Sensory Nerves Regulate Odontoblast Differentiation via the SPP1/ITGA4 Axis During Tooth Root Development

The stem/progenitor cell is crucial for organogenesis. Sensory nerves, as key components of the stem cell niche, secrete various factors to modulate stem/progenitor cell fate decision. Here, we utilised tooth root development as a model to explore the role of sensory nerves in this regulatory process and to elucidate the underlying mechanism.

Spatiotemporal dynamics of nerve innervation were characterised during tooth root development. We treated mouse dental papilla cells (mDPCs) with trigeminal ganglion-conditioned medium (TG-CM) and employed a subrenal co-culture of TG-tooth germ to evaluate sensory nerve function in odontoblastic differentiation and tooth root formation. A subrenal co-culture of TG-tooth germ was employed to detect sensory nerve function in tooth root formation. Integrated analysis of scRNA-seq from the TG and molar at post-natal day 3.5 (PN3.5) and PN30 identified potential nerve-derived factors, which were further assessed through subrenal transplantation with recombinant protein-loaded or neutralising antibody-loaded beads. CellChat was used to analyse cell-cell communication between TGs and molars. Co-immunoprecipitation (Co-IP) and proximity ligation assays (PLA) were used to confirm the interaction between secreted phosphoprotein 1 (SPP1) and integrin alpha 4 (ITGA4). The siRNA-mediated Itga4 knockdown assessed its role in odontoblastic differentiation.

Sensory nerve fibres localized to the apical papilla and follicle at PN3.5 and extended toward the crown. TG-CM and subrenal co-culture of TG-tooth germ enhanced odontoblast differentiation and root elongation, demonstrating the indispensable role of sensory nerves for proper root development. Integrated scRNA-seq analysis of TG and molar at PN3.5 and PN30 uncovered various sensory nerve-derived factors, including SPP1, calcitonin gene-related polypeptide (CGRP) and kit ligand (KITL), whose function in tooth root development was validated in vivo. Furthermore, CellChat analysis revealed SPP1-ITGA4 as a critical ligand-receptor interaction, which was confirmed by Co-IP and PLA. Itga4 was specifically expressed in the apical papilla and upregulated during odontoblastic differentiation. Itga4 knockdown impaired odontoblastic differentiation and abolished SPP1-promoted odontogenesis.

Collectively, our findings elucidate a novel mechanism whereby sensory nerves orchestrate tooth root development by regulating progenitor cell fate through the SPP1/ITGA4 axis. This neuro-mesenchymal crosstalk provides insights for stem cell therapies and tooth root regeneration.