Cells were plated in 12-well plates at a density of 2 × 104 cells/well and incubated for 7 or 14 days. For ALP staining, after being fixed for 15 min at 4°C with 10% formalin, BMSCs were treated with a BCIP/NBT solution (Beyotime, Shanghai, China) in the dark, and areas stained purple were regarded as positive. Photomicrographs of each group were captured with a light microscope (Olympus). ALP activities were determined using p-nitrophenyl phosphate (sigma) as described previously [25 (link)].
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