Bmscs

BMSCs at passage 2 (ZQ0682) were purchased from Zhongqiaoxinzhou Biotech (Shanghai, China). Cells were cultured with low-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (Gibco, USA) and 1% (v/v) penicillin–streptomycin solution (Solarbio, China). Cells at passage 3 were used in this study: 2 × 10⁴ BMSCs were seeded in the lower chamber of a 24-well Transwell plate (pore size: 0.4 μm; Corning, USA). After 4 h of inoculation, most of the cells adhered to the plate and gels were placed in the upper chamber. Cells cultured without gels were labeled Blank. The medium was changed every 2 days. A Cell Counting Kit-8 (CCK-8; Dojindo, Japan) test was performed according to instructions after culturing for 1, 3 and 5 days.

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.