Tgfbr2 Deletion in Dental Pulp Reduces Tertiary Dentin After Injury

Objectives: Innervation and neuronal signaling are vital for many tooth functions, including development and repair. We previously established that transforming growth factor beta (TGFβ) signaling from the dental mesenchyme regulated mineralization and innervation of developing mouse molars. In this study, we investigated mesenchymal TGFβ signaling in pulpal healing of a shallow dentin injury. Previous research demonstrated calcitonin gene-related peptide (CGRP) positive axon outgrowth toward a shallow dentin injury, suggesting these axons are integral to healing. A recent follow-up study demonstrated that CGRP+ axon irritation resulted in pulp chamber hypercalcifcation. We hypothesized that mesenchymal conditional deletion of TGFβ receptor 2 (Tgfbr2) using an Osterix-Cre recombinase (Tgfbr2^cko) that reduced neurite outgrowth during development would similarly reduce neurite outgrowth in response to injury and reduce tertiary dentin formation by odontoblasts.
Methods: The Tgfbr2^cko mice were fed a doxycycline-enhanced diet from in utero until two weeks prior to surgery to inhibit the tetracycline-responsive Cre element and allow normal skeletal and dental development. A shallow dentin injury was made on the mesial side of the first mandibular molar of 3-month-old control and Tgfbr2^cko mice, and hemi-mandibles were collected after 4, 8, 21, and 56 days post injury (dpi) (n=6-8).
Results: In situ hybridization for Sp7 (Osterix) transcripts confirmed Osterix-Cre expression and inferred Tgfbr2 deletion at 4dpi. Immunofluorescence and confocal imaging of CGRP+ axons did not demonstrate differences in axon outgrowth toward the injury in Tgfbr2^cko molars compared to controls. However, microcomputed tomography and histology indicated visibly lower tertiary dentin in Tgfbr2^cko molars.
Conclusions: These results suggest that pulpal signaling downstream of Tgfbr2 does not regulate neurite outgrowth in response to injury, and peptidergic signaling from CGRP+ axons may not compensate for reduced mineralization capacity in Tgfbr2-deficient odontoblasts. Further investigations into the multicellular signals involved in pulp healing are planned to develop the foundation for precision-based, vital pulp therapies.