Alizarin red s reagent

ARS was performed to evaluate cell mineralization. Briefly, Alizarin Red S reagent (#ST1078-25 g; Beyotime Biotech) was used to stain the cells for 5 min at 37°C, according to the manufacturer’s instructions. The images were obtained using a microscope (Olympus, Tokyo, Japan), and quantification was performed with ImageJ software.

The expression of alkaline phosphatase (ALP) in human BMSCs was detected using the BCIP/NBT Alkaline Phosphatase Color Development Kit (#C3206; Beyotime Biotech) according to the manufacturer’s instructions. Briefly, cell slides were washed three times with PBS solution, fixed in 4% paraformaldehyde, and washed again three times with PBS solution. The slides were then incubated with BCIP/NBT staining solution for 1–2 h at room temperature in darkness, washed twice with distilled water for 3 min. Finally, ALP expression in human BMSCs was observed by gross scanning (Bio-Rad, GS-800, Hercules, CA, USA) and light microscopy (OPTEC, TP510, Chongqing, China). At least three biological replicates were conducted for comparison. To evaluate osteogenic differentiation, BMSCs were also stained with Alizarin Red S reagent (#ST1078-25g; Beyotime Biotech) for 1–5 min at room temperature, as outlined in the manufacturer’s instructions. Quantification of ALP and Alizarin Red S were conducted using ImageJ software (National Institutes of Health, Bethesda, MD, USA).