Importance of In Vitro Microenvironment on Differentiation and Translational Applications of Dental Pulp Stem Cells

Abstract

Dental Pulp Stem Cells (DPSCs) are adult stem cells found in dental pulp tissue, and possess the capacity for self-renewal and the potential for multipotent differentiation. DPSCs depend on regulating in vitro microenvironment, and can readily differentiate into osteoblasts, odontoblasts, neurocytes, adipocytes, chondrocytes, myocytes, fibrocytes and many others. In addition, DPSCs play a crucial role in tooth regeneration, bone and nerve repair in current studies of regenerative medicine and tissue engineering. Among them, the influences of stem cell microenvironment or niche on the biological activity of DPSCs are critical and hamper its progress. Herein, we review the influence of culture condition, tissue source, growth factor requirements, and cellular organizational scaffolds and how these features influence the biological characteristics and translational research of DPSCs.

Keywords: Dental pulp stem cells; Differentiation; Microenvironment; In Vitro

Introduction

Dental pulp is an integral part of tooth tissue and comprises connective tissue, mesenchymal cells, nerve fibers, blood vessels, lymph, and other biological components. Dental Pulp Stem Cells (DPSCs), which are also known as post-natal dental pulp stem cells, are adult stem cells located in dental pulp tissues. DPSCs were first isolated from the pulp tissues of the third molar impacted teeth by Gronthos et al. [1]. DPSCs are characterized as high clonogenic cells and proliferative capacity that can generate densely calcified colonies and occasional nodules. Furthermore, DPSCs are comprised predominantly of stem cells that are separated from dental pulp tissues of permanent teeth and from human exfoliated deciduous teeth (SHED). In 2003, Miura and colleges [2] isolated stem cells from the pulp tissue of SHED and found that SHED represented a population of post-natal stem cells that were capable of extensive proliferation and multipotent differentiation. Under different in vitro microenvironmental conditions, DPSCs can differentiate into many cell types like odontoblasts [3,4], neural cells [5,6], osteoblasts [7,8], chondrocytes [9,10], adipocytes [11], hepatic cells [12,13], myoblasts [14], fibroblasts [15] and islet-like cells [16,17]. The cell culture environment also poses significant challenges in stem cell research, especially when there is a need to guarantee the undifferentiated state and directional differentiation of the cultured stem cells. Therefore, cell culture conditions are mostly defining by the requirements of the desired target cells. The specific cultural microenvironment such as inflammatory, acidic, and high glucose could affect differentiation of DPSCs through complex mechanisms. Moreover, the surrounding microenvironment also regulates DPSCs differentiation and proliferation. In addition, cell culture method applied, the tissue source of stem cells, supplementation with defined growth factors and cellular scaffolds could differentially influence the differentiation of DPSCs

Influence of Culture Condition on DPSCs Differentiation

DPSCs had the ability to differentiate into various cell types under specific culture conditions. Several studied have revealed great potential for DPSCs in the repair and regeneration of various tissues, including bone [18], teeth [19], and muscles [20]. Kanafi and colleges [21] applied the alginate acid sodium salt microspheres to fix DPSCs three weeks, they founded that more than 80% of immobilized DPSCs remained viable and proliferation compared with the control group. On day 21, osteocalcin and calcium deposition were found in the microspheres and the expression of the osteogenic genes Alkaline Phosphatase (ALP), runt homology domain transcript factor (Runx-2) and Osteocalcin (OCN) were all up-regulated by Reverse Transcriptase Polymerase Chain Reaction Assay (RT-PCR). They conducted that DPSCs immobilized in alginate hydrogels could enhance osteogenic potential differentiation which was merit to bone tissue regeneration. Umemura and colleges [22] applied hyaluronic acid sodium salt to fix DPSCs, they founded that hyaluronan induced odontoblastic differentiation and the expression of the odontoblastic differentiation markers Dentin Matrix Protein 1 (DMP-1) and Dentin Sialophosphoprotein (DSPP) were up-regulated using western blot analysis. Nakayama and colleges [19] isolated DPSCs from mature teeth and mobilized them by Granulocyte Colony- Stimulating Factor (G-CSF), they founded that mobilized DPSCs had higher differentiation, migration and regeneration potential ability compared to DPSCs. The differentiation procedure was in part mediated by the function of osteoblasts to secrete cytokines or soluble proteins. Kanafi and colleges [23] investigated the influence of hypoxia, high glucose and low serum concentration on the differentiation potential of human SHED. They founded that SHED retained their phenotypic expression and differentiation potential in different cultural conditions described above. They demonstrated that SHED possessed strong capacity of proliferation and differentiation even in harsh culture conditions.

Influence of Tissue Source on DPSCs Differentiation

Obviously, fresh teeth or healthy teeth alone will not satisfy clinical needs in the future application of DPSCs in cell-based therapy. Some studies have shown that inflamed pulp tissue of human functional deciduous teeth (iSHFD), carious deciduous teeth and cryopreserved teeth have similar differentiation capabilities to fresh tissue. Kim and colleges [24] reported that they successfully isolated Mesenchymal Stem Cells (MSCs) from iSHFD and the characteristics of stem cells were similar to that of healthy DPSCs. In addition, Fibroblast Growth Factor 2 (FGF-2) promoted the proliferation and migration of stem cells derived from inflamed dental pulp. It means that iSHFD could be used as seed cells in tissue repair engineering. A study by Werle and colleges [25] has shown that the success rate of isolation were 70% and 80% from stem cell from the pulp of carious deciduous teeth (SCCD) and sound deciduous teeth (SHED-stem cells from human exfoliated deciduous teeth) separately and SCCD had the similar pattern of proliferation and differentiation ability as those obtained from sound deciduous teeth. They concluded that SCCD could represent a feasible source of stem cells. Malekfar and colleges [26] successful isolated human DPSCs from fresh and cryopreserved pulp tissues with irreversible pulpitis respectively. They found that there was no significant difference in osteogenic and adipogenic differentiation potential of DPSCs between the fresh and frozen dental pulp tissues derived stem cells. They concluded that cryopreservation did not lose their normal characteristics and differentiation potential.

Influence of Growth Factors on DPSCs Differentiation

Influence of growth factors on DPSCs osteogenic differentiation

Several studies have shown that some kinds of growth factors such as Enamel Matrix Derivative (EMD), Vascular Endothelial Growth Factor (VEGF), insulin et al. can have positive effects on DPSCs osteogenic differentiation. Ajlan and colleges [27] added EMD, Mineral Trioxide Aggregate (MTA) and Platelet-Derived Growth Factor (PDGF) into DPSCs, respectively. It was shown that EMD significantly increased the expression of Alkaline Phosphatase (ALP) and mineralization compared to the other groups (P<0.05) by computer analysis of ALP-stained chambers and spectrophotometric analysis of alizarin red S-strained mineralized nodule. It suggested that EMD possessed the capacity to promote osteogenic differentiation of human DPSCs. VEGF is another kind of growth factor the can influence the proliferation and osteogenic differentiation of DPSCs which has been demonstrated by D’ Alimonte and colleges [28]. They incubated DPSCs in osteogenic culture medium for three weeks and found that the expression of osteogenic markers (Runt- Related Transcription Factor 2 (RUNX-2), and ALP) had been increased, as did the expression of extracellular calcareous deposition. The influence of Endothelial Cells (ECs) on the osteogenic differentiation of DPSCs had been investigated by several studies. Dissanayaka et al. [29] co-incubated different ratios of Endothelial Cells (ECs) with DPSCs, and found that direct co-culture of DPSCs and ECs enhances the in vitro differentiation toward osteo-/odontogenic and angiogenic phenotypes. When the ratio was set at 1:1, the increase in ALP activity was the most significant. When the ratios were set at 1:1 and 1:5, the mineralization was stronger than was observed at other ratios (P <0.01). It suggested that ECs is able to enhance the osteogenic differentiation of human DPSCs. It is wellknown that insulin is a powerful and important hormone involved in the proliferation and differentiation of osteogenic. Lauritano and colleges [30] treated DPSCs in insulin (100ng/μg) for 24h and 48h, and compared the gene expression profiles in DPSCs in the insulintreated group and the non-treated group. It had founded that after 24 hours treatment, the expression of bone morphogenetic protein family members Bone Morphogenetic Proteins 3 (BMP3), BMP4, and their receptor BMPR1A were increased, and after 48 hours treatment, BMP1 was over-expressed. However, 24 hours after therapy, the expression of BMP7 and Transforming Growth Factor Beta Receptor (TGFBR) 2 had decreased. They conducted that insulin had positive effect on osteogenic differentiation of DPSCs. Several studied have reported that Valproic Acid (VPA) is a Histone Deacetylase (HDAC) inhibitor, which promotes osteoblastic differentiation of HPSCs. Paino and colleges [7] found that low concentration of VPA increased the expression of osteopontin and bone sialoprotein, and further significantly improved bone mineralization. However, the osteocalcin level decreased, which was significant related to the presence of an HDAC2 inhibitor. Accordingly, they concluded that VPA did not induce terminal differentiation of osteogenic, but just stimulated the generation of less mature cells, and specific suppression of the individual HDAC by RNA interference could only a single aspect of osteogenic differentiation. It had proved that Tumor Necrosis Factor-a (TNF-a), a major pro-inflammatory cytokine, with low concentration could promote osteogenic differentiation of DPSCs [31]. While in higher concentration which could decreased the capability of osteogenic differentiation [32], which may be affected by nuclear factor kappa-light-chain- enhancer of activated B cell (NFkB)/ p38 mitogen-activated protein kinases (p-38 MAPK)/Wnt/β- caternin signal pathway [33,34]. The exact underlying molecular mechanisms are need to further study.

Influence of growth factors on DPSCs odontoblastic differentiation

It previously reported that under the induction of a certain factor, DPSCs possessed the ability to migrate, and stimulated the proliferation and differentiation of DPSCs to form dentin, which is an important biological pathway in the regeneration of teeth and dental pulp after dental pulp-dentin damage. The Transforming Growth Factor (TGF)-β family is composed of polypeptide growth factors, including BMPs, TGF-β, activin and growth differentiation factor. TGF-β plays important roles in embryonic development, extracellular matrix formation, bone remodeling and formation. Besides, it also influences cell proliferation and differentiation, and TGF-β is closely related to odontoblast differentiation and dentin matrix formation. Wiesen et al. [15] evaluated the expression of periostin with different concentration of TGF-β1 by RT-PCR, western blot and Enzyme-Linked Immunosorbent Assay (ELISA) in the activity of DPSCs, Dental Pulp Fibroblasts (DPFs), and rat odontoblast-like cells (MDPC-23). After 48 hours of TGF-β1 treatment, the expression of periostin mRNA increased in the DPSCs group and decreased in the DPFs group. These observations indicated that TGF-β1 could promote DPSCs odontoblastic differentiation. Semaphorin was identified by Kolodkin et al. in 1992, and is a type of secretory or transmembrane family member, which contains the Sema structural domain [35]. Sema3A expression (originally referred to as Collapsin), belongs to a subset of the semaphorin family, and was first discovered by Luo et al. [36] in 1993. In 1999, the Semaphorin Nomenclature Committee divided this family into eight sub-types, and twenty members were identified in the mouse and human [37]. Yoshida and colleges [38] applied immunohistochemistry to detect the expression of Semaphorin 3A (Sema 3A) and its receptor (neuropilin 1) in rat DPSCs model. Sema3A induced DPSCs migration, proliferation and odontoblastic differentiation and increased nuclear accumulation of β-catenin and expression of the FARP2 gene (FERM, RhoGEF, and pleckstrin domain protein 2), and improved Rac1 activity. They conducted that Sema3A played a crucial role in tooth regeneration and the possible molecular mechanism might be the Wnt/β-catenin signaling pathway. The differentiation inhibitor which also referred to as the inhibitor of differentiation/DNA binding (Id) belongs to one of the Helix-Loop-Helix (HLH) transcription factor family members. There are four Id molecules in mammalian cells (i.e., Id1-Id4) [39]. Maciejewska and colleges [40] reported that Id1 over-expression could influence the differentiation of human DPSCs. Over-expression of Id1 increased the expression of the Dentin Sialophosphoprotein (DSP) and the gene expression of Dentin Matrix Protein 1 (DMP- 1), and further increased odontoblastic differentiation, while Id1 silencing showing opposite effect to DPSC differentiation. The results suggest that as a vital modulator Id1 plays important role during DPSC differentiation. A study by Liu and colleges has shown the ECs have influence on the odontogenic differentiation of DPSCs [41]. Liu and colleges co-cultured Human Umbilical Vein Endothelial Cell (HUVEC) to DPSCs and the expression of DSP and DMP-1were detected by RT-PCR, Alizarin Red S staining, immunofluorescence and western blotting analysis at day 4, 7, 14 and 21. They founded that HUBEC group exhibited higher odontoblastic differentiation compared to the controls groups and the highest expression levels of DSP and DMP-1 were at day 7. However, recently a kind of growth factor named Casein Kinase 2 Interacting Protein 1 (CKIP- 1) was reported that can suppress odontoblastic differentiation of DPSCs by Song and colleges [42]. They co-cultured DPSCs and CKIP-1 in odontoblastic differentiation medium for 21 days and the expression of CKIP-1, BMP2, Neuropilin-1 (NRP1) were detected by RT-PCR, western blot, ALP straining, alizarin red S staining and immunoprecipitation. The CKIP-1 expression significantly decreased along with cell odeontoblastic differentiation, while CKIP-1 over expression had a negative effect on odentoblastic differentiation of DPSCs. It provides a new perception on odontoblastic differentiation of DPSCS and may be of great significance to the repair of dental caries.

Influence of growth factors on DPSCs neurogenic differentiation

DPSCs derived from the ectoderm, and their capacity of differentiating into the neural lineage is higher than that of cells derived from other blastodermal development sites [43]. Several studies have shown that Basic Fibroblast Growth Factor (bFGF) had positive effects on neurogenic differentiation of DPSCs. A study by Kanafi and colleges [44] was shown that DPSCs in their naïve state spontaneously expressed early and mature neuronal markers like nestin, musashi12, β tubulin III and on exposure to bFGF, DPSCs expressed high level of dopaminergic neuron-specific transcription factors Nuclear Receptor related protein 1 (Nurr1), Engrailed 1 (En1) and paired-like homeodomain transcription factor 3 (Pitx3) as revealed by RT-PCR. Another study by Osathanon and colleges [45] confirmed that bFGF could promote proliferation and neurogenic differentiation of DPSCs. The other growth factor can influence neurogenic potential differentiation of DPSCs is p75. Pan and colleges [46] investigated DPSCs from eight donors, and found that p75 neurotrophin receptors (p75NTR) were expressed at low levels (<10%) in DPSCs and p75 (+) DPSC exhibited higher expression levels of SOX1 (a neural precursor cell marker), SOX2 (a cell pluripotency marker) and nestin (a neuronal stem cell marker) as compared to p75(-) DPSCs. It suggested that p75 (+) human DPSCs possessed greater neurocytic differentiation potential.

Influence of growth factors on DPSC myogenic differentiation

MicroRNA can be considered as a type of endogenous, single stranded non-coding RNA with an average length of approximately 18~24 nt. It plays vital roles in gene regulation and is involved in many biological processes, including cell proliferation, differentiation, aging and apoptosis [47]. Li and colleges [14] applied miR-143 and miR-135 inhibitors to observe their influence on DPSC myogenic differentiation by RT-PCR and western blotting. They founded that the expression of miR-135 and miR-143 markedly decreased in DPSCs and part of the DPSCs treated with miR-135 or miR-143 inhibitors displayed apparent myocytic properties, and formed myotubes. In addition, in complementary studies, miR-135 and miR- 143 inhibitors were transfected into DPSCs, following which half the DPSC population formed myotubes. It indicated that miR-135 and miR-143 inhibitors could induce myogenic differentiation of DPSCs.

Influence of Different Scaffolds on DPSC Differentiation

Different scaffolds provide different microenvironmental niches for DPSCs, which can provoke differential influences on DPSC differentiation, as described in the examples provided below.

Scaffold influencing osteogenic differentiation

Annibali et al. [48] seeded DPSCs on two molecular scaffolds, Granular Deproteinized Bovine Bone (GDPB) and Beta-Tricalcium Phosphate (β-TCP). The repair and regenerative functions of both molecular scaffolds on the damaged cranium were comparing in a rat model. After 2, 4, 8 and 12-week post-implant, Bone Mineral Density (BMD) and the Standard Uptake Value (SUV) of tracer incorporation were measured, respectively. The repair of bone formation mediated by the interplay of GDPB in the damaged cranium was better than β-TCP. It suggests that GDPB scaffolds could improve bone regeneration of DPSCs. Niu et al. [49] investigate the effects of Intrafibrillar-Silicified Collagen Scaffolds (ISCS) on the osteogenic differentiation of human DPSCs by RT-PCR, western blotting, ALP staining and alizarin red S assay. They co-incubated ISCS with DPSCs, and founded that ISCS could promote DPSCs proliferation and significantly increased expression of osteogenesis-related genes and proteins during DPSCs osteoblastic differentiation. It is more important that there is no adverse effect on human DPSCs. Lobo et al. [50] co-incubated Biphasic Calcium Phosphate (BCP) biological ceramics scaffolds with DPSCs. The number of surviving DPSCs in the co-culture group was higher than that in the control group. BCPs with a small pore size promoted cell proliferation, and those with a larger pore size increased expression of osteogenic-related genes and proteins during osteogenic differentiation, which showed that BCPs might possess a bone inductive potential. Farea et al. [51] proved that the combination of a chitosan scaffold and TGF-β1 could increase proliferation and osteoblastic differentiation of SHED. The combination of the chitosan scaffold and TGF-β1 improved bone tissue regeneration with SHED in vivo. It has shown that Hydroxyapatite (HAp) can support alginate as inorganic reinforcement and osteoconductive component of alginate/HAp composite scaffolds. A study by Sancilio and colleges [52] co-incubated Alg/HAp scaffolds to human DPSCs and they founded that when growing onto Alg/HAp scaffolds DPSCs expressed osteogenic differentiation markers and promoted calcium depositing and biomineralization. In summary, all kinds of scaffold above mentioned have positive effects on the proliferation and osteoblastic differentiation of DPSCs to a certain extent, and future research in animal model and clinical experience should confirmed to guarantee the safety and effectiveness.

Scaffold influencing odontogenic differentiation

Conde et al. [53] used WST-1 assay and RT-PCR to detect the influence of poly -L-lactic acid scaffolds with different pore sizes (i.e., 150-250 μm or 251-450 μm) on the proliferation and differentiation of DPSCs. They found that after 21 days, DPSCs showed odontogenic gene expression of DMP-1, DSPP, and Matrix Extracellular Phosphoglycoprotein (MEPE). It concluded that the poly-L-lactic acid scaffold promoted cell proliferation and odontoblastic differentiation. It is well known that the differentiation and regeneration potential of stem cell including DPSCs depends on their environment. A study by Soares and colleges [54] seeded lipopolysaccharide (LPS) or simvastatin and/or nanofibrous poly-l-lactic acid to human DPSCs, they founded that in vitro studies using simvastatin and nanofibrous poly-l-lactic acid scaffolds significantly repress the expression of proinflammatory mediators and reverted the negative effects of LPS on expression of odontoblastic markers in vivo in mouse model. It may provide a new strategy for DPSCS to treat inflammation and dental tissue regeneration. Cavalcanti et al. [55] co-incubated DPSCs with 0.05-0.25% Puramatrix^TM DPSCs survived and proliferated well in Puramatrix™ at least three weeks and kept healthy morphology, and part of the cytoplasm lengthened. After 21 days, DMP-1 and DSPP were expressing in DPSCs with Puramatrix^TM. The peptide hydrogel scaffold could be used as a form of gene therapy and provided support in tooth tissue regeneration.

Scaffold influencing neurogenic differentiation

Chitosan is a natural macromolecule and a kind of biodegradable material that has been widely used in clinics. In terms of having similar structure and composition to Glycosaminoglycans (GAGs) chitosan elicits minimal immune response when implanted in human body. Therefore, chitosan is able to use as an ideal biomedical scaffold material. Feng et al. [56] seeded DPSCs on a three-dimensional (3-D) porous chitosan scaffold, and incubated them with neural induction medium in vitro and they founded that the neurogenic differentiation markers were significantly increased. Zhang et al. [57] incubated DPSCs with chitosan scaffold in neural induction medium for 14 days, and they founded that compared the control rats, the DPSCs/ chitosan group showed the higher cell neural differentiation and the expression of Brain-Derived Neurotrophic Factor (BDNF), Glial Cell Line-Derived Neurotrophic Factor (GDNF), b-Nerve Growth Factor (b-NGF) and Neurotrophic-3 (NT-3) were significantly upregulated by western blotting assay and RT-PCR. Transplantation of DPSCs with chitosan scaffolds into the injured rat model resulted in the significant recovery of hind limb function. It suggested that the scaffold provided a conducive and favorable microenvironment for the survival and neuronal differentiation of DPSCs.

Application of DPSCs in Tissue Engineering and Regenerative Medicine

DPSCs have acceptable efficacy in the repair and regeneration of teeth, bone and nerve tissues, and can achieve significant progress in tissue biology and regenerative medicine, possessing the potential for a wide array of applications.

Animal experiment

Several animal experiments have approved that DPSCs had excellent efficacy in neural repair. Ullah et al. [58] transplanted a total of 1 × 10⁶ human DPSCs with fibrin glue scaffold and collagen tubulization into the sciatic nerve resection (5mm gap) of rat models. After 12 weeks, human DPSCs groups showed notably increased behavioral activities and higher muscle contraction forces compared with control group and regenerated nerve specimens, specific markers for angiogenesis, axonal fiber, and myelin sheath increased by immunohistochemical analysis in DPSCs transplantation groups. They demonstrated that human DPSCs could be an excellent stem cell source for nerve regeneration. Zhang et al. [59] evaluated the therapeutic potency of DPSCs and BDNF in focal cerebral ischemia using rat models. They transplanted DPSCs and injected BDNF into the rats via the tail vein. Proliferation of intravenously transplanted DPSCs and the expression of neural differentiation markers such as nestin, Doublecortin (DCX) and Neuronal Specific Filament (NFH) were founded in the peripheral ischemic regions of the Middle Cerebral Artery Occlusion (MCAO) models. They conducted that treatment with DPSCs combined with BDNF promoted the recovery of neurological function after stroke which suggesting a novel therapeutic strategy against cerebral ischemia. A study by Pisciotta et al. [60] has shown that human DPSCs had potential efficacy in skeletal muscle repair in mice. They co-cultured human DPSCs with myogenic cell line (C2C12 cells) and preliminary demethylation treatment with 5-Aza-2’-deoxycytidine (5-Aza), respectively in vitro for 2 weeks. Then the cells were injected into the dystrophic gastrocnemius muscles of mdx/SCID mice. After 1, 2, and 4 weeks, the gastrocnemius muscles were taken for immunofluorescence and histological analyses. They founded that the populations of cells engrafted within the host muscle of mdx/SCID mice improved, angiogenesis promoted and fibrosis reduced through a paracrine effect, which eventually led to an improvement of the histopathology of the dystrophic muscle. During that last decade, a number of experiments showed the successful application of DPSCs in achieving large bone volume regeneration. Perczel-Kovach et al. [61] implanted DPSCs on osseointegration in rat tail vertebrae and they founded that undifferentiated DPSCs inserted between the implant and the bone slow down the osseointegration process and pre-differentiated DPSCs did not have the similar adverse effect. They suggested that the success of DPSCs application to promote implant osseointegration was highly dependent on the applied conditions, particularly on the parallel application of scaffolds and osteogenic components. Accordingly, efficient and safety scaffolds should be further studied in the future. It had shown DPSCs could be used for stem cell therapy in diabetes. Kanafi et al. [17] found that both SHED and DPSCs could generate Islet-Like Cell Clusters (ICCs), but SHED was superior to DPSCs. Streptozotocin (STZ)-induced diabetic mice alone and mice transplanted with empty macro-capsules both exhibited hyperglycemia throughout the experiment. Whereas mice transplanted with macro-capsules containing ICCs restored to normoglycemia within 3 to 4 weeks, which persisted for more than 60 days. In summary, we think that DPSCs play an important role in the repair and regeneration of tissues including nerves, bones, and muscles, further research should studied.

Clinical applications

There are few clinical applications of DPSCs in practice and just a small number of bone repair treatments trial. Manimaran et al. [62] applied autologous DPSCs and Stromal Vascular Fraction (SVF) with the intent of evaluating bone repair therapy on the post-operative bone defect of a 14-year-old male patient presenting with mandibular ameloblastoma. Ten month post-surgery, bone formation was observed by CT scanning, and no tumor relapse was reported during the follow-up observation. Li et al. [63] located autologous DPSCs onto the β-tricalcium phosphate scaffolds and engrafted them into the periodontal defect area in the root furcation of defect teeth. Nine-month post-surgery, the effect of regeneration of new bones to repair periodontal defects been verified by clinical assessment and radiological study. Ferrarotti et al. [64] random divided 29 chronic periodontitis patients who presented one deep intrabony defects and tooth had to be removed into DPSCs treatment group (n=15) and the control group (n=14). After 1-year treatment, they founded that test sites significantly improved clinical parameters regeneration in autologous DPSCs group compared to control patients. All of the above are autografts, the following case is an example of allogeneic transplantation. Hernández-Monjaraz et al. [65] applied SHED of a 7-year-old donor with scaffold of lyophilized collagen-polyvinylpyrrolidone sponger to a 61-year-old patient with periodontal disease. Six months later, the effect of regeneration of periodontal defects verified. All these clinical trials of the DPSCs are exciting and meaningful which laid a solid foundation on the further applications.

In summary, DPSCs have acceptable efficacy on repair and regeneration of tooth tissue, bone tissue and nerve tissue, and achieve significant progress in tissue biology and regenerative medicine - thus displaying prospects for their wide clinical application. The cell source, culture method and other in vitro microenvironmental niches (Table 1) may all contribute to differentiation characteristics. Currently, research studies on the mechanism of in vitro microenvironment on DPSC differentiation remain incomplete. With continuous development of research advances in the field, DPSCs will play a more significant role in tissue engineering.

  

    
Name of Niche
    
Factor in Detail
    
Differentiated Direction
    
Involved Molecules or Pathway
    
Reference
  
  

    
Cultural Condition
    
Alginate Microspheres Fixture
    
Osteogeni Differentiation
    
ALP, Runx-2 and OCN
    
[21]
  
  

    
Hyaluronic Acid Sodium Salt
    
Odontogenic Differentiation
    
DMP-1, DSPP
    
[22]
  
  

    
Issue Source
    
Inflamed Dental Pulp
    
Odontogenic Differentiation
    
FGF-2
    
[24]
  
  

    
Carious Deciduous Dental
    
Osteogenic, Chondrogenic and Adipogenic    Differentiation
    
CD29+/CD73+/CD90+/CD14-/CD34-/CD45-
    
[25]
  
  

    
Crypreserved Dental Pulp
    
Osteogenic and Adipogenic Differentiation
    
CD166+/CD105+/CD90+/CD73+/CD45-/CD34-/CD19
    
[26]
  
  

    
Growth Factor Requirements
    
EDM
    
Osteogenic Differentiation
    
CD73+/CD90+/CD105+/CD45-/CD34-/CD14-
    
[27]
  
  

    
VEGF
    
Osteogenic Differentiation
    
RUNX-2, and ALP
    
[28]
  
  

    
ECs
    
Osteogenic Differentiation
    
ALP Activity
    
[29]
  
  

    
Odentogenic Differentiation
    
DSP, DMP-1
    
[36]
  
  

    
Insulin
    
Osteogenetic Differentiation
    
BMP3 ,BMP4 and BMPR1A
    
[30]
  
  

    
VPA
    
Osteogenic Differentiation
    
Osteopontin and Bone Sialoprotein
    
[31]
  
  

    
TNF-a
    
Osteogenic Differentiation
    
NF-KB/p38, Wnt/β-catenin Signaling
    
[32]
  
  

    
TGF-b1
    
Odontogenic Differentiation
    
Periostin
    
[33]
  
  

    
Sema A
    
Odontogenic Differentiation
    
FARP2 gene
    
[34]
  
  

    
Id1
    
Odontogenic Differentiation
    
DSF, DMP-1
    
[35]
  
  

    
CKIP-1
    
Odontogenic Differentiation
    
BMP2 NRP1
    
[37]
  
  

    
bFGF
    
Neurocytic Differentiation
    
Nestin, Musashi12, β tubulin III
    
[44,45]
  
  

    
miR-143,miR-135 inhibitors
    
Myogenic Differentiation
    
Formed Myotube
    
[14]
  
  

    
Organizational Scaffolds
    
GDPB 
    
Osteogenic Differentiation
    
BMD, SUV
    
[48]
  
  

    
ISCS 
    
Osteogenic Differentiation
    
OCN
    
[49]
  
  

    
BCP Biological Scaffold
    
Osteogenic Differentiation
    
BSP, OPN
    
[50]
  
  

    
Chitosan Scaffold
    
Osteogenic Differentiation
    
ALP, COL1, BSP, OCN
    
[51]
  
  

    
Alg/Hap Scaffold
    
Osteogenic Differentiation
    
BMP2, RUNX2, SP7
    
[52]
  
  

    
poly-L-lactic Acid Scaffolds
    
Odontoblastic Differentiation
    
DMP-1, DSPP and MEPE
    
[53,54]
  
  

    
PuramatrixTM
    
Odontoblastic Differentiation
    
MP-1 and DSPP
    
[55]
  
  

    
3-D Porous Chitosan Scaffold
    
Neuroblastic Differentiation
    
Nestin, MSP2, GFA
    
[56]
  
  

    
Chitosan Scaffold
    
Neuroblastic Differentiation
    
BDNF, GDNF, b-NGF and NT-3
    
[57]
  
  

    
DPSCs: Dental Pulp Stem Cells; EDM: Enamel    Matrix Derivative; VEGF: Vascular Endothelial Growth Factor; ECs: Endothelial    Cells; VPA: Valproic Acid; TNF-a: Tumor Necrosis Factor-a; TGF: Transforming    Growth Factor; Sema 3A: Semaphorin 3A; Id: Inhibitor of Differentiation/DNA    Binding; CKIP1: Casine Kanase 2 Interacting Protein 1; bFGF: Basic Fibroblast    Growth Factor; GDPB: Granular Deproteinized Bovine Bone; ISCS: Intrafibrillar-Silicified    Collagen Scaffolds; Alg/Hap: Alginate/Hydroxyapatite. 
  

Table 1: Influence of in vitro microenvironmental niches on DPSCs Differentiation.