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

Abstract

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 Ca2+ 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 Ca2+ 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 Ca2+ and BMP-2 on SMAD1/5, ERK and S6K signalling after 24 hours. More importantly, a cooperative effect between Ca2+ 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, Ca2+ alone favoured the phosphorylation of SMAD1, which correlates with the induction of Bmp2 and Bmp4 gene expression. These data suggest that Ca2+ 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 Ca2+ in bone tissue engineering.

Fig 1. Extracellular calcium induces expression of osteogenic markers on bone marrow mesenchymal stem cells.

Three different culture models were compared (cells in monolayer in plastic surface, cells in monolayer in gelatin-coated dishes and cells in 3D gelatin scaffolds). After 10 days, the mRNA expression of Alpl, Osteocalcin (Bglap2) and Osterix was analysed and normalised to Gapdh levels (n = 3). Differences were considered significant at p values: * p < 0.05,** p < 0.01, and ***p < 0.001.

Fig 2. Extracellular calcium increases the effects of BMP-2 on osteogenic marker expression.

Primary BM-MSCs were cultured on 3D gelatin scaffolds with 7.5 mM of CaSO₄ and/or 2 nM of BMP-2 for 10 days. The mRNA expression of Alpl, Osteocalcin (Bglap2), Runx2 and Osterix was analysed and normalised to Gapdh levels (n = 3). Differences were considered significant at p values: * p < 0.05,** p < 0.01, and ***p < 0.001.

Fig 3. Extracellular calcium increases the effects of BMP-2 on bone regeneration in vivo.

Calvarial critical-size bone defects were generated in mice and implanted with cells cultured in 3D scaffolds pre-treated with 7.5 mM of CaSO₄ and/or 2 nM of BMP-2 for 48 hours. After five weeks, the implanted constructs were retrieved and processed for Masson’s trichrome staining. Scale bars are shown for the different magnifications. Lower panels show the relative quantification of the areas of new bone formation from the 4x magnification stained by thrichrome in red or blue (n = 3).

Fig 4. Early effects of extracellular calcium on cell signalling.

Cells were cultured in 3D gelatin scaffolds with Ca²⁺ (7.5 mM) and/or BMP-2 (2nM) for 24 hours and extracts analysed by Western blot. Data was quantified relative to the levels of α-TUBULIN (n = 3). Differences were considered significant at p values: * p < 0.05,** p < 0.01, and ***p < 0.001.

Fig 5. Late effects of extracellular calcium on cell signalling.

Cells were cultured in 3D gelatin scaffolds with Ca²⁺ (7.5 mM) and/or BMP-2 (2nM) for 10 days. Data was quantified relative to the levels of α-TUBULIN (n = 3). Differences were considered significant at p values: * p < 0.05, ** p < 0.01, and ***p < 0.001.

Fig 6. Extracellular calcium induces Bmp2, Bmp4, Fgf21 and Axin2 mRNA expression.

BM-MSCs were cultured on 3D gelatin scaffolds with different extracellular calcium concentrations for 10 days. The mRNA expression of Bmp2, Bmp4, Fgf21 and Axin2 was analysed and normalised to the levels of Gapdh (n = 3). Differences were considered significant at p values: * p < 0.05,** p < 0.01, and ***p < 0.001.