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.