2.10. Osteogenic differentiation and mineralization assay

To induce osteogenic differentiation of BMSCs, primary BMSCs with or without previous treatments were cultured in 6‐well plates at 2.5 × 10⁶ cells per well with the mesenchymal stem cell osteogenic‐induced medium (10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.1 mmol/L dexamethasone, 10 mmol/L β‐glycerol phosphate and 50 mmol/L ascorbate‐2‐phosphate). The culture medium was changed every 3 days.

After 2 days of osteogenic differentiation, the cell lysates were washed using PBS followed by homogenized for ALP activity assay by spectrophotometric measurement of p‐nitrophenol release using an Alkaline Phosphatase Assay Kit according to the manufacturer's instructions (P0321S, Beyotime).

After 7 days of osteogenic differentiation, alkaline phosphatase staining (ALP staining) was carried out to assess the capacity of mineralization as described before. 8 Briefly, the washed cells were fixed in 10% paraformaldehyde for 5 minutes. Then, cells were incubated in ALP incubation buffer (0.2 g barbital sodium, 0.4 g magnesium sulphate, 0.2 g calcium chloride and 0.3 g beta‐glycerophosphate, 10 mmol/L β‐glycerol phosphate and 50 mmol/L ascorbate‐2‐phosphate) at 37°C for 2 hours. Next, 2% calcium chloride was used to wash the cells and 2% cobaltous nitrate was used to incubate cells for 5 minutes. Then, cells were incubated in 1:80 ammonium sulphate for 10 seconds.

After 21 days of osteogenic differentiation, Alizarin Red staining was performed according to the manufacturer's instructions (MUBMX‐90021; Cyagen Biosciences). Briefly, cells were washed using PBS three times followed by 4% paraformaldehyde for 30 minutes. After washed by PBS for three times, cells were stained in Alizarin red solution at 37°C for 5 minutes. The images were captured by the microscope.