Osteogenic induction, alizarin red, alkaline phosphatase staining, and quantification

Osteogenic differentiation of BMSCs was induced using osteogenic induction medium consisting of a 10-mM β-glycerol phosphate (Sigma, USA), 0.1μM of dexamethasone (Sigma, USA), and 50 mg/mL of ascorbate-2-phosphate (Sigma, USA); the medium was refreshed every 3 days. For alkaline phosphatase (ALP) staining, the BMSCs were fixed in 4% paraformaldehyde, washed with phosphate buffered solution (PBS), and stained according with the Alkaline Phosphatase Staining Kit in accordance with the manufacturer’s instructions (Yeasen, China). Stained cells were observed and photographed by a phase-contrast microscope. ALP activity was quantified by a commercial ALP kit (Jiancheng, Nanjing, China). We also collected supernatant and cell lysates. Cells were incubated with p-nitrophenyl phosphate solution, and then, alkaline phosphatase activity was calculated by detecting absorbance at 520 nm. Quantification was conducted by dividing the absorbance by the protein concentration, as determined by the BCA protein assay (Beyotime, China). After 21 days of culture in osteogenic medium, we used Alizarin Red S solution (Cyagen Biosciences, China) to identify areas of calcium deposition. In brief, BMSCs were fixed by 4% paraformaldehyde, stained in Alizarin Red S solution at room temperature for 10 min, and then washed in phosphate-buffered solution (PBS). Stained cells were then observed and photographed by a phase-contrast microscope. The semi-quantification of Alizarin Red S concentrations was conducted by a quantitative de-staining procedure using 10% cetylpyridinium chloride (CPC) (Sigma, USA) for 15 min at room temperature, and absorbance was measured in a microplate spectrophotometer (Bio-Tek, UK) at 562 nm.