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

AIM: 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. METHODOLOGY: 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. RESULTS: 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. CONCLUSIONS: 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.