Advances of tooth‐derived stem cells in neural diseases treatments and nerve tissue regeneration

Abstract Nerous system diseases, both central and peripheral, bring an incredible burden onto patients and enormously reduce their quality of life. Currently, there are still no effective treatments to repair nerve lesions that do not have side effects. Stem cell–based therapies, especially those using dental stem cells, bring new hope to neural diseases. Dental stem cells, derived from the neural crest, have many characteristics that are similar to neural cells, indicating that they can be an ideal source of cells for neural regeneration and repair. This review summarizes the neural traits of all the dental cell types, including DPSCs, PDLCs, DFCs, APSCs and their potential applications in nervous system diseases. We have summed up the advantages of dental stem cells in neural repair, such as their neurotrophic and neuroprotective traits, easy harvest and low rejective reaction rate, among others. Taken together, dental stem cells are an ideal cell source for neural tissue regeneration and repair.


| INTRODUC TI ON
Nervous system injuries, both central and peripheral, can lead to severe negative outcomes, including hypoesthesia and paralysis.
These outcomes sharply reduce the quality of a patient's life.
Alzheimer's disease, one of the most common causes of dementia, 1 has brought great stress to human beings. Spinal cord injuries (SCI) mainly result in the loss of sensory, motor and autonomic func- Currently, however, there are no effective or efficient treatments for nerve injury to improve patient lives. The current curative effect is quite limited and few patients achieve complete recovery. New treatments to repair and regenerate the nervous system are in urgent need; however, their development has been a great challenge.
In the past decade, advancements in research on mesenchymal stem cells (MSCs) have made great achievements sparked by numerous reports of the application of stem cells in tissue regeneration.
MSCs, first identified in the aspirates of adult bone marrow, are a group of cells that possess the ability to self-proliferate and differentiate into multiple lineages in vitro. 3,4 It has been reported that both bone marrow-derived stem cells (BMSCs) and adipose tissue-derived stem cells (ADSCs) have the ability to differentiate into neuron-like/ Schwann cell-like cells in vitro through the activation of the Notch/ Wnt/SHH pathways. 5 These cells also present a great potential for nerve repair and regeneration when transplanted into an injured sciatic nerve or the spinal cord of mice. 6,7 Recently, dental stem cells, which are derived from teeth, have come to our attention.
Different populations of cells with stem cell properties have been isolated from different parts of the tooth. These cells include dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), stem cells from human exfoliated deciduous tooth (SHED), dental follicle progenitor cells (DFPSCs), stem cells from the apical papilla (SCAP) and so on 8 . All of these cells possess the ability to self-renewal and have multilineage differentiation in vitro. As they originate from neural crest, dental stem cells exhibit characteristics similar to neural cells, such as the high expression of neural markers and protein.
The researches of dental stem cells, one of the most crucial and critical members of the MSCS, have brought out a silver lining in the treatments of diseases using cell therapy, 9 especially for nerve repair and regeneration. Ghazaleh et al 9  In this review, we outline the significant biological traits of the various types of dental stem cells, illustrate examples of research that present the great progress being made in nerve repair and regeneration, highlight the advantages of dental stem cells in neural repair, and sum up the neural repair abilities and mechanisms of dental stem cells. We also point out the major obstacles that need to be conquered in stem cell-based therapy for nerve injuries.

| DENTAL S TEM CELL S
Since the discovery of BMSCs, bone marrow has been the most utilized source of MSCs. However, the method of isolating MSCs from bone marrow is not only complicated but also destructive to donors. Therefore, an alternate source of MSCs is in demand.
The first time that people considered that tooth pulp might contain MSCs was during the observation of severe tooth damage.
This kind of damage penetrates both the enamel and dentin and into the pulp and stimulates a natural repair process by which new odontoblasts are formed. This process produces new dentine to repair the lesion. 8 DPSCs from dental pulp and identified their stem cell features.
Since the discovery of DPSCs, varieties of dental stem cells have been isolated and utilized, respectively, including PDLCs, SCAPs, SHEDs and DFPSCs.
Dental stem cells can be divided into several categories based on where they originate from (Figure 1). These cells share a lot of common attributes; however, they differ in certain aspects such as growth rate, gene expression and inclination for cell differentiation. 8 . However, the mechanisms that contributes to these differences remains unknown.

| Dental stem cells include DPSCs
Dental pulp stem cells are isolated from the dental pulp. DPSCs can be classified into two main groups, including immature DPSCs and mature DPSCs. DPSCs express STRO-1, CD146, CD105, CD73 and CD90. They also possess the capacity for multilineage differentiation. Moreover, immature dental stem cells express embryonic stem cell markers including OCT-4, Nanog and SSEA-3. The expression of the above markers could be maintained in subclones, indicating that DPSCs are a great source for tissue regeneration. 13 Researches have also reported that DPSCs presented a more striking odontogenic capability than BMSCs under inducing environment. 14 Moreover, it has been shown that dental pulp that has been diagnosed with irreversible pulpitis also contains DPSCs. 15 These facts provide a bright future for this cellular resources.
F I G U R E 1 Dental stem cells, including DPSCs, PDLCs, SCAPs, SHEDs and DFPSCs, can be divided into several categories based on where they originate from. DPSCs are isolated from the dental pulp. PDLSCs are a group of cells isolated from the periodontal ligament. SHEDs are obtained from exfoliated deciduous teeth. DFSCs are isolated from the dental follicle of unerupted teeth. SCAPs are isolated from the apical papilla

| Periodontal ligament stem cells
Periodontal ligament stem cells are a group of cells that can be isolated from the periodontal ligament which connects the tooth root with alveolar bone. PDLSCs were firstly proposed for use in new periodontal regenerative therapies. The cell surface markers and differentiation potentiality of PDLSCs are similar to that of BMSCs and DPSCs. 16 However, as the prevalence of periodontitis is relatively high, the ability to acquire of healthy tissue source is quite limited, and this limitation is a great challenge for the culturing of these cells.

| Stem cells from human exfoliated deciduous tooth
SHEDs, which are obtained from exfoliated deciduous teeth, have provided a potential non-invasive source of stem cells. SHED presents relatively rapid expansion and proliferation in vitro. Furthermore, SHEDs express mesenchymal stem cell markers including STRO-1 and CD146, CD105, CD73 and CD90. 13 SHEDs are capable of multilineage differentiation and can differentiate into several types of cells including neural cells, adipocytes and odontoblasts. When transplanted in vivo, SHEDs can induce bone formation, generate dentin, survive in the mouse brain and express neural markers. 17

| Dental follicle stem cells
The dental follicle is a kind of soft tissue that surrounds the developing tooth germ. The dental follicle is thought to contain stem cells that can differentiate into cementoblasts, osteoblasts and periodontal ligament cells. 18 Similar to other dental stem cells, dental follicle stem cells (DFSCs) express similar cell surface marker and have a relatively higher proliferation rate. DFSCs were proven to possess osteogenic differentiation ability under appropriate conditions. The neural differentiation potential of DFSCs has also been shown. 19

| Stem cells from apical papilla (SCAPs)
The apical papilla is the soft tissue that can be found at the apices of developing teeth. The dental papilla is the beginning of tooth formation and ultimately grows into dental pulp tissue. 20 SCAPs present a rapid proliferative rate and a greater inclination for mineralization. SCAPs express typical MSC markers including STRO-1, CD105, CD73, CD90 and CD146. As a member of the dental stem cells, SCAPs can undergo adipogenic, odontogenic and neural differentiation under inducing conditions in vitro, while SCAPs were also shown to possess the capacity for multilineage differentiation in vivo. 20-22

| Characteristics of dental stem cells
As a member of the MSCs, dental stem cells have the typical traits of MSCs. Like other MSCs, dental stem cells can be separated and isolated by collagenase. After approximately 1 week of culture, dental cells are able to be grown out from the dental tissue fragments. [23][24][25][26] Experiments have shown that most of the MSCs are fibroblast-like and dental stem cells are as well. A large number of experiments have shown that cells that have been isolated from dental tissues, including the dental pulp, periodontal ligament and dental follicle, are fibroblast-like and display a spindle structure. 8 All dental stem cells have the ability to proliferate. Compared with other MSCs, such as BMSCs or ADSCs, dental stem cells display an equivalent ability to self-renew. Among all the dental cells, DPSCs, DFCs and SHEDs exhibit higher proliferation rates and greater growth ability than others. 8,23,[27][28][29][30]

| Phenotypical profile of dental stem cells
MSCs can generally be obtained from quite a few tissues including bone marrow, skeletal muscle, adipose tissue and dental pulp. These Phenotypical analyses showed that SHEDs have a higher expression of CD71, CD105, CD117 and CD166 compared to that of DPSCs, implying that SHEDs might be more undifferentiated. 35

| Differentiate ability of dental stem cells
Dental cells originate from ectodermal cells that grow around the neural tube. After migrating into the oral region, they then differentiate into cells with mesenchymal phenotypes. Therefore, they have been defined as "ectomesenchymal". 32,36,37 As a member of the MSCs, dental stem cells have the ability for multilineage differentiation abilities. It has been shown that dental stem cells possess the ability to differentiate into both endodermal, mesodermal and ectodermal tissues. 32 Previous experiments have demonstrated that dental stem cells can differentiate into adipose tissues, such as adipocytes, bone tissues, including osteoblasts and chondrocytes, and nerve and neuronal tissues under suitable conditions, both in vivo and in vitro ( Figure 2).
As the field of stem cell biology has rapidly developed, dental stem cells have become an ideal cellular sources for biological therapies and tissue engineering because of their ability to self-renew and differentiation. DSCs are readily accessible and have less ethical constraints. All of these reasons contribute to dental stem cells being an ideal resource for regenerative medicine.

| USAG E OF DENTAL S TEM CELL S IN NERVOUS SYS TEM IMPAIRMENT
Dental stem cells are thought to be derived from the cranial neural crest. They can express early markers for both mesenchymal and neuroectodermal stem cells. 12,17 Considering their origin, dental stem cells inherit some typical traits of neural cells. It has been reported that dental stem cells can express specific neural markers, such as nestin, s100-beta and GFAP. Compared to BMSCs, a type of stem cell derived from bone tissue, the percentage of neural markers expressed by dental stem cells is relatively high.
Nestin-and GFAP-positive cells make up only approximately 30% of BMSCs. However, those cells make up more than 90% of DMSCs. 38 The high expression of neural markers indicates that dental stem cells can be used as an ideal seed for neural induction and regeneration. In addition to neural markers, the expression of neurotrophic factors also plays an important role. Mallappa et al found that all types of dental stem cells can express neurotrophic factors including brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF) and nerve grow factor (NGF). Conditioned media from dental stem cells can enhance the growth rate of Schwann cells and induce the neurite outgrowth in vitro. 39,40 In a model of rat sciatic nerve injury, dental stem cells presented neurotrophic traits and had the ability to induce axon regeneration. However, among all the dental stem cells, SCAPs have shown the greatest potential for neurotrophic effects, indicating that SCAPs could be an optimal cell source for peripheral nerve repair. 39 Moreover, exosomes or DSCs-conditioned medium containing exosomes is beneficial for diseases recovering. 41 It has been reported that DPSCs exosomes possess the ability to penetrate the blood-brain barrier and reduce or replace neuronal loss in Parkinson's disease. 41,42 In addition to the above described indirect effects on nerve repair, dental stem cells themselves can directly differentiate into neural-like cells and participate in nerve regeneration under specific circumstances. Previous research has indicated that both DFCs and SHEDs can differentiate into neural-like cells and possess the functions of neural cells. 43 DPSCs, which are a significant member of dental stem cells, have been shown to differentiate towards F I G U R E 2 As a member of the mesenchymal stem cells, dental stem cells have the ability for multilineage differentiation abilities. Experiments have demonstrated that dental stem cells can differentiate into adipose tissues, such as adipocytes, bone tissues, including osteoblasts and chondrocytes, and nerve and neuronal tissues under suitable conditions, both in vivo and in vitro functional neurons under appropriate microenvironments. With the contribution of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), B27 supplement and Neurobasal Media, DPSCs could form a bipolar and stellate neuron-like morphology, which is consistent with that of functional neurons. Functional voltage-gated Na + channels are present in DPSCs when exposed to neuronal inducing conditions. 44,45 Additionally, inner neurotrophic factors such as BDNF, NT-3 and GDNF can promote the neural differentiation of DPSCs and SHEDs. The DPSCs and SHEDs differentiated cells showed comparable functional neural activities, indicating that both adult and adolescent teeth could be considered as a cell-based therapy source for neural diseases treatments. 46 Moreover, it has been reported that intravenously administered DPSCs could migrate into ischaemic areas, attenuate stroke-induced inflammation and reduce infarct volumes and cerebral oedema. 47 Based on these above evidences, we hypothesize that DSCs could be ideal stem cell sources for central nerve repair. In conclusion, dental stem cells could be used in both peripheral and central nervous system disease treatments.
A summary of the repair mechanisms of dental stem cells can be found in Table 1. A large amount of research has built a solid foundation for nerve repair treatments in nerve repair based on dental stem cells.

| Spinal cord injury
Spinal cord injury (SCI) is a severe and disabling disease resulting in the impairment of sensory and motor functions. By far, this disease does not have any effective treatments. Due to its complicated pathophysiology, which includes the loss of neurons and glia, researches leading to new treatments have faced severe challenges. 48 The pathophysiological changes in SCI occur in two significant phases. In the acute phase, the tissue homeostasis has been broken down by the outside injurious stimulation. This kind of imbalance in the inner microenvironment induces a cascade of secondary injuries, which lead to the necrotic or apoptotic death of neurons, astrocytes and other neural cells.   48 Research has indicated that not only DPSCs but also SHEDs possess the ability to promote locomotor recovery after SCI. SHEDs can prevent demyelination and axonal loss, which helps to preserve anatomical tissue of spinal cord and contributes to SCI recovery. 58 The neuroregenerative ability of DPSCs and SHEDs, as well as those of human BMSCs, is evaluated by the Basso Beattie Bresnahan locomotor rating scale (BBB scale). The DPSCs-and SHEDs-transfected groups showed a higher BBB score, which indicated that the neural repair ability of dental stem cells was more potent than that of BMSCs. As tissue loss and cell apoptosis mainly happen during the first stage of SCI (the first 8 hours), 59  SHEDs, used as a type of neuroprotective agent, downregulated the expression of GFAP, inhibited glial scar formation, delayed the decrease in S100-beta, upregulated the expression of Kir4.1, induced tissue plasticity and finally improved the functional recovery of spinal cord contusions. 60 Experiments also revealed that grafted SHEDs could reduce the increased level of TNF-alpha during the early phase of SCI, through reducing the overexpression of excitatory amino acid transporter 3 (EAAT3) and of nNOS. All these functions contributed to a lower level of neuronal apoptosis. 61 The dental apical papilla is another available source for SCI therapy. Transplantation of SCAPs to the rat SCI lesion sites improved their gait and reduced cell apoptosis. 62

| Alzheimer's disease
Alzheimer's disease (AD) is an age-related neurodegenerative disease that severely affects a person's normal daily life, with symptoms including memory loss, motor disability, linguistic disorders and cognitive deficits. 66,67 The pathological change in AD is quite complicated and includes the loss of neurons, intracellular neurofibrillary tangles and the deposition of insoluble beta-amyloid peptides in the brain. 67,68 However, many of the pathological mechanisms of AD still remain unknown to us now. The therapeutic effect of currently available treatments for AD is still far below our expectation. DPSCs could reduce amyloid beta peptide-induced neural degeneration and apoptosis. [74][75][76] Furthermore, the DPSCs secretome is highly enriched in neurotrophic factors, amyloid beta-degrading enzymes (NEPs) and anti-apoptotic factors, demonstrating that DPSCs are an ideal candidate for therapy of AD. 74 In addition to dental stem cells themselves, conditioned medium from human dental stem cells can also improve cognitive functions in mouse AD models. SHED-CM may provide several neuro-reparative effects that benefit the treatment of cognitive deficits and AD treatments. 75 PDLSCs have also been shown to have the ability to reconstruct tissues destroyed by the chronic pathology of AD. The levels of apoptosis of okadaic acid-induced SH-SY5Y cells were lower and the expression of pTau protein decreased. These facts suggest that dental stem cells have advantages in AD treatment.

| Peripheral nerve system diseases and treatments
Peripheral nerve injury is mainly caused by traumatic accidents or surgical complications, which may result in sensory disturbances, paralysis and locomotive disability and severely affects a person's normal daily life. Routine surgical solutions for peripheral nerve injury tend to choose the end-to-end/end-to-side neurorrhaphy in order to fix the anatomic structure of damaged nerves.

| Sciatic nerve injury
The sciatic nerve injury model is a classical model that is often used to study peripheral nerve injury. 6 The construction of sciatic nerve crush model is not complicated. After exposing sciatic nerve of rats by blunt dissection, the lesion could be created by a completely cut-off or by vascular clips compression. 88

| Inferior alveolar nerve injury
The IAN is a branch of the mandibular nerve that belongs to the trigeminal nerve. IAN injury mainly happens during mandibular fracture or oral surgery such as the third molar extraction and results in unpleasant complications including dysesthesia, allodynia and hyperalgesia. 5,[91][92][93][94][95] There have been no specific reports illustrating the usage of dental stem cells in IAN injury repair by far. However, the utilization of dental stem cells in the sciatic nerve injury model provides powerful and forceful evidences that dental stem cells may be available for IAN nerve repair.

| Facial nerve injury
The facial nerve (seventh cranial nerve) contains both motor and sensory fibres. 96

| MECHANIS MS UNDERLYING DSC S-MED IATED NEUR AL REPAIR
DSCs, as a brand new source for cell therapy, participate in the neurorestoration in several kinds of neurodegenerative disorders.
The mechanisms of how they mediate repair remain complicated.
However, we could conclude, as described below, that several aspects are involved, including cell replacement, paracrine effects, vasculogenesis, synaptogenesis, immunomodulation and apoptosis. 101 First, DSCs take part in neural repair through cell replacement. In the above context, we point out that DSCs can directly differentiate into neural-like cells, which are positive for early neural markers including nestin. Report also revealed that neural crest subpopulation which can directly differentiate into neural lineage could be isolated from dental tissue. 102 These cells migrate to the lesion place in order to replace the necrotic tissue. 43,44,48 101 Neurotrophic factors secreted by DSCs promote the regeneration of axons. 48 Moreover, the CXCR4/SDF1-alpha pathways, which are known to control synapse formation, were shown to be related to the transplanted DPSCs in ischaemic conditions. 104,105 In conclusion, DSCs participate in neural repair through the above six methods. As biomaterials are greatly used extensively in tissue regeneration, a 3D biomaterial scaffold, embedded with neurotrophic factors and combined with neurally inclined dental stem cell sheets may be an ideal tool for nerve repair, however, the details of this will require in-depth research.

CO N FLI C T O F I NTE R E S T S
The authors declare no conflict of interests.