Osteogenic differentiation

The cells were grown to confluence before CCM was switched for osteoblast differentiation media containing: high glucose DMEM (Gibco, Grand Island, NY), 10% FBS, 1% penicillin/streptomycin, 100 mM dexamethasone, 10 mM sodium-β-glycerophosphate, and 0.05 mM ascorbic acid–2–phosphate. The media were changed every three days until day 20. At this point, the plates were washed with PBS and fixed with 10% neutral buffered formalin for 1 h at room temperature. Von Kossa staining was used to identify the matrix mineralization after osteogenic differentiation. The cells were washed before treated with 5% silver nitrate under ultraviolet light for 90 min. After washing three times with deionized water, the plates were treated with 5% sodium thiosulfate for 5 min, and counterstained with Nuclear Fast Red. The cells were then imaged under an Olympus IX70 microscope.