Introduction

The dental pulp is an indispensable part of the tooth. It locates in the root canal space and communicates with periapical tissue through the apical foramen [1]. Histologically, it is rich in mature pulp cells or progenitors/stem cells and extracellular matrix with abundant collagenous fibers. Functionally, it is responsible for nutrients supply, dentin formation, sensory function, and defensive reaction [2]. However, during deep caries, tooth trauma, and severe periodontitis, the dental pulp usually suffers pulpitis, then traditionally removed under root canal therapy (RCT). But tooth without pulp may exhibit discoloration and increased fragility. Two decades ago, researchers isolated a clonogenic, highly proliferative population of stem cells from human dental pulp. These postnatal cells, defined as dental pulp stem cells (DPSCs), can regenerate dentin/pulp-like tissue [3]. Moreover, stem cells from human exfoliated deciduous teeth (SHED), stem cells from apical papilla (SCAPs), were also identified to share the robust self-renewal and multi-potent characteristics. Further researches on the developmental and regenerative mechanism of the dental pulp have gradually uncovered the specific signaling transductions that regulating the regenerative capabilities in dental stem cells, suggesting the possibility of dental pulp regeneration by mediating signaling pathways. Based on these findings, two burgeoning strategies for pulp regeneration have been developed. The one is the tissue engineering approach based on exogenous cell transplantation with scaffold and growth factors [4]. The other is cell homing approach based on endogenous cell migration controlled by chemokines [5]. At present, dental pulp regeneration has been a front topic in current oral medicine and striking developments have been achieved. Therefore, this review aims to pay attention to the researches about the reactivation of signaling transduction contributing to regenerative potentials in dental stem cells, then comprehensively discuss the challenges and prospects of signaling modulation in clinical translation of pulp regeneration.

Dental Stem Cells for Dental Pulp Repair and Regeneration

Several dental stem cell populations, such as DPSCs, SHED, SCAPs, have been isolated from dental pulp and apical papilla [6,7,8,9]. These dental stem cells share an important prospect for dental pulp repair and regeneration. During deep caries, DPSCs migrated toward the damaged site and differentiated into odontoblasts, which secreted reparative dentin to protect the dental pulp tissue from bacteria [10, 11]. Moreover, transplantation of DPSCs regenerated dentin-pulp like tissue with blood vessels remodeling in preclinical experiments, including ectopic regeneration in nude mice and orthotopic regeneration in dogs or minipigs, even clinical studies [12,13,14,15,16,17,18]. Compared to DPSCs, SHED presents a higher proliferation rate, but lower ALP activity level and odontogenic differentiation markers expression [19]. When in vivo transplanting hydroxyapatite/tricalcium phosphate (HA/TCP) or tooth slices with SHED in the nude mouse, dentin-pulp-like tissues were also observed [20, 21]. Likewise, SHED with puramatrix (peptide hydrogel) or recombinant human Collagen type I in the premolar roots were implanted into immunodeficient mice, which regenerated pulp-like tissues with new tubular dentin [22]. Apical papilla from immature teeth retains a population of SCAPs that presenting the regenerative characteristics of high proliferation, and odontoblastic phenotype differentiation [23, 24]. SCAPs-derived pellet transplantation or co-transplanting with 3D-printed hydroxyapatite scaffold regenerate dental pulp-like tissue with vascularity and dentine-like deposition in ectopic nude mouse models [15, 25]. Moreover, SCAPs can still be recruited into the root canal space as an endogenous cell source for pulp-dentin regeneration [26]. These stem cells colonize and proliferate in the root canal while secreting vasotrophic factors and neurotrophic factors to induce the sprouts of blood vessels and nerves. The stem cells adjacent to the dentin further differentiate into the specific odontoblasts integrating with the native dentin. Collectively, the discovery of dental stem cells makes it possible to postnatally regenerate dental pulp tissue based on their latent regenerative capabilities, including migration, proliferation, odontogenic differentiation, pro-angiogenesis, and pro-neurogenesis, which is largely controlled by the specific signaling pathways (Fig. 1).

Fig. 1
figure 1

Schematic diagram of dental stem cells-based dental pulp regeneration. Activation of the specific signaling pathways drives the migration, proliferation, odontogenic differentiation, pro-angiogenesis, and pro-neurogenesis capabilities of dental stem cells. These regenerative potency of dental stem cells ensures structural and functional pulp regeneration

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Signaling Transduction Regulates the Regenerative Potency of Dental Stem Cells

Epithelial-mesenchymal interaction plays a vital role in the early stage of tooth development. Sonic hedgehog (SHH) signaling controls the tooth morphogenesis by regulating cell proliferation [27]. FGF signaling ensures the odontogenic fate of dental mesenchymal cells [28]. BMP signaling participates in tissue homeostasis by regulating odontogenic differentiation and mesenchymal stem cell (MSC) lineage commitment [29]. Moreover, when exposed to the low-grade or short-termed inflammation, the reparative capacities of DPSCs in adhesion, migration, and proliferation were triggered by the SDF-1/CXCR4 axis, NF-kB pathways, and MAPK pathway [30, 31]. In theory, dental pulp regeneration can be achieved by simulating the signaling pattern that resembled what dental epithelial cells delivered to mesenchyme during pulp formation [32]. It is necessary to understand the mechanism of reactivation of specific signaling pathways contributing to migration, proliferation, odontogenic differentiation potentials of dental stem cells, and angiogenesis, neurogenesis of dental pulp (Fig. 2).

Fig. 2
figure 2

The pivotal signaling pathways and downstream transductions contributing to the regenerative capabilities of dental stem cells. Growth factors such as the WNT, the FGF, the TGF, the VEGF, et al., activate the corresponding pathways and controlling different cellular events. Intracellular downstream PI3K/AKT, MEK/ERK, and MAPK are responsible for multiple upstream signals and perform different functions in regulating regenerative potentials in dental stem cells

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Cell Migration‐related Signaling

Various chemokines share a positive function in promoting the migration of dental stem cells, suggesting the possibility of recruiting endogenous stem cells into the root canal. SDF-1 and CXC chemokine ligand 14 (CXCL14), members of the CXC cytokine family, can potently induce the migration of both DPSCs and SCAPs via activating the phosphorylation of FAK (focal adhesion kinases), PI3K (phosphoinositide 3-kinase), and AKT, while enhancing the expression of CXCR4 and the translocation of cytoplasmic CXCR4 to the cell membrane through the SDF-1(CXCL)-CXCR4 axis [26, 33,34,35,36,37]. Moreover, SDF-1α activates autophagy, which is positively correlated with the enhanced migration of DPSCs [34]. Stem cell factor (SCF)/the tyrosine kinase proto-oncogene Kit (c-Kit) system enhanced migration potential and cytoskeleton rearrangement in dental pulp progenitors through both MEK/ERK and PI3K signaling transduction [38]. Similarly, glutamine (Gln) dose-dependently promoted human dental pulp cells (HDPCs) migration accompanied by the up-regulation of chemokines through MAPKs (ERK, p38, JNK) and PI3K/AKT signaling transduction [39].

The above signaling pathways (CXCL/CXCR4-PI3K/AKT pathways, SCF/c-Kit- MEK/ERK and PI3K/AKT pathways, Gln-MAPK and PI3K/AKT pathways) are promising targets for promoting dental stem cells migration via modulating the adhesion, autophagy, cytoskeleton rearrangement, and upregulating the expression of chemokines and their receptors.

Cell Proliferation‐related Signaling

Cell proliferation is an indispensable factor for the regeneration of dental pulp tissue. Researches have shown that FGF2 enhanced the colony-forming efficiency and proliferation rate of DPSCs in vitro [40, 41]. The FGF/FGFR signaling transduction strongly stimulated the proliferative potential of HDPCs (containing a mixed population of stem and progenitor cells) via initiating cell cycle progression and mitosis and upregulating the expression of cdc2 and cyclin B1. The MEK/ERK signaling transduction was activating in FGF-FGFR mediated proliferation [42]. Also, the canonical WNT proteins enhanced the proliferation of HDPCs by promoting their self-renewing abilities. Overexpression of WNT10a in HDPCs can promote cell proliferation by increasing G2/M and S phases cells via the canonical WNT/β-catenin signaling transduction [43]. Conversely, knockdown of WNT10a inhibited proliferation [44]. Besides, silencing stathmin(soluble tubulin) in DPSCs down-regulated WNT5a and β-catenin, which caused a decreased proliferation rate and cells in S phase due to the inhibition of the WNT/β-catenin signaling. But treatment with lithium (LiCl, an activator of the WNT/β-catenin pathway) rescued these inhibitory effects [45]. Additionally, 0.4-T static magnetic fields (SMFs) surprisingly promoted DPSCs proliferation associated with cytoskeleton reorganization via activating the p38/MAPK signaling pathway [46]. SHH also enhanced HDPCs proliferation via SHH/Gli 1 signaling transduction [47].

To sum up, the FGF/FGFR-MEK/ERK, WNT/β-catenin, SMF-p38/MAPK, and SHH/Gli 1 signaling pathways can effectively improve the proliferation potential of dental stem cells via controlling cell cycle, cytoskeleton reorganization, and self-renewing ability.

Odontogenic Differentiation‐related Signaling

The odontogenic differentiation potential of dental stem cells is the key point of pulp regeneration, which ensures the regenerated pulp tissue to acquire the capability of dentin formation. DPSCs under transforming growth factor-β1 (TGF-β1) treatment exhibited the polarized odontoblasts-like appearance, the ultrastructural changes, and the expressions of specific odontogenic proteins and genes such as dentin matrix protein 1 (DMP-1) and dentin sialophosphoprotein (DSPP) [41]. BMP2, a member of the TGF-β superfamily, promoted the ALP activity, mineralized nodules formation, and odontoblastic differentiation of DPSCs via the phosphorylation and nuclear translocation of Smad 1/5 [48, 49]. BMP7 delivery increased the odontoblastic differentiation and the formation of mineralized nodules in DPSCs as well [33]. Glutamine, mineral trioxide aggregates (MTA), extracellular Ca2+, and Mg2+ also promoted odontogenic differentiation of DPSCs with increased gene expressions of DMP-1 and DSPP by enhancing BMP2 or BMP4 expressions and its downstream Smad1/5/8 phosphorylation [39, 50,51,52].

Besides, Both LiCl and MTA extracts (containing Ca2+) stimulated odontogenic differentiation with nuclear translocation of β-catenin and the expression of WNT3a, β-catenin, and Axin2 in SCAP and HDPCs respectively via the canonical WNT/β-catenin signaling transduction [53, 54]. Besides, glutamine stimulated the phosphorylation of GSK-3β, which indicated that the WNT/β-catenin pathway was involved in Gln-induced odontoblastic differentiation [39]. The high stiffness of scaffolds indirectly activated the canonical WNT/β- catenin pathway, which induced odontogenic differentiation in DPSCs [55]. An electrospun polystyrene nanofibrous (PSF) matrix promoted the expression of WNT3a and β-catenin that leading to remarkable DSPP expression in DPSCs, concomitant with increased alkaline phosphatase activity and alizarin red staining, indicating that the PSF matrix promoted odontoblastic differentiation via the canonical WNT/β-catenin signaling transduction [56]. As for, there is no evidence indicating the function of the non-canonical WNT pathway on odontogenic differentiation of dental stem cells.

FGF2 promoted odontoblastic differentiation of DPSCs with the enhancement of ALP activity, mineralization, and odontogenic markers expression via the MAPK (p38, ERK) and PI3K/AKT signaling transduction [57]. Lipopolysaccharide(LPS), epiregulin(EREG) and parathyroid hormone(PTH), or materials like demineralized dentin matrix (DDM) and ceramic bovine bone (CBB) enhanced DSPP and DMP-1 expressions of DPSCs through activating the MAPK (p38 and ERK) signaling pathways [58,59,60,61]. DPSCs-derived exosomes under odontogenic differentiation conditional medium triggered the MAPK (p38) transduction, which promoted the DSPP expression and odontogenic differentiation of DPSCs [62].

Collectively, the TGF-β, the BMP, the canonical WNT/β-catenin, the MAPK (p38 and ERK) signaling pathways play a critical role in the odontogenic differentiation of dental stem cells.

Angiogenesis Related Signaling

Vasculogenesis vitally ensures dental pulp regeneration. This process involves multiple cellular signals, which regulate endothelial cell migration, proliferation, branching, and tube formation [63]. Pericytes and endothelial cells interact to regulate new vessel formation. A study found that DPSCs can act as pericyte-like cells to secrete proangiogenic factors such as VEGF for vascularization in pulp regeneration [64]. Hinokitiol, Iloprost, MTA, TNF-α, and LPS upregulated the expression of proangiogenic factors (VEGF, hypoxia-inducible factor-1alpha (HIF-1α), Ang-1, vWF) in HDPCs by activating the MAPK (p38 and ERK) pathway [65,66,67,68]. EphrinB2/ EphB4 signaling promoted VEGF secretion in DPSCs via the MEK/ERK1/2 and MAPK (p38) signaling transduction, which induced sprouting angiogenesis of endothelial cells [69]. Under the hypoxia condition, SCAP presented an upregulated expression of VEGF via activation of the HIF-1α signaling, which accelerated the formation of vessel-like structures by human umbilical vein endothelial cells (HUVECs) [70].

Besides, dental stem cells can directly differentiate into the endothelial cells to promote angiogenesis [71]. A study indicated that VEGF induced SHED to differentiate into angiogenic endothelial cells through binding to VEGFR1 and activating downstream ERK and AKT signaling [72]. The Sema4D/PlexinB1 signaling up-regulated the VEGF secretion via the phosphorylation of AKT and ERK1/2, which is contributing to the endothelial differentiation of DPSCs [73]. Moreover, VEGF or Wnt1 treatment upregulated the expressions of endothelial cell differentiation markers (VEGFR2, VE-Cadherin, Tie-2, and CD31) respectively in DPSC and SHED via the canonical WNT/β-catenin pathway, along with the increased expressions of LRP-6 and Fzd-6 (the WNT receptors) and β-catenin [74].

Altogether, dental stem cells can not only act as pericytes secreting pro-angiogenic factors to induce endothelial cells for vascularization, but also directly differentiate into endothelial cells via the VEGF, the HIF-1α, the MAPK (p38 and ERK), the canonical WNT/β-catenin, and the PI3K/AKT signaling transduction.

Neurogenesis Related Signaling

The innervation is crucial for DPSCs proliferation and apoptosis, and tooth homeostasis [75]. Research has revealed that dental stem cells shared the neurotrophic function for neurogenesis [76]. Nerve growth factor (NGF) was upregulated in the odontoblasts adjacent to caries and injury sites. NGF signaling induced axonal migration and growth of Schwann cells during dental pulp repair [77]. Brain-derived neurotrophic factor (BDNF) signaling triggered the neurotrophic effects of dental mesenchymal stem cells (including SCAP, DPSCs) by enhancing neurotrophic factors expressions, such as BDNF, NGF, neurotrophin 3 (NT-3) [78]. Besides, FGF2 signaling transduction also induced HDPCs to release neurotrophic factors to support axonal regeneration [79].

Dental stem cells can differentiate into neural cells for nerve sprouting. Basic-FGF increased neurosphere size of DPSCs and expressions of neurogenic markers via FGF-FGFR and PLCγ intracellular transduction, suggesting neural differentiation of DPSCs [80]. NGF and Basic-FGF together stimulated the expressions of neuronal markers (Nestin, βIII-tubulin), and promoted the neural differentiation of DPSCs via the ERK and AKT signaling transduction [81]. Otherwise, the chitosan scaffold induced neural differentiation of DPSCs attributing to the activation of the WNT/β-catenin signaling pathway with upregulating the levels of BDNF, NGF, and NT-3 expressions [82].

Neurotrophic factors like BDNF, NGF secreted by dental stem cells can promote neurogenesis. Moreover, the FGF-FGFR signaling and the WNT/β-catenin signaling may contribute to both neurotrophic effects and neural differentiation of dental stem cells.

Methods in Regulating Signaling Transduction

The reactivation of signaling transduction can be modulated by multiple methods, which are summarized below (Fig. 3).

Fig. 3
figure 3

Several means in modulating cellular signaling transduction. a The drug delivery method is based on topically delivering some bioactive growth factors, small molecules, or metal ions to modulates cellular activities; b Genetic modification include inputting or deleting target gene fragments from the genome through gene editing or gene transfection to modulate signaling transduction in the host cell; c Physical stimulation such as stiffness of scaffolds, local treatment of low-frequency ultrasound and magnetic field are also possibly applied for specific signaling activation

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Drug Delivery

Growth factors share the role in signaling regulation by binding to their receptors. The activators of the WNT pathway (WNT1, WNT3a), the BMP pathway (BMP2, BMP7), and the FGF pathway (FGF2), et al., can activate the related signaling transduction [42, 57, 83,84,85]. On the contrary, some inhibitory factors, rhDKK1 and noggin, negatively regulate the WNT pathway and the BMP pathway respectively [85, 86]. Besides, amounts of chemically synthesized small molecules as inhibitors blocked the transduction of the BMP signaling (LDN193189), and the TGF-β signaling (SB431542) [85, 87]. CHIR99021 (CHIR), an adenosine triphosphate (ATP)–competitive glycogen synthase kinase-3 (GSK-3) inhibitor, can stimulate the WNT/β-catenin signaling activity [88]. LiCl can also inhibit GSK-3β to indirectly activate the WNT signaling transduction [53, 89]. Moreover, metal ions such as Ca2+ and Mg2+ can activate the BMP pathway [51, 52]. Together, the specific drug delivery by pre-encapsulated into scaffolds is a clinically feasible way to modulate the signaling pathway. But an effective sustained-release system should be established in further studies.

Genetic Modification

Genetic modification refers to the use of biochemical methods to modify DNA sequences including inputting target gene fragments into host cells or deleting specific gene fragments from the genome to change the host cell genotype or strengthen the original genotype [90]. A study showed that overexpressing the platelet-derived growth factor-BB (PDGF-BB) by lentivirus significantly facilitated the migration of DPSCs in vitro and promoted the dentin-pulp regeneration in vivo via activating the PI3K/AKT signaling transduction [91]. When transfected with VEGF and SDF-1 overexpressed lentiviral particles, DPSCs acquired stronger potentials for the vascularized pulp regeneration [4]. Moreover, transfecting the Delta-1 (the Notch pathway ligands) genes by retrovirus enhanced the proliferation and odontogenic differentiation of DPSCs [92]. Compared to drug delivery, gene modification possesses the superiority of stable and persistent effects on activating the signaling. However, the usage of viral vectors has latent adverse effects such as immunoreaction. Genome editing techniques like CRISPR/Cas system may have higher security than viral transfection, but it is also necessary to consider how to terminate the persistent activation of specific signaling pathways.

Physical Stimulation

Physical stimulation influences signaling transduction. Elastic polydimethylsiloxane substrates raised the proliferation and odontogenic differentiation in DPSCs along with the stiffness increasing, which is accompanied by the activation of the WNT/β-catenin pathway [55]. Static Magnetic Fields (SMFs) arouses higher proliferative rate and intracellular calcium ions change of DPSCs via the p38 signaling [46]. Magnetic nanofiber scaffold can also improve odontogenic differentiation and angiogenesis of HDPCs by activating the WNT/MAPK/NF-kB pathways [93]. Furthermore, low-frequency ultrasound treatment promoted VEGF expression in odontoblast-like cells [94]. Therefore, the physical properties of scaffolds (stiffness, magnetic materials) and local treatment of low-frequency ultrasound are possibly applied to improve the regenerative potentials of dental stem cells.

Modulation of Regeneration‐related Signaling in Pulp Regeneration Strategies

Cell Homing Approach

The cell homing approach load chemokines into bioactive scaffolds to recruit periapical stem/progenitor cells, to regenerate pulp tissue in the root canal endogenously (Fig. 4a) [95]. Reinstating regenerative related signals in the scaffold is conducive to pulp regeneration.

Fig. 4
figure 4

Schematic diagram of growth factors in pulp regeneration strategies. a The cell homing approach is based on transplanting bioactive scaffolds loading the chemokines into the root canal to recruit endogenous stem/progenitor cells to regenerate pulp tissue; b The cell transplantation approach is based on in vitro cell culture and in vivo transplantation of exogenous stem cells with growth factors and scaffolds for dental pulp tissue regeneration

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Chemotaxis of cytokines strongly recruited endogenous stem cells for dental pulp regeneration. Implanting collagen gel with Basic-FGF into the endodontically prepared root canals successfully yielded pulp-like tissue with abundant cells [33]. Stem cell factor (SCF) promoted pulp regeneration by increasing cell number and capillaries, as well as collagen sponge remodeling and collagen fiber neogenesis. PI3K/AKT and MEK/ERK signaling were involved in that process [38]. SDF-1 potently enhanced the migration of CXCR4 positive DPSCs and promoted pulp regeneration with dentin, nerves, vasculature formation [96]. Moreover, SDF-1α-loaded silk fibroin scaffolds improved the de novo formation of pulp-like tissues in pulpectomized dog teeth [34]. These findings seemly indicated that CXCL/CXCR signaling transduction is a promising target for the cell homing approach of pulp regeneration.

In a preclinical orthotopic minipig model, researchers infused recombinant human WNT3a with collagen gel into root canals, which successfully recruited endogenous stem cells to regenerate neurovascular stroma and parenchymal odontoblast-like cells embedded into newly formed dentin. Besides, BMP7 or WNT3a + BMP7 delivery both lead to excessive mineralization in the regenerated pulp-like tissue [84]. WNT and BMP pathways may play a critical role in admitting postnatal endogenous stem cells with an innate regenerative capacity to regenerate pulp tissues.

Platelet-rich plasma (PRP) contains many growth factors including PDGF-BB, TGF-β1, IGF-1, VEGF, and Basic-FGF [97]. Transplantation of the PRP into beagle dog teeth regenerated newly formed tissue (cementum-like and periodontal ligament-like), without significant difference compared to combined transplantation of DPSCs and PRP [98]. Besides, canine teeth with preexisting necrotic pulps and periapical lesions were filled with the PRP, which significantly promoted apical development and hard tissue deposition than not using the PRP [99]. A clinical case reported that after injecting the PRP into the root canal of maxillary premolar promoted root development, apical foramen closure with normal response to cold and electric pulp tests [100].

Tissue Engineering Approach

The procedure of the tissue engineering approach is transplanting exogenous stem cells and growth factors with scaffolds into the endodontically prepared root canal to regenerate dental pulp tissue (Fig. 4b) [101]. Currently, exogenous stem cells including DPSCs, SHED, SCAP, et al., have been commonly utilized in vitro and in vivo studies of pulp regeneration [102]. Besides, the scaffold should be biocompatible and biodegradable, which resembles the extracellular matrix. Growth factors exert essential biological functions in regulating cellular events and have been applied to pulp regeneration.

Ectopically transplanting DPSCs with SDF-1α into nude mice recruited host-derived cells. Two types of cells together regenerated pulp-like tissue with vascularity, well-organized fibrous matrix, and dentin deposition [34]. Exosomes (containing multiple growth factors) derived from DPSCs under odontogenic differentiation conditions triggered the p38/MAPK signaling activation. When co-transplanting the exosomes and DPSCs into tooth root slices and implanting subcutaneously in the back of nude mice regenerated dental pulp-like tissue [62]. Besides, downregulation of the RA signaling enhanced the odontogenic differentiation of DPSCs and increased bone-like tissue formation when subcutaneously transplanting with HA/TCP ceramic particles with DPSCs into nude mice [103]. Combined delivery of different growth factors potently enhanced pulp regeneration due to the modulation of different regenerative capabilities of dental stem cells. Transplanting DPSCs with a group of growth factors, TGF-β1, Basic-FGF, and VEGF into self-assembling peptide hydrogel into the back of immunocompromised mice successfully regenerated a vascularized dental pulp-like tissue with odontoblast-like cells embedded into the dentinal tubules [104].

Angiogenesis is essential for the transplanted constructs. PDGF-BB overexpression in DPSCs regenerated dentin-pulp like tissue infiltrated with blood vessels by subcutaneously transplanted with porous calcium phosphate cement (CPC) scaffold in nude mice model [91]. Transplanting DPSCs with Granulocyte Colony Stimulating Factor (G-CSF) into the root canal of the dog yielded a large amount of dentin-pulp like complex with neovascularization [14]. The root fragments seeded with two types of DPSCs (overexpressed SDF-1α and overexpressed VEGF) were co-implanted into nude mice, which produced an increased vessel area density in the regenerated pulp-like tissue [4].

Based on current evidence, the cell homing approach is seemly a more convenient strategy than the cell transplantation approach. it does not require the isolation, culture, and transplantation of stem cells. But this strategy still encounters some hardships about the shortage of endogenous stem cells in defect sites and the effective delivery of chemotactic factors. The tissue engineering approach has achieved considerable pulp regeneration in animal models. Notably, bioactive molecules play an essential role in pulp regeneration via directly or indirectly modulating signaling transduction. However, stem cells and growth factors modulating the key signaling pathways still needs a long run to validate for clinical application.

Challenges of Signaling Modulation in the Clinical Application of Pulp Regeneration

The researches of pulp regeneration are gradually transferring into the clinical phase. Some preclinical studies have demonstrated the safety and efficacy of stem cell transplantation [12, 17]. As mentioned above, pulp regeneration by modulating specific signaling transduction can regulate the regenerative capabilities of dental stem cells, which shares a promising prospect in the clinical application of pulp regeneration strategies. But several issues should be resolved before clinical application.

Controlled Release of Growth Factors

Clinical pulp regeneration will be a long-term procedure. Growth factors can be delivered only once, while gene modification is difficult for clinical use because of its security issues. Therefore, growth factors sustained-release system becomes extremely critical for the long-term biological effects on signaling modulation. Currently, growth factors encapsulated into hydrogels and microspheres, and crosslinked with scaffolds surface are available for sustained-release delivery [105,106,107]. But the sustained-release systems are not suitable for drugs with very short or very long biological half-lives, high effective dose, or low solubility. A novel scaffold with injectable property, biocompatibility, maintenance of growth factors activity, and sustained-release of growth factors is very appropriate for the clinical translation of pulp regeneration.

Single or Combined Deliveries of Growth Factors

Pulp regeneration involves multiple regenerative potentials of dental stem cells. Modulating a single signaling pathway fail to meet the requirements of structural and functional pulp regeneration. The combinations of different growth factors targeting to different signaling pathways may be the more feasible approach for pulp regeneration. However, different growth factors may cause intricate crosstalk. Some pathways promote each other and some antagonize each other. Balancing the effects of growth factors to control the complicated signaling crosstalk is a tough but meaningful job.

Safety of Modulating Signaling Transduction

Safe and effective strategies are necessary for the clinical application of pulp regeneration. Generally, drug delivery can be applied by encapsulating growth factors into scaffolds and then injected into the root canal [108]. However, it is noteworthy that these signaling molecules may regulate a wide range of cell types and cellular events, which probably results in side effects during clinical use. Genetic modification maybe not clinically suitable due to its latent safety problems of viral vectors and gene editing. Perhaps, physical stimulation may unite the drug delivery to enhance the therapeutic effect via synergistically regulating the related signaling. Further researches should target to maintain the safe, stable, and controllable effects of signaling modulation when clinically applied.

The Barriers of Complex Clinical Conditions

Endogenous stem cell migration is the key to cell homing strategy. Treatments of apical foramen and the elimination of local inflammation are necessary during the procedure of pretreating the root canals. Moreover, the senescence of dental stem cells limits their regenerative potentials [109]. It is difficult to obtain vigorous autologous stem cells from aged patients. The stem cells from the elders may also exhibit limited reactivity to signals.

Control the Abnormal Calcification in Regenerated Pulp Tissue

The level of dentinogenesis in regenerated pulp should be similar to that of the normal pulp. But odontogenic differentiation-related signaling may increase the risk of pulp calcification [110]. Extensive calcification is harmful to long-term outcomes. For young permanent teeth with an open apex, the potential of dentinogenesis should be enhanced for the continued apical development. However, in mature permanent teeth, the dentinogenesis should be maintained or moderately inhibited to prevent excessive calcification.

The Future Perspective of the Stem Cell-based Dental Pulp Regeneration

The finding of dental stem cells suggests a possibility of dental pulp regeneration through activation of autogenous stem cells or transplantation of exogenous stem cells. The good manufacturing practices (GMP)-compliant facilities and stem cells provide the safety and quality for cell-transplanted pulp regeneration strategy, which have been confirmed by series of preclinical/clinical trials. Since the functionality largely depends on the signals, understanding the mechanisms of signals (such as the WNT pathway and the BMP pathway) determining the function of dental stem cells is essential to dental pulp regeneration. In the future, transplanting autologous/allogeneic stem cells or recruiting the endogenous stem cells in periapical niches are expected to be applied in dental clinical practice as an ideal therapy instead of root canal therapy (RCT) for pulpitis, periapical inflammation, and other pulp diseases. Meanwhile, precisely activating and controlling the postnatally pivotal signaling pathways will make pulp regeneration encounter big prospects and future.

Conclusions

Signaling transduction controls a series of cellular events. Dental stem cells such as DPSCs, SCAPs, SHED, play an essential role in pulp regeneration. Multiple evidence has shown that the reactivation of specific signaling transduction is vitally critical for the regenerative potency of dental stem cells. Both cell transplantation and cell homing approaches are dependent on the regenerative capacities of dental stem cells. These findings inspire us to consider a novel strategy that synergistically or antagonistically integrating different signaling pathways especially contributing to the migration, proliferation, odontogenic differentiation, pro-angiogenesis, and pro-neurogenesis potentials of dental stem cells via orchestrating gene expression precisely. In the meantime, the methods of modulating signaling must be clinically feasible and effective. Growth factor delivery and physical stimuli approaches are more appropriate for clinical use. Collectively, further researches should focus on the comprehensive mechanism of signaling modulation during pulp regeneration. Postnatally reactivation of pivotal signaling pathways in dental stem cells will share an essential prospect in pulp regeneration strategies and especially clinical translation of strategies based on the cell homing approach.