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Periodontal Ligament Stem Cells: An Overview
J. Oral Biosci. 52 （3）：275−282, 2010
REVIEW
Periodontal Ligament Stem Cells：An Overview Aneesha Acharya§, Sharath Shetty and Vijay Deshmukh Department of Periodontology and Oral Implantology, Dr D. Y. Patil Dental College and Hospital, Pimpri, Pune Sant Tukaram Nagar, Pimpri, Pune, India−411018 〔Received September 25, 2009；Accepted May 8, 2010〕 Key words：stem cell／mesenchymal／periodontal ligament／tissue engineering Abstract：Adult postnatal stem cells are multipotent and can be experimentally induced to differentiate into various specialized cell lineages. This has generated considerable interest in the arena of stem cell− based therapeutics. The identification of stem cells within the periodontal ligament represents a significant development in this regard. Achieving predictable periodontal regeneration has long been a challenge, and it is known that cells involved in the mechanisms of periodontal wound healing are of mesenchymal stem cell （MSC）type. Thus, periodontal ligament stem cell（PDLSC）−based therapeutics may be a step towards predictable periodontal regeneration. Additionally, PDLSC may have alternative potential applications in hard tissue and tooth engineering. PDLSC may be isolated, grown under tissue culture conditions, expanded, optionally genetically modified and then collected and transplanted. This paper aims to overview the current knowledge, recent developments and methodology regarding PDLSC−based applications.
Introduction A stem cell is defined as a cell that can continuously produce unaltered daughter cells and has the ability to generate cells with different and more restricted properties1）. Stem cells can be categorized into two groups―embryonic and adult. Though embryonic stem cells are totipotent, regulatory issues concerning their procurement and use have caused current attention to be directed toward stem cells derived from adult tissues；adult postnatal stem cells. These cells are slow cycling, and are ‘self− renewing’ cells or can generate large numbers of progeny. They are multipotent and differentiate into intermediate cell types, called precursor or progenitor ；the differentiating cells with‘clonogenic potential’2） §
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ability into cell types of the tissue in which they reside3）. Having more restricted differentiation potential than totipotent embryonic stem cells, postnatal stem cells can thus be classified depending on their origin and differentiation potential. Common examples are hematopoietic and mesenchymal stem cells 4） first described （MSC）. Friedenstein et al. （1970） MSC in bone marrow. They described MSC as fibroblast−like cells with the ability to assume an osteogenic phenotype in diffusion chamber cell suspensions when isotransplanted in to mice. Although MSC have recently been isolated from a variety of adult and embryonic tissues5）, presently, bone marrow represents the major source of postnatal MSC and is the most studied. However, bone marrow aspiration is an invasive procedure and, therefore, the search for alternative sources of MSC, and research regarding their potency is of significant value.
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Stem Cells in Dental Tissues
Identification and Characterization of PDLSC
Five different sources of MSC in human postnatal dental tissue have been identified：dental pulp, periodontal ligament, exfoliated deciduous teeth, dental follicle and root apical papilla. MSC populations have been identified in the adult human dental pulp and are thought to reside in a perivascular niche6,7）. Postnatal MSC have also been isolated from soft tissue surrounding apices of developing human permanent teeth or root apical dental papilla8）, the dental follicle （the connective tissue sheath surrounding developing teeth） in developing human third molar and rodent teeth9―11） and from the dental pulp of exfoliated human deciduous teeth12）. With regard to dental tissues, epithelial stem cells （EpSC）are the precursors of ameloblasts. Interestingly, postnatal EpSC have been demonstrated only in the‘cervical loop’ present at apices of rodent incisor teeth13,14）. Rodent incisors continually erupt through the life of the animal as most apical cells retain the ability to proliferate and differentiate into different dental tissues, including enamel14）. In humans, EpSC have not been isolated from postnatal dental tissues, as ameloblasts and their precursor cells do not remain viable after eruption, although recently, evidence of the presence of ‘EpSC−like cells’ in dental pulp of deciduous teeth has been demonstrated15）.
No specific shared definitive means of characterization or a single antigenic marker has been recognized for adult stem cell identification, unlike embryonic stem cells, which are defined by their blastocyst origin. Thus, for MSC, combinations of cell surface antibodies must be utilized as identification profiles. Presently, MSC are defined as plastic adherent, multipotential fibroblast−like cells expressing CD73, CD105, and CD13, and negative for hematopoietic markers CD14, CD34, CD 38 and CD4520）, although some of these properties and markers are also shared by stromal cells, such as fibroblasts21）. Stromal precursor cell marker （STRO 1）, other stromal markers including CD146（MUC 18） , pericyte−associated antigen；CD106 （Vascular cell adhesion molecule−1 or VCAM−1）, pericyte associated antigen；3G5, along with CD44, alkaline phosphatase, and α−smooth muscle actins have been used to identify MSC in bone marrow7,22―25）. Also, as the properties of MSCs may be altered by culture conditions26）, identification of specific MSC markers for their prospective isolation from primary tissues is an area of research. Although there is no consensus on the best characterization method to prospectively isolate MSC from the various source tissues, MSC in the periodontal ligament may be isolated in vitro using these available markers. However, caution needs to be maintained while extrapolating data regarding CD antigen expression patterns in rodent and other animal models to human cells as significant differences may exist between the two. Human PDLSC were first isolated by Seo et al. in 200427）. Cell lines from the periodontal ligament have been shown to express mesenchymal stem cell markers in various studies in both human27―29）and animal models30―32）, including CD146, STRO−1, CD105, 32） , along with a CD16629,33）, and CD106 （VCAM−1） high level of a tendon−specific marker, Scleraxis （SCX）32,34）, and other stromal cell markers, CD90, CD29, CD44, CD105, and CD1335）. Cells denoted as PDLSC have been shown as lacking hematopoietic markers, CD14, CD45, and CD3132）.
Stem Cells in the Periodontal Ligament （PDLSC） The periodontal ligament’s known ability to establish new attachment fibers between the cementum and bone to achieve regeneration16） implies that progenitor cells, and possibly stems cells, exist within the periodontal ligament cell populations 17）, as opposed to gingival connective tissue, which lacks this potential18）. As the cell population in the periodontal ligament is heterogeneous19）, in order to specifically identify and isolate the subset of stem cells within the periodontium, marker identification that can be used to distinguish these types of cells is essential.
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An area of concern is that cultured cell lines designated as putative PDLSC on the basis of marker expression, such as Sro−1, are actually heterogeneous subpopulations, including progenitor cells at various stages of differentiation along other specific lineages29）. In a significant development in this regard, a recent study reported a highly purified clonal isolation of a homogeneous single cell−derived PDLSC subpopulation, which was obtained from a primary non−transformed human periodontal ligament36）. Molecular gene markers facilitate more precise means of distinguishing unique cell subsets and generation of a proteomic profile will help identify markers very specific to PDLSC. Messenger RNA levels of molecular gene markers, apolipoprotein D, major , histocompatibility complex−DR−alpha（MHC−DR α） and major histocompatibility complex−DR−beta （MHC−DR β）, have been shown to distinguish PDLSC from bone marrow MSC（BMSC）37）. Recently, a specific gene for F−spondin, indicative of an early stage dental follicular cell type with clonogenic potential has been proposed as a marker for PDLSC, along with negativity for Tenascin−N；a gene indicative of terminally differentiated periodontal ligament cells38,39）. Such data will help determine the underlying molecular mechanisms that mediate the regenerative potential of stem and progenitor cells and have important ramifications for increasing the purity of preparations and monitoring their development during ex vivo expansion. Developmental Origins of PDLSC Interestingly, a common perivascular origin has been suggested for MSC detected in various tissues, including bone marrow, based on immunophenotypical characteristics shared with cultured perivascular cells40,41）. In agreement with this hypothesis, PDLSC have been found to be concentrated in paravascular areas and inflamed samples33）and express some such shared markers with pericytes, including CD146, CD44, CD10529,33）. Also notable is that, developmentally, the periodontal ligament originates from migrating cranial neural crest cells contained in the dental follicle, by a series of progressive restrictions, and a
subset of neural crest cells is considered to retain multipotent42,43）. Cultures of multipotent rat PDLSC with neural crest features have been demonstrated44）. Substantiating evidence is also provided by recent reports that human PDLSC subpopulations express markers of neural crest lineage, including Nestin, and other markers of undifferentiated neural crest cells, such as HNKI, p7545,46）. Slug and Sox1046）. It may thus be envisioned that the periodontal ligament area contains stem and progenitor cell populations that are ontologically derived from native host tissue of neural crest origin, as well as those of mesodermal origin derived from surrounding vasculature and bone marrow. Differentiation Potential of PDLSC The multilineage differentiation potential of PDLSC, including adipogenic, ostoegenic and chondrogenic potential, has been demonstrated in various ；however, the extent to which in vitro studies27,28,31,32） differentiation capacity is physiologically relevant remains unclear and, at present, is a key issue for the field. More recently, neurogenic differentiation from murine44） and human PDLSC subsets demonstrating neural crest markers has been demonstrated46）. Osteogenic Potential PDLSC are shown to have osteogenic potential, similar to BMSC, as suggested by in vitro studies demonstrating mineralized nodule formation under appropriate culture conditions27,32,47,48）, although in vitro mineralization cannot be considered a definitive indicator of in vivo osteogenic differentiation potential in the true sense. In this regard, it is interesting to note that many studies have reported PDLSC isolates to have lower osteogenic potential than BMSC28,35,49,50） and also dental pulp−derived stem cells27,49）. Seo et al.27） showed the inability of PDLSC to form bone in vivo, as opposed to reports by others, including Kim et al.50）, who recently reported new bone formation by PDLSC in a peri−implant defect model, albeit at lower levels than BMSC. Some of the variations amongst reports could be attributed to different
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marker profiles, culture techniques and scaffold adoption. Thus, although the potential use of PDLSC for generating graft biomaterials for bone tissue engineering in regenerative dentistry can be envisioned, as these cells are more routinely accessible, it is however necessary to delineate more refined isolates of pluripotent progenitors using genomic and proteomic marker characterization. In periodontal ligament cell lines, homeobox protein Msx2 expression has been shown to coincide with the suppression of osteoblastic differentiation and mineralization51）. More recently, the molecular marker periodontal ligament−associ／asporin has been identified ated protein−1（PLAP−1） by Yamada et al.52,53） as being specific to periodontal ligament phenotype and to inhibit mineralization. Periodontal Regeneration by PDLSC PDLSC represent a novel stem cell population, in terms of in vivo capacity to develop into cementoblast−like cells, and cementum／periodontal ligament− like tissue, as evidenced positively in preclinical studies27）. Seo et al.27）, using a rodent model, demonstrated a cementum／PDL−like complex generated in surgically created periodontal defects by transplanting in vitro expanded human PDLSCs in a ceramic particle scaffold. There is a similar report of histologic periodontal regeneration in vivo by expanded autologous PDLSC in a swine model30）. Another porcine model study reports transplanting autologous swine PDLSCs, which lead to the generation of a root／periodontal complex capable of supporting a porcelain crown, resulting in normal tooth function54）. Besides periodontal regeneration, another potential application of PDLSCs is in the area of hybrid ‘tooth engineering’55） in combination with other stem and progenitor cell populations and scaffolds. BMSC have been shown to form de novo organized dental tissue with a regenerated periodontal complex when used in an animal model56）. In a recent study, Ma et al.56） showed that in vitro induction of PDLSC with dentin noncollagenous proteins increased cell differentiation along the cementoblast lineage, denoting a potential inductive role of root surface in the activation of PDLSC differentiation, which can be utilized for bio−
engineering applications. Periodontal tissue engineering using PDLSC conventionally needs 3−D biomaterial scaffold technology that can closely mimic the effect of extracellular matrix（ECM）derived signals for optimal differentiation；however, there are inherent shortages in current scaffold technology. This has led to the development scaffold−free methodology for PDLSC transplantation, such as cell sheets57） and recently, a promising novel 3D human PDLSC cell pellet, which self−secretes ECM and has favorable fabrication and handling, demonstrated the formation of a cementum／PDL−like complex on transplantation into immunocompromised mice58）. It is of interest to note that cryopreserved PDLSC may be collected and saved for future use through preservation techniques such as freezing in liquid nitrogen. Seo et al.59） reported that periodontal ligament, preserved frozen in liquid nitrogen, generated high proliferative PDLSC, although the number of PDLSC colonies derived was decreased in comparison with freshly isolated tissue samples. Thus, in future, use of cryopreserved PDLSC could widen the application arena. Microenvironment and Ageing Influences Stem cell properties are highly sensitive to external influences. Ageing adversely affects the mitogenicity of stem cells. In a recent sudy, Zheng et al.60） exposed aged PDLSC to young periodontal ligament cell−conditioned medium（PLC−CM）and vice versa. It was observed that aged PDLSC induced by young PLC−CM showed enhanced cemental tissue−regenerative capacity but young PDLSC induced by aged PLC−CM mainly formed connective tissues. Such findings further stress the key role of the extrinsic microenvironment in modulating PDLSC activity. For example, addition of dexamethasone substantially enhances osteoblastic differentiation in human periodontal ligament cells by decreasing endogenous collagenase expression, which is likely to be a regulator of the differentiation phenotype61）. The elucidation of appropriate local environmental signaling molecules and pathways is essential for fine− tuning potential regenerative applications. Currently,
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the onus is on human clinical trials to assess periodontal regenerative outcomes by implantation of ex vivo cultured PDLSC and other progenitor cells, and the modulating factors involved, such as microenvironment characteristics.
Immunomodulatory Properties of PDLSC There is much in vitro evidence indicating that various MSC are hypoimmunogenic, and also modulate the T cell response, independent of major histocompatibility complex （MHC） expression62,63）. In vitro, human MSCs express intermediate levels of human leukocyte antigen（HLA）, MHC class I molecules, are negative for MHC class II molecules （but can be induced to express MHC class II by interferon gamma）, and lack co−stimulatory molecules B7−1, B7−2, CD40, and CD40 ligand64）. Consequently, these cells may escape recognition by alloreactive T cells62,63）. In agreement, Wada et al. in 200949）showed that ex vivo expanded PDLSCs demonstrated in vitro immunosuppressive ability by inhibiting the proliferation of mononuclear cells, partly attributed to induced soluble factors such as hepatocyte growth factor and indoleamine 2, 3−dioxygenase（IDO）production. However, very significantly, contradictory in vivo findings have been noted, showing a strong cellular immune response to transplanted MSC, indicating a possible alteration of antigen expression in vivo65,66）. Hence, the in vivo utility of allogeneic PDLSC remains highly questionable, in line with earlier reports showing that human allogeneic tooth transplantations cause rejection due to immune−mediated osteoclast activation67）. Although a recent animal study showed evidence that, after allogeneic tooth transplantation, the donor periodontal tissue was replaced and regenerated by host cells without exhibiting a MHC−mediated host immune response68）, findings from in vitro and inbred animal studies need to considered with caution and validated in appropriate preclinical trials. Further studies are needed to investigate any potential value of PDLSC purported immunomodualory activity in actual clinical settings.
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61）Hayami, T., Quin, Z., Kapila, Y. and Kapila, S.： Dexamethasone’s enhancement of osteoblastic markers in human periodontal ligament cells is associated with inhibition of collagenase expression. Bone 40： 93―104, 2007. 62）Jones, B. J., Brooke, G., Atkinson, K. and McTaggart, S. J.：Immunosuppression by placental indoleamine 2, 3−dioxygenase：A role for mesenchymal stem cells. Placenta 28：1174―1181, 2007. 63）Aggarwal, S. and Pittenger, M. F.：Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 105：1815―1822, 2005. 64）Majumdar, M. K., Keane−Moore, M., Buyaner, D., Hardy, W. B., Moorman, M. A., McIntosh, K. R. and Mosca, J. D.：Characterization and functionality of cell surface molecules on human mesenchymal stem cells. J. Biomed. Sci. 10：228―241, 2003. 65）Prigozhina, T. B., Khitrin, S., Elkin, G., Eizik, O., Morecki, S. and Slavin, S.：Mesenchymal stromal cells lose their immunosuppressive potential after allotransplantation. Exp. Hematol. 36：1370―1376, 2008. 66）Camp, D. M., Loeffler, D. A., Farrah, D. M., Borneman, J. N. and LeWitt, P. A.：Cellular immune response to intrastriatally implanted allogeneic bone marrow stromal cells in a rat model of Parkinson’s disease. J. Neuroinflam. 6：17, 2009. 67）Schwartz, O., Frederiksen, K. and Klausen, B.：Allotransplantation of human teeth. A retrospective study of 73 transplantations over a period of 28 years. Int. J. Oral Maxillofac. Surg. 16：285―301, 1987. 68）Kim, E., Cho, S. W., Yang, J. Y., Cai, J., Lee, S. L., Ohshima, H. and Jung, H. S.：Tooth survival and periodontal tissues healing of allogenic−transplanted teeth in the mice. Oral Dis. 12：395―401, 2006. 69）Bartold, P. M., McCulloch, C. A., Narayanan, A. and Pitaru, S.：Tissue engineering：a new paradigm for periodontal regeneration based on molecular and cell biology. Periodontology 2000 24：253―269, 2000. 70）Godara, P., McFarland, C. D. and Nordon, R. E.： Design of bioreactors for mesenchymal stem cell tissue engineering. J. Chem. Tech. Biotech. 83：408― 420, 2008.
REVIEW
Periodontal Ligament Stem Cells：An Overview Aneesha Acharya§, Sharath Shetty and Vijay Deshmukh Department of Periodontology and Oral Implantology, Dr D. Y. Patil Dental College and Hospital, Pimpri, Pune Sant Tukaram Nagar, Pimpri, Pune, India−411018 〔Received September 25, 2009；Accepted May 8, 2010〕 Key words：stem cell／mesenchymal／periodontal ligament／tissue engineering Abstract：Adult postnatal stem cells are multipotent and can be experimentally induced to differentiate into various specialized cell lineages. This has generated considerable interest in the arena of stem cell− based therapeutics. The identification of stem cells within the periodontal ligament represents a significant development in this regard. Achieving predictable periodontal regeneration has long been a challenge, and it is known that cells involved in the mechanisms of periodontal wound healing are of mesenchymal stem cell （MSC）type. Thus, periodontal ligament stem cell（PDLSC）−based therapeutics may be a step towards predictable periodontal regeneration. Additionally, PDLSC may have alternative potential applications in hard tissue and tooth engineering. PDLSC may be isolated, grown under tissue culture conditions, expanded, optionally genetically modified and then collected and transplanted. This paper aims to overview the current knowledge, recent developments and methodology regarding PDLSC−based applications.
Introduction A stem cell is defined as a cell that can continuously produce unaltered daughter cells and has the ability to generate cells with different and more restricted properties1）. Stem cells can be categorized into two groups―embryonic and adult. Though embryonic stem cells are totipotent, regulatory issues concerning their procurement and use have caused current attention to be directed toward stem cells derived from adult tissues；adult postnatal stem cells. These cells are slow cycling, and are ‘self− renewing’ cells or can generate large numbers of progeny. They are multipotent and differentiate into intermediate cell types, called precursor or progenitor ；the differentiating cells with‘clonogenic potential’2） §
Corresponding author E−mail：[email protected]
ability into cell types of the tissue in which they reside3）. Having more restricted differentiation potential than totipotent embryonic stem cells, postnatal stem cells can thus be classified depending on their origin and differentiation potential. Common examples are hematopoietic and mesenchymal stem cells 4） first described （MSC）. Friedenstein et al. （1970） MSC in bone marrow. They described MSC as fibroblast−like cells with the ability to assume an osteogenic phenotype in diffusion chamber cell suspensions when isotransplanted in to mice. Although MSC have recently been isolated from a variety of adult and embryonic tissues5）, presently, bone marrow represents the major source of postnatal MSC and is the most studied. However, bone marrow aspiration is an invasive procedure and, therefore, the search for alternative sources of MSC, and research regarding their potency is of significant value.
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Stem Cells in Dental Tissues
Identification and Characterization of PDLSC
Five different sources of MSC in human postnatal dental tissue have been identified：dental pulp, periodontal ligament, exfoliated deciduous teeth, dental follicle and root apical papilla. MSC populations have been identified in the adult human dental pulp and are thought to reside in a perivascular niche6,7）. Postnatal MSC have also been isolated from soft tissue surrounding apices of developing human permanent teeth or root apical dental papilla8）, the dental follicle （the connective tissue sheath surrounding developing teeth） in developing human third molar and rodent teeth9―11） and from the dental pulp of exfoliated human deciduous teeth12）. With regard to dental tissues, epithelial stem cells （EpSC）are the precursors of ameloblasts. Interestingly, postnatal EpSC have been demonstrated only in the‘cervical loop’ present at apices of rodent incisor teeth13,14）. Rodent incisors continually erupt through the life of the animal as most apical cells retain the ability to proliferate and differentiate into different dental tissues, including enamel14）. In humans, EpSC have not been isolated from postnatal dental tissues, as ameloblasts and their precursor cells do not remain viable after eruption, although recently, evidence of the presence of ‘EpSC−like cells’ in dental pulp of deciduous teeth has been demonstrated15）.
No specific shared definitive means of characterization or a single antigenic marker has been recognized for adult stem cell identification, unlike embryonic stem cells, which are defined by their blastocyst origin. Thus, for MSC, combinations of cell surface antibodies must be utilized as identification profiles. Presently, MSC are defined as plastic adherent, multipotential fibroblast−like cells expressing CD73, CD105, and CD13, and negative for hematopoietic markers CD14, CD34, CD 38 and CD4520）, although some of these properties and markers are also shared by stromal cells, such as fibroblasts21）. Stromal precursor cell marker （STRO 1）, other stromal markers including CD146（MUC 18） , pericyte−associated antigen；CD106 （Vascular cell adhesion molecule−1 or VCAM−1）, pericyte associated antigen；3G5, along with CD44, alkaline phosphatase, and α−smooth muscle actins have been used to identify MSC in bone marrow7,22―25）. Also, as the properties of MSCs may be altered by culture conditions26）, identification of specific MSC markers for their prospective isolation from primary tissues is an area of research. Although there is no consensus on the best characterization method to prospectively isolate MSC from the various source tissues, MSC in the periodontal ligament may be isolated in vitro using these available markers. However, caution needs to be maintained while extrapolating data regarding CD antigen expression patterns in rodent and other animal models to human cells as significant differences may exist between the two. Human PDLSC were first isolated by Seo et al. in 200427）. Cell lines from the periodontal ligament have been shown to express mesenchymal stem cell markers in various studies in both human27―29）and animal models30―32）, including CD146, STRO−1, CD105, 32） , along with a CD16629,33）, and CD106 （VCAM−1） high level of a tendon−specific marker, Scleraxis （SCX）32,34）, and other stromal cell markers, CD90, CD29, CD44, CD105, and CD1335）. Cells denoted as PDLSC have been shown as lacking hematopoietic markers, CD14, CD45, and CD3132）.
Stem Cells in the Periodontal Ligament （PDLSC） The periodontal ligament’s known ability to establish new attachment fibers between the cementum and bone to achieve regeneration16） implies that progenitor cells, and possibly stems cells, exist within the periodontal ligament cell populations 17）, as opposed to gingival connective tissue, which lacks this potential18）. As the cell population in the periodontal ligament is heterogeneous19）, in order to specifically identify and isolate the subset of stem cells within the periodontium, marker identification that can be used to distinguish these types of cells is essential.
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An area of concern is that cultured cell lines designated as putative PDLSC on the basis of marker expression, such as Sro−1, are actually heterogeneous subpopulations, including progenitor cells at various stages of differentiation along other specific lineages29）. In a significant development in this regard, a recent study reported a highly purified clonal isolation of a homogeneous single cell−derived PDLSC subpopulation, which was obtained from a primary non−transformed human periodontal ligament36）. Molecular gene markers facilitate more precise means of distinguishing unique cell subsets and generation of a proteomic profile will help identify markers very specific to PDLSC. Messenger RNA levels of molecular gene markers, apolipoprotein D, major , histocompatibility complex−DR−alpha（MHC−DR α） and major histocompatibility complex−DR−beta （MHC−DR β）, have been shown to distinguish PDLSC from bone marrow MSC（BMSC）37）. Recently, a specific gene for F−spondin, indicative of an early stage dental follicular cell type with clonogenic potential has been proposed as a marker for PDLSC, along with negativity for Tenascin−N；a gene indicative of terminally differentiated periodontal ligament cells38,39）. Such data will help determine the underlying molecular mechanisms that mediate the regenerative potential of stem and progenitor cells and have important ramifications for increasing the purity of preparations and monitoring their development during ex vivo expansion. Developmental Origins of PDLSC Interestingly, a common perivascular origin has been suggested for MSC detected in various tissues, including bone marrow, based on immunophenotypical characteristics shared with cultured perivascular cells40,41）. In agreement with this hypothesis, PDLSC have been found to be concentrated in paravascular areas and inflamed samples33）and express some such shared markers with pericytes, including CD146, CD44, CD10529,33）. Also notable is that, developmentally, the periodontal ligament originates from migrating cranial neural crest cells contained in the dental follicle, by a series of progressive restrictions, and a
subset of neural crest cells is considered to retain multipotent42,43）. Cultures of multipotent rat PDLSC with neural crest features have been demonstrated44）. Substantiating evidence is also provided by recent reports that human PDLSC subpopulations express markers of neural crest lineage, including Nestin, and other markers of undifferentiated neural crest cells, such as HNKI, p7545,46）. Slug and Sox1046）. It may thus be envisioned that the periodontal ligament area contains stem and progenitor cell populations that are ontologically derived from native host tissue of neural crest origin, as well as those of mesodermal origin derived from surrounding vasculature and bone marrow. Differentiation Potential of PDLSC The multilineage differentiation potential of PDLSC, including adipogenic, ostoegenic and chondrogenic potential, has been demonstrated in various ；however, the extent to which in vitro studies27,28,31,32） differentiation capacity is physiologically relevant remains unclear and, at present, is a key issue for the field. More recently, neurogenic differentiation from murine44） and human PDLSC subsets demonstrating neural crest markers has been demonstrated46）. Osteogenic Potential PDLSC are shown to have osteogenic potential, similar to BMSC, as suggested by in vitro studies demonstrating mineralized nodule formation under appropriate culture conditions27,32,47,48）, although in vitro mineralization cannot be considered a definitive indicator of in vivo osteogenic differentiation potential in the true sense. In this regard, it is interesting to note that many studies have reported PDLSC isolates to have lower osteogenic potential than BMSC28,35,49,50） and also dental pulp−derived stem cells27,49）. Seo et al.27） showed the inability of PDLSC to form bone in vivo, as opposed to reports by others, including Kim et al.50）, who recently reported new bone formation by PDLSC in a peri−implant defect model, albeit at lower levels than BMSC. Some of the variations amongst reports could be attributed to different
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marker profiles, culture techniques and scaffold adoption. Thus, although the potential use of PDLSC for generating graft biomaterials for bone tissue engineering in regenerative dentistry can be envisioned, as these cells are more routinely accessible, it is however necessary to delineate more refined isolates of pluripotent progenitors using genomic and proteomic marker characterization. In periodontal ligament cell lines, homeobox protein Msx2 expression has been shown to coincide with the suppression of osteoblastic differentiation and mineralization51）. More recently, the molecular marker periodontal ligament−associ／asporin has been identified ated protein−1（PLAP−1） by Yamada et al.52,53） as being specific to periodontal ligament phenotype and to inhibit mineralization. Periodontal Regeneration by PDLSC PDLSC represent a novel stem cell population, in terms of in vivo capacity to develop into cementoblast−like cells, and cementum／periodontal ligament− like tissue, as evidenced positively in preclinical studies27）. Seo et al.27）, using a rodent model, demonstrated a cementum／PDL−like complex generated in surgically created periodontal defects by transplanting in vitro expanded human PDLSCs in a ceramic particle scaffold. There is a similar report of histologic periodontal regeneration in vivo by expanded autologous PDLSC in a swine model30）. Another porcine model study reports transplanting autologous swine PDLSCs, which lead to the generation of a root／periodontal complex capable of supporting a porcelain crown, resulting in normal tooth function54）. Besides periodontal regeneration, another potential application of PDLSCs is in the area of hybrid ‘tooth engineering’55） in combination with other stem and progenitor cell populations and scaffolds. BMSC have been shown to form de novo organized dental tissue with a regenerated periodontal complex when used in an animal model56）. In a recent study, Ma et al.56） showed that in vitro induction of PDLSC with dentin noncollagenous proteins increased cell differentiation along the cementoblast lineage, denoting a potential inductive role of root surface in the activation of PDLSC differentiation, which can be utilized for bio−
engineering applications. Periodontal tissue engineering using PDLSC conventionally needs 3−D biomaterial scaffold technology that can closely mimic the effect of extracellular matrix（ECM）derived signals for optimal differentiation；however, there are inherent shortages in current scaffold technology. This has led to the development scaffold−free methodology for PDLSC transplantation, such as cell sheets57） and recently, a promising novel 3D human PDLSC cell pellet, which self−secretes ECM and has favorable fabrication and handling, demonstrated the formation of a cementum／PDL−like complex on transplantation into immunocompromised mice58）. It is of interest to note that cryopreserved PDLSC may be collected and saved for future use through preservation techniques such as freezing in liquid nitrogen. Seo et al.59） reported that periodontal ligament, preserved frozen in liquid nitrogen, generated high proliferative PDLSC, although the number of PDLSC colonies derived was decreased in comparison with freshly isolated tissue samples. Thus, in future, use of cryopreserved PDLSC could widen the application arena. Microenvironment and Ageing Influences Stem cell properties are highly sensitive to external influences. Ageing adversely affects the mitogenicity of stem cells. In a recent sudy, Zheng et al.60） exposed aged PDLSC to young periodontal ligament cell−conditioned medium（PLC−CM）and vice versa. It was observed that aged PDLSC induced by young PLC−CM showed enhanced cemental tissue−regenerative capacity but young PDLSC induced by aged PLC−CM mainly formed connective tissues. Such findings further stress the key role of the extrinsic microenvironment in modulating PDLSC activity. For example, addition of dexamethasone substantially enhances osteoblastic differentiation in human periodontal ligament cells by decreasing endogenous collagenase expression, which is likely to be a regulator of the differentiation phenotype61）. The elucidation of appropriate local environmental signaling molecules and pathways is essential for fine− tuning potential regenerative applications. Currently,
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the onus is on human clinical trials to assess periodontal regenerative outcomes by implantation of ex vivo cultured PDLSC and other progenitor cells, and the modulating factors involved, such as microenvironment characteristics.
Immunomodulatory Properties of PDLSC There is much in vitro evidence indicating that various MSC are hypoimmunogenic, and also modulate the T cell response, independent of major histocompatibility complex （MHC） expression62,63）. In vitro, human MSCs express intermediate levels of human leukocyte antigen（HLA）, MHC class I molecules, are negative for MHC class II molecules （but can be induced to express MHC class II by interferon gamma）, and lack co−stimulatory molecules B7−1, B7−2, CD40, and CD40 ligand64）. Consequently, these cells may escape recognition by alloreactive T cells62,63）. In agreement, Wada et al. in 200949）showed that ex vivo expanded PDLSCs demonstrated in vitro immunosuppressive ability by inhibiting the proliferation of mononuclear cells, partly attributed to induced soluble factors such as hepatocyte growth factor and indoleamine 2, 3−dioxygenase（IDO）production. However, very significantly, contradictory in vivo findings have been noted, showing a strong cellular immune response to transplanted MSC, indicating a possible alteration of antigen expression in vivo65,66）. Hence, the in vivo utility of allogeneic PDLSC remains highly questionable, in line with earlier reports showing that human allogeneic tooth transplantations cause rejection due to immune−mediated osteoclast activation67）. Although a recent animal study showed evidence that, after allogeneic tooth transplantation, the donor periodontal tissue was replaced and regenerated by host cells without exhibiting a MHC−mediated host immune response68）, findings from in vitro and inbred animal studies need to considered with caution and validated in appropriate preclinical trials. Further studies are needed to investigate any potential value of PDLSC purported immunomodualory activity in actual clinical settings.
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