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Understanding the molecular events that regulate osteoblast differentiation is essential for the development of effective approaches to bone regeneration. In this study, we analysed the osteoinductive properties of extracellular calcium in bone marrow-derived mesenchymal stem cell (BM-MSC) differentiation. We cultured BM-MSCs in 3D gelatin scaffolds with Ca 2+  and BMP-2 as osteoinductive agents. Early and late osteogenic gene expression and bone regeneration in a calvarial critical-size defect model demonstrate that extracellular Ca 2+  enhances the effects of BMP-2 on  Osteocalcin ,  Runx2  and  Osterix  expression and promotes bone regeneration  in vivo . Moreover, we analysed the molecular mechanisms involved and observed an antagonistic effect between Ca 2+  and BMP-2 on SMAD1/5, ERK and S6K signalling after 24 hours. More importantly, a cooperative effect between Ca 2+  and BMP-2 on the phosphorylation of SMAD1/5, S6, GSK3 and total levels of β-CATENIN was observed at a later differentiation time (10 days). Furthermore, Ca 2+  alone favoured the phosphorylation of SMAD1, which correlates with the induction of  Bmp2  and  Bmp4  gene expression. These data suggest that Ca 2+  and BMP-2 cooperate and promote an autocrine/paracrine osteogenic feed-forward loop. On the whole, these results demonstrate the usefulness of calcium-based bone grafts or the addition of exogenous Ca 2+  in bone tissue engineering.

Extracellular calcium promotes bone formation from bone marrow mesenchymal stem cells by amplifying the effects of BMP-2 on SMAD signalling