Fig. 5

SCUBE3 pre-treatment enhances cell survival, odontoblastic differentiation, cell polarization, and pulp-like tissue formation. A Haematoxylin and eosin images of the control, SCUBE3, and SCUBE3 + LDN-193189 groups (A1, 3, 5, respectively), and their higher magnifications (A2, 4, 6). B Masson’s trichrome staining images of control, SCUBE3, and SCUBE3 + LDN-193189 groups (B1, 3, 5, respectively), and their higher magnifications (B2, 4, 6, respectively). Black arrowheads show the cells attached to dentin wall. Green arrowheads show new-born blood vessels. C–E Quantification of new-born pulp-like tissue, attached cell layer, and new-born blood vessels, respectively. F Confocal immunofluorescence of α-tubulin and DSPP. Odontoblastic processes with a length of about 20 μm that extended in the dentin tubules were observed in the SCUBE3 pre-treatment group. G Human mitochondria immunohistochemistry. Black arrowheads show the human mitochondria-positive cells. H Quantification of polarized cells observed in (F). I Quantification of human mitochondria-positive stained cells. J Overall schematic diagram of SCUBE3 in the regulation of odontoblastic differentiation. SCUBE3 is produced and secreted into the ECM by Epi via a paracrine pathway. As a co-receptor of BMP2, secreted SCUBE3 can bind to BMP2 to form a complex. The SCUBE3–BMP2 complex then recruits BMPR1A into raft microdomains, augments the specific interactions between BMP2 and BMPR1A, and activates BMP2/Smad-mediated odontoblastic differentiation in Mes. Additionally, as a ligand of TGFβR1, SCUBE3 can promote cell proliferation and migration via TGF signalling. SCUBE3 also improves endogenous SCUBE3 expression and triggers the autocrine/paracrine secretion of SCUBE3 and BMP2 during differentiation, which can positively promote odontoblastic differentiation of the developing tooth germ in response to epithelium–mesenchyme interactions. n = 3 independent biological samples. *P < 0.05, **P < 0.01, ***P < 0.001. rPulp, regenerative pulp. Epi, tooth germ epithelial cells. Mes, tooth germ mesenchymal cells