Stem Cells and Periodontal Regeneration; Present and Future Potential Implications

Stem Cells and Periodontal Regeneration; Present and Future Potential Implications
Noreen Haseeb*, Khalid Almas**

How to CITE:

J Pak Dent Assoc 2010;19(2):78 - 76



Abstract

The aim of this review is to highlight the recent developments in stem cell therapy in periodontal regeneration. The review contains recent developments and progress of knowledge about adult and mesenchymal stem cell research and their clinical implications. An effort has been made to review the classifications of stem cells, their sources from various body tissues and their potential use for periodontal regeneration. The cellular sources of periodontal wound healing, regenerative concepts and techniques are highlighted. Recent developments in understanding of biologics, various growth factors and clinical relevance of stem cell therapy (implications) have been incorporated. It is hoped that the review would develop interest among the dental professionals, to be aware of the recent advancements in stem cell therapy especially in periodontal regeneration. Future trends have been highlighted.

KEYWORDS:

Stem cells, periodontal regeneration, mesenchymal stem cells, wound healing, repair and bone regeneration.

Introduction

stem cell refers to a clonogenic, undifferentiated cell that is capable of self-renewal and multi-lineage differentiation.1

The term stem cell first appeared in the literature during the 19th century and since then the concept has expanded greatly with identification of novel sites and functions. A stem cell is capable of propagating and generating additional stem cells, while some of its progeny can differentiate and commit to maturation along multiple lineages giving rise to a range of specialized cell types.2

Stem cells divide to form one daughter cell that goes on to differentiate and one daughter cell that retains its stem cell properties. Classification of stem cells is based on their species of origin, tissue of origin, or differentiation capability of ≥1 specific type of the mature cells.3 Some stem cells are more pluripotent than others. For example, all cells within the early embryo are totipotent up until the 16-cell stage or so and are thought to be the only single cells capable of differentiating into any cell type.4

Stem cells exist in both embryonic and adult tissues, concept is supported by descriptive and experimental studies. Depending on the site, stage of development or cell culture induction environment, human stem cells can be classified as being of pluripotent, multipotent, totipotent or inducible pluripotent potential. Six types of stem cells have been isolated in humans.

 

  1. Embryonic Stem Cells: Are derived from inner cell mass of pre-implantation blastocyst-stage embryo and are classified as pluripotent stem cells.

 

  1. Embryonic Germ Cells: Are derived from primordial germ cells isolated from embryonal gonad and are pluripotent stem cells in nature.

 

3. Embryonal Carcinoma Cells: Are derived from primordial germ cells in embryonic gonad but usually found as components of testicular tumors in adults and are also classified as pluripotent stem cells

4. Adult Stem Cells: Are derived from many ectodermal and mesodermal organs in adults. Adult stem cells are pluripotent but have more limited differentiation ability and thus, are considered multipotent

5. Adult cells undergone nuclear transformation (clonal) and are totipotent in nature.

  1. Adult cells induced to an embryonic stem cell phenotype and have an inducible pluripotent potential.5-8

Stem cells are self renewing and thus can generate any tissue for a life time. This is a key property for a successful therapy. The capacity to expand stem cells in culture is an indispensable step for regenerative medicine, and a considerable effort has been made to evaluate the consequences of the cultivation on stem cell behavior

Cell sources for the development of teeth in vitro or ex vivo

Epithelial-mesenchymal interactions results in tooth formation so two different populations of stem cells have to be considered: epithelial stem cells (EPSC), which will give rise to ameloblasts, and mesenchymal stem cells (MSC) that will form the odontoblasts, cementoblasts, osteoblasts and fibroblasts of the periodontal ligament. Thus, tooth engineering using stem cells is based on their isolation, association and culture as recombinants in vitro or ex vivo conditions to assess firstly tooth morphogenesis and secondly cell differentiation into tooth specific cells that will form dentin, enamel, cementum and alveolar bone. Various approaches could be used according to the origin of stem cells.9 Mesenchymal stem cell (MSC) populations have been isolated from human dental tissues such as the periodontal ligament10,11 and the dental follicle12 in addition to the dental pulp.13

Adult stem cells and mesenchymal stem cells:

Adult stem cells, also known as somatic stem cells, are undifferentiated cells found in specialized tissues and organs of adults. All specialized tissues with renewal capacity throughout life probably contain adult stem cells in very small numbers that probably help replenish cell loss during normal senescence or tissue injury.14

Hematopoietic stem cells from bone marrow were the first type of adult stem cells to be identified and are currently used therapeutically.15 Another population of adult non-hematopoietic stem cells also resides in the bone marrow microenvironment.16,17 These are termed bone marrow stromal stem cells (BMSSCs) or mesenchymal stem cells

MSCs) and their biological properties are less well understood. Mesenchymal stem cells (MSCs), also termed multipotent mesenchymal stromal cells, are a phenotypically and functionally heterogeneous cell population. In culture, they are defined as plastic-adherent, fibroblast-like cells which are able to self-renew and differentiate into bone, adipose and cartilage tissue.18

 

Although traditionally isolated from bone marrow, more recent reports have detailed the isolation of cells with MSC characteristics from a variety of tissues including cord blood, peripheral blood, fetal liver and lung, adipose tissue, skeletal muscle, amniotic fluid, synovium and the circulatory system.16

 

Mesenchymal stem cells have been characterized both morphologically and immuno-phenotypically using various surface markers. The broad expression of surface

markers (CD49a /CD29, CD44, STRO-1,

CD90,CD105, CD106, CD140b, CD166, CD271 ) suggests a common link between different cellular types since most of these markers are expressed by all MSC.16,17,19 The ability to form colonies is a particular feature of human stem cells and under special conditions, these cells can differentiate along numerous lineages including those for osteoblasts, adipocytes, myelosupportive stroma chondrocytes and neuronal cells.20 MSC possess a high self-renewal capacity and the potential to differentiate into mesodermal lineages thus forming cartilage, bone, adipose tissue, skeletal muscle and the stroma of connective tissue.21 The potential of dental MSC for tooth regeneration and repair has been extensively studied in the last years. The ability of MSCs to give rise to multiple specialized cell types along with their extensive distribution in many adult tissues including those of dental origin have made them an attractive target for use in periodontal regeneration. Mesenchymal progenitors that have been assessed for tooth engineering purposes, such as progenitors derived from teeth and bone marrow are being discussed below:

Stem cells from human exfoliated deciduous teeth (SHED):

Recent findings demonstrated the isolation of mesenchymal progenitors from the pulp of human deciduous incisors. These cells were named SHED and exhibited a high plasticity since they could differentiate into neurons, adipocytes, osteoblasts and odontoblasts.13

Adult dental pulp stem cells (DPSC):

Dental pulp is involved in reparative process termed as dentinogenesis in case of dental injury. In dentinogenesis cells elaborate and deposit a new dentin matrix for the repair of the injured site.22 Dental pulp progenitors have not been clearly identified but some data suggest that pericytes, which are able to differentiate into osteoblasts, could also differentiate into odontoblasts.23 Tooth repair is a lifetime process thus suggesting that MSC might exist in adult dental pulp. The in vivo therapeutic targeting of these adult stem cells remains to be explored.

Stem cells from the apical part of the papilla

(SCAP):

Stem cells from the apical part of the human dental papilla (SCAP) have been isolated and their potential to differentiate into odontoblasts was compared to that of the periodontal ligament stem cells (PDLSC).24 SCAP exhibit a higher proliferative rate and appears more effective than PDLSC for tooth formation. Importantly, SCAP are easily accessible since they can be isolated from human third molars.

Stem cells from the dental follicle (DFSC):

DFSC have been isolated from follicle of human third molars and express the stem cell markers Notch 1, STRO-1 and nestin.12 Immortalized dental follicle cells are able to re-create a new periodontal ligament (PDL) after in vivo implantation.25

Periodontal ligament stem cells (PDLSC):

The PDL is a specialized tissue which has a role in the maintenance and support of the teeth. Its continuous regeneration is thought to involve mesenchymal progenitors arising from the dental follicle. PDL contains STRO-1 positive cells that maintain certain plasticity since they can adopt adipogenic, osteogenic and chondrogenic phenotypes in vitro.26 It is thus obvious that PDL itself contains progenitors, which can be activated to self renew and regenerate other tissues such as cementum and alveolar bone.10

<43>Bone marrow derived mesenchymal stem cells (BMSC):

BMSC have been tested for their ability to recreate periodontal tissue. These cells are able to form in vivo cementum, PDL and alveolar bone after implantation into defective periodontal tissues. Thus, bone marrow provides an alternative source of MSC for the treatment of periodontal diseases.27

Epithelium-originated dental stem cells (EpSC):

No information is available for dental EpSC in humans although progress has been made in the MSC. The major problem is that dental epithelial cells such as ameloblasts and ameloblasts precursors are eliminated soon after tooth eruption. Therefore, epithelial cells that could be stimulated in vivo to form enamel are not present in human adult teeth. Stem cell technology appears to be the only possibility to re-create an enamel surface.9

Several studies describe the use of EpSC isolated from newborn or juvenile animals, usually from third molar teeth. Dental EpSC can be isolated from post-natal teeth but exhibit complex problems that strongly limit their clinical application in humans. Other sources along with the association of epithelial and mesenchymal stem cells are required as teeth are formed from two different tissues. The recombination of dissociated dental epithelial and mesenchymal tissues leads to tooth formation both in vitro and in vivo.28

Periodontal Regeneration:

Periodontal regeneration can be defined as the complete restoration of the lost tissues to their original architecture and function by recapitulating the crucial wound healing events associated with their development.29 Periodontal regeneration represents the ultimate goal of periodontal therapy and entails the reformation of all components of the periodontium: gingival connective tissue, periodontal ligament, cementum and alveolar bone.30

Regenerative periodontal therapy comprises procedures which are specially designed to restore those parts of the tooth-supporting apparatus which have been lost due to periodontitis. Periodontal regeneration has been reported following a variety of surgical approaches involving root surface biomodification, often combined with coronally advanced flap procedures, the placement of bone grafts or bone substitute implants, or the use of organic or synthetic barrier membranes (guided tissue regeneration).31 Successful regeneration is assessed by periodontal probing, radiographic analysis, direct measurements of new bone, and histology.32

Periodontal Wound Healing:

Regeneration of the periodontium include the formation of new cementum by inserting collagen fibres on the previously periodontitis-involved root surfaces and the regrowth of the alveolar bone. The fibrous attachment may exist without opposing bone in a normal dentition, not affected by periodontitis, in the presence of bone dehiscences and fenestrations. In 1976, Melcher suggested that the type of cell which repopulates the root surface after periodontal surgery determines the nature of the attachment that will form. After flap surgery the curetted root surface may be repopulated by four different types of cell:33

 

  1. Epithelial cells

 

  1. Cells derived from the gingival connective tissue

 

  1. Cells derived from the bone

 

  1. Cells derived from the periodontal ligament

Regenerative concepts

To obtain new attachment, scaling and root planning combined with soft tissue curettage (i.e. mechanical removal of the diseased root cementum and the pocket epithelium ) was one of the first methods. Most of the methods have been based on surgical procedures. Please see table 1

Root Surface Conditioning:

In an early approach, root surfaces were conditioned in early attempts either by demineralization of root surfaces, or by coating root surfaces with chemical agents such as fibronectin, or both. The demineralization procedure was believed to reverse periodontitis-induced root surface hypermineralization and to expose collagen fibres with which newly-formed fibres could interdigitate. Exposed collagen fibres were also expected to discourage the attachment of unwanted epithelial cells. However, this procedure did not yield predictable regeneration, and often caused ankylosis and root resorption as side effects instead. The advantage of using fibronectin root surface coating was also unclear because serum contains high fibronectin levels and providing additional protein is unlikely to have any beneficial effect.36

Grafting Procedures:

Another approach with the aim of stimulating periodontal regeneration involved the flap procedure combined with the placement of bony grafts or implant materials into the curetted bony defects. The following are the various graft materials

  1. Autogenous grafts: grafts transferred from one position to another within the same individual e.g., cortical bone

 

  1. Allogenic grafts: grafts transferred between genetically dissimilar members of the same species e.g., freeze dried bone.

 

  1. Xenogeneic grafts: grafts taken from a donor of another species.

 

  1. Alloplastic materials: synthetic or inorganic implant materials which are used as substitutes for bone grafts.37

Although utilization of such grafting materials for periodontal defects may result in some gain in clinical attachment levels and radiographic evidence of bone fill, careful histologic assessment usually reveals that these materials have little osteoinductive capacity and generally become encased in a dense fibrous connective tissue.38

Guided Tissue Regeneration (GTR):

The procedure involves draping a barrier membrane over the periodontal defect from the root surface and onto adjacent alveolar bone prior to replacement of the mucoperiosteum flap. The barrier membrane prevents unwanted epithelium and gingival connective tissue from entering the healing site while promoting re-population of the defect site by cells from the periodontal ligament.34

Various studies reported that only cells of the periodontal ligament possessed regenerative capacity, and that exclusion of gingival tissues from the wound site allowed periodontal ligament cells to re-populate the site, making regeneration biologically possible.39

Barrier membranes are either resorbable or non-resorbable and require some criteria for their design to be effective. Resosbable membranes are made of collagen, polylactic acid and polyglycolic acid as well as autograft and allograft materials.40 Non-resorbable membranes are commonly made of expanded polytetrafluoroethylene (ePTFE).41 Resorbable membranes have the advantage of bio-disintegration and are used in one stage procedure where as non-resorbable membranes are used in a two-stage procedure involving removal of the membrane 6-8 weeks after initial placement. The clinical outcomes of GTR are most frequently evaluated by changes in clinical attachment levels (CAL), bone levels (BL), probing pocket depth (PPD), and the position of the gingival margin. However, evidence of true regeneration of periodontal attachment can only be provided by histologic means.37

Growth Factors:

Growth factor is a regulatory term to denote a class of polypeptide hormones that stimulate a wide variety of cellular events such as proliferation, chemotaxis, differentiation, and production of extracellular matrix proteins.42 Proliferation and migration of periodontal ligament cells and synthesis of extracellular matrix as well as differentiation of cementoblasts and osteoblasts is a prerequisite for obtaining periodontal regeneration. Therefore it is conceivable that growth factors may represent a potential aid in attempts to encourage regeneration of the periodontium.37

Among the growth factors currently characterized and available, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-ß) have been proposed to be potential use in relation to their regulatory effects on immune function, cells of epithelium, bone and soft connective tissues. Two of these growth factors, PDGF and IGF-I, enhance regeneration in beagle dogs and monkeys with periodontal disease.43,44

Transforming growth factor-ß3 also induces periodontal tissue regeneration. For regenerative procedures and tissue engineering of bone in clinical contexts, the inductive activity of recombinant transforming growth factor-ß3 is enhanced by the addition of minced fragments of autogenous rectus abdominis muscle tissue. Thus adding responding stem cells for further tissue induction and morphogenesis by the recombinant transforming growth factor-ß3 isoform. This is shown by the partial restoration of the bone-induction cascade when minced fragments of rectus abdominis muscle are added to osteogenic preparations of the recombinant transforming growth factor-ß3 osteogenic devices implanted in nonhealing calvarial defects of P.ursinus.45

Enamel Matrix Proteins (EMD):

Emdogain EMD is a protein extract purified from porcine enamel and has been introduced in clinical practice to obtain periodontal regeneration.46 EMD is mainly composed of amelogenin 90% and the remaining 10% is composed of non-amelogenin enamel matrix proteins such as enamelins, tuftelin, amelin and ameloblastin.47 Enamel matrix proteins, produced by Hertwigs epithelial sheath play a role in regeneration of periodontal tissues after periodontal therapy.48 As no specific growth factors have been identified in EMD preparations49 it is postulated that EMD acts as a matrix enhancement factor, creating a positive environment for cell proliferation, differentiation and matrix synthesis.50

Bone Regeneration:

Bone is a unique organ that has the ability to regenerate and repair itself.51 Periosteum is a source of mesenchymal stem cells MSCs that can be transformed into osteoblasts.52 The fibroblasts from the outer layer of the periosteum and the osteoblasts originating from the inner layer provide a collagen matrix. When the periosteum is inverted, the fibroblastic layer is in contact with bone and the periosteal layer is in contact with soft tissue. The two layers contain an ample supply of primitive stem cells and have the ability to proliferate and differentiate into osteoblasts adjacent to the bone. The local growth factors from the adjacent tissue guide the path of differentiation of the primitive stem cells. These cells secrete growth factors that stimulate progenitor cells to proliferate and differentiate into endothelial cells. These cells vascularize the site, bringing nutrients, additional cells, and cytokines needed for bone regeneration.53 MSCs are normally quiescent. They are activated by injury, local infection, hypoxia, and surgical trauma, including the manipulation involved in releasing the periosteum.54

During osseous surgery , the manipulation of the periosteum is responsible for most of the postoperative pain and swelling. Conversely, surgical trauma triggers the body’s natural healing response that release mast cells and other inflammatory cells. Cytokines and other growth factors secreted by this interaction of cells stimulate stem cells to proliferate, differentiate, and migrate to the site of injury. The reversed periosteal flap provides a 2-layer closure, pedicled release of periosteum from the mucosa, coverage and stabilization of the graft site, and closure of the mucoperiosteal flap without tension of the incision.53

The success of the reverse periosteal flap is possible because it is a source of stem cells and connective tissue progenitor cells needed for bone modeling and remodeling. The surgical technique requires a pedicle split thickness dissection of the periosteum from the mucosal base. The inverted pedicled flap advances the vascular stem cell rich periosteal layer over the graft and provides direct contact with the adjacent tissue. The technique is recommended when augmenting sites with 3-or 4- wall defects, in conjunction with implant placement or in combination with bone graft surgery.53

Clinical Relevance of Stem Cell in Periodontal Therapy:

The clinical introduction of stem cell regenerative periodontic treatment can only be delivered to patients after federal efficacy and safety assurance requirements have been addressed. The ideal design of the periodontal constructs is to be the same shape as general periodontal barrier membranes, and its benefits include the replacement of diseased and traumatized periodontal tissues.55 The recent findings indicate that the control of morphogenesis and cytodifferentiation is a challenge that necessitates a thorough understanding of the cellular and molecular events involved in development, repair and regeneration of teeth.9

The identification of several types of epithelial and mesenchymal stem cells in the tooth and the knowledge of molecules involved in stem cell fate is a significant achievement. In vitro and in vivo experiments using these cells have provided promising results illustrated by the generation of a complete tooth with all dental structures including cells and extracellular matrix deposition.56

The regeneration and replacement of dental tissues has been a goal of dentists for many decades, however many problems remain to be addressed before considering the clinical use of the technologies available. The engineering of tri-dimensional matrices (either polylactic acid polymers or collagen sponges) which a composition more or less similar to that of the organs to reconstruct, and the addition of growth factors such as FGF, bone morphogenetic protein (BMP) or PDGF might facilitate the transplantation and the differentiation of stem cells. Advances in stem cell purification, cell culture technology, and the design of biomaterial scaffolds may allow the de novo creation of tissues in the laboratory. These tissue-engineering advances have the potential to create pulp and periodontal constructs, which may follow future dentists to replace or regenerate diseased, traumatized, or missing pulp and periodontal tissues.57

However, the engineering of tooth substitutes is hard to scale up, costly, time-consuming and incompatible with the treatment of extensive tooth loss. Scientific knowledge is not enough and the main challenge in stem-cell therapy is to find a compromise between the benefits to the patients, regulatory agencies, increased stem cell requirements, costs, coverage by health insurance and the role of pharmaceutical companies.9

Regenerative medicine using MSCs is a minimally invasive approach compared to medical implantation

Hematopoietic cell transplantation (HCT) is now widely used as a potentially curative procedure for hematologic malignancies as well as number of other diseases. HCT can serve as a rescue procedure to reconstitute the hematopoietic system when damaged by high-dose chemo / radiotherapy for treatment of malignancy, because hematopoietic stem cells possess the capacity for self-renewal as well as differentiation into blood cell lineages. In addition, allogenic stem cells or their progeny can be used to deliver anticancer immunotherapy.58

Biologically, the matrix scaffold should have good biocompatability for the cellular and molecular components normally found in regenerating tissues.59

There is evidence to suggest that cultured human PDLSCs in a suitable scaffold and implanted into surgically-created periodontal defects can result in the formation of a periodontal ligament-like structure.11 However, the optimal mechanism of propagation and incorporation of these cells into a carrier scaffold still needs further refinement.60 Thus, refinement of current techniques and clinical research to facilitate laboratory handling of these cells is required, if these cells are to be used in clinical periodontics.

Future Implications:

Based on our current understanding of graft healing and the prerequisites for optimal bone regeneration, tissue-engineering research has focused on providing the necessary cellular machinery, i.e., the mesenchymal stem cells (MSCs), directly in sites that require bone regeneration. Stem cell therapy has a broad base of current applications under investigation that include the repair and regeneration of heart muscle, cartilage, and bone tissues. Pluripotential MSCs have the unique capability to differentiate into a variety of cell types based on the inducing signals received from the recipient tissue. Of great interest to osseous reconstruction for implant dentistry is the appropriate stimulation of implanted MSCs that can differentiate along the osteoprogenitor cell lineage with osteogenic properties that would result in bone formation for reconstructive implant therapy.61

The evolution and refinement of techniques for harvesting, ex vivo culture expansion, and in vivo reimplantation of adult stem cells have led to the production of biomaterials for clinical application. Stem cells are characterized by their ability to renew themselves through cell division and differentiate into a diverse range of specialized cell types. In adult organisms, stem cells give rise to progenitor cells that act as a repair system for the body, replenishing specialized cells and tissues.61

Conclusion

Based on the literature reviewed it can be concluded that:

1- Despite biological evidence showing that regeneration can occur in humans, complete and predictable regeneration still remains an elusive clinical goal, especially in advanced periodontal defects. Periodontal regeneration, based on replicating the key cellular events that parallel periodontal development, has not been possible because of our incomplete understanding of the specific cell types, inductive factors and cellular processes involved in formation of the periodontium.62

2- Furthermore, most basic discoveries on periodontal stem cells have 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 directly extrapolated to humans. In addition, the molecular pathways that underlie self-renewal and differentiation are also largely unknown.63

3- In addition to biological and technical challenges, several clinical risk factors in MSC-based therapy requires an understanding and management. Immune rejection, tumour growth and efficacy of cell transplantation are the main complications that needs a thorough understanding.34

 

4- The identification of stem cells in human dental tissues in recent years have develop effective approaches to periodontal regeneration and reconstructive therapy. Periodontal ligament PDL contains stem cells that have the potential to generate cementum / PDL like tissue in-vivo. Transplantation of these cells, (tissue resource) and expanded ex-vivo, might hold promise as a therapeutic approach for reconstruction of tissues destroyed by periodontal diseases.

5- The regeneration of whole tooth organs like teeth is certainly much more demanding than the regeneration of individual tissues like bone or dentin. However, this goal may not be as distant as it appeared to be a few years ago. The accumulation of molecular information will contribute to a more complete understanding of the mechanisms that regulate tooth morphogenesis and the roles that growth and differentiation factors play in these processes

6-Perhaps it is possible that tooth development could be initiated in-vivo by applying specific growth and differentiation factors. So ideally, we would require only to introduce the signal that starts tooth formation and then let nature run its course but much research is required before tooth regeneration in dental practice is a reality.64

7- Moreover, a contribution in bone regeneration, at least in part by the host cells from the surrounding tissue, the number of cells attached to carriers and transplanted in defects must be considered. Presently available materials for periodontal regeneration through stem cells induction is Osteocel, a bone matrix product containing stem cells has recently been launched by Ace surgical, USA. Case series has been published for sinus augmentation procedure.61

An expanded role of stem-cell based regeneration including various body tissues, is on the horizon. Quantitative studies are needed in the future. Education in basic aspects of underlying healing processes of stem cells in periodontal regeneration is required. Further research is required to have a better and improved understanding of the role of mesenchymal stem cells in the periodontal regeneration and to evaluate the safety, efficacy and predictability of their clinical applications

ACKNOWLEDGEMENT

The authors report no conflicts of interest related to this review. The authors got inspired by the clinical and research work of Drs. Muna Soltan and Dennis Smiler, California, USA. Interested readers may look at an interesting website www.store-A-tooth.Com

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