Colgate Australian Clinical Dental Research Centre, The University of Adelaide, South Australia.Mesenchymal Stem Cell Group, Division of Haematology, Institute of Medical and Veterinary Science Hanson Institute, Adelaide,South Australia.

ABSTRACT

Periodontitis is an inflammatory disease which manifests clinically as loss of supporting periodontal tissues includingperiodontal ligament and alveolar bone. For decades periodontists have sought ways to repair the damage which occurs

during periodontitis. This has included the use of a range of surgical procedures, the use of a variety of grafting materialsand growth factors, and the use of barrier membranes. To date periodontal regeneration is considered to be biologicallypossible but clinically unpredictable. Recently, reports have begun to emerge demonstrating that populations of adult stemcells reside in the periodontal ligament of humans and other animals. This opens the way for new cell-based therapies forperiodontal regeneration. For this to become a reality a thorough understanding of adult human stem cells is needed. Thisreview provides an overview of adult human stem cells and their potential use in periodontal regeneration.

Key words:Mesenchymal stem cells, periodontal ligament stem cells, periodontal regeneration.

Abbreviations and acronyms: BMP = bone morphogenetic proteins; BMP-7 = bone morphogenetic protein-7; BMSSCs = bone marrowstromal stem cells; EGF = epidermal growth factor; ePTFE = expanded polytetrafluoroethylene; ES = embryonic stem; FGF = fibroblastgrowth factor; IGF = insulin-like growth factor; HATCP = hydroxyapatitetricalcium phosphate ceramic; MHC = major histocompat-ibility; MSCs = mesenchymal stem cells; PDGF = platelet-derived growth factor; PDLSCs = periodontal ligament stem cells.

(Accepted for publication 5 December 2007.)

INTRODUCTION

Periodontitis is a disease of the periodontium charac-terized by irreversible loss of connective tissue attach-ment and supporting alveolar bone.1 These changesoften lead to an aesthetically and functionally compro-mised dentition. For many decades, periodontists havebeen interested in regenerating tissues destroyed byperiodontitis. Periodontal regeneration can be definedas the complete restoration of the lost tissues to theiroriginal architecture and function by recapitulating thecrucial wound healing events associated with their

development.2 Conventional open flap debridementfalls short of regenerating tissues destroyed by thedisease,3,4 and current regenerative procedures offer alimited potential towards attaining complete periodon-tal restoration.59 Recently, the isolation of adult stemcells from human periodontal ligament has presentednew opportunities for tissue engineering.10,11 Clearly,in order for such therapies to be successful, a thoroughunderstanding of stem cells and their role in regener-ating periodontal tissues is required.

The aim of this review is to discuss the current stateof our understanding of adult human stem cells in

dental tissues and their potential application in regen-erative periodontal therapy. Current regenerative pro-cedures, in particular guided tissue regeneration, arecritically assessed. Furthermore, potential clinical impli-cations of dental stem cells as well as the challenges forfurther research are also highlighted.

Definition and types of stem cells

The term stem cell first appeared in the literatureduring the 19th century. Like many other terms inbiology, the concept of a stem cell has expanded

greatly with identification of novel sites and functions.A stem cell refers to a clonogenic, undifferentiatedcell that is capable of self-renewal and multi-lineagedifferentiation.12 In other words, a stem cell is capableof propagating and generating additional stem cells,while some of its progeny can differentiate andcommit to maturation along multiple lineages givingrise to a range of specialized cell types. Depending onintrinsic signals modulated by extrinsic factors in thestem cell niche, these cells may either undergoprolonged self-renewal or differentiation.13 A pluri-potent stem cell can give rise to cell types from all three

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germ layers of the body (i.e., ectoderm, mesoderm andendoderm) whereas a multipotent stem cell can producecell types from more than one (but not all) lineages.Descriptive and experimental studies support thenotion that stem cells exist in both embryonic andadult tissues.14 To date, six types of stem cells havebeen isolated in humans1517 and these are depicted inFigure 1.

Embryonic stem cells

In 1998, Thomson and co-workers derived the first

human embryonic stem (ES) cell line from the inner cellmass of 4- to 7-day-old blastocyst-stage embryosdonated by couples undergoing fertility treatment.16 Alist of essential characteristics that define human EScells is presented in Table 1. The capacity of human EScells to form teratomas containing derivatives of allthree germ layers highlights their potential to differen-tiate into a range of cell types. To date, human ES cellshave not been tested for their ability to participate inhuman embryonic developmentin vivoor to contributeto germ lines because of ethical concerns. The use ofembryonic stem cells for clinical therapies is a relatively

Sperm Ovum

Germ cells

Fertilization

Blastocyst Embryonic Stem Cells

Embryonic Germ Cells

(derived from inner cell mass of pre-implantation embryo)

Embryonal Carcinoma Cells

PLURIPOTENT STEM CELLS

MULTIPOTENT STEM CELLS

TOTIPOTENT

INDUCIBLE PLURIPOTENT

Adult Stem Cells

(derived from many ectodermal and

Adult cells undergone nuclear transformation (clonal)

Adult cells induced to an embryonic stem cell phenotype

mesodermal organs in adults)

(derived from primordial germ cells inembryonic gonad but usually found as

components of testicular tumours in adults)

(derived from primordial germ cells isolated from

embryonal gonad)

Foetus

Adult

Fig 1. Sources and derivation of stem cell populations. Depending on the site, stage of development or cell culture induction environment humanstem cells can be classified as being of pluripotent, multipotent, totipotent or inducible pluripotent potential.

Table 1. Defining properties of embryonic stem cells

1. Derived from the inner cell massepiblast of the blastocyst ofpre-implantation or peri-implantation embryo.

2. Capable of undergoing unlimited proliferation in anundifferentiated state.

3. Exhibit and maintain a stable, diploid normal complement ofchromosomes.

4. Can give rise to differentiated cell types that are derivatives ofall three embryonic germ layers (ectoderm, mesoderm andendoderm) even after prolonged culture.

5. Capable of integrating into all foetal tissues during development.6. Capable of colonizing the germ line and giving rise to egg or

sperm cells.7. Clonogenic, i.e. a single ES cell can give rise to a colony

of genetically identical cells or clones, which have the sameproperties as the original cell.

8. Expresses the transcription factor Oct-4, which then activatesor inhibits a host of target genes and maintains ES cells in aproliferative, non-differentiating state.

9. Can be induced to continue proliferating or to differentiate.10. Lacks the G1 checkpoint in the cell cycle. ES cells spend most of

their time in the S phase of the cell cycle, during which theysynthesize DNA. Unlike differentiated somatic cells, ES cells donot require any external stimulus to initiate DNA replication.

11. Do not show X inactivation. In every somatic cell of a femalemammal, one of the two X chromosomes becomes permanentlyinactivated but this does not occur in undifferentiated ES cells.

Not shown in human ES cells. All of the criteria have been met bymouse ES cells.

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new endeavour and currently this development hasbeen hampered by ethical concerns.

Adult stem cells and mesenchymal stem cells

Adult stem cells, also known as somatic stem cells, are

undifferentiated cells found in specialized tissues andorgans of adults.12 Compared to the pluripotent andalmost immortal nature of embryonic stem cells, adultstem cells appear more mature with a finite lifespan andonly multipotent differentiation capacity. It appearsthat all specialized tissues with renewal capacitythroughout life probably contain adult stem cells invery small numbers that probably help replenish cellloss during normal senescence or tissue injury.1820

Haematopoietic stem cells from bone marrow were thefirst type of adult stem cells to be identified.21 Overthe years these cells have been extensively studied andare currently used therapeutically. Another population

of adult non-haematopoietic stem cells also resides inthe bone marrow microenvironment.2226 These aretermed bone marrow stromal stem cells (BMSSCs) ormesenchymal stem cells (MSCs) and their biologicalproperties are less well understood.

In recent years, human MSCs have been identified inmany tissues throughout the adult body. However, theprimary source of MSCs is the bone marrow where theyexist at a low frequency (one per 34 000 nucleatedcells), which declines with age.25,27 MSC-like cellpopulations have also been identified in other tissues,including adipose tissue, muscle, peripheral blood,

foetal pancreas and liver.

2833

Because of their wide-spread distribution, it has been proposed that MSCsarise from a perivascular stem cell niche26,33,34 where ithas been suggested that MSC exhibit a phenotypecharacteristic of pericytes.34,35

Mesenchymal stem cells have been characterizedboth morphologically and immuno-phenotypicallyusing various surface markers. While the morphologyof MSCs typically falls into one of two types (largeand flat or elongated and fibroblastic), this is not adefining or distinguishing feature of these cells. Ofmore relevance to their identification is the expressionof a number of phenotypic characteristics of osteo-

blasts, endothelial, perivascular cells, neural or mus-cle cells and a range of surface markers (includingCD49aCD29, CD44, STRO-1, CD90, CD105,CD106, CD146, CD140b, CD166, CD271). Thisbroad expression of cell surface molecules suggests acommon link between different cellular types sincemost of these markers are expressed by allMSC.25,26,3638 However, the heterogeneous nature ofthese cells is highlighted in clonal studies demonstratingfunctional differences between MSCs, based on theirproliferative potentials and developmental capacitiesin vitro and in vivo.25,26,3943 A particularly

distinguishing feature of human MSCs is their abilityto form colonies (i.e., they are clonogenic). In addition,under special inductive culture conditions, these cells candifferentiate along numerous lineages including those forosteoblasts, adipocytes, myelosupportive stroma, chon-drocytes and neuronal cells.26,4244 The ability of MSCs

to give rise to multiple specialized cell types along withtheir extensive distribution in many adult tissues(including those of dental origin) have made them anattractive target for use in periodontal regeneration.

Goal of periodontal therapy

Following disease control, periodontal regenerationrepresents the ultimate goal of periodontal therapyand entails the re-formation of all components of theperiodontium: gingival connective tissue, periodontalligament, cementum and alveolar bone.45 Periodontalregeneration aims to restore these lost tissues to their

original form and function by recapitulating the crucialwound healing events associated with periodontaldevelopment.45,46 Hence an understanding of theprocesses involved in the development of the periodon-tium is necessary in order to appreciate the cellular andmolecular events that might occur during periodontalregeneration.

Development and formation of periodontium

Periodontal development commences at the end ofcrown stage, when cells of the inner and outer enamel

epithelium proliferate from the cervical loop of theenamel organ to form Hertwigs epithelial root sheath(Fig 2). The root sheath separates cells of the dentalpapilla from the dental follicle, and initiates thedifferentiation of odontoblasts from the dental papillato form root dentine. After root dentine is deposited,cells of the root sheath secrete a fine matrix of proteinsonto the dentine surface, known as the hyaline layer ofHopewell Smith.47,48 It is thought that subsequentfragmentation of the root sheath allows cells of thedental follicle to attach to this protein matrix andsubsequently differentiate into cementoblasts, althoughthe exact events are unclear.49,50 Apical development of

the root continues with continuing cementum deposi-tion. Coronal to the developing root front, collagenfibres become embedded in the newly-formed cemen-tum, known as Sharpeys fibres. This process initiatesthe formation of the periodontal ligament through theactivities of the periodontal ligament fibroblasts whichare also derived from the dental follicle.51,52 At thisstage of root development, bundle bone (i.e., theportion of the alveolar process that lines the toothsocket) is also formed and this is derived fromosteoblasts, also originating in the dental follicle.53

Insertion of Sharpeys fibres into this newly-forming


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bone completes the development of the attachmentapparatus of the periodontium. The formation ofcementum, periodontal ligament and alveolar bone ina spatially and temporally coordinated manner, alongwith the transformation of reduced enamel epitheliumto sulcular and junctional epithelium during tooth

eruption, give rise to a complete periodontal attach-ment apparatus.54 To facilitate regeneration of lostconnective tissue attachment, further research is neededto fully elucidate the developmental processes of theperiodontium as well as the wound healing eventsfollowing periodontal therapy.

Periodontal wound healing and regeneration

Wound healing is the process by which an injured tissuerepairs itself. This consists of three interdependent,sequential phases that overlap with each other: inflam-

mation, granulation tissue formation and remodellingof the newly-formed tissue (Fig 3).55 The duration ofeach phase varies depending on a range of local andsystemic factors, particularly wound morphology andthe condition of adjacent tissues.55,56

Immediately following injury, a complex cascade of

molecular and cellular events occurs to initiate healing.An inflammatory response is mounted and a blood clotfills the site to provide tensile strength to the wound,and to serve as a reservoir of growth factors and aprovisional matrix for cell migration. Infiltrates ofneutrophils and monocytes enter the wound site tophagocytose dead or damaged tissue and foreignmatter. Epithelial cells originating from wound marginsalso migrate to close the epithelial breach. The fibrinclot is subsequently organized into granulation tissue asnew capillaries form and fibroblasts differentiate intomyofibroblasts to contract the wound. In the final

phase, the granulation tissue is remodelled into eitherrepaired (scar) tissue or regenerated tissue. Healing byrepair results in a tissue that does not completely restorethe architecture or function of the original tissue,whereas healing by regeneration produces a new tissuethat is identical in both structure and function to theoriginal tissue.57

Healing following conventional periodontal therapyis more complicated than simple soft tissue healingbecause of the involvement of mineralized tissues (i.e.,cementum and bone) in addition to soft tissue compo-nents. Most mechanical and surgical periodontal pro-cedures (e.g., scaling, debridement and flap procedures

of various types) favour the healing of anatomicaldefects produced by periodontitis. This largely involvesrepair of the gingival connective tissues and the coro-nal portion of the periodontal ligament with virtuallyno repair of the cementum or alveolar bone. Theseevents, by definition, do not constitute regenerationof the periodontium.45 Indeed, healing after flapsurgery is mediated by a range of reparative responses(Table 2), none of which can be considered to be tissueregeneration.

For regeneration to occur, healing events shouldprogress in an ordered and programmed sequence both

E

D

DP

Tooth

Epithelium

Crown

Ameloblasts

Enamel

Dentin

Odontoblasts

Hyaline Layer

Epithelial Root

Epithelial Cell

Epithelial Diaphragm

Sheath

Rests

Fig 2. Schematic representation of root development. At the end ofcrown stage, root formation and periodontal development commence.The inset illustrates deposition of root dentine by odontoblasts and

subsequent fragmentation of epithelial root sheath. Enamel (E),dentine (D), and dental papilla (DP) (reproduced, with permission

from reference 45).

Clot lysisInflammationEpithelialization

AngiogenesisMatrixSynthesisOrganizationWoundContaction

RemodellingApoptosisCollagenSynthesis

Days After Injury

0 1 10 30 100

Tissue Repair& Maturation

Fig 3. A time course of wound repair events that overlap in time andvary in duration depending on local and systemic factors (adapted

from reference 45).




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temporally and spatially, replicating the key events inperiodontal development.46,58 The course of healingis dependent on the availability of appropriate cells,inductive factors and extracellular matrix secreted bythese cells.59 Although the exact events are unclear,appropriate progenitor cells must migrate and attach to

the denuded root surface, and then proliferate andmature into all of the tissue components whichconstitute a functional attachment apparatus. Thecorrect proliferation, migration and maturation ofthese cells is dependent partly on the presence ofinductive factors and the contact with extracellularmatrix, controlling gene expression and release ofspecific inductive factors.59,60 In the absence of appro-priate cellular, molecular or matrix components, heal-ing may be compromised and occur by repair ratherthan regeneration.

Progenitor and stem cells are of particular interest in

periodontal wound healing and regeneration as theyare most likely the parental cells of synthetic cells(e.g., osteoblasts, cementoblasts and fibroblasts) respon-sible for the restoration of lost periodontal tissues(Fig 4).6163 Whether progenitor cells arise from bloodvessels in the periodontal ligament or surrounding bonestroma has been debated.64 However, one recent studyhas clearly demonstrated the presence of periodontal

ligament stem cells in perivascular niches of periodontalligament.65 Cells cultured from bone have the capacityto form cementum-like material in vitro66 and thesecells appear to be transferred to the periodontalligament via numerous vascular channels in the alveolarbone.67 Recent evidence shows that some clonal celllines isolated from the periodontal ligament havecharacteristics of stem cells.6871 A better understand-ing of these cells in the periodontal tissues will facilitateuse of these cells for regenerative periodontal therapy.

Periodontal regeneration

For decades attempts have been made to developclinical procedures which might lead to predictableperiodontal regeneration (Table 3). Most of these havebeen based on surgical solutions to the problem.However, until recent times many of the approacheswere not based on sound biological principles and thuswere eventually found to be of limited value.

Root surface conditioning

In an early approach, root surfaces were conditionedin early attempts either by demineralization of rootsurfaces, or by coating root surfaces with chemicalagents such as fibronectin, or both. The demineraliza-tion procedure was believed to reverse periodontitis-induced root surface hypermineralization and to exposecollagen fibres with which newly-formed fibres couldinterdigitate. Exposed collagen fibres were alsoexpected to discourage the attachment of unwantedepithelial cells. However, this procedure did not yieldpredictable regeneration, and often caused ankylosisand root resorption as side effects instead.72 Theadvantage of using fibronectin root surface coating

was also unclear because serum contains high fibronec-tin levels and providing additional protein is unlikely tohave any beneficial effect.73

Bone filling materials

Another approach to periodontal regenerationinvolved the introduction of a filler material intoperiodontal defects in the hope of inducing boneregeneration. Various types of bone grafts have beeninvestigated to determine their ability to stimulate newbone formation. Of these, the following have been

Table 2. Possible healing responses followingconventional periodontal therapy

Repair Control of inflammationLong junctional epitheliumConnective tissue attachment to the root surface

(reattachment or new attachment)New bone separated from the root surface by long

functional epitheliumNew bone with root resorption or ankylosis or bothRegeneration New functional attachment apparatus with formation

of cementum periodontal ligament and alveolar bone

(Adapted from reference 45.)

Progenitor cells inparavascular spaces

Cementoblasts

Fibroblasts

Osteoblasts

Growth Factors

Extracellular MatrixCell Adhesion Molecules

Fig 4. The role of stem cells in periodontal regeneration. Cells in theparavascular areas of mature periodontal ligament have the potential

to differentiate into mature osteoblasts, periodontal ligamentfibroblasts and cementoblasts.

Table 3. Phases of periodontal regeneration

Surgical techniques 19501970Root surface conditioning 19701980Guided tissue regeneration 19801990Growth factors 19902000Tissue engineering 2000????Stem cells ????


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studied in detail: (1) alloplastic materials which aregenerally synthetic filler materials; (2) autografts whichare grafted tissue from one site to another in the sameindividual; (3) allografts of tissue between individualsof the same species but with different genetic compo-sition; and (4) xenografts which consist of grafted

materials between different species. Although utiliza-tion of such grafting materials for periodontal defectsmay result in some gain in clinical attachment levelsand radiographic evidence of bone fill, careful histo-logic assessment usually reveals that these materialshave little osteoinductive capacity (let alone cemento-genic capacity) and generally become encased in a densefibrous connective tissue.74

Guided tissue regeneration

In recent years guided tissue regeneration has come tobe considered the gold standard upon which to

compare regenerative technologies. This procedureinvolves draping a barrier membrane over the peri-odontal defect from the root surface and onto adjacentalveolar bone prior to replacement of the mucoperio-steal flap. The barrier membrane prevents unwantedepithelium and gingival connective tissue from enteringthe healing site while promoting re-population of thedefect site by cells from the periodontal ligament(Fig 5). The concept of using barrier membranes inperiodontal regeneration is based on findings of a seriesof experiments carried out to address the regenerativecapacity of different periodontal tissues.7577 From

these studies it was reported that only cells of theperiodontal ligament possessed regenerative capacity,and that exclusion of gingival tissues from the wound

site allowed periodontal ligament cells to re-populatethe site, making regeneration biologically possible.78

Barrier membranes require at least seven essentialcriteria for their design to be effective (Table 4).7981

Resorbable membranes are made of collagen, polylacticacid and polyglycolic acid82 as well as autograft andallograft materials.83 Non-resorbable membranes arecommonly made of expanded polytetrafluoroethylene

(ePTFE).

84

Resorbable membranes have the advantageof bio-disintegration which negates the need for asecond surgical procedure to retrieve the membrane. Incontrast, non-resorbable membranes are used in a two-stage procedure involving removal of the membrane68 weeks after initial placement.

Histological analysis of guided tissue regeneration-mediated healing shows that new connective tissueattachment to the root surface forms with minorcontributions from new cementum and bone which,by definition, is not true regeneration.5 Guided tissueregeneration procedures represent an attempt toachieve concordance between biological principles and

clinical practice. However, their limited clinical andhistological success, particularly in advanced periodon-tal defects,85,86 has led researchers to further explorebiologically-basedregenerative therapy strategies involv-ing growth factors, gene therapy and replacementtherapy with stem cells.

Growth factors

In another approach to induce periodontal regenera-tion, polypeptide growth factors have been locallyapplied to the root surface in order to facilitate the

Barrier membrane

Excluded gingival tissues

Space for regenerative

processes

Alveolar bone

Periodontal ligament

Fig 5. Schematic representation of guided tissue regeneration. Themembrane physically excludes gingival tissues from the wound site

while providing space to allow cells of the periodontal ligamentto migrate into the site and promote regeneration (reproduced,

with permission from reference 45).

Table 4. Essential design criteria for guided tissueregeneration membranes

1. Tissue integration An open microstructure to encouragetissue integration and limit epithelialmigration, while creating a stablesite for wound healing

2. Cell occlusivity Separate all cell types so that the desired

cells can repopulate the defect area3. Clinical manageability Easy to cut and shape to fit particularperiodontal defects

4. Space provision Resist collapse from the pressure ofoverlying tissue so that they canmaintain adequate space during thehealing period

5. Biocompatibility Non-toxic, non-antigenic and induceminimal inflammatory response fromthe host

6. Membrane stability* Remainin situto allow progenitor cellsadequate time to repopulate the defectsite without interference from gingivalconnective tissue or epithelium

7. Membrane resorption* Be degraded, replaced, or incorporatedinto the healing flap after cell selectionis complete

*Apply only to resorbable membranes. (Adapted from references 79,80 and 81.)




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cascade of wound healing events that lead to newcementum and connective tissue formation. Among themyriad growth factors currently characterized andavailable, epidermal growth factor (EGF), fibroblastgrowth factor (FGF), insulin-like growth factor (IGF),PDGF and TGF-bhave been proposed to be of potential

use in relation to their regulatory effects on immunefunction, cells of epithelium, bone and soft connectivetissues. Two of these growth factors, PDGF and IGF-I,enhance regeneration in beagle dogs and monkeys withperiodontal disease.87,88 Another promising group ofpolypeptide growth factors is the bone morphogeneticproteins (BMP) which offer good potential for stimu-lating bone and cementum regeneration.89

Enamel matrix proteins

Enamel matrix proteins, produced by Hertwigs epithe-lial sheath, are known to play an important role in

cementogenesis, as well as in the development of theperiodontal attachment apparatus.90,91 There is someevidence that these proteins also play a role inregeneration of periodontal tissues after periodontaltherapy.92 In vitro studies have demonstrated thatEMD addition to cultures of periodontal fibroblastsresults in enhanced proliferation, protein and collagenproduction as well as promotion of mineralization.93,94

As no specific growth factors have been identified inEMD preparations,93 it is postulated that EMD actsas a matrix enhancement factor, creating a positiveenvironment for cell proliferation, differentiation and

matrix synthesis.

95

New perspectives for periodontal regeneration

There is no doubt that the desired clinical endpoint ispredictable regeneration of the periodontal tissuesdamaged by inflammation to their original form andfunction. To date, this has been elusive. In order forregeneration to occur there will need to be thecoordinated recruitment of specialized cells to the areaand deposition of specific matrix molecules consistentwith both soft and hard connective tissue formationand this will be largely driven by soluble cytokines and

growth factors.To understand the rational basis of regenerative

procedures, more information is needed on the varietyof molecular and cellular processes associated with theformation of each periodontal component. In particu-lar, very little is known about cementogenesis and themechanisms necessary for reattachment. While the useof growth factors shows some promise in this area,these suffer from being very broad in their range ofactivity and thus lack a degree of tissue specificity.Therefore, it seems reasonable to continue to probelocal factors (both cellular and molecular) which may

be specific for the regeneration of the periodontaltissues. To this end efforts to characterize stem cellpopulations in the periodontal ligament have becomeimportant.

Stem cells in human periodontal ligament

The presence of multiple cell types (fibroblasts,cementoblasts and osteoblasts) within the postnatalperiodontal ligament has led researchers to speculatethat these cells may share common ancestors. Thepossibility that progenitor cells might exist in thepostnatal periodontal ligament has been recognizedfor some time but until recently had never beenformally proven.96 These cells are believed to providea renewable cell source for normal tissue homeostasisand periodontal wound healing.61,97

Cell kinetic studies in mice have indicated that agroup of progenitor cells exhibiting some classical

features of stem cells exist in the periodontal liga-ment.67,98,99 Recently, multipotent stem cell popula-tions, termed periodontal ligament stem cells (PDLSCs),have been isolated from the periodontal ligament ofextracted human third molar teeth.10 These PDLSCsgive rise to adherent clonogenic clusters that resemblefibroblasts and are capable of developing into adipo-cytes, osteoblast- and cementoblast-like cells in vitro,and demonstrate the capacity to produce cementum-and periodontal ligament-like tissues in vivo.10,70,100

PDLSCs express an array of cementoblast and osteo-blast markers as well as the BMSSC associated markers,

STRO-1 and CD146 antigens, which are also presenton dental pulp stem cells.101,102 The similarity betweenPDLSCs, dental pulp stem cells and BMSSCs suggeststhat PDLSCs represent another MSC-like population.Further work is now focusing on identifying markersuniquely expressed by PDLSCs to discriminate thesecells from other types of MSC-like cells identified indental tissues.65 However, this is likely to be a complextask as earlier studies have indicated that there isconsiderable heterogeneity amongst cells of the peri-odontal tissues with regenerative capacity.103

The first reported isolation and identification ofmesenchymal stem cells in human periodontal ligament

was in 2004.10 Since then, there has been considerableactivity trying to understand the function of these cellpopulations and their interactions with each other witha view to laying the fundamental groundwork forclinical applications in regenerative periodontics. Anumber of studies have now been carried out to confirmthe presence of MSC-like cells in the periodontalligament. These have not been limited to human butalso include mouse, rat and sheep.6971,102105 All ofthese studies have confirmed the multipotent natureof periodontal ligament stem cells, and while theinitial studies indicated this to include an ability to


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differentiate into osteoblast, cementoblast or lipido-genic phenotypes at least one recent study has indicatedan ability of these cells to differentiate into neuronalprecursors.105 Importantly cryo-freezing does not seemto alter the properties of PDLSCs68 and this will havesignificant relevance should banking of these cells

become a clinical necessity.

Potential applications of stem cells in periodontaltherapy

Identification of stem cells in postnatal dental tissueshas presented exciting possibilities for the applicationof tissue engineering as well as gene and cell-basedtherapies in reconstructive dentistry. The use of stemcells with these technologies may constitute novelstrategies for regenerative periodontal therapy.

Tissue engineeringTissue engineering is a specialized field of science basedon principles of cell biology, developmental biology andbiomaterials science to fabricate new tissues to replacelost or damaged tissues.106,107 Successful tissue engi-neering requires an appropriate extracellular matrix orcarrier construct which contains regulatory signals andresponsive progenitor cells. A potential tissue engineer-ing approach to periodontal regeneration involvesincorporation of progenitor cells and instructive mes-sages in a prefabricated three-dimensional construct,which is subsequently implanted into the defect site

(Fig 6).

58

This strategy eliminates some of the limita-tions associated with conventional regenerative proce-dures because of direct placement of growth factors andprogenitor cells into the defect site overcomes thenormal lag phase of progenitor cell recruitment tothe site.

The technical requirements for successful cell-basedtissue engineering can be divided into two maincategories (Table 5): engineering issues related tomaintenance of an in vivo cell culture in the defect(e.g., biomechanical properties of the scaffold) andbiological functions of the engineered matrix (includingcell recruitment, neovascularization and bioavailability

of growth factors).108With respect to the biochemical features of the

matrix scaffold, these compounds should act in amanner consistent with the principles of membrane-based guided tissue regeneration and have similardesign features as listed in Table 4.80 In particular,these properties should include: ease of handling,rigidity to withstand soft tissue collapses into thedefect, and ability to maximize cell colonization andtissue ingrowth of desired type.109 It is also importantthat unwanted epithelium is not totally excluded, butrather encouraged to form a biological seal over the

scaffold and onto the tooth in the vicinity of thecemento-enamel junction, protecting the regeneratingevents occurring beneath.110

The concept of cell transplantation into periodontaldefects was first described over 15 years ago.111 Sincethen, other studies have attempted to induce periodon-tal regeneration using implantation of cultures ofperiodontal ligament fibroblasts and alveolar bonecells.112 While these investigations met with somesuccess, overall the treatment strategies were somewhatlimited due to the heterogeneous nature of the crudecell preparations used in these studies. More recently,the use of purified stem cells for tissue engineering

approaches to facilitate periodontal regeneration hasbeen investigated. Transplantation of autologousbone marrow MSCs in combination with atelocollageninto class III defects in dogs has been shown toregenerate cementum, periodontal ligament and alveo-lar bone.113 Ex vivoexpanded PDLSCs co-transplantedwith hydroxyapatitetricalcium phosphate ceramic(HATCP) particles into nude rats are capable offorming cementumperiodontal ligament-like struc-tures.68 One novel report has shown that stem cellsisolated from the root apical papilla of human teethand PDLSC can be combined to regenerate the

Progenitor cells

Progenitor cellscultured in vitro

Scaffold with seeded Regeneration of lostconnective tissueattachment

cells implanted intothe defect site

seeded onto a scaffold

Fig 6. Schematic representation of periodontal tissue engineering. Anengineered matrix (left) with necessary cells and instructive messagesseeded in vitro, and then (right) transferred into a periodontal defectto promote regeneration. Rapid formation of an epithelial seal should

be encouraged to minimize salivary and microbial contaminationduring wound healing.

Table 5. Requirements for successful tissueengineering

Biochemical features Space maintenance Barrier or exclusionary features

Biological functions Biocompatibility

Incorporation of cells (with appropriate phenotype for ongoingperiodontal regeneration) Incorporation (and bioavailability) of instructive messages

(Adapted from reference 58.)




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rootperiodontal structure respectively.114 In this studya root-shaped scaffold structure was prepared intowhich stem cells isolated from the root apical papillafrom porcine teeth were seeded. Gelfoam containingporcine PDLSC was then wrapped around the artificialroot construct and then placed into a prepared bony

socket in the mandible of mini-pigs. After a three-month healing phase this biologically created rootwas restored with a porcelain crown. Collectively, thesefindings demonstrate the feasibility (and potential) ofusing a combination of MSC-like cell populations forfunctional tooth regeneration.

Gene and cell-based therapy

The inherent proliferative and pluripotent capabilitiesof stem cells may offer lifelong opportunities fortreatment of some important human diseases, includingperiodontitis, by repairing, replacing or regenerating

damaged tissues. Stem cells may act as suitable vehiclesfor the delivery of therapeutic genes in gene therapy,and as therapeutic agents per se in cell-based therapy.

Gene therapy is a new approach for the treatment ofhuman diseases. It relies on genetic engineering, whichinvolves molecular techniques to introduce, suppress ormanipulate specific genes, thereby directing an individ-uals own cells to produce a therapeutic agent. In thecontext of periodontal regeneration, gene therapy seeksto optimize the delivery of agents such as growthfactors to periodontal defects so that the limitationsassociated with topical application (e.g., short duration

of action) can be overcome.

115

Two major strategies fordelivering therapeutic transgenes into human recipientsare: (1) direct infusion of the gene of interest using viralor non-viral vectors in vivo; and (2) introduction ofgene into delivery cells (often a stem cell) outside thebody ex vivo followed by transfer of the delivery cellsback into the body.30

The use of both in vivo and ex vivo gene deliverystrategies via adenoviral (Ad) vectors encoding growthpromoting molecules such as platelet-derived growthfactor (PDGF) and bone morphogenetic protein-7(BMP-7) has been investigated for its potential inperiodontal regeneration by Giannobile and col-

leagues.116119 Recent findings in rats have revealedsustained transgene expression for up to 10 days atAd-BMP-7 treated sites,119 and enhanced bone andcementum regeneration at Ad-BMP-7 and Ad-PDGFtreated sites beyond that of control vectors.118 Theintroduction of transgenes into dental stem cells mayoffer an alternative to conventional methods becausestem cells have the potential to provide a sustainedsource of growth factors for regeneration. However,much work is still needed to optimize the number of cellsthat are virally transduced to express specific genes,in order to maximize the duration and extent of gene

expression, and ultimately to determine the success ofgene transfer techniques in periodontal regeneration.Further research is also needed to address potentialrisks of viral recombination and immune responsestowards viral antigens which could potentially hinderthe progress of gene therapy in treating periodontal

diseases.

120

Current challenges and future directions for research

In view of the gaps and deficiencies in our knowledge ofperiodontal development and its applications to peri-odontal therapy, many challenges need to be overcomebefore stem cell-based treatment can become a clinicalreality. This section highlights the main biological,technical and clinical challenges in the area, whichcould form the basis for further research.

Biological challengesDespite biological evidence showing that regenerationcan occur in humans, complete and predictable regen-eration still remains an elusive clinical goal, especiallyin advanced periodontal defects. Periodontal regenera-tion, based on replicating the key cellular events thatparallel periodontal development, has not been possiblebecause of our incomplete understanding of the specificcell types, inductive factors and cellular processesinvolved in formation of the periodontium.58 Further-more, most basic discoveries on periodontal stem cellshave emerged from cell culture and animal models

which does not always translate to the human situation.Thus, not all findings in animal models can be directlyextrapolated to humans. In addition, the molecularpathways that underlie stem cell self-renewal anddifferentiation are also largely unknown.121 Furtherresearch is needed to elucidate the cellular and molec-ular events involved in restoring lost periodontal tissuesbefore a reliable biologically-based therapy can bedeveloped. In light of these concerns, the isolation andcharacterization of stem cells from periodontal tissuesmay provide a good starting point to investigate the roleof stem cells in periodontal wound healing and theirpotential applications in regenerative therapy, including

tissue engineering.

Technical challenges

Biologically, the matrix scaffold should have goodbiocompatibility for the cellular and molecular compo-nents normally found in regenerating tissues.122,123

There is evidence to suggest that cultured humanPDLSCs in a suitable scaffold and implanted intosurgically-created periodontal defects can result in theformation of a periodontal ligament-like structure.68

However, the optimal mechanism of propagation and


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incorporation of these cells into a carrier scaffold stillneeds further refinement.124,125 In addition, furtherstudies are needed to understand the conditions thatinduce lineage-specific differentiation and efficacy ofin vitro expanded stem cells derived from regeneratingperiodontal defects.7,126 Possible karyotypic instability

and gene mutations can limit the usefulness of cell linesafter prolonged culture.127 There are also difficulties inproviding clinical-grade stem cell lines using animal-free media to prevent cross-infection in humans.128

Thus, refinement of current techniques to facilitatelaboratory handling of these cells and to maximize theirregenerative potential represents a long-term endeavourif these cells are to be used in clinical periodontics.

Clinical challenges

In addition to biological and technical challenges, thereare a number of clinical barriers in MSC-based clinical

therapy that must be understood and overcome:immune rejection, tumour growth and efficacy of celltransplantation.

Firstly, it is important to understand how theimmune system will respond to human stem cellderivatives upon transplantation. Generally, the immu-nogenicity of a human cell depends on its expression ofclass I and II major histocompatibility (MHC) antigens,which allow the body to distinguish its own cells fromforeign cells. Human ES cells express a low level of classI MHC antigens, but this expression is up-regulatedwith differentiation.129 The use of patient-specific

(autologous) adult stem cells from redundant thirdmolar teeth should overcome potential immune rejec-tion.130,131 However, this approach may be redundantif recent reports are considered which indicate thatMSC can suppress the immune system and thus allowsthe use of either autologous or allogeneic MSCpreparations.132

Secondly, the prevention of tumour formation fol-lowing MSC implantation is a major safety consider-ation as current studies lack sufficient statistical powerand long-term follow-up to draw firm conclusions.133 Itis likely that the more specific and extensive thetherapeutic application, the longer the stem cells may

have to remainin vitroto obtain sufficient numbers fortherapeutic use. Thus during this extended period inculture there could be a greater likelihood that geneticor epigenetic changes will accumulate. If such changesare not accompanied by an overt phenotypic transfor-mation, they may go undetected and harm the patient.Therefore, it is critical to have a thorough understand-ing of the rate of genetic change and the type ofselective pressures that allows this change to dominate aculture.

Thirdly, it is unclear whether human stem cellderivatives can integrate into the recipient tissue and

fulfil the specific functions of lost or injured tissues.134

Delivery of appropriate cells and molecules to the targetsite without inducing ectopic tissue formation is ofparamount importance for the safety and effectivenessof tissue engineering-based periodontal regeneration. Itis hoped that, as our knowledge on progenitor cells,

growth factors and delivery systems improves, it willeventually lead to the development of regenerativetherapy based on sound scientific principles.

CONCLUSIONS

Regeneration of tissues destroyed by periodontitis haslong been an altruistic goal of periodontal therapy.Periodontal regeneration requires consideration ofmany features that parallel periodontal development,including the appropriate progenitor cells, signallingmolecules and matrix scaffold in an orderly temporaland spatial sequence. It is clear that current regenera-

tive procedures are less than ideal but the identificationof stem cells in human dental tissues in recent yearsholds promise to the development of novel, moreeffective approaches to periodontal regeneration andreconstructive therapy. One way forward is to embracethe field of stem cell-based tissue engineering and adoptan interdisciplinary approach to periodontal regenera-tion. However, before this is feasible, many biological,technical and clinical hurdles need to be overcome anda thorough understanding of underlying healing pro-cesses in periodontal regeneration is required.

ACKNOWLEDGEMENTS

The authorswork cited in this review has been fundedvariously by the National Health and Medical ResearchCouncil of Australia, the Australian Dental ResearchFoundation and the Australian PeriodontologyResearch Foundation.

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Address for correspondence:Professor P Mark Bartold

Colgate Australian Clinical Dental Research CentreSchool of Dentistry

The University of AdelaideFrome Road

Adelaide, South Australia 5005Email: [email protected]



Stem Cells and Periodontal Regeneration

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