MSC osteogenesis and Alizarin Red S staining

The MSCs (2 × 10⁵ /well) were cultured with 0.1% gelatine in six‐well plates. When the ratio of fusion reached between 60% and 70%, the medium was discarded and osteogenic differentiation culture medium (2 mL) was added. The cells were fixed in 4% paraformaldehyde 21 days after the induction of differentiation for 15 min [21, 23] and then stained with 1% Alizarin Red (AR; Sigma‐Aldrich Corp., St. Louis, MO, USA) in 10% cetylpyridinium chloride at pH 4.2 for 5 min. Thereafter, optical density was assessed at 562 nm using a spectrometer (Thermo Fisher Scientific Inc.) [24].