Keywords

1 Introduction

Stem cell-based therapy as a major field in regenerative medicine has attracted scientists in the field of tissue engineering [1]. Stem cells possess a remarkable capability for proliferation and for differentiation into various cell types, and such capabilities can be useful for regenerative therapies. Nowadays, although multipotent mesenchymal stem cells derived from bone marrow (BMMSC) are among the best-known and best-characterized cells for tissue engineering [2], the invasive procedure needed to isolate these from bone marrow, and variations in their potency according to the age of the donor [3, 4] impede their usefulness. Therefore, several alternative tissue origins for stem cells have been explored including skeletal muscle [5], dental tissues [6,7,8,9], and adipose tissue [10,11,12].

In clinical dentistry, teeth are extracted due to various reasons, such as impacted third molars for orthodontic reasons. Collecting these extracted teeth does not require additional procedures. In addition, deciduous human teeth naturally exfoliate. Hence, harvesting MSCs from dental tissues of such exfoliated teeth is convenient. Dental stem cells (DSCs) have demonstrated the capability to differentiate into various cell lineages, including osteogenic, odontogenic, neurogenic, and adipogenic pathways [13, 14].

Although because of their origin DSCs seem to be more efficient for developing odontogenic structures than other osseous tissues when compared with BMMSCs [15], the precise differences between DSCs and BMMSCs, and among each of the different types of DSCs remain unclear. These cells are derived from the neural crest (i.e., they have an ectomesenchymal origin) [16], which gives them a superior capability for regenerating a wide variety of tissues [17], when compared to BMMSCs that originate from mesoderm [18]. Adding to this greater usefulness, DSCs can be obtained without any major ethical concerns [19, 20].

The first report of MSCs in the dental pulp was in 1985 by Yamamura [21, 22]. To date, five main types of dental tissue-derived stem cells have been reported. The first type was termed as “postnatal dental pulp stem cell” (DPSC) by Gronthos et al. [23] in 2000. These can regenerate a “dentine–pulp complex like” tissue in vitro. Subsequently, in 2003, Miura et al. reported the isolation and characterization of stem cells from exfoliated deciduous teeth which had proliferated from remnants of living DPSCs. These are known as stem cells from human exfoliated deciduous teeth (SHED) [24]. Morsczeck et al. [25] and Kemoun et al. [26] reported undifferentiated mesenchymal progenitor cells from the human dental follicle, in the connective tissue sac surrounding developing teeth. They named these dental follicle stem cells (DFSCs). The dental follicle forms the tooth and the supporting structures, and the follicle develops into the periodontal ligament when tooth is erupting. Stem cells have also been reported as being present in the periodontal ligament [27]. In 2006, Sonoyama et al. described unique undifferentiated stem cells from the dental apical papilla of human immature permanent teeth (SCAP) with the ability to generate osteoblasts, odontoblasts, and adipocytes in vitro [28, 29].

Figure 1 schematically shows the potential sources of these various dental tissue-derived stem cells. Although these DSCs have been investigated in many studies [13, 14, 30, 31], only limited work has been done to characterize their biological properties and compared their in vivo applications. In this chapter, we have compiled information regarding the isolation, characterization, and potential applications of DSCs in tissue regeneration and tissue engineering.

Fig. 1
figure 1

Schematic view of various sources of stem cells from dental tissues

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2 Stem Cells Obtained from Human Permanent Teeth or Exfoliated Deciduous Teeth

As mentioned earlier, the first isolated DSCs were dental pulp stem cells (DPSCs). DPSCs were isolated initially from permanent third molar teeth. They demonstrated colony formation and a high proliferation rate, as well as the ability to form calcified nodules [23]. DPSCs derived from impacted third molars at the stage of root development can differentiate into active migratory odontoblast-like cells, which are able to produce a three-dimensional dentine-like mineralized structure [32]. The stem cell properties of DPSCs vary according to the donor’s age when the specimens are obtained (Nakamura et al. 2009); i.e., the cells show a reduction in stem cell features as donor age is increased.

The transition of deciduous teeth in the primary dentition to the permanent dentition is a dynamic and distinctive process, in which the developing permanent teeth buds gradually resorb the roots of the overlying deciduous teeth [33]. As mentioned above, a unique population of DSCs can be harvested from the remaining living pulpal cells of exfoliated teeth, and cultivated in the laboratory. These cells, known as SHED, provide an easily accessible source of stem cells that can be preserved for future applications [24]. These cells differ from regular DPSCs because of their greater proliferation rate and enhanced population doubling rate, and their capability to form sphere-like cell clusters [34]. SHED express the minimum essential markers for stem cells defined by the International Society for Cellular Therapy criteria [15, 35]. They also express some embryonic stem cell markers [24], as listed in Table 1.

Table 1 Sources, surface markers, multipotentiality, and in vivo applications of dental stem cellsa
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While SHED have been isolated using similar methods to those used to isolate DPSCs, there are two major differences between SHED and DPSCs. Firstly, the source of SHED is the dental pulp tissue of deciduous teeth, rather than permanent teeth. Secondly, the isolated cells do not grow as proliferative cells, but instead grow as clustered fibroblast-like cells [36]. It has been shown that 69.8% of SHED are in the S and G2 stages of the cell cycle; however, 56% of DPSCs are in those cell cycle phases. This explains the different proliferative capacity of these cells [37]. In the ensuing sections, the isolation, characterization, and various applications of DPSCs and SHED will be discussed.

2.1 Isolation and Characterization

There are many approaches to the collection and isolation of DPSCs. A key consideration is to maintain the quality and safety of the isolated cells. Generally, enzymatic digestion of related tissues (such as the dental pulp of an adult tooth or the remaining dental pulp of an exfoliated tooth) or an outgrowth of a tissue explant [38] are the major approaches that have been used for DPSCs and SHED. Enzymatic digestion usually involves placing the source tissues into an appropriate mixture of enzymes such as collagenase I and dispase for 30–60 min at 37 °C, to break the tissue down in order to obtain single-cell suspensions. The tissue remnants are filtered through a sieve or strainer. This is followed by colony-based cultivation of the stem cells, or by cell sorting using magnetic or fluorescent markers [39].

On the other hand, in the outgrowth method, the harvested tissue is diced into 1–2 mm pieces and placed into culture plates, to allow outgrowth of cells from the tissue pieces [40]. Comparing these two methods of isolation, several studies have shown that DPSCs isolated by enzymatic digestion have a higher proliferation rate, higher expression of stromal precursor antigen (STRO-1) and CD 34, and higher rates of osteogenic or odontogenic differentiation [41,42,43].

After the isolation of DPSCs and SHED, the next step is to cultivate the cells to expand their number to reach the amount required for cell-based therapy. In addition, the pathway of differentiation can be adjusted by the choice of parameters used in the culture system [39, 44, 45]. Various culture systems have been used including serum-free versus serum-rich culture media, sphere-forming culture, and co-culture systems. Most commonly, a 10–20% concentration of fetal bovine serum (FBS) is included in the culture media for both isolation and expansion of DPSCs and SHED, to accelerate cell adhesion during the first stage of culture. Because of the risk of contamination of serum by bovine pathogens (and the presence of bacterial endotoxin) and an altered possibility of malignant transformation of the stem cells in a serum-containing culture system, the use of a chemically defined serum-free culture system has been recommended [46,47,48,49]. In this regard, a serum-free medium (Table 2) has been reported to be successful for obtaining DPSCs [50]. Cells from the adherent population appeared to be more engaged in the odontoblastic lineage than the adherent cells. The neural stem cells that are derived from various sources can proliferate in sphere-forming culture systems [51, 52], which are also recommended for differentiation of DSCs [53, 54].

Table 2 Serum-free medium for DPSC culture
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Phenotypic markers expressed by DPSCs are summarized in Table 1. Expression of these markers relates to the unique capability of these cells [28, 55]. For example, STRO-1 positive cells show greater odontogenic or osteogenic differentiation, while CD34 and CD117 positive DPSCs show a greater ability for cell renewal and for mineralization [56]. Low expression of Class II HLA-DR surface antigen indicates that these cells may be immunologically privileged, which reduces concerns around antigenicity in the setting of tissue matching between the donor and the recipient. If the cells truly are not immunogenic then this raises the possibility of creating banks for DPSCs. This aspect is discussed in the last section of this chapter.

The phenotype of SHED differs from that of DPSCs. Pluripotency markers such as Pou5f1, Sox2, Oct3/Oct4, and Nanog are expressed more strongly in SHED than in DPSCs [57]. Nestin as a neuroepithelial marker is expressed less in SHED cells compared to DPSCs [58], which explains the reduced ability of SHED cells in comparison to DPSCs for neuronal regeneration [58]. However, due to the greater proliferative capability of SHED cells, they form sphere-like clusters in a neurogenic culture medium [24].

DPSCs have shown odonto/osteogenic, neurogenic, and adipogenic differentiation in preclinical studies [23, 59, 60] (as listed in Table 1). More recently, in vitro investigations have revealed osteogenic, chondrogenic, and myogenic differentiation of DPSCs [61,62,63]. Similar to DPSCs, SHED cells have also demonstrated the ability to undergo odonto/osteogenic, chondrogenic, neurogenic, adipogenic, and myogenic differentiation [13, 14, 30, 31] (Table 1).

2.2 Regenerative Applications

The concept of harvesting and banking dental tissue MSCs has opened up a new window for stem cell-based regenerative treatments. As already mentioned, DPSCs and SHED can differentiate effectively to various cell lineages. In this section, the regenerative capacity of these cells will be discussed further.

2.2.1 Dentine–Pulp Complex Regeneration

Dental pulp is a specialized connective tissue, which consists of odontoblasts, endothelial cells, neurons, fibroblasts, and other cells. The peculiar internal anatomy of teeth and the unique blood flow may influence the survival of stem cells [64]. When the dental pulp is infected by bacterial pathogens, it is hard to remove or inactivate these pathogens through antibiotics alone. For a number of clinical reasons, extirpation of the whole pulp may need to be undertaken [65].

For regeneration, dental pulp stem cells can be expanded in the laboratory. When seeded onto hydroxyapatite/tricalcium phosphate (HA/TCP), the cells have demonstrated formation of a dentine-pulp-like complex in mice [23]. In addition, SHED implanted into mice can generate odontoblast-like cells and dentine-like mineralized nodules. However, SHED cells do not seem to be able to form a complete dentine-pulp-like complex like that seen with transplantation of DPSCs [24]. In contrast, DPSCs can form mineralized nodules, which are covered by a layer of dentine-like reparative tissue in vivo [66]. DPSCs that have been seeded onto calcium phosphate [67], hexafluoropropanol silk [68], and polylactic acid [69] scaffolds have demonstrated dentine–pulp complex formation in animal models.

In addition to dentine–pulp complex regeneration, DPSCs have also been used in periodontal regeneration [31]. DPSCs transplanted into immunocompromised mice have been shown to differentiate into collagen forming cells, with the capacity to form a cementum-like tissue [70]. The expression of particular phenotypic markers such as SCD-1, STRO-1, CD44, and CD146 on DPSCs and SHED cells sensibly linked to their role in periodontal regeneration [71, 72]. In addition, both SHED and DPSCs are capable of bone regeneration [31]. This aspect will be discussed further in the next section.

2.2.2 Bone Regeneration

Bone formation, including the aggregation of osteoprogenitor cells, is similar in some ways to tooth bud development, but without the epithelial invagination aspect. Intramembranous and endochondral bone formation are the two main processes of bone regeneration. In endochondral regeneration, the aggregated MSCs first undergo chondrogenesis followed by ossification of the cartilage into bone tissue [73]. In intramembranous bone formation, MSCs first become osteoprogenitor cells and then further differentiate into osteoblasts, that create extracellular matrix containing collagen fibrils and other bone components, and this is called osteoid.

Bone tissue possesses the intrinsic capability of regenerating itself through adulthood [74]. However, when bone defects or fractures are larger than the regenerative capacity of the bone, other interventions are required to rehabilitate the defect site in the bone tissue [74]. One alternative is using stem cell-based therapy with stem cells of dental origin [75]. In vitro studies have demonstrated the osteogenic and chondrogenic multipotency of both DPSCs and SHED [75,76,77]. An in vivo investigation showed that DPSCs can regenerate osteoblasts and endotheliocytes that eventually formed bone in immunocompromised rats [78].

The application of SHED in combination with a hydroxyapatite (HA)/tricalcium phosphate scaffold has been shown to regenerate calvarial bone defects in animal models [79]. These studies have also shown that a large amount of bone and bone marrow-like structures are formed in the regenerated mineralized matrix. Other investigations have reported that the application of DPSCs in conjunction with fibroin/collagen scaffolds can significantly enhance bone formation after 4–8 months in rats [80,81,82]. The application of DPSCs loaded onto a HA-based hydrogel showed superior bone healing in rat calvarial defects compared to the scaffold only and to untreated control groups [83]. In addition, DPSCs cultured for 13 days on poly(lactide-co-glycolide) scaffolds caused significant bone regeneration in the same types of defects [84]. Other studies have reported the successful application of DPSCs and SHED in combination with HA/TCP in rats and mice in the treatment of calvarial defects [85].

The research team of d’Aquino et al. demonstrated that administration of DPSCs loaded onto a collagen sponge resulted in higher mineralization after 1 month and complete bone regeneration with a large amount of cortical bone formed after 3 months, in comparison to a scaffold only group [78]. Moreover, an increased level of clinical attachment was found in the gingiva overlying the defect site after transplantation of DPSCs loaded onto a collagen scaffold [86].

Several studies have investigated the use of DPSCs or SHED for bone regeneration in maxillary or mandibular bone defects [87,88,89,90,91]. Alkaisi et al. created a mandibular defect (from the first premolar to mental foramen) in rabbits, and assessed the formation of bone after applying SHED without using a scaffold [87]. Radiographic evaluations revealed that in SHED transplanted animals, partial bone defects were bridged after only 2. In addition, the regenerated bone in this group showed a bony ridge with higher radiopacity than insights treated with a scaffold only after 4 weeks. Finally, after 6 weeks, clear corticalization and strong radiodensity were observed in the defect gap in sites treated with SHED group. Histomorphometric analysis showed that the regenerated bone was significantly higher in the SHED group compared with the scaffold control.

Paino et al. created tissue-engineered woven bone tissue using DPSCs in vitro, and then transplanted the manufactured bone tissue into the mandibular defects in rats [88]. Their findings confirmed the remodeling capacity of the implanted tissue, and its integration into the surrounding normal bone with the passage of time. Lamellar bone tissue with vital osteocytes and vascularized Haversian canals were found after bone remodeling.

SHED have been administered intravenously in order to treat osteoporosis in animal models of this condition [79, 92]. Ma et al. have found that this type of administration route for SHED can ameliorate problems of low bone mineral density, and can improve the radiopacity of the trabecular bone structures [79]. Moreover, Liu et al. showed that systemic administration of SHED was able to enhance bone volume, and promote trabecular number, thickness, and density [92]. In general, SHED transplantation has been found to improve cortical bone parameters, including bone area, thickness, and cortical bone fraction [93]. Bone that has been regenerated using SHED has been found to express higher levels of Runx2, alkaline phosphatase, and osteocalcin than controls [94]. At the same time, systemic application of SHED markedly downregulates genes associated with bone resorption such as RANKL and C-terminal telopeptide, and upregulates osteoprotegrin (OPG) [79, 92, 95]. This is a powerful demonstration of the immunomodulatory effects of SHED.

According to a recent systematic review by Leyendecker et al., transplantation of DPSCs into cranial, maxillary, and mandibular bone defects gives superior regenerative outcomes compared to controls [93]. However, in one study Annibali et al. found no difference in bone regeneration between DPSCs and control groups [96], while in another study by Behnia et al. there was no difference after the application of SHED in combination with a collagen scaffold compared to a scaffold only for the treatment of mandibular defects in dogs [97].

The type of scaffold material that is used as the carrier for DSCs plays an important role in bone repair. For example, Zhang et al. did not find ectopic bone regeneration in DPSCs loaded on HA/TCP [98]. However, Kuo et al. demonstrated that DPSCs in combination with alpha-calcium sulfate hemihydrate/amorphous calcium phosphate could efficiently promote bone formation in comparison to DPSCs + calcium sulfate dihydrate or DPSCs + calcium sulfate dihydrate/TCP [90].

2.2.3 Neural Regeneration

Neural regeneration is a challenging objective for two major reasons. The first is the lack of neural progenitor cells, while the second reason is that the local microenvironment may impede regenerative processes [99]. DPSCs express markers of neural progenitors such as nestin and Pax6, due to the neural crest origin of these cells [100, 101]. Human DPSCs are able to form spheroids under serum-free neuronal stimulating culture conditions [102]. In addition, even without neural induction, DPSCs express neural markers such as CDH2, TUBB3, and NFM [103].

Transplantation of DPSCs and SHED into defect sites in the central nervous system (CNS) has been shown to enhance neural recovery [56, 101, 104]. DPSCs can coordinate axonal regrowth by secreting CXCR-4 and stimulating the SDF-1/CXCL12 axis that induces neuroplasticity [105]. It has been reported that the implantation of DPSCs into the hippocampus of immunocompromised mice can accelerate cell recruitment and proliferation, and the maturation of endogenous existing neural cells [118]. In fact, taken together all these findings suggest that DPSCs may have an application as a modulator and stimulator in neural recovery in the CNS [143].

In spinal cord trauma and cerebral ischemia models, DPSCs can significantly enhance neurological dysfunctions [57, 161]. Furthermore, in the case of a peripheral nerve injury, DPSCs can mediate neural tissue engineering with artificial nerve conduits, to regenerate myelinated neural fibers [122]. Luo et al. demonstrated that transplantation of a heparin–poloxamer hydrogel and DPSCs with fibroblast growth factor could significantly regenerate neurons in spinal cord injuries, and functionally repair nerve injury defects in rats after 28 days [101].

Despite several studies which have demonstrated that both DPSCs and SHED cells can (1) differentiate into functional neural cells which were voltage-sensitive in vitro, (2) express neural markers, and (3) migrate into the CNS in animal models [24, 105, 106], many in vivo investigations have reported that DPSCs and SHED are unable to differentiate into functional neurons that the sites of injuries to nerves [102]. The neural regenerative capacity of DPSCs and SHED seems to occur because of their production of neurotrophic products [107]. Furthermore, these cells impede axon growth inhibitor signals and improve the microenvironment for regeneration [108]. Further studies are necessary to clarify the neural regeneration capability of DPSCs and SHED.

2.2.4 Other Regenerative Applications

Muscle Regeneration

Arminan et al. reported the differentiation of DPSCs into cardiomyocytes in rats [109], while Yang et al. documented that DPSCs can differentiate into dystrophin-producing muscle cells in a mouse model of cardiac muscle injury [110]. Based on the observation that DPSCs infused into cardiac defect sites express dystrophin and myosin [111], it has been proposed that DPSCs can potentially be applied for muscle regeneration [110].

Corneal Regeneration

Some experimental evidence indicates that DPSCs are more similar to epithelial stem cells than BMMSCs [112, 113]. In fact, DPSCs can differentiate into keratinocytes and can express keratinocyte markers [114]. Gomez et al. reported that eye transparency in a rabbit corneal defect model was improved by the implantation of a sheet of tissue-engineered DPSCs into the defect sites [115]. In another study, DPSCs delivered by soft contact lenses in a clinical application were shown to promote corneal epithelial regeneration [116]. DPSCs transferred from the contact lenses to the corneal surface expressed the keratinocyte markers cytokeratin 3 and 12. Moreover, DPSCs can impede conjunctival cells from growing into the center of the cornea [117].

Cartilage Regeneration

As mentioned earlier, DPSCs can develop into dentine, cartilage, and bone tissues [118]. Yu et al. showed cartilage formation after 14 days after the implantation of pellets containing DPSCs into the renal capsule of rats [119]. In addition, Morito et al. reported similar results after the subcutaneous implantation of DPSCs in combination with FGF in immunocompromised mice [120].

Hair Follicle and Blood Vessel Regeneration

DPSCs have been transplanted to the surgically compromised hair follicles, where they have been shown to cause the formation of new head bolts and the regeneration of hair fibers [121].

DPSCs can promote angiogenesis and vasculogenesis in sites where there is peripheral nerve injury [122]. DPSCs can differentiate into endotheliocytes in rat models [78].

Endocrine Regenerative Potential

Previous studies have documented the possibility of using DPSCs and SHED in the treatment of diabetes mellitus using a regenerative approach, due to their multipotent capabilities, since they can differentiate into insulin-producing cells [13]. DPSCs are capable of differentiating into pancreatic cells and also into insulin-producing islet-like cells [123, 124]. Kanafi et al. implanted islet-like cell aggregates derived from DPSCs or SHED cells into diabetic mice, and found that the SHED group was superior to the group treated with DPSCs in terms of maintaining normal blood glucose levels [50].

SHED have also been studied for the treatment of injuries to the kidney, where they influence the proliferation of tubular epithelial cells [125], through a paracrine effect that induce cell migration and facilitates the repair of acute kidney damage.

Regenerative Therapy of Various Systemic Disease

Previous investigations demonstrated the positive impact of DPSCs and SHED when used in the treatment of various systemic diseases [126]. The enormous capacity of these DSCs makes them an attractive source for regenerative therapies in medicine. Both DPSCs and SHED can differentiate into hepatic cells [127, 128], which makes them promising in the treatment of liver cirrhosis [129].

As already discussed, due to their myogenic multipotency, DPSCs and SHED cells can be of value in the treatment of muscle injuries, including to the myocardium. Both cell types have been used experimentally to treat myocardial infarction [111] and Duchene muscular dystrophy [130, 131]. Moreover, the immunomodulatory and anti-inflammatory effects exerted by these cells have been applied successfully in the treatment of a range of inflammatory and autoimmune diseases including systemic lupus erythematosus [132], rheumatoid arthritis [133], autoimmune encephalomyelitis [134], Alzheimer’s disease, and Parkinson’s disease [135, 136].

There have been few systematic comparisons between BMMSCs and DSCs in terms of their immunophenotype, gene expression profile, and regenerative potentials [34]. Collectively, in vitro investigations show that DPSCs share a similar pattern of gene expression with BMMSCs [15]. In addition, signaling pathways of odontoblastic differentiation of DPSCs are similar to the pathways whereby bone marrow-derived stem cells take on osteoblastic features [15].

Shi et al. evaluated gene expression in DPSCs and BMMSCs, and showed that more than 4000 known human genes were similar between these cells [137]. However, they have found that collagen type 18, insulin-like growth factor-2, and cyclin-dependent kinase 6 were much more highly expressed in DPSCs, while insulin-like growth factor binding protein-7 and collagen types I and II were expressed more in BMMSCs [137].

Yamada et al. characterized and compared DPSCs and BMMSCs using a cDNA microarray system, including 12814 genes and a clustering algorithm [138]. They demonstrated that after osteoinduction DPSCs expressed alkaline phosphatase, DSPP, and DMP-1 at levels that were higher than BMMSCs. However, in the clustering assessment, it became apparent that both cells share similar gene regulation pathways for signaling, cell metabolism, and communication [138].

Despite DPSCs and BMMSCs having many similarities in regulating roles, in signaling factors, and in their expression profile, these two types of stem cells are very different in their proliferative capacity and their differentiation potential, and this has led to distinct patterns of use for tissue regeneration in preclinical studies [15]. For instance, the chondrogenic potential of DPSC, and the adipogenic capacity of both SCAP and DPSCs are weaker than those of BMMSCs. Conversely, the neurogenic capabilities of DSCs are far more potent than those of BMMSCs. In addition, SHED and PDLSCs have a much higher growth potential compared to BMMSCs [139]. These differences may be due to the neural crest origin of DSCs.

3 Stem Cells from the Apical Papilla

The apical part of dental papilla is a cell-rich zone containing stem cells (Fig. 1). The apical papilla can be harvested from an immature extracted tooth, and used for isolation of SCAP [140]. The main difference between SCAP and DPSCs is that SCAP is the precursor of the radicular pulp. The characteristic differences between these cells are listed in Table 1. In general, SCAP derives from the developing dental tissues, which consists of early progenitor cells that are distinctive from the cells of mature tissues (DPSCs) [141].

3.1 Isolation and Characterization

After harvesting the apical papilla of a developing root, the tissue is diced into smaller pieces and subjected to enzymatic digestion with collagenase and dispase [140] as already described for the isolation of DPSCs and SHED.

Cultivated SCAP possess low immunogenicity, as seen by lymphocyte assays in the laboratory [142]. Flow-cytometry analysis shows that SCAP express typical cell markers including CD73, 90, and 105 (Table 1) [143]. In addition, the perivascular location of SCAP reflected by their expression of STRO-1 and CD146, which gradually fades with extended passaging of the cells [144,145,146]. Although expressed at a relatively low amount CD24 seems to be exclusively positive in SCAP compared to other DSCs and other MSCs [147, 148]. Expression of CD24 expression reduces to zero after the 10th passage [149, 150]. If CD 24 is a specific marker for determining the “stemness” of SCAP, these findings imply that loss of stemness in SCAP occurs after the 10th passage.

3.2 Regenerative Applications

SCAP can differentiate into various cell lineages [149], which make these cells an attractive source for tissue engineering.

3.2.1 Pulp Regeneration and Angiogenesis

Regenerative endodontic therapy includes the process of regenerating the dentine–pulp complex [151]. SCAP are one of the most promising stem cell sources for such therapy due to their known odontogenic potency and their expression of dentine-related differentiation markers such as dentine sialphosphoprotein (DSPP) [152].

Nowadays, with the application of scaffolds and the inclusion of growth factors such as vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF), there is increasing optimism regarding the possibility of regeneration of dentin-pulp complex [153,154,155]. Cell homing therapies have determined that chemotactic factors for SCAP include SDF-1, TGF-β, and granulocyte-colony stimulating factor (G-CSF). These not only can improve the migration of SCAP, but they also can promote their differentiation [156].

Sonoyama et al. reported that human SCAP can develop into a functional root in an animal model [29]. De novo dentine–pulp complex regeneration by SCAP begins with the migration of these cells onto the dentine surface, followed by odontoblastic differentiation and the expression of phenotypic markers of dentinogenesis [157].

Hikens et al. demonstrated that SCAP are able to express a number of markers of angiogenesis including VEGF), thrombospondin-1, angiopoietin-1, endostatin, matrix metalloproteinases, and FGF [153]. Revascularization is a critical requirement for pulpal tissue engineering [158]. SCAP cells appear to be a good candidate for promoting revascularization because of their original niche in the perivascular location [159].

Moreover, in repairing dentine–pulp complex, the administration of scaffolds can be challenging, as some of the biomaterials that are used (e.g., HA and TCP) are osteoinductive, and thus there is a risk of generalized calcification occurring in the pulp space [157]. Thus, the properties of the scaffold need to be adjusted to suit the requirements of pulp tissue regeneration. Amirkia et al. used a three-dimensional silk fibroin as a natural scaffold, and found that this improved the attachment of SCAP and their differentiation [160]. In addition, chitosan-based scaffolds seem to be an appropriate carrier for SCAP as they improve their odontogenic potential [161].

3.2.2 Neural Regeneration

The neurogenic potential of SCAP has been exploited for neural tissue engineering [158]. SCAP originated from the neural crest, and they express high level of the neural marker nestin [152]. In addition, SCAP can drive a neuroprotective mechanism, by decreasing inflammation and inducing differentiation of oligodendrocytes [155]. SCAP also express various markers (genes) for neurogenesis (Table 1). In vitro and in vivo investigations show SCAP can participate in neurite outgrowth and in axonal induction [158].

Under neurogenic induction, SCAP start to mimic spindle-shaped neurocytes, with long cellular process [152, 162, 163]. In a study by De Berdt et al., the entire apical papilla was transplanted into an area of artificial spinal cord damage in an animal model. The apical papilla functioned as a scaffold for SCAP to regenerate neural tissue in the original niche of the cells [164]. Interestingly, this study revealed that hypoxic conditions could stimulate SCAP to express neural-specific genes and to secrete growth factors [164].

3.2.3 Bone Regeneration

The osteogenic potency of SCAP has been confirmed by studies which have shown differentiation of these cells into osteoblasts, as determined by alizarin red staining of calcium deposition and the expression of osteogenic markers, including bone sialoprotein, alkaline phosphatase, gamma-carboxyglutamate protein, runt-related transcription factor-2 (RUNX2), and bone morphogenetic proteins (BMPs) [32]. The osteogenic differentiation capability of SCAP is comparable to that of BMMSCs [148].

The cultivation of SCAP in an osteogenic medium results in the formation of osteoblast-like cells that are able to produce mineralized nodules [165]. Nada et al. observed that SCAP isolated from different teeth showed different differentiation patterns. For instance, SCAP isolated from third molars tended to produce diffuse mineralization, whereas SCAP isolated from premolar teeth showed a localized pattern of calcific deposits [165]. To date, no in vivo investigations have been conducted to evaluate the bone regeneration capacity of SCAP.

3.2.4 Other Regenerative Applications

The chondrogenic potential of SCAP has been assessed using Alcian Blue staining of the chondrocytes that have formed in vitro [166], but to date no study has been carried out to evaluate the molecular evidence for the chondrogenic potential of SCAP, such as the expression of chondrogenic genes in SCAP under chondrogenic induction. Thus far, no in vivo study of cartilage formation by SCAP has been reported.

Several studies have demonstrated the adipogenic capacity of SCAP [165]. SCAP can express lipoprotein and lipase, suggesting the adipogenic capability of these cells [152]. However, in comparison to BMMSCs, their adipogenic potential is low [148, 153, 167].

4 Dental Follicle Stem Cells

The dental follicle is a loose connective tissue sac surrounding the tooth bud. It plays an important role in tooth development and eruption. The dental follicle is involved in tooth eruption by controlling osseous remodeling through the timely production of various secreted mediators [168]. Stem cells have been harvested from dental follicle of different species in various developmental stages [169, 170]. DFSCs are multipotent, and can differentiate to form periodontium, bone, and cementum [169, 171].

4.1 Isolation and Characterization

Human extracted third molar teeth are the major tissue source that has been used to isolate human DFSCs. The method for DFSCs isolation is similar to that described for other DSCs.

In culture, DFSCs have a typical fibroblast-like morphology. They express CD9, CD10, CD13, notch-1, and nestin, but they do not express CD31, CD34, CD45, and HLA-DR [172, 173] (Table 1). DFSCs also express cementum attachment protein and cementum protein-23 (CP-23), which are two putative cementoblast markers [15]. DFSCs also express STRO-1 and BMP receptors in vivo [26].

4.2 Regenerative Applications

DFSCs can differentiate into osteoblasts, cementoblasts [30], and adipocytes in the appropriate inducing culture media [170]. Although experimental investigations have revealed mineralized tissue formation by DFSCs, DPSCs have a greater capacity for all hard tissue formation. Yagyuu et al. reported hard tissue formation of DFSC at preclinical studies [176], while Yokoi et al. demonstrated that DFSCs are capable of regenerating soft tissue and PDL in vivo [174].

4.2.1 Bone Regeneration

DFSCs have the capacity to create calcification, as seen both in cell culture [25] and in animal [175] studies. Several investigations have reported the osteogenic differentiation of DFSCs in appropriate osteogenic medium [176,177,178,179]. In vivo bone formation by DFSCs has been demonstrated in critical size bone defects in rat calvaria [180]. Moreover, in vitro investigations have indicated that BMP-6 and BMP-9 promote the osteogenic differentiation of DFSCs [176].

Rezai Rad et al. showed that a temperature of 37–40 °C was optimal for inducing osteogenesis of DFSCs in vitro [181]. Honda et al. reported the results of two different animal studies to evaluate the bone regenerative capacity of DFSCs [180, 182]. The findings of both studies suggest that DFSCs supported bone regeneration, although it was not clear whether the transplanted stem cells had in fact differentiated into osteoblasts.

4.2.2 Periodontal Regeneration

DFSCs are capable of generating osteoblasts, cementoblasts, and PDL [14]. Honda et al. isolated DFSCs from the third molar teeth of 6-month-old pigs [180]. DFCSs were seeded in the bottom of a tube and DPSCs and the enamel organ epithelium (which originated from same pigs) were added in order to produce a recombination which mimicked the tooth primordia. The whole mixture was then transplanted into the omentum of immunocompromised rats (Fig. 2). A thick layer of dentine with viable odontoblasts and a layer of cementum-like tissue were found at 24 weeks post-surgery. Moreover, collagen fibers were present in a pattern that resembled the PDL, and they were attached to the cementum-like layer.

Fig. 2
figure 2

Schematic diagram of procedure used to regenerate engineered dental root analogue in Honda et al. study [180]

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Another histological evaluation also confirmed the possibility of whole periodontium regeneration via expanded DFSCs [182]. A further investigation reported that DFSCs could differentiate into PDL cells, and could regenerate PDL-like structures including cementum-like tissues [183]. Other researchers have indicated that DFSCs used in combination with a treated dentine matrix could regenerate root-like tissues with a dentine–pulp complex [184].

5 Stem Cells from the Periodontal Ligament

Although early studies provided some evidence to support the differentiation capability of periodontal ligament (PDL) cells, such as the ability to differentiate into cementum-forming cells and osteoblasts [185, 186], Seo et al., conclusively identified a population of MSCs within the periodontal ligament that can express stem cell markers and that have the capability to differentiate into various cell lines [27].

5.1 Isolation and Characterization

To isolate PDLSCs, extracted teeth with an intact PDL are immersed into a digestion solution consisting of collagenase and trypsin [187]. The resultant cells from the enzymatic digestion can then be cultured in various media such as serum-containing media and neurosphere-forming medium, according to the intended therapeutic purpose [53].

PDLSCs possess low immunogenicity, and they can modulate behavior of peripheral blood mononuclear cells by secreting TGF-β and HGF [188]. PDLSCs isolated from inflamed periodontium can significantly reduce the activity and proliferation of T lymphocytes [189]. They express high levels of interleukin (IL)-10, and IL-17 in comparison to PDLSCs derived from healthy tissues [190].

PDLSCs express MSC-related markers and tendon specific transcription factor (scleraxis) (Table 1). Scleraxis expression is significantly higher in PDLSCs than in DPSCs and in BMMSCs [15]. PDLSCs do not express hematopoietic markers such as CD14, CD19, CD34, and CD45 [15], or markers associated with hematopoietic cells including CD40, CD80, and CD86 [188].

5.2 Regenerative Applications

The current literature report that PDLSCs can differentiate into cementum-like structures, and along osteogenic, adipogenic, and chondrogenic cell lineages (Table 1).

5.2.1 Periodontal Regeneration

PDLSCs were firstly applied in animal models and used to reconstruct cementum-PDL-like structures [27]. Subsequently, several studies investigated the capability of PDLSCs for periodontal regeneration [191,192,193]. Ninomiya et al. implanted a HA scaffold loaded with PDLSCs into the dorsal muscle of rats, and demonstrated bone-like tissue formation [194]. PDLSCs seeded on HA/TCP successfully formed PDL and cementum-like tissues surrounding the scaffold [195, 196]. Complete PDL regeneration was achieved, with formation of Sharpey’s fibers between the newly formed cementum and the fibers of the PDL [197].

Likewise, when transplanting PDLSCs that were treated with recombinant human plasminogen activator inhibitor-1, and then seeded on HA/TCP scaffold, into the dorsal region of immunodeficient mice, cementum-like tissue surrounded by PDL-like tissue was observed after 10 weeks [198].

Using stem cell all sheets derived from PDLSCs for tissue engineering has been attempted. HA/TCP wrapped with PDLSCs cell sheets has been shown to generate PDL/cementum-like structures in rats and mice [199, 200]. Moreover, adding platelet-rich fibrin (PRF) to a HA/TCP scaffold wrapped with PDLSCs sheets was shown to regenerate not only cementum and PDL, but also blood vessels [201].

Transplantation of human PDLSCs sheets into infra-bony mandibular defects in dogs showed new cementum regeneration, with the formation of collagen and nerve fibers around the roots of the teeth after 8 weeks [202]. When PDLSCs and gelatin sponge scaffolds were grafted onto fenestration defects in rats, complete healing of bone, cementum, and PDL was seen after only 3 weeks [203]. These findings suggested that there is considerable potential for regeneration of the periodontium using PDLSCs, and that this approach is likely to be successful when used in the clinic to manage real bony defects [204].

However, a significant point is that the regenerative capability of PDLSCs is affected considerably by the presence of inflammation and by the age of the PDL tissue from which the cells are harvested [200, 204, 205]. Gao et al. compared various PDLSCs from donors of different ages [200]. They found that PDLSCs from younger donors had greater cementum/PDL formation potential than those from older donors. Other studies have reported that PDLSCs derived from donors with periodontitis have a significantly lower capacity to form bone than PDLSCs derived from healthy sites or healthy donors [205].

5.2.2 Bone Regeneration

The osteogenic potential of PDLSCs has been shown in several in vitro studies, which have reported the formation of mineralized nodules [27, 206,207,208]. Although PDLSCs typically form cementum-like structures [15], PDLSCs implanted into periodontal defects of animal models appear to accelerate the regeneration of trabecular bone next to PDL-like structures, thus demonstrating their capacity for alveolar bone regeneration [27, 209].

Transplantation of human PDLSCs encapsulated in a RGD-modified alginate has been shown to enhance bone formation in critical-sized calvarial defects in rats [210]. Several studies have observed a lower bone regenerative potential of PDLSCs compared to BMMSCs. By way of comparison, BMMSCs were reported to be more effective for alveolar bone repair in canine models [211]. Another study confirmed the lower osteogenic capability of PDLSCs compared to BMMSCs [212]. The reason for this may be the presence of more end-differentiated cells in the PDLSCs population [213]. However, there are a few contrary reports in the literature suggesting similar or even better osteogenic potentials of PDLSCs compared to BMMSCs [214], and other DSCs [80, 215]. This point needs further research to resolve it.

5.2.3 Tendon and Cartilage Regeneration

Gronthos et al. showed that PDLSCs can express a tendon-specific marker (scleraxis) in vitro [206]. A combination of human PDLSCs with an RGD-coupled alginate could form a tendon-like tissue in mice. When used for tendon regeneration, compared with BMMSCs, PDLSCs gave a more highly organized tissue with a greater amount of collagen fibers [216].

Moshavernia et al. showed that cartilage healing could be achieved by applying encapsulated PDLSCs in an alginate hydrogel [216]. Moreover, several animal studies have successfully used PDLSCs for cartilage tissue engineering. Ectopic cartilage formation was observed at PDLSC transplantation sites [102, 217, 218]. It is well-known that cartilage has a very restricted ability for self-renewal and regeneration [219]. In this regard, the chondrogenic potential of PDLSCs is noteworthy, and it is likely that they will be of interest for cartilage repair [102].

5.2.4 Other Regenerative Applications

Recently, it has been shown that PDLSCs have both neurogenic and angiogenic differentiation potentials [53]. PDL-derived spheres are able to differentiate into mesodermal and neural cells. MSCs originated from the PDL can form Schwann cells by inducing the Erk1 signaling pathway [220]. Furthermore, these cells can regenerate retinal ganglion-like cells via functional synapses and respond to calcium [221]. Cen et al. demonstrated that human PDLSCs ameliorate ganglion cells and axonal regeneration when used in the retina of animals with a traumatized optic nerve [222].

Another novel application of PDLSCs is its possible use for cardiogenic differentiation. PDLSCs express some cardiac cell markers, such as sarcomeric actin and cardiac troponin T [223].

6 Banking of DSCs

According to the diversity of DSCs and their beneficial aspects, the potential uses of stem cell-based treatments using DSCs in both dentistry and medicine are significant. The administration of a patient’s own DSCs during therapy may not often be practical, since they may have more conditions requiring treatment in their later years, but have higher numbers of stem cells in their tissues when they are in their childhood years. Banking of DSCs is one way of easily maintaining a suitable supply of DSCs to meet the needs of patients later in their life. Such an approach can pave a new road for progress in healthcare by maintaining a promising source of autologous cells for personalized regenerative treatments.

Given these considerations, the ability to harvest and safely preserve DSCs becomes more important. Nowadays, DSCs can be cryopreserved for a long period of time [224,225,226]. In a number of developed countries, licensed tooth banks have been founded (Table 3) [30, 34, 227]. When such banks have been established there are a number of ethical controversies, as well as social, and legal issues that need to be considered. Consistent and well-documented laboratory procedures are needed to evaluate and preserve the cells. There is also a need for appropriate regulations or legislation regarding stem cell banking.

Table 3 Licensed dental tissue-derived stem cells bank all around the worlda
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7 Limitations

Although stem cell-based therapeutic approaches have shown much promise with an appealing path to their use in tissue regeneration and functional repair, multiple factors need to be optimized. This will require thorough clinical investigations as well as cell culture studies to enhance methods to grow up and maintain a large quantity of cells. One must bear in mind that the availability of the dental tissues over a lifetime will alter, and the time of tooth extraction may not match the time when the patient needs therapy with dental stem cells. Banking of dental stem cells may partially address this issue, but more work is needed to optimize preservation methods, and make such banks less expensive and easier to use for clinical applications. Cell banks must address quality and safety issues such as the stability of the cell phenotype over time, and the possible risks of contamination with endotoxins or with pathogens [30, 228].

8 Conclusions and Future Direction

DSCs as one of the more versatile MSCs have been used widely utilized in preclinical studies for tissue engineering purposes. DSCs exert a range of immunomodulatory activities, and these have not yet been characterized fully. DSCs have a low immunogenicity and may also have immunosuppressive actions. This makes dental tissues a promising source for stem cells for the repair of bone, dental pulp, periodontium, nerves, and other tissues. The various recognized DSCs not only have the potential to differentiate into different cell lines and undergo self-renewal, but their collection could be done as part of normal dental treatment, harvesting them from extracted teeth. Isolation of DSCs usually does not require additional surgical procedures, as exfoliated and extracted teeth are often discarded as medical waste. The collection of DSCs does not pose any major ethical concerns, unlike the use of embryonic stem cells. Nonetheless, in future, it is important to optimize DSC cryopreservation protocols, and address issues including donor-related diversity, and the influence of cell culture conditions [34, 229, 230]. Because of their potential use in a range of cell-based therapies, the ability to expand stem cells of dental origin while also maintaining their original stemness properties is critical. Thus, development of safe and efficient cell expansion strategies should be a focus for research in the future.