Introduction
Stem cells are primitive cells with self-replicating and multi-directional differentiation potentials. Moreover, they can be differentiated into various functional cells or tissues and organs under certain conditions, and are thus known as “universal cells.” Stem cell therapy is the use of the multi-directional differentiation characteristics of stem cells in order to repair diseased cells or reconstruct normal functioning of cells and tissues [1]. Therefore, stem cell therapy has provided new hope for tissue and organ regeneration.
In recent years, new ideas for tooth and tissue regeneration have been proposed secondary to the rapid developments in theories and technologies in tissue engineering. Various types of stem cells and new biological methods such as those that use bioactive factors, have been widely applied in tooth regeneration research [2]. The teeth are highly differentiated chewing organs formed by the development of tooth germ tissue located in the jaw and consist of the enamel, dentin, cementum, pulp, and periodontal tissue. Moreover, the teeth have a complicated regulatory mechanism, special histologic origin, diverse structure, and important function. These characteristics, to a certain extent, greatly complicate the research in tooth regeneration.
Feasible availability is one of the superior properties of dental stem cells. Dental stem cells can be easily obtained from premolar and wisdom teeth and are thus extracted for orthodontics use; moreover, dental stem cells are increasingly becoming the source for regenerative medicine research. Healthy dental tissues contain large amounts of normal stem cells that are needed to maintain normal function, whereas inflamed or traumatized tissues have a low amount of robust stem cells, which leads to failure in tissue repair [3,4]. Thus, ex vitro expansion/manipulation of stem cells is seen to be an important source when it comes to supplementing host cells and promoting tissue regeneration [5]. The transplanted cells not only participate in the regeneration directly, but also produce building blocks and secrete trophic factors to regulate regeneration procedures [6]. These developments and findings in stem cells will broaden the regeneration medicine research fields, as well as become the basis for translation research. In this paper, the research background, research status, and several common dental stem cells are reviewed. Moreover, the prospect of future applications is discussed.
Stem cells of dental origin
Stem cells from pulp tissue
Dental pulp stem cells (DPSCs), located in pulp tissue, were the first stem cells isolated from adult human dental pulp [7]. DPSCs, which show strong ability to proliferate and self-renew and eventually differentiate into odontoblast-like cells and osteoblasts to form dentin and bone, are best sourced from the third molar [7]. DPSCs composited with hydroxyapatite/tricalcium phosphate (HA/TCP) produced dentin-like structures with pulp tissue inside and an odontoblastic cell lining after being transplanted into immunocompromised mice [7]. Moreover, stem cells isolated from the human pulp of exfoliated deciduous teeth (SHED) not only generated bone and dentin from the odontoblasts, but also differentiated into other mesenchymal and non-mesenchymal derivative stem cells in vitro, such as adipocytes and neural cells [8]. In addition to forming mineralized tissues, DPSCs and SHED expressed neural markers and had the capacity to differentiate into adipocytes. SHED displayed a faster rate of proliferation than DPSCs, but showed noticeable defects in forming a complete dentin/pulp-like complex in vivo.
Moreover, DPSCs and SHED can generate bone-like tissues in vivo [9]. A comparison on the genetic profiles between DPSCs and normal osteoblasts with microarrays was done by Carinci et al., and they identified several up- and downregulated genes. Therefore, several functional activities were differentially regulated between DPSCs and normal osteoblasts, including cell differentiation, adhesion, developmental maturation, and production of cytoskeletal elements [10]. Furthermore, SHED have not only the ability to form bone-like tissue directly, but also the unique potential of osteo-inductive ability. When SHED and DPSCs were respectively mixed with HA/TPA, odontoblast-like cells can differentiate from recipient murine cells only with SHED but not DPSCs transplanted into immunocompromised mice [11,12].
Because of the above characteristics, DPSCs and SHED were considered potential dental stem cells for pulp, dentin, and periodontal regeneration. As a result, when transplanted subcutaneously into the immunodeficient mice, functional dental pulp was developed by the SHED injected into the roots of human premolars with either PuramatrixTM (BD Biosciences, Bedford, MA, USA) or rhCollagen type I (human recombinant BDTM Fibrogen Collagen Type I; BD Biosciences) [13]. Additionally, under certain environments, DPSCs and SHED can be induced into neural cells, and thus may become useful for treating neurological deficits and mortality [14,15]. DPSCs have already been initially and successfully applied for clinical alveolar bone reconstruction in a clinical study [16]. This study showed that DPSC/collagen sponge biocomposites can restore human mandibular bone defects completely, indicating that DPSCs can be used for tissue and organ reparation/or regeneration [16]. Thus, stem cells derived from pulpal tissue are an alternative and available source for regenerating pulp and periodontal tissue.
Periodontal ligament stem cells
The periodontal ligament is located between the teeth and the inner wall of the alveolar fossa and is surrounded by fibrous connective tissue. The presence of multiple types of cells indicates that the periodontal ligament contains stem cells, which are responsible for maintaining homeostasis and building periodontal tissue. Periodontal ligament stem cells (PDLSCs) were first discovered in 2004 by Professor Songtao Shi using mesenchymal stem cell-related markers. PDLSCs can differentiate along the mesenchymal cell lineage to produce osteoblast-like cells, adipocytes, and collagen-forming cells in vitro. Moreover, PDLSCs express an increased level of tendon-specific transcription factors, suggesting that PDLSCs belong to the unique postnatal mesenchymal stem cell population [17]. Transplantation of PDLSCs into immunocompromised mice can regenerate the cementum/PDL tissue, which contains dense collagen I-positive tissue [17]. These collagen-positive tissues mimicked the physiologic attachment of Sharpey’s fibers. Therefore, PDLSCs can form osteoblast-like cells, cementum/PDL tissue, and Sharpey’s fibers. Moreover, human PDLSCs restored defects and migrated into the PDL compartment after being transplanted into defects created surgically at the periodontal area of mandibular molars, thus suggesting that PDLSCs can be used in periodontal tissue regeneration. PDLSCs also successfully regenerated periodontal tissue in surgically periodontal defect areas in swine or canine [18,19]. PDLSCs exist not only in the root surface, but also in the alveolar bone surface with stronger ability of differentiation and proliferation [20]. Furthermore, the root of human deciduous teeth will gradually become a new source for PDLSCs that are highly purified by magnetic cell sorting [21]. To promote periodontal regenerative treatment technology, various immortalized clonal human PDLSC cell lines have been established [22,23]. This series of studies have confirmed that PDLSCs are crucial cells for maintaining dynamic balance and defect restoration in periodontal tissue, and can form new periodontal structures in the animal body, thereby providing basis for PDLSC applications in periodontal regeneration, with further focus on safety and stability of PDLSC cell lines.
PDLSCs have been used in clinical studies to observe periodontal regeneration. A clinical trial showed the regeneration potential of PDLSCs in three patients with severe periodontal disease: two of these patients regained healthy periodontal tissue with reasonable clinical regeneration; and the loosening and probing depth degree of another patient was significantly reduced and the periodontal attachment was stabilized. This study summarizes the significant effects of autologous PDLSCs in periodontal disease [24]. Host immune rejection can be avoided with the application of autologous PDLSCs. However, the shortage of PDLSCs collected from the same donor limits the use of PDLSCs as a treatment for periodontal disease. Recently, PDLSCs have been shown to have the same low immunogenicity as BMMSCs and have important immunomodulatory functions [25]. When transplanted into periodontal bone defects in a pig model, allogeneic PDLSCs reversed periodontitis without immunological rejection [18]. Therefore, PDLSCs has become the first choice for periodontal regeneration [26], with a successful protocol proposed for clinical trials. Recent clinical studies have reported the use of autologous PDLSCs to repair periodontal defects, suggesting that autologous PDLSCs therapy is safe and efficient to cure periodontal defects [27].
Microenvironment around the stem cells has a profound impact on their function. PDLSCs differentiate along the cementoblastic lineage to form cementum/PDL tissue when cultured with conditioned medium of apical tooth germ cells to provide a highly cementogenic microenvironment [28]. Moreover, recent data has shown that inflamed microenvironments regulate PDLSC osteogenesis via epigenetic modification and endoplasmic reticulum stress [29,30]. Further elaboration of molecular mechanisms can further increase our understanding of chronic inflammatory diseases and provide theoretical basis for PDLSC-based stem cell therapy of periodontitis [2].
Root apical papilla stem cells
The apical papilla is located only in growing teeth before completion of root development. The dental papilla contributes to the formation of teeth, and stem cells from apical papilla (SCAPs) are mesenchymal stem cell-like cells isolated from human immature permanent apical papilla [31]. Like DPSCs and SHED, SCAPs are positive for STRO-1 and CD146, and negative for CD34 and CD45. However, CD24 is only present in SCAP and not in DPSCs and SHED. The expression level of CD24 is downregulated when SHED undergoes odontogenic differentiation in vitro. The clonal cell population could be obtained via culturing by using type I collagenase and neutral protease to digest the apical papilla tissue and prepare a single cell suspension. The cells are induced in vitro to be transformed into odontoblast-like cells, adipocytes cells, and neuron-like cells. Moreover, cultured SCAPs show positive staining even in the absence of neurogenic stimuli.
SCAP, which has high proliferative potential, is suitable for cell-based tooth regeneration. Moreover, SCAP appears to be more suitable for tissue regeneration than DPSCs because SCAP has higher proliferative capacity and mineralization potential [32]. SCAP appears to be the source of primary odontoblasts when forming root dentin, whereas DPSCs are the likely source of replacement odontoblasts to form reparative dentin. Compared with PDLSCs, SCAPs show a higher rate of proliferation than PDLSCs at 3rd passages [33]. To construct the bio-root with SCAPs, PDLSCs and SCAPs were co-implanted around the root into the alveolus of minipigs. The results showed that the combined use of these stem cells can produce a root/periodontal complex to repair root function [31]. Furthermore, recent data have reported that stem cells derived from the apical end of developing roots displayed unique “embryonic” characteristics [34] and formed a typical cementum/PDL-like complex in vivo.
Dental follicle stem cells
The dental follicle is a loose connective tissue capsule derived from ecto-mesenchyme, and surrounds the developing tooth germ. The dental follicle contains progenitor cells that can differentiate into periodontal ligament cells and osteoblasts [35,36]. Human dental follicle cells (DFCs) are the most common cells extracted from the sac of the third molar. Their ability to adhere on plastic surface makes DFCs easy to be isolated from human dental follicles during culturing [37]. Moreover, DFCs have putative markers of mesenchymal stem cells, such as Nestin and Notch-1, and other markers of Stro-1, namely, CD105 and CD90. Furthermore, DFCs displays multipotency similar to BMMSCs and PDLSCs. Characteristics of the different dental stem cells are concluded in Table 1.
The DFC can differentiate into PDL cells, osteoblasts, and cementoblasts because of its heterogeneity. After being transplanted into nude mice, DFCs differentiated into PDL fibroblasts that secrete collagen and interact with fibers on the surface of adjacent bone and cementum to further generate cementum/PDL-like tissue [38]. In addition, the morphology, proliferation, immune profile, and mineralization characteristics of DFCs can still be observed after more than 30 passages, and finally result in cementum/PDL-like tissue formation [39]. Furthermore, DFCs combined with treated dentin matrix (TDM) regenerated root-like tissue with pulp-dentin complex and a PDL-connecting cementum-like layer after being transplanted into rat alveolar fossa, and this phenomenon suggested that DFCs can form tooth roots [39]. The heterogeneity of DFCs and the potential of multidirectional differentiation suggested that targeting their differentiation into the desired cell lineage may be a strategy for root regeneration. Moreover, bone morphogenetic protein (BMP) and enamel matrix derivatives (EMD) may be important in cementoblast and osteoblast differentiation of DFCs [40,41]. Recent studies have reported that DFC sheets induced by Hertwig’s epithelia root sheath cells can produce periodontal tissue through epithelial-mesenchymal interaction [42]. The critical factors that induce DFC differentiation remain unclear; however, DFCs are still worth considering for tooth regeneration.
Tab.1 Characteristics of different types of dental stem cells |
| Types | Tissue sources | Markers | Differentiation potency |
|---|---|---|---|
| DPSC | Adult human dental pulp | STRO-1, CD146 | Odontoblast-like cells, osteoblasts, adipocytes, neural cells |
| SHED | Pulp of exfoliated deciduous teeth | STRO-1, CD146/MUC18, CD90, CD29, CD44, CD166, CD105, CD13 | Odontoblasts, osteoblasts, adipocytes, neural cells |
| PDLSC | Periodontal ligament | STRO-1, CD146, CD73, CD90, CD105 | Osteoblast-like cells, adipocytes, collagen-forming cells |
| SCAP | Apical papilla | STRO-1, CD146,CD24 | Odontoblasts |
| DFC | Dental follicle | STRO-1, CD105, CD90, nestin, notch-1 | Periodontal ligament cells, osteoblasts, cementoblasts |
Stem cells and dentin/pulp regeneration
Conventional endodontic treatments use biocompatible materials to fill in roots without any regeneration of dentin and pulp tissue. Given the impact of the loss of pulp vitality on the prognosis of the tooth [43], scientists are devoted to regenerating dentin pulp complex in endodontic treatments [44]. Recent advances in stem cell therapy have paved the way for dentin and pulp regeneration. Moreover, only implantable scaffoldings cannot guide the functional regeneration of the pulp-dentin complex harmonically and consistently. Appropriate microenvironment for dentin-pulp complex regeneration is produced by the interaction between scaffolds and cells.
The dentin slices were prepared in vitro, seeded with stem cells, and then implanted into the immunocompromised mice subcutaneously [45]. Our group developed a treated dentin matrix (TDM), which contained a rich extracellular matrix that exists in dentin [46]. Combination of TDM and DFCs were capable of regenerating complete and prefabricated shaped dentin tissues [46]. Moreover, the pulp-like tissue in full-length human roots was regenerated by injectable scaffolds mixed with SHED [13].
Pulp-like tissue can be formed after implantation of dental stem cells onto the matrix scaffold; however, present scaffolds do not fully possesses the characteristics of the ideal material. Many currently available scaffolds fail to mimic essential functions of natural ECM. Compared with the traditional scaffold-mediated method, self-assembly of monodisperse cells into 3D structures produce more extracellular matrix and promote intercellular communication. The use of scaffold-free microtissue DPSC spheroids that are pre-vascularized by suspending with human umbilical vein endothelial cells showed regeneration with good vascularization and pulp-like tissue [47]. Cell aggregates mimic the cell condensation and thus provide appropriate stimulation for organ development and regeneration. We used autogenous stem cells from pig deciduous teeth (SPED) to form cell aggregates, which contained cells and extracellular cell matrix, and regenerated pulp-like tissue in the root canal of miniature pig (Fig. 2). The pulp tissue was removed before implantation with calcium hydroxide (control) and cell aggregates of SPED (regenerated). Cell aggregates have succeeded in avoiding the major drawbacks of scaffolding methods, such as the inability to mimic natural extracellular matrix, lack of intercellular cross-talk, selective degradation, and remodeling [48,49].
Fig.1 Cell aggregates of SPED regenerated pulp-like tissue in root canal of miniature pig. (A) Full-length root canal of pigs inserted with calcium hydroxide or cell aggregates. (B) H&E and Masson staining showed that pulp tissue was regenerated by SPED implantation after 3 months. In the control group, calcium hydroxide was inserted into young permanent incisors in miniature pigs (n = 3). After 3 months, no pulp tissue was regenerated and only calcium hydroxide was observed. |
Stem cells and periodontal regeneration
The effectiveness of periodontal therapy based on stem cells has been strongly supported in recent years. Numerous animal studies have reported that implantation of stem cells into periodontal wounds contributes to the periodontal regeneration process. Moreover, cell sheet engineering has recently been developed as a scaffold-free strategy for cell delivery [50]. To generate cell sheets, cells are grown to confluence in a cell culture dish and develop cell-to-cell interactions and ECM over time. The monolayer is then removed without the use of proteolytic enzymes to preserve the sheet structure. To facilitate cell metabolism, motivation, and communication in native tissues, establishing a microenvironment similar to that in the real regeneration process is important. Cell sheet engineering aims to prevent ECM degradation using enzymes that are usually used for isolating cells, so that the ECM can be retained completely and finally ensure normal cell function. Numerous types of scaffold have been reported, and these types have prospects in view of functional layers of cells produced in cell sheet engineering [51,52]. In addition to providing support for cells and tissues, ECM contributes a 3D sub-structure for cell adhesion and movement. Furthermore, ECM contains growth factors and facilitates signal delivery, which are necessary for morphogenesis and differentiation.
PDLSCs may be the first choice for periodontal regeneration in regenerating typical cementum/PDL structures with HA/TCP as a carrier. Moreover, cell sheets of PDLSCs have been developed successfully. The cement/periodontal ligament complex has been observed by grafting monolayered or layered PDLSC in vivo [53,54]. Several pilot studies have shown that PDLSC sheet transplants can regenerate periodontal tissues in experimental deficiencies in rats, dogs, and pigs [16,52,53]. In a mesial dehiscence model in a immunodeficient rat, the dentin surface showed a thin layer of cementum-like tissue formation and new fibrous structure insertion after being restored by PDLSC sheets, indicating that cell sheet transplantation can be used for periodontal regeneration [55]. In addition, PDLSCs sheets promoted the potential of periodontal tissue regeneration in a beagle dog in an experimental group, wherein the specimens showed newly generated cementum and periodontal ligament, as well as new blood supply of collagen fibers that were inserted vertically into the new bone and cement [52]. In the mini pig periodontitis model, obvious periodontal regeneration was seen in the autologous and allogeneic PDLSC transplantation groups with no significant differences [18].
In addition to animal models, autologous periodontal ligament progenitor cells (PDLPs) with bone grafting material have shown their therapeutic benefit in humans when implanted into bone defects, as demonstrated in a retrospective pilot study [24]. Periodontal defects were reconstructed, and clinical trials and basic studies proved the security and efficiency of autologous PDL cells in periodontitis therapy [24]. Moreover, our recent clinical trial showed that autologous PDLSC therapy is safe and effective for treating periodontal intrabony defects [27]. PDLSCs have a low chance of immune rejection, and have become a good resource for periodontal tissue therapy; furthermore, PDLSCs have greater potential for periodontal regeneration than other mesenchymal stem cells.
Challenges and prospects
Research and clinical application of dental stem cells have reached several breakthroughs; however, they are still in infancy. Moreover, many theoretical mechanisms still need to be studied for successful clinical applications and real tissue regeneration. Many technical problems have not yet been overcome. However, its incomparable advantages in self-renew ability, potential for multidirectional differentiation, availability, and low autologous transplant rejection result in a broad prospect of its application in dental tissue engineering. With rapid developments in science and technology, people will be able to induce dental stem cells and genetically modify them, thus opening up a new way for tissue regeneration and replacement therapy.
Conclusions
The purpose of this review was to illustrate the types and characteristics of the commonly applied dental stem cells, namely, DPSCs, SHED, PDLSCs, SCAPs, and DFCs. Stem cells isolated from teeth are readily available and can be applied to stem cell therapy. Moreover, cell aggregates and cell sheets continue to be practical biotechnological materials for scaffold-free tissue reconstruction. However, given the clinical application of dental stem cells, a comprehensive global strategy is needed to be developed to address the multiple issues involving safety, harmonization, privacy, and transparency. Stem cell-based tooth regeneration has shown potential clinical use in the near future. With the rapid developments in science and technology, the research in dental stem cells will open up broader application prospects for tissue engineering and regenerative medicine.