Primary mouse BMSC was collected from the bone marrow of 6-week-old, male C57BL/6 mice as described previously [36 (link)]. Briefly, mice were executed and the bone marrow were isolated from the femoral and tibial. The obtained cell suspension was then cultured in the α–MEM with 1% penicillin/streptomycin with 10%FBS. Upon cells grew into about 90% confluence, the cell supernatant was removed and cell were passaged. The cells at the second passage were selected for osteoblast differentiation.
As for the osteoblast differentiation, BMSC was cultured in the osteogenic medium containing 10 mM β-glycerophosphate, 0.1 μM dexamethasone and 0.05 mM ascorbic acid. After cells were cultured for 7 and 14 days, ALP staining (CWBIO, Beijing, China) and ALP activity (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was carried out to detect the osteogenic effect according to the previous study [37 (link)]. And Alizarin red staining (Sigma–Aldrich, St Louis, MO) was carried out when cells were cultured for 14 days.
As for the osteoblast differentiation, BMSC was cultured in the osteogenic medium containing 10 mM β-glycerophosphate, 0.1 μM dexamethasone and 0.05 mM ascorbic acid. After cells were cultured for 7 and 14 days, ALP staining (CWBIO, Beijing, China) and ALP activity (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was carried out to detect the osteogenic effect according to the previous study [37 (link)]. And Alizarin red staining (Sigma–Aldrich, St Louis, MO) was carried out when cells were cultured for 14 days.