In order to facilitate the observation of staining, 104 BMSCs were seeded in 24-well plates and cultured with an osteogenic medium (OM). After a week of culture, the cells were fixed by 4% paraformaldehyde. Sirius Red (Sigma, 365548, USA) staining was used to determine the collagen deposition while ALP was stained with BCIP/NBT alkaline phosphatase Color Development Kit (Beyotime, C3206, China). In addition, 5 × 103 BMSCs were seeded in 24-well plates and stained with Alizarin Red (Cyagen, China) after 2 weeks of culture. Staining positive area fraction calculated as staining positive area/total area was obtained using ImageJ software (n = 6).

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