5.3. Osteogenic Differentiation Protocol

Undifferentiated hBMSC were cultured in Mesenchymal Stem Cell Basal Medium (BM) supplemented with 7% FBS, 100 IU/mL penicillin/streptomycin, 2.4 mM, 125 pg/mL FGF-b and 15 ng/mL IGF-1, at a density of 5 × 10³ cells/cm² and incubated for 24 h at 37 °C under 5% CO₂. To induce osteogenic differentiation hBMSC were cultured in OM, (DMEM with 10% FBS, 100 IU/mL penicillin/streptomycin, 2 mM L-glutamine, 10 mM β-glycerophosphate, 100 nM dexamethasone, 100 μM ascorbic acid 2-phosphate), or cultured in BM with CGF supplemented with 7% FBS, 100 IU/mL penicillin/streptomycin (CGF) (Figure 1), or with CGF supplemented with 7% FBS, 100 IU/mL penicillin/streptomycin, 10 mM β-glycerophosphate (BGP), 100 μM ascorbic acid 2-phosphate (AA) (CGF + BGP + AA) for 21 days. The medium was replaced at a rate of 50% every 3 days.