Epithelium-derived SCUBE3 promotes polarized- odontoblast differentiation of dental mesenchymal stem cells and vascularized pulp regeneration

Abstract: Background Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3), a secreted multifunctional glycoprotein, whose transcript expression is restricted to the tooth germ epithelium (Epi) during the development of embryonic mouse teeth, has been demonstrated to play a crucial role in the regulation of tooth development. Based on this, we hypothesized that epithelium-derived SCUBE3 contributed to bio-function in dental mesenchymal cells (Mes) via epithelium-mesenchyme interactions. Methods Immunohistochemical staining and a co-culture system were performed to reveal the temporospatial expression of SCUBE3 protein during mouse tooth germ development. In addition, human dental pulp stem cells (hDPSC) were used as a Mes model to study the proliferation, migration, polarized-odontoblast capacity and mechanism of rhSCUBE3. Novel pulp-dentin-like organoid models were constructed to further confirm the polarized odontoblast-induction function of SCUBE3. Finally, semi-orthotopic animal experiments were performed to explore the clinical application of rhSCUBE3. Results The epithelium-derived SCUBE3 translocated to the mesenchyme via paracrine during mouse embryonic development, and the differentiating odontoblasts in postnatal tooth germ could subsequently secrete SCUBE3 protein via autocrine. In hDPSCs, exogenous SCUBE3 promoted cell proliferation and migration via TGF-β signalling and accelerated odontoblastic differentiation via BMP2 signalling. In the semi-orthotopic animal experiments, we found that SCUBE3-pretreatment induced polarized odontoblast-like cells attached to the dental walls and better angiogenesis performance. Conclusion SCUBE3 protein expression is transferred from the epithelium to mesenchyme during embryonic development and elaborated the function of epithelium-derived SCUBE3 in Mes, including proliferation, migration, and polarized-odontoblastic differentiation, and their mechanisms. These findings shed light on exogenous SCUBE3 application in clinic dental-pulp regeneration.