Keywords: Mesenchymal stem cells; Dental mesenchymal stem cells; Periodontal ligament stem cells; Cell sheet engineering
Several procedures have been attempted to achieve periodontal regeneration, which include root surface conditioning agents, bone grafts, guided tissue regeneration and growth factor application [5-11]. Ramsier, et al. [12] suggested that surgical regenerative therapy is best suited for deep infrabony defects. There are several drawbacks associated with conventional regenerative techniques which have been described in the Table 1 [13-21].
Tissue engineering as proposed by Langer, et al. [22] comprises of multiple progenitor cells, signaling molecules and conductive extracellular matrix scaffold, along with an adequate blood supply [22-25]. Scaffolds act as the extracellular matrix creating an environment suitable for cell proliferation and differentiation for a limited period of time [26]. Hence scaffolds should fulfill certain requirements such as shape, pore size, rate of porosity to provide a viable extracellular matrix to the cells [27]. Signaling molecules also referred to as immunomodulatory polypeptides are essential to enhance cellular activities such as cell proliferation, differentiation, migration and apoptosis [28,29]. Finally progenitor cells or stem cells thriving within the scaffolds process the signals and carry out tissue regeneration. According to Hynes, et al. [30] the most critical component of tissue engineering is the choice of stem cell population [30]. Currently studies on stem cells have made an achievable progress as a potential application in regenerative periodontal therapy
Bone Grafts
|
autogenous bone grafts-donor site morbidity and complications; limited graft availability [13-15] |
xenografts and alloplasts associated with only osteoconductive property and prone to fibrous encapsulation [16] |
|
EMD |
limited predictability and high degree of variability in results [17,18] |
PRP |
limited predictability and high degree of variability in results [17,18] |
GTR
|
resorbable membrane are collapsible hence placed alongwith bone grafts [19] |
non-resorbable membrane prone to infection and and require surgical reentry procedure [19] |
|
significant results in case of narrow 2/3 walled defects, circumferential defects, class2 molar furcations [20] |
|
no effect in class 3 molar furcations [21] |
are classified as:
Embryonic stem cells
Adult stem cells
Induced pluripotent stem cells
A bioengineered tooth was developed by Nakao, et al. [37] using murine embryonic stem cells derived from epithelium and mesenchyme, which was able to erupt from the oral cavity of the mouse and develop into a fully functional tooth.
Embryonic stem cells are ideal for periodontal regeneration. However, their use in clinical therapy has been hampered by ethical concerns [38]. Another important disadvantage is that, their implantation in the human body has been associated with the occurrence of rare cancers [36].
Bone Marrow derived Mesenchymal Stem Cells (BMMSC): The main source of adult stem cells is the bone marrow in proportion of 1 in 34,000 of nucleated cells. MSCs derived from the bone marrow are referred to as bone marrow derived mesenchymal stem cell (BMMSC) and have been the most studied amongst mesenchymal stem cells [42-44].
They are characterized by embryonic stem cell markers Oct-4 and Nanog, and also express MSC markers CD73, CD90, CD105, CD106 and CD166 [45]. BMMSCs have been found to be capable of differentiating into various cell lineages like osteoblasts, chrondocytes and adipocytes [36,44,46]. BMSSCs have been shown to form cementum, periodontal ligament and alveolar bone, suggesting that bone marrow may be a useful source for periodontal regeneration [46].
They have been incorporated in class III furcation defects in canine models resulting in successful results [47]. BMMSCs were present in the defect even after 1 month, suggesting that they ultimately form various periodontal cells needed for regeneration [47]. Also, Yamada, et al. [48] managed to successfully carry out periodontal regeneration using autologous BMMSCs and plateletrich plasma.
Since harvesting BMMSCs is associated with certain limitations such as pain, morbidity and decreased number of cells obtained, therefore alternate sources for obtaining MSCs to carry out periodontal regeneration have been sought.
Dental MSCs: MSCs has been found in various dental tissues. These are easier to harvest and associated with lesser patient related complications. The various dental MSCs that has been found till date to be useful in periodontal regeneration as illustrated in Table 2 and Figure 1 include:
Dental pulp stem cells (DPSCs): In an investigation, Gronthos, et al. [49] isolated from adult human dental pulp a clonogenic, rapidly proliferative population of cells which were found to be similar to BMSCs. In vitro characterization reveals mesenchymal stem cell markers such as STRO-1, CD31 and CD146 and also embryonic stem cell markers Oct-4 and Nanog [41]. They also express a mesenchymal marker vimentin [50,51].
Human derived DPSCs along with hydroxyapatite or beta tricalcium phosphate have been reported to be capable of forming bone and cementum [49,50,52]. However, some authors are skeptical about the role of these cells in periodontal regeneration. Carinci and coworkers isolated a subpopulation within the
Periodontal Ligament stem cell (PDLSC): Periodontal ligament is a specialized connective tissue that connects cementum and alveolar bone, to maintain and hence support the teeth in sight also preserve tissue homeostasis. Multipotent stem cells from human periodontal ligament were isolated for the first time by Seo, et al. [56]. He reported that PDLSCs exhibited some characteristic features similar to BMMSCs [56]. The peculiar features were multipotency, clonogenic ability, high proliferation and expression of putative stem cell marker such as STRO-1 and perivascular cell marker CD 146 [49,57]. Like the BMMSCs they also express CD44, CD90, CD105, CD166. Scleraxis which is a transcription factor specific to tendon was found to be highly expressed by PDLSCs as compared to BMMSCs and DPSCs [58]. Hence it was concluded that periodontal ligament derived MSC are one of the most effective source for periodontal regeneration.
Park JY et al. [55] carried out a study in beagle dogs comparing PDLSCs with stem cells obtained from other dental sources and concluded that PDLSCs are most predictable in carrying out regeneration. Han et al. [59] created fenestration defects in murine models and placed allogeneic PDLSC to assess the time required for the defect to be mineralized. They observed that by day 14 and 21 significant amounts of mineralized tissue and bony bridge was formed. Ji, et al. [60] investigated the effect of PDLSCs derived from retained deciduous teeth (DePDLSC) in periodontal regeneration and compared it with PDLSCs derived from permanent teeth (PePDLSC). DePDLSCs were found to be comparatively immature hence readily differentiated into osteoblasts in osteogenic medium. Also, they were found to have the higher colony forming ability and increased proliferation rate as compared to PePDLSCs. Further DePDLSC cell sheets when combined with dentin blocks resulted in the formation of PDL and cementum like tissue on the dentin block however PePDLSCs resulted in formation of no cementum. Numerous other studies carried out in periodontal defect models in animals have reported positive results with application of PDLSC [61-64].
Stem cells from apical papilla (SCAP): Apical papilla is the soft tissue present at the apices of developing roots of permanent teeth. It is responsible for the formation of the radicular pulp hence SCAP resemble DPSCs however, they are comparatively more immature hence superior for tissue regeneration [65]. They are isolated from tips of developing roots, hence can be harvested easily during extraction of impacted third molars. In vitro characterization reveals MSC markers STRO-1, CD146 and CD24 which seem to be a unique feature of these cells [66]. They have been incorporated along with periodontal ligament stem cells in extraction sockets of miniature pigs, resulting in the successful formation of root and supporting periodontal structures [67]. They have been considered crucial in root formation which might be partly because, SCAP are the source of primary odontoblasts responsible for formation of root dentin [68].
Dental Follicle Stem Cells (DFSC): Dental follicle is an ectomesenchyme derived loose connective tissue sac surrounding the developing tooth bud from which arises the alveolar bone, cementum and periodontal ligament. DFSCs are relatively easy to harvest as can be procured from the follicles of unerupted third molars. In vitro characterization reveals stem cell markers Nestin, Notch-1 and STRO-1 [69]. They express vimentin (mesenchymal marker) and cementoblast markers (Cementum protein-23, Cementum attachment protein). Guo, et al. [70] has reported that DFSCs have the potential for regenerating the entire root of the teeth.
In support of the role of DFSCs in forming various periodontal structures, the authors discovered a crucial role played by Wnt5a [71]. Wnt5a proteins follow the non- canonical pathway involving tyrosine kinase like orphan receptor (ROR) proteins. These proteins have been found to play an important role in stimulating and regulating the role of DFSCs in forming non-mineralizing PDL and mineralized alveolar bone and cementum [71].
Stem cells from Human Exfoliated Deciduous teeth (SHED): In a study by Miura M, et al, [72] they found that multipotent stem cells are found in exfoliated human deciduous teeth. Hence, these cells can be readily harvested. They have a higher proliferation rate as compared to BMMSC and PDLSC, also result in increased bone formation [72]. SHED instead of directly forming the specific cells, create a special template for recruiting host cells, resulting in inducing the new tissue formation [72]. In vitro characterization reveals early MSC markers STRO-1 and CD146. Studies by Kerkis, et al. [73] revealed that they are basically immature DPSC and contain cell markers like Oct-4, Nanog which are present in embryonic stem cells.
Fu, et al. [74] investigated the role of allogeneic SHED in the swine periodontitis model, and found that they resulted in predictable periodontal regeneration similar to PDLSCs. SHED have been found to elevate regulatory T cells and downregulate T-helper 17 cells, hence have significant immunomodulatory capacity [75]. Also, Yamada, et al. [76] used SHED obtained from puppies and placed them in mandibular osseous defects created in parent canines. At 8 weeks the defect was completely filled with mature bone. Hence SHED derived from a child can be successfully used as graft in the parent.
Gingival Mesenchymal stem cells (GMSC): Oral MSCs derived from human gingiva (GMSCs) also have been considered as a promising alternative cell source for periodontal regeneration [77]. In addition to physical characteristics of gingival fibroblasts they exhibit adherence to plastic and multilineage differentiation potential [76]. In vitro characterization reveals cell surface markers CD44, CD73, CD90 and CD105 also stem cell markers such as SSEA-4, STRO-1, CD146, CD166, CD271 and vimentin which is a mesenchymal marker [78]. In a canine model with class III furcation defects, the transplanted GMSCs significantly enhanced the regeneration of the damaged periodontal tissue, including the alveolar bone, cementum, and functional periodontal ligament [79].
Epithelial Cell Rests of Malassez (ECRM): These are remnants of the Hertwig's epithelial root sheath from which arise, all the periodontal structures. Since ECRM is normally present within the periodontium, hence studies have been carried out to research the stemness of ECRM [80-82]. They express MSC markers CD29, CD44, HSP-90β also embryonic stem cell markers Oct-4, Nanog and SSEA-4. Also Xiong J, et al. [82] has reported their capacity to carry out epithelial mesenchymal interactions. Following which numerous studies by the same authors have revealed that ECRM can differentiate into bone, cementum and periodontal ligament [80-82].
Hence, dental MSCs is a promising tool for periodontal regeneration. They have been found with similar characteristics as embryonic stem cells and other MSCs. Also, they are in many cases harvested from dental tissues which are to be discarded. Harvesting these cells is associated with minimal donor morbidity. However, there are a few limitations associated with their application as described in Table 3 [83].
Biological
|
Technical
|
Clinical |
Molecular pathways responsible for stem cell proliferation and differentiation are unknown
|
Culture mediums are not well developed enough to mimic in vivo conditions to ensure safe and consistent stem cell proliferation and differentiation. Stem cell line production for human trials could be hampered by the use of xenogenic products in culture mediums as they could be a potential source of pathogens. Mesenchymal stem cells have a limited life span unlike embryonic stem cells which are immortal An ideal biocompatible scaffold and transport mechanism is still under research.
|
Integration of the human stem cell derivatives with the recipient tissue and their ability to carry out the desired functions in humans is still under speculation
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Okano, et al. [87] incorporated a temperature responsive polymer poly(N-isopropylacrylamide) (PIPAAm) in the culture dishes to detach the cell sheets [88,89]. Since this polymer is hydrophilic at temperatures greater than 32°C and hydrophobic when temperature is reduced below 32°C, also cells adhere to hydrophobic surfaces, therefore it is a useful aid in detaching the cells from the culture dishes.
Cell sheet engineering with the help of temperature responsive dishes can act as an effective means for periodontal regeneration. These temperature responsive cell sheets can be grafted to recipient site without suturing [89,90]. Figure 2 describes the technique of carrying out cell sheet engineering in temperature responsive culture dishes.
To increase the strength and number of cells, a 3D culture model of multilayered cell sheet have so been developed [91,92]. Also more recently cell sheet fragments and cell sheet pellets have been developed to increase the efficacy of the cells transplanted especially in cases where the target sit is too small for the entire cell sheet [93,94]. Further Co-culturing and micro-patterning of different types of cells are under trials for creation of more tissue-like materials which would give better results than single cell-sheets [95]. Also several researchers have attempted to incorporate biocompatible scaffolds like hyaluronic acid, fibrin gel and ceramic bovine bone to the fragile cell sheets also referred to as scaffold based cell sheet technology to improve the results following cell sheet engineering [95,96]. Hence cell sheets of the following types have been manufactured till date-
Multi-Layered cell-Sheet (MLS)
Cell Sheet Fragments (CSF)
Cell Sheet Pellets (CSP)
Co-culturing and micropatterning
Scaffold based CST
Author |
Cell sheets |
Target site |
Results |
King GN, et al. 2001 [97] |
Human periodontal ligament cell sheet (MCS)in osteogenic differentiation medium |
Athymic rat mandible (King's method) |
A layer of cementum & new attachment of collagen fibers to cementum. |
Akizuki T, et al. 2005 [98] |
PDLSC cell sheets(MCS) using hyaluronic acid as carrier from temperature responsive culture dish |
Mesial dehiscence model beagle dogs |
Newly formed periodontal ligaments with a rich capillary supply found between alveolar bone & cementum. |
Flores MG, et al. 2008 [91] |
PDLSC cell shhets(MLS) |
Athymic rats |
Cementum like hard tissue on dentin surface & Collagen fibres resembling periodontal & sharpey's fibers were inserted into tissue.
|
Iwata, et al. 2009[92] |
PDLSC cell sheets(MLS) alongwith TCP |
Surgically created three walled defects in dogs |
New bone and Cementum was formed with well-oriented collagen fibres |
Ding, et al. 2010 [99] |
Autologous and allogenic PDLSC cell sheets(MLS) alongwith HA/TCP |
Periodontal defects in miniature pigs |
Significant periodontal regeneration in12 weeks using both autogenous and allogenic PDLSC |
Bai, et al. 2011 [100] |
DFSCs (MCS) alongwith HERS |
Omenta of adult male rats |
DFSCs in presence of HERS formed tissues resembling cementum and periodontal ligament |
Xei, et al. 2012 [95] |
Co-cultured human PDLSC and BMMSC alongwith ceramic bovine bone powder (scaffold based CST) |
Athymic rats |
Cementum and periodontal ligament like tissue was formed alongwith neovascularization |
Na, et al. [87] |
CSP of SCAP |
In vitro |
Increased expression of alkaline phosphatase, bone sialoprotein and runt related gene-2 (RUNX2) mRNA as compared to cell sheets |
Zhao, et al. 2013 [86] |
CSF of PDLSCs and PRF granules |
Reimplantation of teeth in dogs |
Better periodontal regeneration and reduced Ankylosis and inflammation |
Dan, et al. 2014[88] |
MLS composed of cells of gingival connective tissue, alveolar bone and Periodontal progenitors in a calcium phosphate- coated melt electrospun polycaprolactone scaffold |
Athymic rats |
Scaffold promoted bone formation in 4 weeks. Significant periodontal tissue formed by alveolar bone cells and periodontal progenitors |
Careful investigations need to be carried out to guarantee that the MSC would not form neoplasms.
Periodontium is a complex tissue comprising of 2 hard tissues (alveolar bone and cementum) and 2 soft tissues (periodontal ligament and gingiva). Complete regeneration implies the simultaneous production of all these tissues. To be able to achieve this extracellular matrix in the target site should generate the correct signals at the appropriate time for all the tissues to form.
Some authors have suggested the use of allogeneic MSC. However, further investigations need to be carried out to assess the feasibility of this approach, considering that in the clinical environment, complete regeneration needs to occur in a diseased environment containing inflammatory cytokines.
Further on various interactions between the different types of cells in the periodontium need to be assessed and also the effect of mechanical stress on periodontal regeneration needs to be examined in detail.
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