CGRP and Shh Mediate the Dental Pulp Cell Response to Neuron Stimulation

Abstract

Dental pain is a persistent, detrimental public health issue that requires a better understanding of the mechanisms of tooth pain and inflammation in order to develop more effective treatments. Calcitonin gene-related peptide (CGRP) and dental pulp cells are promising candidates for mediating tooth pain and generating reparative dental tissues, respectively, but their behavior in the context of pulpitis remains elusive. The mouse incisor requires Sonic hedgehog (Shh) secreted from sensory nerves to continuously regenerate. However, it is unknown whether sensory nerves also regulate the comparatively nonregenerative mouse molar through CGRP and Shh. This is an important knowledge gap to fill since mouse incisors differ biologically from human teeth, while mouse and human molars are similar. In this work, we identified that molar pulp cells express CGRP receptor and Gli1, a Hedgehog (Hh) signaling protein found to label a dental stem cell population in the mouse incisor. We also observed in a mouse molar injury model that Hh signaling was activated and Shh expression was upregulated in vivo. We then determined in vitro that Shh and CGRP regulate differentiation of primary mouse molar and incisor pulp cells and a human dental pulp stem cell line. Furthermore, conditioned media from stimulated sensory neurons induced Hh signaling activation and inflammatory gene expression in primary molar pulp cells, which was abolished by inhibition of either Shh or CGRP. Our results suggest that CGRP and Shh signaling may promote an inflammatory response after injury in the molar and that activated sensory nerves secrete CGRP and Shh to regulate molar pulp cell expansion and differentiation into odontoblast-like cells for dentin repair. Thus, CGRP/Shh signaling should be considered for new strategies that seek to manage pain or dentin regeneration in the molar.

Introduction

The currently available clinical solutions for pulpal-mediated dental pain in response to deep caries involve pulp removal and replacement with synthetic materials or extraction of the tooth entirely with a subsequent need for tooth replacement. These treatments can be expensive and are often not accessible to everyone. Thus, there is need for alternative pulp-preserving treatments that demand a deeper understanding of the cellular and molecular mechanisms underlying dental pulp cell behavior during inflammatory injury, clinically known as pulpitis.

Sensory neurons penetrate the molar pulp and are thought to be critical regulators of stem cell and immunological reaction during dental disease processes (Huang et al. 2010; Rosa et al. 2013). The importance of sensory afferents was observed in human teeth when the onset of dental pulp necrosis occurred noticeably faster with surgical denervation (Byers and Taylor 1993). Similarly, ablating sensory neurons via capsaicin treatment severely enhanced periapical bone destruction in a rat pulpal disease model (Austah et al. 2016). However, the role of interactions between sensory nerves and dental pulp cells, including dental pulp stem cells (DPSCs), in dentin repair and inflammation during pulpitis remains unknown.

Hedgehog (Hh) signaling is important for DPSC differentiation and pulpitis, and cells expressing one of its downstream targets, Gli1, are thought to be responsible for murine incisor regeneration (Zhao et al. 2014; Ma et al. 2018). Specifically, Sonic hedgehog (Shh) ligand released from the sensory neurons innervating the pulp maintains Gli1⁺ cells, which differentiate into cells that generate new dentin and enamel (Zhao et al. 2014). Furthermore, Shh was also found to be secreted by trigeminal ganglion (TGG) sensory neurons expressing calcitonin gene-related peptide (CGRP), a neuropeptide involved in orofacial pain and neurogenic inflammation (Schou et al. 2017). CGRP activates CGRP receptors (CGRPr) expressed in ameloblasts and odontoblasts, participates in crown and root development, and increases expression of key factors associated with dentin formation (Calland et al. 1997; Vandevska-Radunovic et al. 2003). These studies suggest that both CGRP and Shh secreted by sensory neurons may facilitate dentin repair. The continuously regenerating murine incisor is a useful model for studying tooth regeneration, but it is not representative of the repair response in human teeth. The murine molar is similar to the human molar in both developmental and repair processes, making it an optimal model for recapitulating inflammation and repair in human teeth. We therefore explored interactions between sensory neurons and molar pulp cells by investigating the role of Shh and CGRP in differentiation and inflammation in this work.

Materials and Methods

Animals and Tamoxifen Injections

Wild-type, Gli1CreER (Stock #007913), and Rosa26^tdTomato (Stock #007914) mice were obtained from The Jackson Laboratory. Gli1CreER males were bred with Rosa26^tdTomato females to generate Gli1CreER;Rosa26^tdTomato offspring. Mice received injections of 100 mg/kg tamoxifen (Sigma-Aldrich, T5648) + 10% ethanol in corn oil at postnatal day 30 (P30), P32, and P34 to induce tdTomato expression to track Gli1-expressing cells and their progeny. All mice were on a C57BL/6 background and were housed, cared for, and monitored for pain in accordance with Institutional Animal Care and Use Committee (IACUC) standards in an American Association for Accreditation of Laboratory Animal Care accredited facility. Two nonbred male and female mice were used to address potential sex differences for a total n = 4 for each group to confirm reproducibility. This work conforms to the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.

Primary Cell Isolations and Cell Culture

Trigeminal ganglia (TG) from P30 to P35 wild-type mice were collected and cell dissociation was performed in accordance with a published protocol (Malin et al. 2007). After enzymatic and mechanical cell dissociation, an additional step of neuron purification was added using a 2-layer Percoll gradient (60% and 35%). After centrifugation for 20 min at 800 × g at 4°C, trigeminal neurons at the interface of the 2 Percoll solutions were collected, washed, and seeded in a 6-well plate. Two hours after plating, a final step of neuronal isolation was performed by shaking the 6-well plate for 15 min at 100 rpm to detach neurons, leaving the remaining nonneuronal cells attached to the plate. Neurons in supernatant were seeded onto 6-well plates and cultured in F12 + 5% fetal bovine serum (FBS) (Sigma, F2442) + 1% PenStrep (Gibco, 15140-122) for 24 h at 37°C and 5% CO₂ prior to conditioned media preparation. Neurons were not passaged prior to experiments.

Commercially available human DPSCs (hDPSCs; Lonza) were cultured as described in our previous publication (Michot et al. 2020). hDPSCs were plated at a density of 5,000 cells/cm² and maintained at 37°C and 5% CO₂.

Primary incisor cells were isolated in accordance with a published protocol (Chavez et al. 2014). The extracted region contains cells from the dental pulp, labial cervical loop, and lingual cervical loop, but for simplicity, we refer to this population as “incisor pulp” throughout the article. A scalpel was used to pry mandibular and maxillary molars from the socket, scrape off surrounding tissue, and bisect at the root/crown interface to expose the molar pulp. Molar pulp was isolated from both root and crown segments. Incisor and molar pulp were digested in 3 mg/mL Collagenase I (Millipore, MM_NF-SCR103) + 4 mg/mL Dispase II (Sigma-Aldrich, D4693) in a low-attachment 6-well plate (Corning, 3471) for 90 min at 37°C and 5% CO₂. All primary dental cells were isolated from P30 to P35 mice and cultured in minimum essential media (MEMα) + 10% FBS + 1% PenStrep. Cell passages 1 to 2 were used for experiments.

Differentiation Media and Alizarin Red Staining

hDPSCs and primary dental cells were treated for 3 wk with odontogenic differentiation media (DM) (MEMα + 5 mM β-glycerophosphate + 100 nM dexamethasone) and 5 nM Shh (Tonbo Biosciences, 21-8679), 10 nM CGRP (AnaSpec, AS20682), 100 ng/µL Shh neutralizing monoclonal antibody (Shh_i) (Developmental Studies Hybridoma Bank, 5E1), or 50 ng/µL CGRP antagonist (CGRP_i) (GenScript, RP11089), then fixed with 10% formalin for 15 min. Alizarin red staining was performed and quantified as previously described (Michot et al. 2020).

Conditioned Media Experiments

Primary neurons were incubated in media with or without 0.01 µM capsaicin (Sigma-Aldrich, M2028) for 5 min followed by fresh culture media for 20 min at 37°C and 5% CO₂. Conditioned media (CM) were transferred to hDPSCs and primary dental cells cultured to 80% confluence, which were also treated with 5 nM Shh, 10 nM CGRP, 5 nM Shh + 10 nM CGRP, 100 ng/mL Shh_i, 50 ng/mL CGRP_i, or 100 ng/mL Shh_i + 50 ng/mL CGRP_i for 24 h at 37°C and 5% CO₂.

Colony-Forming Units

Primary dental cells isolated from wild-type mice were plated onto 12-well plates at a density of 10³ cells/cm² and incubated for 14 d in culture media containing 5 nM Shh, 10 nM CGRP, or both 5 nM Shh + 10 nM CGRP. Media were changed every 3 d. Cells were fixed with 4% paraformaldehyde and stained with 4% w/v crystal violet (Sigma-Alrich, C0775) for 20 min at ambient temperature. Colony-forming units were manually counted and images were acquired using an all-in-one fluorescence microscope (Keyence, BZ-X710).

Quantitative Reverse Transcriptase Polymerase Chain Reaction

Cells were lysed and RNA was extracted using RNAzol RT solution (Sigma-Aldrich, R4533) per company protocol. RNA was converted to complementary DNA (cDNA) using the HiFi cDNA Synthesis Kit (CoWin Biosciences, CW2569M). Quantitative polymerase chain reaction (PCR) was performed using Power SYBR Green Master Mix (ThermoFisher Scientific, 4368577) and a StepOnePlus Real-Time PCR System (Applied Biosciences). Values were normalized to GAPDH to account for variability in messenger RNA (mRNA) expression between samples. Experimental groups are expressed as a fold change in relation to controls.

Pulp Exposure and Dentin Exposure Injury Models

Gli1CreER;Rosa26^tdTomato mice were injected with tamoxifen at P30, P32, and P34 and injury surgeries were performed at P37. Mice were anesthetized via intraperitoneal injection of 40 mg/kg ketamine (Henry Schein) + 5 mg/kg xylazine (Akorn) in sterile PBS, and the exposures were generated in accordance with published protocols (Gibbs et al. 2013; Lee et al. 2017). Briefly, the left maxillary first molar was drilled with a ¼ round bur at low speed until the dentin of the first pulp horn was exposed in the dentin exposure model. For the pulp exposure model, the middle pulp horn was drilled until the pulp was exposed. The contralateral maxillary first molar served as the uninjured control in both models. The area was washed with sterile PBS to remove any debris. All surgeries were conducted using sterile technique and animals were monitored per IACUC regulations.

Histology and Immunostaining

The hard palate and maxilla were dissected together 3 and 7 d post-injury (dpi) for the pulp and dentin exposure models, respectively. Tissue was fixed in 4% paraformaldehyde (Sigma-Aldrich, P6148) at 4°C overnight, decalcified in 15% EDTA (VWR, BDH4616) for 3 wk, cryosectioned (10–30 µm), and mounted with media containing a nuclear stain (Electron Microscopy Sciences, 17985-50). For immunohistochemistry, cryosections were permeabilized in 0.5% Triton X-100 (Sigma-Aldrich, T8787) for 5 min, blocked in 10% goat serum (MP Biomedicals, 0219135680) for 1 h at ambient temperature, and incubated in primary antibody overnight at 4°C. Primary antibodies were used at a 1:500 dilution in PBS, including mouse monoclonal Shh (DSHB, 5E1), mouse monoclonal CGRP (Sigma-Aldrich, C7113), and rabbit polyclonal CGRPr (Bioss, bs-1860R). For immunocytochemistry, cells were fixed in 4% paraformaldehyde for 5 min and incubated in the CGRPr antibody for 1 h at ambient temperature. Following primary incubation, slides or cells were washed in PBS for 30 min and incubated at 1:1,000 in one of the following fluorescent secondaries for 2 h at ambient temperature: Alexa Fluor 568 goat anti-rabbit (Invitrogen, A11011), Alexa Fluor 488 goat anti-rabbit (Invitrogen, A32731), and Alexa Fluor 488 goat anti-mouse (BioLegend, 405319). Images were acquired using a fluorescence microscope (Keyence, BZ-X710) and a confocal microscope (Leica, Stellaris). For images obtained with confocal microscopy, 40 Z-stacks were merged, and image processing was performed using Fiji (Schindelin et al. 2012).

Statistics

Animals were randomly assigned to groups depending on genotype, and researchers were blinded to all data analysis. No sex-dependent differences were identified according to a 2-way analysis of variance (ANOVA) so males and females were grouped together for analysis. Differences between control and experimental groups were determined using a 1-way ANOVA followed by a post hoc Tukey test. Values are reported as mean ± SEM, with P < 0.05 considered statistically significant. The sample size was selected to achieve a power of at least 80%. Statistical analysis was conducted using GraphPad Prism (GraphPad Software).

Results

We used murine molar injury models to interrogate whether there is interaction between sensory nerves and molar pulp cells. We first examined the presence of CGRP and Hh pathway components in the pulp cells since sensory nerves are known to secrete CGRP and Shh. Cells expressing CGRPr were observed in molar and incisor pulp cells cultured in vitro, as well as in the murine molar pulp in vivo (Appendix Fig. 1), indicating these cell populations can respond to secreted CGRP. We then sought to determine whether Hh signaling is activated in vivo in response to injury using dentin and pulp exposure injury models. Gli1CreER;Rosa26^tdTomato reporter mice were selected to visualize Gli1-expressing cells and their progeny (Gli1⁺) as a marker of Hh signaling activation. In the pulp exposure model, tissue necrosis at the site of the injury was prominent and caused loss of CGRP⁺ fibers (Fig. 1A, B). At the noninjured pulp horns where CGRP⁺ fibers were present, we observed an increase in Gli1⁺ cells (Fig. 1B). In the dentin exposure model (Appendix Fig. 2A), we also observed an increase in Gli1⁺ cells (Appendix Fig. 2B), and CGRP⁺ fibers were preserved at the injury site (Appendix Fig. 2C). Using this model of dentin exposure, we further observed that CGRP and Shh expression was increased in the pulp horn region where Hh signaling was upregulated (Fig. 1B, C).

Figure 1.

Hedgehog (Hh) signaling and expression of calcitonin gene-related peptide (CGRP) and Sonic hedgehog (Shh) in murine pulp and dentin exposure injury models. (A) Noninjured molar pulp horn (gray arrow) with CGRP⁺ fibers (green) intact and Gli1⁺ cells (red). A higher magnification view of the noninjured pulp horn with intact CGRP⁺ fibers is depicted in the right panel. (B) Pulp exposure model where the molar pulp horn has been shaved down to expose the pulp (gray arrow) 3 d post-injury (dpi). Gray dashed lines indicate where the pulp and CGRP⁺ fibers should be. A higher magnification view of an intact pulp horn (white boxed region) containing Gli1⁺ cells is depicted in the right panel. (C) Gli1⁺ cells and CGRP expression in the dentin exposure model 7 dpi. (D) Gli1⁺ cells and Shh expression in the dentin exposure model 7 dpi. Nuclei are depicted in blue. Scale bars in A, B represent 100 µm and scale bars in C, D represent 50 µm.

To determine the function of CGRP and Shh in dentin regeneration, we first examined their effects on dental pulp cell proliferation. We isolated cells from murine incisor pulp (known regenerative potential) and molar pulp (unknown regenerative potential) and compared their ability to form colonies when treated with CGRP and/or Shh. Interestingly, cells isolated from the molar pulp were consistently more efficient in forming colonies (Appendix Fig. 3A), and CGRP treatment uniquely increased the number of colony-forming units (CFUs) of the molar pulp cells (Fig. 2A). Since inflammation occurs after injury, we also determined expression of inflammation marker genes in the isolated pulp cells treated with CGRP/Shh. Both cell types exhibited increased expression of inflammatory genes when treated with CGRP, and a further increase was found when treated with both CGRP and Shh (Fig. 2B). We then asked if this behavior in the heterogeneous mouse molar pulp cells was consistent with a purified population of hDPSCs by treating hDPSCs with CGRP/Shh. hDPSCs proliferated too rapidly to enable comparisons between CFUs, but the pattern and magnitude of changes in the expression of relevant genes were consistent with findings in mouse molar cells (Fig. 2B). Since Shh alone did not promote proinflammatory gene expression, we examined changes in Hh signaling targets and confirmed that Shh ligand indeed activated their expression (Appendix Fig. 3B), indicating that Shh by itself does not regulate inflammation. Interestingly, we also found that Hh signaling activation was mitigated with dual treatment of Shh and CGRP, suggesting antagonistic roles for these ligands on Hh core signaling.

Figure 2.

Effects of Sonic hedgehog (Shh) and calcitonin gene-related peptide (CGRP) treatment on dental pulp cell colony-forming units (CFUs) and messenger RNA (mRNA) expression. (A) Number of CFUs as a result of treatment with 10 nM CGRP, 5 nM Shh ligand, or both (Shh + CGRP). (B) Changes in mRNA expression of inflammatory markers with Shh/CGRP treatment. *P < 0.05, ***P < 0.001, ****P < 0.0001 compared to no treatment group, Tukey’s test; n = 3–5 independent cell cultures.

To explore the role of sensory neurons in producing CGRP and Shh that regulate pulp cells, we used a conditioned media (CM) experiment. Primary neurons isolated from the murine TGG were treated with capsaicin to stimulate secretion from a nociceptive population of afferent neurons, and the resulting CM was transferred to the pulp cell cultures (Fig. 3A). CM from nonstimulated neurons altered inflammatory gene expression compared to regular culture media, so we used this as our control group (Appendix Fig. 4). CM from capsaicin-stimulated neurons (Cap CM) enhanced expression of inflammatory markers in each cell type, but this response was severely attenuated when either CGRP or Shh was inhibited (Fig. 3B). We also evaluated Hh signaling activation and found that Hh target expression was similarly increased by Cap CM, but this effect was mitigated when Shh was inhibited (Fig. 3C). These data indicate that when sensory neurons are stimulated, as would occur after dentin injury, they secrete factors such as CGRP and Shh to regulate dental pulp cells.

Figure 3.

Changes in dental pulp cell messenger RNA (mRNA) expression due to conditioned media (CM) from stimulated neurons and odontogenic differentiation media. (A) Illustration of how CM from primary trigeminal ganglia neurons was generated and transferred to dental pulp cells. Neurons were treated with 0.01 µM capsaicin for 5 min to induce an inflammatory response. Capsaicin-containing media was replaced with fresh culture media for 20 min to collect released factors from the stimulated neurons and then transferred to the primary dental cells. Media from neurons not stimulated by capsaicin served as the control. (B) Changes in mRNA expression of inflammatory markers and (C) Hedgehog (Hh) signaling targets 24 h after incubation in CM from nonstimulated (CM) or stimulated neurons (Cap CM) and treatment with 50 ng/mL calcitonin gene-related peptide (CGRP) antagonist (CGRP_i) and/or 100 ng/mL Sonic hedgehog (Shh) antibody (Shh_i). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to no treatment group, Tukey’s test; n = 4–6 independent cell cultures.

We previously observed that CGRP treatment alone does not promote odontogenic differentiation, so we investigated whether CGRP and Shh enhance dental cell differentiation using odontogenic DM supplemented with CGRP and/or Shh ligand (Michot et al. 2020). Expression of odontogenic differentiation markers increased after a week of incubation in DM for all 3 cell types (Appendix Fig. 5). These increases were enhanced with the addition of Shh ligand or CGRP inhibitor and decreased with inhibition of Shh (Fig. 4). Hh signaling is known to regulate cell differentiation in many contexts, so we also examined Hh target expression to determine if activating this pathway was indicative of odontogenic differentiation in our cells of interest. Indeed, Hh target gene expression was similarly increased by the addition of Shh ligand or CGRP inhibitor and decreased with CGRP ligand or Shh inhibitor. Supplementing with both ligands and inhibitors resulted in no change compared to the untreated groups, further suggesting conflicting roles for CGRP and Shh. To evaluate the long-term effects on dental cell differentiation, we observed calcium nodule formation. Nodules formed in all 3 cell types after incubating in DM for 3 weeks (Appendix Fig. 6). Nodule formation was uniquely enhanced in the murine incisor pulp cells when either Shh or CGRP ligand was added, but this effect was lost with the addition of Shh or CGRP inhibitors (Fig. 5). Molar pulp cells produced large, distinct nodules, whereas mineralization was evenly distributed in the hDPSCs.

Figure 4.

Sonic hedgehog (Shh) and calcitonin gene-related peptide (CGRP) treatment influence changes in dental pulp cell messenger RNA (mRNA) expression with odontogenic differentiation media. (A) Changes in mRNA expression of odontogenesis markers and (B) Hedgehog (Hh) signaling targets 1 wk after incubation in odontogenic differentiation media (DM) and treatment with 10 nM CGRP, 0.5 nM Shh ligand, 50 ng/mL CGRP_i, and/or 100 ng/mL Shh_i. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to no treatment group, Tukey’s test; n = 4–6 independent cell cultures.

Figure 5.

Effects of Sonic hedgehog (Shh) and calcitonin gene-related peptide (CGRP) on odontogenic differentiation of dental cells. (A) Alizarin red staining depicting calcium nodule formation in incisor pulp, molar pulp, and human dental pulp stem cells (hDPSCs). (B) Quantitative analysis of alizarin red staining. Cells were treated for 3 wk with standard culture media, differentiation media (DM), DM + CGRP, DM + CGRP + CGRP_i (CGRP antagonist), DM + Shh, or DM + Shh + Shh_i (Shh antibody). *P < 0.05, **P < 0.01 compared to DM treated group, Tukey’s test; n = 3–5 independent cell cultures. Black scale bars indicate 90 µm.

Discussion

Collectively, our results show for the first time that CGRP and Shh produced from stimulated nociceptive neurons play distinct roles in murine molar pulp cell proliferation, differentiation, and inflammatory gene expression. Our results suggest that pain sensation may act through CGRP and Shh to regulate dentin repair and inflammation. We found that murine molar pulp cells respond to both Hh and CGRP signaling, and Hh signaling was upregulated by Shh expression induced in the molar pulp in a dentin exposure model recapitulating reactionary dentin formation. We also found that Shh enhanced odontogenic differentiation of molar pulp cells, while CGRP appeared to maintain a proliferative state. Shh signaling has been well characterized in the continuously regenerating incisor, but our work adds another critical player, CGRP, in a more relevant molar injury model for human tooth disease or regeneration (Zhao et al. 2014).

Our CFU, CM, and DM studies provide evidence that Shh and CGRP have both synergistic and antagonistic roles as neuron-secreted factors. Dual treatment with Shh + CGRP produced the greatest increase in inflammatory marker expression, and neuron CM-induced inflammatory marker expression was equally disrupted when either Shh or CGRP inhibitor was introduced. Our differentiation studies indicate Shh uniquely drives odontogenic differentiation since differentiation was enhanced when Shh ligand or CGRP antagonist was introduced. In addition, molar pulp cells treated with CGRP produced more CFUs, which is indicative of promoting cell proliferation. Thus, we propose that CGRP and Shh work synergistically to enhance inflammation and antagonistically to balance proliferation and differentiation of dental pulp cells into odontoblast-like cells. The effects of these pathways and other potential factors secreted by activated neurons on pathological processes within the pulp warrant further investigation in vivo.

We conducted in vitro experiments in primary murine pulp cells and an hDPSC cell line in order to compare the response of murine and human cells to CGRP and Shh. In general, the murine cells and hDPSCs exhibited similar responses to neuron CM and odontogenic DM, with some notable differences indicating cell and species specificity in regulation. Moreover, the noted discrepancies emphasize the suitability of modeling human tooth regeneration with the murine molar. Differences in the uniform mineralization pattern exhibited by hDPSCs compared to the distinct patches of mineralization produced by primary murine cells likely derive from cell heterogeneity in the pulp cultures. In other words, the primary cultures contain some cells capable of differentiating to produce patches of mineral, whereas all hDPSCs can differentiate and produce mineral. Therefore, there is a need to further characterize molar pulp stem cells to study their regenerative potential.

We selected the Gli1CreER;Rosa26^tdTomato mouse model to explore Hh-responsive cells in general. While Gli1⁺ cells have been identified as stem cells in the mouse incisor, periodontal tissues, and alveolar bone, Gli1⁺ molar pulp cells have not been characterized perhaps due to a previous report that these cells were absent in the molar pulp of P30 mice (Seidel et al. 2010; Zhao et al. 2014; Seidel et al. 2017; Hosoya, Shalehin, Takebe, Fujii, et al. 2020; Men et al. 2020). However, we and another group have identified Gli1⁺ cells in the mature molars of mice using a fluorescent reporter, and our data suggests potential expansion with injury (Hosoya, Shalehin, Takebe, Shimo, et al. 2020). Whether the Gli1⁺ molar pulp cell population contains stem cells warrants further investigation.

Our results indicate that elements for CGRP and Hh signaling are present in the murine molar, and expression of CGRP and Shh is upregulated in response to injury. Our data further suggest sensory neurons secrete Shh and CGRP, which play antagonistic roles in dental cell differentiation into odontoblast-like cells. We conclude that Shh/CGRP signaling mediates dentin formation in the molar, and the extent of its influence perhaps varies with cell type. Further studies are required to elucidate specific interactions between Shh and CGRP, as well as the specific dental cell populations affected by their signaling.

Author Contributions

E.R. Moore, B. Michot, O. Erdogan, J.L. Gibbs, Y. Yang, contributed to conception or design, data acquisition, analysis, or interpretation, drafted and critically revised the manuscript; A. Ba, contributed to acquisition data analysis, or interpretation, critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.