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The periodontium is an extraordinarily intricate tissue composed of two hard and two soft tissues. The hard tissues are cementum and bone; the soft issues are gingiva and the periodontal ligament. The periodontium has a limited capacity for regeneration once it has been damaged.16
During orthodontic tooth movement, new cementum formation, periodontal ligament remodeling, and new bone formation have been observed. However, researchers consider this to be a physiological response rather than regeneration or true repair of pathologically damaged tissue. Some minor regeneration of the periodontium may be seen during the early phases of periodontal disease. Only therapeutic intervention has the potential to induce regeneration once periodontitis becomes established.16
The multifaceted chain of events related to periodontal regeneration includes recruiting progenitor cells that are derived locally to the site where they can then differentiate into periodontal ligament-forming cells, mineral-forming cementoblasts, or bone-forming osteoblasts. Up to the present time techniques to restore damaged or diseased periodontal tissues have been dependent on the use of implantation of structural substitutes, which unfortunately have had little to no reparative potential. The focus has generally been directed exclusively to the regeneration of lost alveolar bone.16
A variety of materials have been used including autografts (cortical/cancellous bone, bone marrow), allografts (demineralized freeze-dried/freeze-dried bone) and alloplastic materials (ceramics, hydroxyapatite, polymers and bioglass). However, investigators have challenged the suitability of these materials for use in periodontal regeneration due to their variability in safety, clinical effectiveness and stability over time.16
Emerging more recently as prospective alternatives to conventional treatments are biological approaches that are based on principles of tissue-engineering. Included in these approaches are gene therapy and the local administration of biocompatible scaffolds with or without the presence of selected growth factors. Since these new approaches are based on knowledge of the cell and molecular biology of the developing and regenerating periodontium, they provide interesting alternatives to existing therapies for repair and regeneration of the periodontium.16
A highly fibrous and vascular tissue, the periodontal ligament has one of the highest turnover rates in the body. Included in the many cells present in the periodontal ligament are cementoblasts, osteoblasts, fibroblasts, myofibroblasts, endothelial cells, nerve cells and epithelial cells. Additionally, in vivo cell kinetic studies have identified a smaller population of ‘progenitor cells’.16
Progenitor cell populations within the periodontal ligament have been reported to be enriched in locations adjacent to blood vessels, exhibiting some of the classical cytological features of stem cells, including small size, responsiveness to stimulating factors and slow cycle time.16 Thus, Bartold et al.16 investigated the theory that there are cells within this group that possess characteristics of mesenchymal stem cells competent to sustain renewal and tissue regeneration.
Nearly 20 years ago, Melcher was the first to propose that stem cells may reside in the periodontal tissues. He questioned whether cementoblasts, alveolar bone cells and periodontal ligament fibroblasts (the three cell populations of the periodontium) were derived ultimately from a single population of ancestral cells or 'stem cells'.16
Henceforth, in the literature, researchers have repeatedly referred to the proposed presence of stem cells within the periodontal ligament. Yet, researchers have provided only a small amount of direct evidence to support this theory. The in vivo and histological studies of McCulloch and coworkers have provided the most compelling evidence that stem cells are present within periodontal tissues.16
Periodontal regeneration can be considered a recreation of the developmental process including morphogenesis, cytodifferentiation, extracellular matrix production and mineralization. These processes affirm the theory that some mesenchymal stem cells remain within the periodontal ligament and thus promote tissue homeostasis; they provide a source of renewable progenitor cells which generate cementoblasts, osteoblasts and fibroblasts during adult life.16
If injury occurs in the periodontium, the mesenchymal stem cells may be stimulated to initiate terminal differentiation and regeneration or repair of tissues. A great number of cells of differing phenotype have been isolated from the periodontal ligament and regenerating periodontal tissue with the use of cloning techniques. Various clonal cell lines were reported to have characteristics of stem cells based on the results of preliminary investigations, thus justifying further studies of these properties and their application for cell-based periodontal regenerative therapies.16
Bartold et al.16 reported, from unpublished results, the identification and characterization of cell populations derived from adult human and sheep periodontal ligament that have the morphological, phenotypic and proliferative characteristics consistent with adult mesenchymal stem cells. The identification of reputed mesenchymal stem cell populations located in the periodontium has inspired interest in the future clinical utility of stem cell-based therapies to treat tissue injury resulting from trauma or periodontal disease.16
Friedenstein and co-investigators were the first to identify mesenchymal stem cells in aspirates of adult bone marrow “based upon their ability to form clonogenic clusters of adherent fibroblastic-like cells or fibroblastic colony-forming units with the potential to undergo extensive proliferation in vitro and to differentiate into different stromal cell lineages”.16 Bartold et al.16 used this same criteria to identify cells classified as mesenchymal stem cells, also obtained from the adult periodontal ligament (periodontal ligament stem cells). When plated under the same growth conditions as described for bone marrow stromal stem cells, these periodontal ligament stem cells demonstrated the ability to produce clonogenic adherent cell colonies.16
Fibroblastic colony-forming units were defined as aggregates of 50 cells or more.16 Bartold et al.16 reported the incidence of fibroblastic colony-forming units obtained from periodontal ligament (170) was greater than that that reported for bone marrow stromal stem cells (14) per 105 cells plated. It remains to be determined whether this is indicative of a propensity for stem cells to be present within periodontal ligament tissue or whether it is a reflection of a difference in stromal tissue turnover in periodontal ligament vs. bone marrow.16
Cloning of periodontal ligament fibroblasts in the past has largely been unsuccessful. Thus researchers resorted to using the technique of viral transfection to immortalize clonogenic cell lines.16 Transfection is a molecular biology term defined as “the introduction of DNA into a recipient eukaryote cell and its subsequent integration into the recipient cells chromosomal DNA. Only about 1% of cultured cells are normally transfected. Transfection is analogous to bacterial transformation but in eukaryotes transformation is used to describe the changes in cultured cells caused by tumor viruses”.3
Bartold et al.16 used specific culture conditions to establish some clones that manifested a high proliferative capacity. However, they found that many of the individually isolated colonies (> 80%) were incapable of proliferating beyond 20 population doublings. Thus, highly proliferative periodontal ligament stem cells constitute only a small number of the cells that are capable of being expanded in vitro over successive cell passages.16
They reported that periodontal ligament stem cell cultures demonstrated about 30% higher rates of proliferation in comparison to the growth of cultured bone marrow stromal stem cells. Apparently these cells retain their capacity for higher growth potential beyond the 100 population doublings before in vitro senescence is detected. Whereas, in vitro senescence occurs after approximately 50 population doublings for bone marrow stromal stem cells.16
What remains to be established is whether this proliferative characteristic of periodontal ligament stem cells is similar to that of dental pulp stem cells; because researchers have found elevated levels of the cell cycle activator, cyclin-dependent kinase 6, and the mitogen, insulin-like growth factor-2, and both are recognized mediators of cell cycle progression from G1 to the start of DNA synthesis.16
Nonetheless, these periodontal ligament stem cells go through senescence and thus have a finite lifespan, even though they demonstrate a high proliferative potential. Undergoing senescence seems to be a characteristic of most postnatal stem cells that particularly distinguishes them from embryonic stem cells, which are essentially immortal. Yet investigators recently reported that compared to bone marrow stromal stem cells, human peripheral fat-derived mesenchymal stem cells have a higher proliferation rate and demonstrate a tendency toward spontaneous immortalization following extensive cell passage.16
Enzyme telomerase is thought to play a significant role in maintaining telomere lengths and chromosomal stability during cellular division. Embryonic stem cells are known for their high expression of the enzyme telomerase, and it is associated with their immortal nature. In many mesenchymal stem cells, telomerase activity is absent. This absence may contribute to prolonging cellular senescence by controlling a number of key cell cycle regulators, which subsequently allow progression within the cell cycle from G1 to S phase, resulting in increased proliferation potential and survival rate. The lifespan of bone marrow stromal stem cells increases almost threefold when they are induced to express active telomerase.16
Therefore it is likely that scientists may develop techniques to genetically manipulate ex vivo expanded mesenchymal stem cells, such as periodontal ligament stem cells, to improve and control their growth properties in preparation for clinical applications. Restraint is advised in view of recent study results indicating that overexpressing-telomerase bone marrow stromal stem cell clones can develop into tumors.16
During embryogenesis, cells residing within the dental follicle form the periodontal ligament. It is believed that these cells are derived from the ectomesenchyme. It is unclear whether these cells are similar to the mesenchyme from which bone marrow stromal stem cells are derived.16
Scientists use the alleged stem cell marker, STRO-1, to isolate and purify bone marrow stromal stem cells. This same marker is also expressed by human periodontal ligament stem cells and dental pulp stem cells. Scientists can therefore utilize this expression to isolate human mesenchymal stem cells using immunomagnetic or fluorescence activated cell selection.16
Additionally, periodontal ligament stem cells share expression of the perivascular cell marker CD146 in common with bone marrow stromal stem cells. Researchers have also reported the coexpression by these cells of alpha-smooth muscle actin and/or the pericyte-associated antigen, 3G5.16 Such findings support the theory of a perivascular origin for these cells. Indeed, earlier scientific research demonstrated that progenitor cells reside within the perivascular spaces of mouse periodontal ligament. Even though these bone marrow stromal stem cells and periodontal ligament stem cells arise from different embryonic origins as independently unique stem cell populations, they inhabit a common environment in the perivascular niches in their respective tissues.16
Researchers have examined the phenotypic characteristics of batch cultures, single clone cultures of cells and mesenchymal stem cells expanded in vitro from dental pulp, periodontal ligament, cementum, alveolar bone and bone marrow. Unfortunately there was no evidence in periodontal ligament stem cells or bone marrow stromal stem cells of the hematopoietic markers CD14 (monocyte/macrophage), CD45 (common leukocyte antigen) and CD34 (hematopoietic stem/progenitor cells/endothelium). In spite of this significant absence, these cells do express many mature mineralized tissue markers, including alkaline phosphatase, type I collagen, osteonectin, osteopontin, osteocalcin and bone sialoprotein.16
Tendon and periodontal ligament tissues share some morphological and functional features such as dense collagen bundles and the capacity to absorb mechanical forces of stress and strain. Bartold et al.16 used semiquantitative reverse transcription-polymerase chain reaction to evaluate the expression levels of scleraxis, a tendon-specific transcription factor, in cultured human periodontal ligament stem cells. The results indicated that periodontal ligament stem cells expressed quantitatively higher levels of scleraxis transcripts compared to bone marrow stromal stem cells. From this they concluded that periodontal ligament stem cells probably symbolize a unique population of postnatal stem cells that are different from bone marrow-derived mesenchymal stem cells.16
Using cDNA microarray and proteomic technologies to achieve improved characterization of periodontal ligament stem cells, scientists are conducting studies to determine the genotypic and protein expression profiles of the periodontal ligament stem cells. Their goal is to identify key molecules that characterize these cells as well as other molecules which might play a role in the development of bone, cementum and periodontal ligament.16
Previous studies have indicated that, in the presence of inductive media containing ascorbic acid, dexamethasone and an excess of inorganic phosphate, human bone marrow stromal stem cells can be induced to form mineralized deposits in vitro, which are similar physiologically to hydroxyapatite in vivo. Researchers have confirmed that a subpopulation of cells derived from primary explants of periodontal ligament have demonstrated the ability to form mineralized deposits in vitro.16 An explant is a living tissue that has been transferred from an organism to an artificial medium for culture.3
More recent reports indicate that, under the same conditions, human periodontal ligament stem cells exhibited a similar capacity to form Alizarin Red-positive mineralized deposits in vitro. The fact that periodontal ligament stem cells form Oil-red O-positive lipid-containing clusters of fat cells when cultured in the presence of adipogenic inductive medium, further confirms their multipotential capabilities.16
Determining their capacity to form an organized, functional tissue following implantation in vivo will be the next step in the characterization of periodontal ligament stem cells. In order to achieve the induction of bone, dentin, and cementum formation in vivo, it appears that these cells generally require a suitable scaffold. Investigators have reported the formation of a typical cementum/periodontal ligament-like structure when periodontal ligament stem cells are incorporated into a hydroxyapatite/tricalcium phosphate scaffold and implanted subcutaneously into immunocompromised mice. Additionally, these xenografts formed a type I collagen-positive periodontal ligament-like tissue, morphologically similar to Sharpey's fibers, within the transplants connecting with the newly formed cementum.16
Researchers have clearly identified the cells responsible for the regeneration of these tissues in the xenografts as being of human origin by using human-specific antimitochondria antibodies. Of further interest is the degree of hetereogeneity in morphological characteristics, differentiation potential and proliferative capacities demonstrated by ex vivo expanded periodontal ligament-derived fibroblastic colony-forming unit clones.16
This finding suggests that there is a mixture of stromal progenitor cells at various stages of development within the total fibroblastic colony-forming unit population, and that they are probably maintained by a minor population of multipotential, mesenchymal stem cells with the capacity for self-renewal, that was previously described for the bone marrow stromal stem cell system.16
Once the periodontal tissues have been destroyed, the regeneration of affected tissues to their original architecture and function becomes one of the major goals of periodontal therapy. While various surgical procedures have been recommended to achieve periodontal regeneration, the most recent is the use of synthetic barrier membranes to encourage appropriate progenitor cell population of the wound site. To its credit, this method has demonstrated the potential for regeneration of the root surface cementum, alveolar bone and periodontal ligament. However, the clinical results can be unpredictable and demonstrate great variation.16
Research has led to an increased understanding of the molecular processes associated with tissue repair and regeneration. Consequently, application of polypeptide growth factors to root surfaces has been used to facilitate periodontal regeneration, including epidermal growth factor, fibroblast growth factor, insulin-like growth factor, platelet-derived growth factor, tumor-derived growth factor and bone morphogenetic proteins. Also useful in promoting periodontal regeneration are combinations of growth factors such as those present in platelet-rich plasma preparations. However the body of literature remains negligible that documents the clinical outcomes of using such combinations.16
Another approach was being generated based on the current knowledge of tooth root formation and, in particular, cementum formation at the same time that polypeptide growth factors were being evaluated for periodontal regeneration. Although the exact molecular mechanisms that produce cementum remain ambiguous, a new theory proposes that a special matrix deposited on the newly formed dentin surface enables the attachment and differentiation of progenitor cells into cementoblasts. However, that theory has yet to be widely accepted.16
In an attempt to recreate the molecular process of cementogenesis, researchers have applied extracts of this matrix to root surfaces at the time of periodontal surgery with the goal of inducing periodontal regeneration. They are not sure yet whether these proteins act like growth factors as instructional messengers which stimulate cells to undergo regeneration, or if they simply act as a scaffold allowing regeneration to occur.16
Although the regeneration of periodontal tissues in response to these proteins has not been entirely predictable or consistent, they do appear capable of promoting periodontal tissue regeneration with encouraging clinical results.16 According to Bartold et al.16 the critical factors needed to achieve successful periodontal regeneration are the accurate recruitment of correct cells to the site and the generation of an appropriate extracellular matrix consistent with the periodontal tissues.
Since cell seeding has been used successfully to promote regeneration of other tissues (skin, cartilage, bone, cardiovascular components, pancreas, etc.), Bartold et al.16 proposed that it is reasonable to expect “that autologous periodontal ligament stem cells cultured within a suitable delivery scaffold, in conjunction with the growth and differentiation factors present in an autologous blood clot, will lead to new periodontal tissue attachment via a tissue engineering approach”.
The principles of cell biology, developmental biology, and biomaterials are the cornerstones of tissue engineering, whose goal is the development of techniques for the production of new tissues to restore damaged or diseased tissues. Successful tissue engineering of cartilage, bone and other tissues has resulted in part due to recent advances in growth factor biology and biodegradable polymers; it is believed that periodontal tissues are a potential primary candidate for such techniques.16
Current studies demonstrated that there are no adverse immunologic or inflammatory sequelae when periodontal ligament cells are transplanted into periodontal defects. More recently investigators have used periodontal tissue engineering models to examine cementoblasts and various other periodontal cells transfected with vectors for overexpression of various growth factors.16
A strategy that utilizes tissue engineering for periodontal regeneration by taking advantage of the regenerative capacity of stem cells residing within the periodontium is an appealing argument. Such an approach eliminates the need for cell recruitment to the site and might possibly enhance the predictability of the outcome.16
For a number of years, science has used mobilized peripheral blood stem cells as an accepted form of therapy for hematopoietic bone marrow reconstitution in cancer patients being treated with myeloablative therapy.16 Myeloablative agonists are agents that destroy bone marrow activity. They are used to prepare patients for bone marrow or stem cell transplantation.3
Based on the success of this therapy, scientists have investigated other stem cell populations, such as bone marrow stromal stem cells, for their potential as promising novel cellular-based therapies for a number of diseases and congenital defects of neural, bone, cartilage and muscle tissues. Collectively, these studies illustrate the clinical potential of bone marrow stromal stem cells and other mesenchymal stem cells for various tissue engineering techniques in the regeneration of tissue. Studies have revealed the presence of different mesenchymal stem cells residing in dental or craniofacial tissues. This discovery invites the use of these cells in future clinical investigations for the regeneration of tissues of the orofacial region, including the periodontium.16
Bartold et al.16 originated a number of studies to investigate the use of these cells for periodontal regeneration to determine if periodontal ligament stem cells possess a tissue regenerative capacity comparable to that of bone marrow stromal stem cells. They implanted cultured human periodontal ligament stem cells into surgically created periodontal defects in nude rats. They reported that that the periodontal ligament stem cells attached to both the alveolar bone and cementum surfaces resulting in the formation of a periodontal ligament-like structure.16
More recently, Bartold et al.16 reported in unpublished data the identification of “ovine counterparts of human bone marrow stromal stem cells and periodontal ligament stem cells, which demonstrate similar functional properties when transplanted into immunocompromised mice with the hydroxyapatite/tricalcium phosphate carrier particles”. The term ovine means relating to, affecting, resembling or derived from a sheep.3
Presently, they are attempting to regenerate defects created in alveolar bone, periodontal ligament and cementum. In an established ovine preclinical model, they are utilizing the selective implantation of autologous bone marrow stromal stem cells and periodontal ligament stem cells combined with different biocompatible materials/scaffolds.16
Encouraged by new reports that adult human stem cell populations reside in the periodontal ligament, researchers must further determine if the cells can be utilized clinically. The next step is to determine how useful ex vivo expanded stem cells are in repairing periodontal defects. They also need to develop techniques to identify and maintain multipotential stem cells in vitro.16
Furthermore, they must establish the growth and differentiation conditions that induce lineage specific commitment. In addition it is necessary to develop appropriate carriers and inductive factors capable of promoting implant integration into the surrounding environment for the reconstruction of functional complex organ systems. Success will depend on the cooperation of cell biologists, matrix biologists, pharmacologists, biomaterials scientists/engineers and nanotechnologists to resolve the multiplicity of issues associated with tissue engineering.16