BMSC culture and BMSC sheet preparation

Sprague-Dawley rats 6–8 weeks old (male, 60–100 g) were obtained from the Laboratory Animal Center of Tongji Hospital of Hubei province in China. All experimental procedures followed the Guidelines of Animal Care and Use Committee for Teaching and Research of Huazhong University of Science and Technology. Rat BMSCs were isolated according to the process described previously [25]. Briefly, BMSCs were collected by flushing the bone marrow outside the femurs and tibias of rats with an 18-gauge sterile needle. The bone marrow was suspended in growth medium (GM) consisting of DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS; Gibco, NY, USA), 100 U/ml penicillin, and 100 U/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA). The isolated cells were then washed twice with phosphate-buffered saline (PBS, pH 7.4), resuspended in GM, plated at a density of 1 × 10⁶ cells/cm² in 25-cm² flasks, and cultured at 37 °C in 5% CO₂. After every passage, nonadherent cells were removed. The second or third passage was used for subsequent experiments.

For osteogenic differentiation, adipogenic differentiation, and chondrogenic differentiation, BMSCs were cultured with inductive medium respectively according to the protocol from Cyagen Biosciences.

For BMSC sheet harvest, third-passage cells were seeded at 1 × 10⁴ cells/cm² onto 10-cm dishes. Cells were cultured with GM and the GM was refreshed every 3 days. After approximately 14 days, the cells reached hyperconfluence and were lifted as a cell sheet using a scraper.