Osteogenic Differentiation of HBMSCs

HBMSCs were passaged in 24-well plates and cultured in an osteogenic induction medium for 14 d. Cells were fixed in 4% paraformaldehyde (Beyotime, Shanghai, China) for 20–30 min at room temperature and then rinsed three times with PBS. Alizarin Red S solution (Beyotime) was then added and incubated at room temperature for 15 min followed by rinsing with PBS three times. ARS quantification was performed as previously described [45 (link)]. The stain was incubated with 10% cetylpyridinium chloride for 1 h at room temperature, and the solutions were collected and plated in a 96-well plate and read at 560 nm with a microplate reader (ELX808; BioTek). The results were normalised to those of the control group.