Tgfbr2 in Dental Pulp Cells Guides Neurite Outgrowth in Developing Teeth

Abstract

Transforming growth factor β (TGFβ) plays an important role in tooth morphogenesis and mineralization. During postnatal development, the dental pulp (DP) mesenchyme secretes neurotrophic factors that guide trigeminal nerve fibers into and throughout the DP. This process is tightly linked with dentin formation and mineralization. Our laboratory established a mouse model in which Tgfbr2 was conditionally deleted in DP mesenchyme using an Osterix promoter-driven Cre recombinase (Tgfbr2cko). These mice survived postnatally with significant defects in bones and teeth, including reduced mineralization and short roots. Hematoxylin and eosin staining revealed reduced axon-like structures in the mutant mice. Reporter imaging demonstrated that Osterix-Cre activity within the tooth was active in the DP and derivatives, but not in neuronal afferents. Immunofluorescence staining for β3 tubulin (neuronal marker) was performed on serial cryosections from control and mutant molars on postnatal days 7 and 24 (P7, P24). Confocal imaging and pixel quantification demonstrated reduced innervation in Tgfbr2cko first molars at both stages compared to controls, indicating that signals necessary to promote neurite outgrowth were disrupted by Tgfbr2 deletion. We performed mRNA-Sequence (RNA-Seq) and gene onotology analyses using RNA from the DP of P7 control and mutant mice to investigate the pathways involved in Tgfbr2-mediated tooth development. These analyses identified downregulation of several mineralization-related and neuronal genes in the Tgfbr2cko DP compared to controls. Select gene expression patterns were confirmed by quantitative real-time PCR and immunofluorescence imaging. Lastly, trigeminal neurons were co-cultured atop Transwell filters overlying primary Tgfbr2f/f DP cells. Tgfbr2 in the DP was deleted via Adenovirus-expressed Cre recombinase. Confocal imaging of axons through the filter pores showed increased axonal sprouting from neurons cultured with Tgfbr2-positive DP cells compared to neurons cultured alone. Axon sprouting was reduced when Tgfbr2 was knocked down in the DP cells. Immunofluorescence of dentin sialophosphoprotein in co-cultured DP cells confirmed reduced mineralization potential in cells with Tgfbr2 deletion. Both our proteomics and RNA-Seq analyses indicate that axonal guidance cues, particularly semaphorin signaling, were disrupted by Tgfbr2 deletion. Thus, Tgfbr2 in the DP mesenchyme appears to regulate differentiation and the cells’ ability to guide neurite outgrowth during tooth mineralization and innervation.

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

“…We previously reported that Tgfbr2 in dental pulp fibroblasts and odontoblasts regulates the postnatal neuro-pulpal development of mouse molars [3,7,9]. A shallow dentin injury without pulp exposure has been shown to initiate the expression of neurotrophic factors, such as nerve growth factors (NGF), by odontoblasts [5][6][7].…”

“…In this report, we sought to determine whether the signaling downstream of Tgfbr2 in the developing dental pulp mesenchyme also regulates reactionary dentinogenesis and odontoblast secretion of neurotrophic signals that promote axon sprouting. Since conditional deletion of Tgfbr2 in the Osteocalcin-Cre model results in pulpal obliteration [5], and deletion in the Osterix-Cre model results in late postnatal death [3,[7][8][9], we utilized the tetracycline-responsive element in the Osterix-Cre mouse model to isolate conditional deletion of Tgfbr2 to the time period immediately before the dentin injury through the studies on the injury response. Our ISH of Sp7 indicated that there was Tgfb2 deletion in the dental pulp, confirming the validity of our model.…”

“…Dentinogenesis occurs throughout the lifetime of an animal, with primary dentin laid down during tooth development, secondary dentin slowly secreted throughout adulthood, and tertiary dentin secreted in response to injury or infection [1,2]. The secretion and mineralization of dentin involves the TGF superfamily, predominantly mediated through TGFβ receptors I and II, which are expressed in both odontoblasts and dental pulp cells [3][4][5][6][7]. We previously reported that Tgfbr2 promotes the proliferation and differentiation of mineralizing cells required for bones and teeth to grow and mineralize [3,[7][8][9].…”

“…The secretion and mineralization of dentin involves the TGF superfamily, predominantly mediated through TGFβ receptors I and II, which are expressed in both odontoblasts and dental pulp cells [3][4][5][6][7]. We previously reported that Tgfbr2 promotes the proliferation and differentiation of mineralizing cells required for bones and teeth to grow and mineralize [3,[7][8][9]. If Tgfbr2 is deleted in mature odontoblasts, the cells lose their polarity, resulting in ectopic matrix formation, osteodentin 2 formation, and even pulpal obliteration [5,10].…”

“…Several studies reported that CGRP moderates the neuroinflammatory response to dentin injury [11,14,16], yet the roles of CGRP in pulp repair, particularly in the absence of pulp exposure, are not yet fully understood. Since Tgfbr2 signaling in the dental pulp cells promotes axon sprouting during tooth development [3,17], we hypothesized that this could be recapitulated during dentin repair. In the present study, we evaluated whether Tgfbr2 deletion in the odontoblasts and dental pulp cells during a shallow dentin injury would impact CGRP+ axon sprouting and tertiary dentin secretion.…”