Bone Marrow Stromal Cells Produce Two Molecular Weight Forms of Semaphorin <scp>3C</scp> in Response to Titanium Surface Topography That Differentially Regulate <scp>BMSCs</scp> , Osteoblasts and Osteoclasts

RNAseq analysis showed that human bone marrow stromal cells (BMSCs) express neurogenic factors, including semaphorins 3A and 3C (sema3A; sema3C) when grown on titanium substrates. Sema3A causes BMSCs to differentiate into osteoblasts in vitro and promotes osseointegration in vivo, suggesting that sema3C may also contribute to peri-implant bone formation. Human BMSCs were cultured on smooth/hydrophobic (PT), microrough/hydrophobic (SLA), and microrough/hydrophilic (modSLA) Ti substrates. Production of sema3C was sensitive to surface characteristics and was mediated by integrins α2, and β1, BMP2, and Wnt5A, but not Wnt11 or sema3A. Sema3C did not affect differentiation of hBMSCs, but inhibited osteoclast differentiation and activity. Conditioned media from hBMSCs grown on modSLA reduced osteoclast differentiation of RAW264.7 cells. Blocking sema3C with anti-sema3C antibodies partially mitigated its inhibitory effect on resorption. However, sema3C did not affect macrophage polarization. Treatment of human umbilical vascular endothelial cells (HUVECs) with modSLA conditioned media increased angiogenesis at 24 h. BMSCs produced sema3C as two variants; the 95 kDa protein was stored in the ECM, whereas the 65 kDa protein was released to the media. Treatment with 65 kDa sema3C inhibited substrate-dependent osteoblastic differentiation of hBMSCs; 95 kDa sema3C had no effect. In conclusion, this study showed that BMSCs produce sema3C in a surface-dependent manner. The 65 kDa sema3C inhibits osteoblast differentiation and the 95 kDa sema3C directly inhibits osteoclastogenesis, resorption, and angiogenesis. Our findings indicate that sema3A and 3C modulate peri-implant bone formation through differential actions on multiple cell types present in the peri-implant environment to promote new bone formation.