Induction and identification of osteogenic differentiation

When the cells reached 80% confluence, they were incubated (37˚C, 5% CO₂) with osteogenic induction medium containing 80 ng/ml IGF-1 (PeproTech, Inc.), Dulbecco's modified Eagle's medium [(DMEM)/F12 supplemented with 10% FBS, 0.25 mmol/l ascorbic acid, 10 mmol/l sodium β-glycerophosphate, 10^-7 mol/l dexamethasone], and the medium was changed once every 2 days on average. Osteogenesis was monitored continuously, and 3 weeks after induction, the cells were fixed with 4% paraformaldehyde for 15 min, stained with 0.1% Alizarin Red (Cyagen Biosciences, Inc.), and observed under a fluorescence microscope. Images were captured at 400 X magnification.