Keywords

1 Introduction

Teeth are composed of hard tissues including outer layers of enamel of the crown/cementum of the root and an inner layer of dentin which enclose the soft pulp tissue containing blood vessels and nerves, etc. Tooth-supporting structures consist of gingival, periodontal ligament and alveolar bone.

Various dental stem cells have been identified from different teeth and tooth-supporting tissues which shared similar in vitro properties with bone marrow mesenchymal stem cells (BMMSCs) such as dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), stem cells from apical papilla (SCAPs) and dental follicle cells (DFCs) (Sharpe 2016) (Fig. 1). Current treatments with artificial materials for tooth defect and tooth loss can restore the esthetic and function of tooth to a certain extent, still several complications following the treatments can be a big headache for dentists. Thus, tooth regeneration with dental stem cells has been studied for many years and achieved great progress. A better understanding of the properties of different dental stem cells and their possible application in tooth regeneration is necessary. This review provides an overview of key findings and advances of dental stem cells and tooth regeneration.

Fig. 1
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Location and origin of dental stem cells

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2 DPSCs and Dental Pulp Regeneration

Dental pulp tissue consists of odontoblasts, fibroblasts, nerves, immune cells and stem cells, etc. which work as a pulp-dentin complex and hold the function of tooth development, nutrition supply, dentin mineralization, sensory and immune response (Ajay Sharma et al. 2015). It is a very vulnerable soft tissue to different stimulations such as infection and trauma which requires effective clinical treatments. Conventional endodontic treatments including dental pulp capping and root canal therapy merely maintain the structure and function of teeth for prolonged periods of time. However, they fail to sustain the vitality of dental pulp and bring about complications such as lack of capacity of forming reparative dentin, vulnerability to mastication and discoloration, etc. (Zhang and Yelick 2010). Therefore, maintaining dental pulp vitality would be the aim and challenge of future endodontic treatments.

2.1 DPSCs Isolation and Identification

Human dental pulp stem cells (DPSCs) were first isolated and identified from impacted third molar in 2000 by Gronthos et al. with clonogenic and dentin-like structure forming capacity (Gronthos et al. 2000). Human deciduous teeth can also be a resource of dental pulp stem cells and these cells are known as SHED (stem cells of human exfoliated deciduous teeth) (Miura et al. 2003). Explant culture and enzymatic digestion methods of isolating DPSCs have been applied and compared and results indicated that both methods are efficient to yield stem cell populations capable of colony formation and muti-differentiation (Hilkens et al. 2013). Several markers of DPSCs have been reported and used to identify DPSCs including STRO-1, CD29, CD44, CD73, CD90, CD105 and CD146, etc. as positive and CD34, CD45 and CD71,etc. as negative (Suchanek et al. 2009; Kawashima 2012). Different resources of DPSCs have been investigated intensely. DPSCs can be obtained from both permanent teeth and primary teeth, especially impacted third molar and exfoliated deciduous teeth, also supernumerary tooth has been used (Gronthos et al. 2000; Miura et al. 2003; Huang et al. 2008). Growth rate and differentiation capacity of DPSCs and SHEDs have been compared and it showed that SHEDs hold higher proliferation and differentiation capacity while DPSCs possess higher inflammatory cytokines levels which suggested SHED might represent a more proper source for tooth regeneration (Kunimatsu et al. 2018). Extensive expansion in vitro of DPSCs and SHED can alter stem cell properties such as proliferation and differentiation, thus proper passages of DPSCs and SHED shall be carefully chosen before being applied in clinics (Wang et al. 2018a). Long-term cryopreservation have been proved to be an effective way to preserve tissue and stem cells as stem cells from dental pulp after 2 years’ cryopreservation still express stem cell surface antigens and hold their differentiation capacity and cryopreserved dental pulp tissues from exfoliated deciduous teeth owned similar stem cell properties (Papaccio et al. 2006; Ma et al. 2012). Therefore, cells and tissues after long-term cryopreservation can be a useful and reliable resource for regenerative medicine.

2.2 DPSCs Properties and Pathways

Numerous pathways are involved in DPSCs differentiation thus regulating their regenerative capacity. DNA microarray was performed to analyze the gene expression profile of DPSCs and SHEDs and results showed that genes that participate in pathways related to cell proliferation and extracellular matrix were expressed higher in SHEDs than DPSCs (Nakamura et al. 2009). Canonical Wnt signaling inhibited odontoblast differentiation capacity of DPSCs (Scheller et al. 2008). IGF-1 could enhance proliferation and osteogenic differentiation of DPSCs and mTOR pathway was involved (Feng et al. 2014). Odonto/osteogenic differentiation of DPSCs can be regulated by estrogen level, LPS stimulation, TNF-α stimuation via NF-κB pathway (Wang et al. 2013; He et al. 2015; Feng et al. 2013). Biological materials hold the capacity of regulating DPSCs properties via different pathways. Natural mineralized scaffolds promote odontogenic differentiation and dentinogenic potential of DPSCs via MAPK pathway (Zhang et al. 2012). With better understanding of DPSCs molecular mechanisms especially pathways involved in their proliferation and differentiation, methods to increase DPSCs regenerative capacity would be chosen more wisely.

2.3 Dental Pulp Regeneration

As DPSCs hold the ability to differentiate into odontoblasts, they have been used directly for dental pulp regeneration or in vitro study for optimizing biocompatible materials. Dental pulp regeneration research and clinical trial have been the focus for years to replace the conventional treatments.

Cell-based therapy has been widely used in both animal studies and clinical trials which is isolation and ex vivo expansion of stem cells and transplantation into dental pulp. Studies indicated that vascularized pulp-like tissue was generated by transplantation of DPSCs or SHEDs seeded in biodegradable scaffolds in immunodeficient mice (Cordeiro et al. 2008; Prescott et al. 2008). Following studies showed that both DPSCs and SHEDs seeded onto some scaffolds, were able to form vascularized pulp/dentin-like tissue in an emptied human root canal which had been subcutaneously transplanted into immunodeficient (SCID) mice (Huang et al. 2010; Rosa et al. 2013). In large animals, reparative dentin was formed after autologous transplantation of DPSCs pellets stimulated by BMP-2 onto the amputated pulp of dog teeth (Iohara et al. 2004). Autologous transplantation of DPSCs mobilized by granulocyte colony-stimulating factor (G-CSF) in dog pulpectomized tooth was taken and proved to be able to regenerate complete pulp/dentin tissue with an apical opening of 0.6 mm (Iohara et al. 2013). With animal studies above, DPSCs application in endodontic treatment is quite promising and of great potential. In the first clinical trial of dental pulp regeneration in 1961, scientists intentionally induced blood from apical into root canal by over-instrumenting which led to mineralization along the root canal walls (Ostby 1961). In the following years, various improvements including disinfection of root canal have been explored and successfully applied. The blood clot induction presumably induced stem cells from apical papilla (SCAPs) into dental pulp for pulp regeneration. A pilot clinical study showed that human DPSCs of passage 9 or 10 with G-CSF in atelocollagen successfully formed tooth pulp tissue after being transplanted in human pulpectomized teeth and some patients even formed functional dentin after 24 weeks (Nakashima et al. 2017). Our latest study demonstrated very successful outcomes in clinical trial applying autologous DPSCs in premature teeth with crown facture with regeneration of three-dimensional dental pulp tissue, consisting of whole dental pulp with odontoblast layer, blood vessels and nerves (unpublished data). No transplantation rejection and inflammation response was observed during the treatment which indicates that this method could be a potential and effective way for dental pulp diseases (unpublished data). Thus, using DPSCs holds great potential for endodontic treatment and extensive clinical trials to evaluate efficacy and safety and optimize the treatment are required.

Manipulation of DPSCs using different methods to enhance its regeneration capacity has been studied. DPSCs from human third molars cultured in 3-dimensional (3-D) scaffold materials including a spongeous collagen, a porous ceramic, and a fibrous titanium mesh were proved to benefit DSPP-expressing tissue formation both in vitro and in vivo (Zhang et al. 2006). Also it has been reported that three-dimensional pellet culture system of dental pulp progenitor/stem cells stimulated by BMP2 effectively promoted dentin formation (Iohara et al. 2004). Application of DPSCs, collagen as scaffold and DMP1 as growth factor on mice by subcutaneous transplantation could induce dental pulp-like tissue (Prescott et al. 2008). Optimization of DPSCs’s application in clinics is necessary and crucial to improve the therapeutic efficacy and more optimization work would be the focus of future study.

3 PDLSCs and Periodontal Regeneration

Periodontitis is a multifactorial inflammatory disease characterized by destruction of tooth-supporting tissues including the periodontal ligament (PDL), alveolar bone and root cementum (Pihlstrom et al. 2005). As a prevalent disease, periodontitis not only causes periodontal attachment and bone loss which finally leads to tooth loss but also is closely related to systemic diseases (Winning and Linden 2017). Conventional interventions and treatments including bone grafts (Hjorting-Hansen 2002), enamel matrix derivate (EMD) (Miron et al. 2016), platelet rich plasma (PRP) (Needleman et al. 2006) and guided tissue regeneration (GTR) (Andrei et al. 2018) are effective in partially restoring periodontal tissue but failed to regenerate the whole functional periodontal tissue. Periodontal tissue repair and regeneration in clinics is of great difficulty. Therefore, a better understanding of tissue specific stem cell-based regeneration seems to be crucial for periodontal tissue remodeling or repair.

3.1 PDLSCs Isolation and Identification

Periodontal ligament stem cells (PDLSCs) are a small population of mesenchymal stem cells isolated periodontal ligament that have self-renewal capacity and hold the capacity of differentiating to osteoblasts, adipocytes and chondrocytes under specific differentiation inductions (Seo et al. 2004). Periodontal ligament obtained from normal impacted third molars or extracted orthodontic teeth are most frequently used for PDLSCs isolation following established explant culture or enzymatic digestion methods. In addition, residual periodontal ligament on retained deciduous teeth has been proposed to be a new resource of PDLSCs (Silverio et al. 2010). Apart from comparative osteogenic differentiation capacity, PDLSCs derived from deciduous teeth showed higher self-renewal ability compared to PDLSCs obtained from permanent teeth (Ji et al. 2013). Moreover, it has also been reported that PDLSCs can be provoked from cryopreserved human periodontal ligament and maintain tissue specific stem cells features, including the expression of surface markers, colony formation capacity, pluripotent differentiation ability and specialized tissue regeneration, thereby providing another access for PDLSCs isolation using frozen tissues (Seo et al. 2005).

Surface markers similar to BMMSCs and DPSCs have been also applied to identify PDLSCs, containing both positive (CD13, CD29, CD44, CD49d, CD73, CD90, CD105, CD166, etc.) and negative (CD19, CD34, CD45, etc.) markers (Trubiani et al. 2005). Recent evidence also suggests that highly osteogenic subpopulations of PDLSCs incline to express ascending levels of integrin b1 (ITGB1), intercellular adhesion molecule 1 (ICAM1) and telomerase reverse transcriptase (TERT) (Sununliganon and Singhatanadgit 2012). Although PDLSCs express an array of alkaline phosphatase (ALP), osteocalcin (OCN), matrix extracellular phosphoglycoprotein (MEPE) and bone sialoprotein (BSP) after osteogenic induction, the newly formed mineralized nodules are much fewer compared to BMMSCs and DPSCs, which credits to a lower calcium content in extracellular matrix (Seo et al. 2004). However, a higher expression of tendon specific scleraxis highlights the unique identity of PDLSCs to regenerate periodontal tissues among various postnatal mesenchymal stem cells (Seo et al. 2004). In addition, PDLSCs rarely express MHC class II antigen and co-stimulatory molecules (CD40, CD80 and CD86) which indicate low immunogenicity of PDLSCs (Wada et al. 2009). Although PDLSCs exhibit stem cell properties with colony formation and pluripotent differentiation, the property disorders during long-term in vitro expansion cannot be ignored.

3.2 PDLSCs’ Properties and Pathways

Numerous mechanisms related to PDLSCs’ degenerative properties under periodontitis have been reported, which is commonly regarded as a chronic inflammatory microenvironment. TNFα and IL-1β have been acknowledged as crucial inflammatory factors to destroy periodontal tissues and to block functions of PDLSCs (Xue et al. 2016). WNT pathway exerts its critical role in periodontal homeostasis, and dysregulation of β-catenin is largely related to the disorders of PDLSCs in inflammatory microenvironments (Napimoga et al. 2014). Dickkopf 1 (DKK1), a specific WNT inhibitor, could improve function of PDLSCs in periodontitis with diabetes mellitus by mediating WNT signaling (Liu et al. 2015). NF-κB signaling, MAPK signaling and BMPs signaling are also involved in inflammation induced PDLSCs dysfunction (Mao et al. 2016). In recent years, microRNAs such as miR-17 and miR-21 have been frequently reported to regulate PDLSCs functions at posttranscriptional level, whereas mechanisms mediated by microRNAs remain poorly understood (Liu et al. 2011; Yang et al. 2017). Epigenetically, histone acetyltransferase GCN5 has been proved to be able to regulate PDLSCs’ osteogenesis through WNT signaling and Osthole could restore function of PDLSCs from inflammatory tissue via epigenetic regulation (Li et al. 2016; Sun et al. 2017a). Moreover, abnormality of subcellular structures has been verified to affect PDLSCs functions. Autophagy and edoplasmic reticulum stress were both reported to be involved in periodontitis-associated chronic inflammation and proper manipulation of such pathways could alleviate inflammatory condition of periodontitis (Xue et al. 2016; An et al. 2016).

3.3 Periodontal Regeneration

As PDLSCs exhibit multi-potency with differentiation into osteoblasts, fibroblasts and tooth cementoblasts, they have been used alone or combined with biomaterials for periodontal tissues regeneration.

When the PDLSCs were discovered, a typical cementum/PDL-like structures regenerated by PDLSCs-aggregate, which are different from specialized structures generated by BMMSCs and DPSCs, have been verified using a subcutaneous transplantation assay (Seo et al. 2004). Meanwhile, newly formed collagen fibers were also observed to connect with regenerated cementum/PDL-like structures, mimicking physiological attachment of Sharpey’s fiber (Seo et al. 2004). Furthermore, PDLSCs transplanted into artifical periodontal defects in immunocompromised rats were observed to integrate into the surfaces of alveolar bone and teeth roots, bi-directionally (Seo et al. 2004). Additionally, it has been reported that PDLSCs can effectively generate periodontal tissue in a swine or canine model of periodontitis (Liu et al. 2008; Ding et al. 2010), and combination of stem cells from apical papilla (SCAPs) and periodontal ligament stem cells has successfully formed root/periodontal structure (Sonoyama et al. 2006). Transplantation of PDLSCs and BMMSCs was able with to form alveolar bone in a canine peri-implant defect model (Kim et al. 2009). Besides of animal researches, human studies have also been conducted. Recently, a randomized clinical trial has been designed to repair periodontal intra-bony defects on patients using autologous PDLSCs, resulting in a marked elevation of alveolar bone height with high biological safety (Chen et al. 2016). However, the therapeutic effects showed no statistically differences between the therapies using and not using PDLSCs (Chen et al. 2016). Therefore, further studies are needed to develop modified strategy for advancement of PDLSCs based periodontal regeneration. Addition of exogenous protein signalings has been verified to promote PDLSCs regenerative capacity. When treated with dentin noncollagenous proteins (DNCPs) or bone morphogenetic proteins (BMPs), PDLSCs presented an improved proliferation, adhesion capability and cementoblastogenesis, which are indicated by changes of morphology, enhancement of ALP activity, improvement of matrix mineralization and upregulation of osteogenic genes (Ma et al. 2008; Wang et al. 2017). Although autologous PDLSCs are tolerated by hosts’ immune system and safe for therapy, the limited resource restricts their large scale clinical application. Thus, it is urgently needed to research and develop allogeneic PDLSCs based regeneration medicine, whereas their therapeutic safety has not been totally defined. Recent studies have demonstrated that allogeneic PDLSCs engaged in immune-modulatory function similar to BMMSCs and finally reconstructed the experimental periodontal bone defects, indicating that allogeneic PDLSCs based therapy might be an efficacious and safe alternative for the treatment of periodontal diseases (Ding et al. 2010; Han et al. 2014). Furthermore, extracellular matrix (ECM) derived from periodontal ligament cells has been reported to induce the differentiation of induced pluripotent stem cells (iPS) to PDLSC-like cells, suggesting a novel approach to obtain enough seed cells for periodontal bioengineering (Hamano et al. 2018).

For these results, PDLSCs are generally regarded as the optimum selection of seed cells for periodontal repair and regeneration, not only because of pluripotent stem cell features, but also due to their unique potential to organize three-dimensional periodontal tissues. More studies involving in the underlying mechanism of PDLSCs and periodontal regeneration are greatly required.

4 Other Dental Stem Cells and Tooth Regeneration

4.1 SCAPs and Tooth Regeneration

Stem cells from apical papilla (SCAPs), a type of dental stem cells essential for the developing dental pulp-dentin complex, alveolar bone and tooth root (Sonoyama et al. 2008; Bakopoulou et al. 2011), have been isolated from root tips of growing teeth, and are similar to DPSCs but with a markedly higher proliferative capacity and mineralization potential. SCAPs express high level of STRO-1, CD-146, and negatively express CD34 and CD45 (Bakopoulou et al. 2011). Studies showed that SCAPs had a greater capacity for dentin regeneration compared to DPSCs (Bakopoulou et al. 2011). Furthermore, SCAPs also exhibit a higher proliferation and better tooth regeneration capacity compared to PDLSCs (Han et al. 2010). However, it has been reported that SCAPs and PDLSCs with a HA/TCP carrier can produce a functional biological tooth root in a swine model and finally resemble a functional tooth with an artificial crown (Sonoyama et al. 2006). In addition, besides of healthy SCAPs, SCAPs derived from inflamed root tips also exhibit high proliferation and multipotency. Further researches are essential to identify regenerative properties of inflammation derived SCAPs. Complex molecular mechanisms underlying SCAPs differentiation and proliferation have been investigated extensively. bFGF has been reported to enhance stemness of SCAPs and differentiation capacity under certain conditions (Wu et al. 2012). Canonical WNT signaling also participate in osteo/odontoblastic differentiation of SCAPs (Zhang et al. 2015). MicroRNAs play vital roles in regulateing odonto/osteogenic differentiation capacity of SCAPs (Sun et al. 2014; Wang et al. 2018b). MAPK pathway, NF-κB pathway, etc. are involved in this process as well (Li et al. 2014a, b). As stem cells from developing stage, SCAPs hold superior potential for regenerative medicine, and more mechanism study and clinical trial are expected in the future in order to make better use of them.

4.2 DFCs and Tooth Regeneration

The dental follicle, a loose ectomesenchyme origined connective tissue, surrounds tooth germ during tooth development and plays important roles in tooth eruption and tooth root development. Undifferentiated ectomesenchymal cells known as dental follicle stem cells or dental follicle cells (DFCs) can be obtained from impacted third molars or ectopic impacted teeth, and express high level of STRO-1, CD44, CD105, Nestin and Notch-1 (Yao et al. 2008; Morsczeck et al. 2005). DFCs are multipotent stem cells dental follicle cells are precursor cells of periodontal fibroblasts, osteoblasts and cementoblasts during the process of periodontal tissues development. It has been reported that DFCs hold the properties similar to MSCs, which were able to form a connective tissue-like structure with mineralized clusters after being induced in osteogenic differentiation medium (Sowmya et al. 2015). After transplantation of DFCs with treated dentin matrix scaffold, root-like tissues stained positive for markers of dental pulp and periodontal tissues were found in the alveolar fossa (Guo et al. 2012a). Also data showed that rat DFCs formed a tooth root when seeded on scaffolds of a treated dentin matrix (TDM) and transplanted into alveolar fossa (Sun et al. 2017b). Apart from generating periodontium alone, DFCs have also been observed to improve regenerative capacity of healthy PDLSCs and even rescue degeneration of inflamed PDLSCs, indicating that DFCs could assist PDLSCs to regenerate periodontal tissues via ameliorating local microenvironment (Liu et al. 2014). Additionally, human dental follicle tissue after cryopreservation has been proven to be a reliable resource for regenerative medicine (Park et al. 2017). As DFCs support bone regeneration in defect models of the calvaria of immunocompromised rats, they are also a promising cell medication for bone regeneration (Guo et al. 2012b).

5 Dental Stem Cells Banking

Although dental stem cells have been reported to well regenerate dental tissues, a long period procedure of tooth extraction, primary culture and in vitro cell expansion limits their usage at the time of clinical requirements. Therefore, long-term storage and timely application of dental stem cells remain to be settled. Recently, dental stem cell banking has been emerging to cryopreserve dental stem cells, which highlights the potential to realize a novel approach to support large scale of dental stem cells based regenerative medicine. Several banks provided dental stem cells have been prepared, such as BioEDEN (Austin, USA, http://www.bioeden.com/), Store-A-ToothTM (Lexington, USA, http://www.store-a-tooth.com/), Teeth Bank Co., Ltd., (Hiroshima, Japan, http://www.teethbank.jp/), Advanced Center for Tissue Engineering Ltd., (Tokyo, Japan, http://www.acte-group.com/) and Stemade Biotech Pvt. Ltd., (Mumbai, India, http://www.stemade.com/). Recently, a National Dental Stem Cells Bank (http://www.kqgxb.com/) has been established in People’s Republic of China, which is the first high-tech organization of dental stem cells research, storage and translational medicine development according to Good Manufacturing Practice (GMP) around the world.

Apart from evaluating therapeutic effects of dental stem cells on tooth regeneration, it might be crucial to formulate legislation, industry standard, quality control, bio-insurance, checks and audits for dental stem cells banking development. With these problems solved, dental stem cells banking will be a prospective industry in regenerative medicine.

6 Conclusion

This review concentrated on stem cells from dental tissues and how their current advancement in tooth and periodontal tissues regeneration. Although dental stem cells possess colony formation, proliferation and multipotent differentiation capacity to generate osteogenic, adipogenic and chondrogenic lineages in vitro similar to BMMSCs under certain conditions, they also displayed their own distinctive regenerative potential different from each other in vivo, suggesting that tissue specific stem cells might be the optimal choice for self-tissues repair and regeneration. Basic researches and clinical pilot studies in regenerative medicine highlight the promise of dental stem cells dependent translational medicine. Although the frame of dental stem cells dependent translational medicine has been primarily and successfully constructed, a proper quality control and efficacy in the clinic, and a better understanding of underlying mechanisms regulating dental stem cells regenerative capacity are generally regarded as problems remaining to be urgently solved.