Impaired Tertiary Dentin Secretion after Shallow Injury in Tgfbr2-Deficient Dental Pulp Cells Is Rescued by Extended CGRP Signaling

Abstract

The transforming growth factor β (TGFβ) superfamily is a master regulator of development, adult homeostasis, and wound repair. Dysregulated TGFβ signaling can lead to cancer, fibrosis, and musculoskeletal malformations. We previously demonstrated that TGFβ receptor 2 (Tgfbr2) signaling regulates odontoblast differentiation, dentin mineralization, root elongation, and sensory innervation during tooth development. Sensory innervation also modulates the homeostasis and repair response in adult teeth. We hypothesized that Tgfbr2 regulates the neuro-pulpal responses to dentin injury. To test this, we performed a shallow dentin injury with a timed deletion of Tgfbr2 in the dental pulp mesenchyme of mice and analyzed the levels of tertiary dentin and calcitonin gene-related peptide (CGRP) axon sprouting. Microcomputed tomography imaging and histology indicated lower dentin volume in Tgfbr2^cko M1s compared to WT M1s 21 days post-injury, but the volume was comparable by day 56. Immunofluorescent imaging of peptidergic afferents demonstrated that the duration of axon sprouting was longer in injured Tgfbr2^cko compared to WT M1s. Thus, CGRP+ sensory afferents may provide Tgfbr2-deficient odontoblasts with compensatory signals for healing. Harnessing these neuro-pulpal signals has the potential to guide the development of treatments for enhanced dental healing and to help patients with TGFβ-related diseases.

Keywords: calcitonin gene-related peptide; dental pulp cells; fibrosis; neuropeptide; pulp biology; tertiary dentin; transforming growth factor beta.

Figure 1

Experimental timeline for dentin injury and model validation. (A) Timeline of dentin injury activities, beginning with doxycycline removal for Tgfbr2^cko mice 14 days prior to dentin injury and the collection of tissue on specific days post-injury (dpi). (B–D) Creation of the dentin injury. Pre-injury (B), injury in process with a #1/16 carbide round bur (C), and post-injury (D), with an inset showing a higher magnification image of the injury (red arrows). (E) Percent weight change post-dentin injury in male and female WT and Tgfbr2^cko mice at 4 dpi and 56 dpi. There was no significant difference in weight pre- to post-dentin injury in either genotype, sex, or dpi. (F,G) Sp7 (Osterix) in situ hybridization of 4 dpi Tgfbr2^cko control (F) and injured (G) mice to confirm Osterix-Cre expression (N = 4). Osterix was being actively transcribed in both control and injured Tgfbr2^cko sections (black arrows). Insets show lower magnification of sections, with dotted lines outlining the main images of (F) and (G). Dentin injury is marked with the yellow * in the inset of (G). Od = odontoblasts, DP = dental pulp, D = dentin. Scale bars = 10 μm (F) and 100 μm (F inset).

Figure 2

Histological analyses of 4 and 8 dpi M1s. No tertiary dentin was present at 4 or 8 dpi in WT (A–C,G–I) or Tgfbr2^cko (D–F,J–L) M1s as depicted by H&E (A–F) and Masson’s trichrome staining (G–L). The yellow asterisks indicate the area drilled due to dentin injury. Scalebars in (A,G) = 50 µm. N = 6/genotype/time point.

Figure 3

Histology of tertiary dentin formation. (A–F) H&E and (G–L) Masson’s trichrome stained coronal sections (7 µm thick) of control, 21 dpi, and 56 dpi M1s. Tertiary dentin formation was not demonstrated at 21 dpi in the Tgfbr2^cko mice (E,K) compared to WT mice (B,H). Comparable tertiary dentin was formed by 56 dpi in both the Tgfbr2^cko (F,L) and WT (C,I) mice. The yellow asterisks indicate the area drilled due to dentin injury. White dotted lines demarcate the tertiary dentin border. Scale bar (shown in (A)) = 10 µm. N = 6-8/genotype/time point.

Figure 4

Micro-CT analysis of tertiary dentin formation. (A–G) Micro-CT analysis (6 µm resolution) of control, 21 dpi, and 56 dpi M1s from all groups. Yellow dotted lines indicate inset images in (A–F) that represent the ROIs used for quantifications. There was a significantly lower dentin volume in Tgfbr2^cko mice at 21 dpi compared to WT mice (B,E,G), but at 56 dpi, the tertiary dentin volume was comparable between genotypes (C,F,G). Scale bars = 1 mm. In (G), * indicates p < 0.05 and *** indicates p < 0.001 by mixed-effects 2-way ANOVA.

Figure 5

CGRP+ axon sprouting in response to dentin injury. (A–J) Representative maximum projections of confocal images (20 μm thick) showing the CGRP+ (red) axon outgrowth in control mice and WT and Tgfbr2^cko M1s collected at 4–56 dpi (N = 4–8/group). DAPI is labelled in blue. Dentin is visible with Differential Interference Contrast (DIC). The injury is visible as a half-moon on the right of each frame (B–E,G–J) via DIC imaging. In some images, gingiva is apparent in the injured area. Tgfbr2^cko CGRP+ axon sprouting increased significantly between 4 and 21 dpi compared to that in the WT mice (B,D,G,I). At 56 dpi, there were no significant differences in sprouting between the genotypes (E,J). Scalebar in (A) = 50 μm. (K) Prediction of CGRP pixels in response to injury for each genotype fit from a generalized estimating equation model.