Bmsc culture medium

The mouse BMSCs were separated from the bone marrows of bilateral femur in 8-week-old C57BL/6 J mice in accordance with the previous study [51 ]. BMSCs were maintained in BMSC culture medium (Cyagen Biosciences, China) and placed in a standard culture environment. The fresh culture medium was replaced every other day. When the final density of BMSCs was closed to 90%, the cells were digested under the action of 0.25% trypsin (Cyagen Biosciences, China) and then sub-cultured for further analysis.

Primary C3H10T1/2 BMSCs were purchased from Cyagen Biosciences Inc. Then, 1 × 10⁵ cells/cm² BMSCs were seeded into 6-well plates and cultured with BMSC culture medium (Cyagen Biosciences, USA) supplemented with 100 U/mL penicillin-streptomycin, 10% fetal bovine serum (FBS), 10 nmol/L dexamethasone, 1% glutamine, 10 mmol/L β-glycerophosphate, 0.2 mmol/L l-ascorbic acid, and bone morphogenetic protein- (BMP-) 2 (Changzhou Kangfulai Medical, China). Next, the BMSCs were collected to eliminate the thrombus and seed into 25 cm² flasks (Corning, USA) followed by incubating at 37°C in a humidified atmosphere with 95% air and 5% CO₂ (Thermo, USA). After the cells were cultured to 80%~90% confluence, the culture medium was discarded, and 2 mL of BMSC osteoblastic differentiation medium containing 1% glutamine, 10% FBS, 1% penicillin-streptomycin, 1% b-glycerophosphate, 0.2% ascorbic acid, and 0.01% dexamethasone was added. The medium was replaced every 3 days. Finally, cells were detached with 0.25% trypsin (Cyagen Biosciences, USA) and collected for ARS staining, ALP staining, qRT-PCR, or western blot after being cultured with osteoblastic differentiation culture medium for 14 days. BMSCs between the third and fifth passages were used in this study. All cell experiments were repeated three times.