Alizarin red

EMSCs were cultured with the osteogenic induction medium. On days 7 and 21, the cells were stained using an alkaline phosphatase (ALP) staining kit (Beyotime, China) or Alizarin Red (Sangon, China) separately, according to the previous study.23

Bone marrow aspirates extracted from New Zealand White rabbits (2.5 ± 0.2 kg, 12 weeks old) were used to isolate and culture BMSCs, as previously described [17 (link)]. Mononuclear cells were gathered in Ficoll–Hypaque gradient (Sigma) after centrifugation and then suspended in cell culture medium containing 10% fetal bovine serum (FBS, Gibco). As the culture medium changed, the suspended cells were removed after culture at 37°C in 5% CO₂ for 72 h. When the adherent cells reached 70–80% confluence, subculture was performed. After culture for 2 weeks, a homogenous BMSC population was obtained, and the third passage was collected and seeded on the silk–collagen scaffold. BMSCs adherent on the scaffold were observed by SEM after being seeded for 72 h. The osteogenic, adipogenic, and chondrogenic differentiation abilities of passage 3 cells were identified after culture with special inducing media (Gibco) for 3 weeks. Finally, alizarin red (Sangon), oil red O (Sangon), and alcian blue staining (Sangon) were performed according to the manufacturer's protocols.

Each group cells were cultured with the osteogenic induction medium. On days 14 and 21, the cells were fixed with 4% paraformaldehyde and stained using an alkaline phosphatase (Alp) staining kit (Beyotime, Shanghai, China) or alizarin red (Sangon Biotech, Shanghai, China) staining kit separately, according to the manufacturer’s instructions and the previous study^{1 (link)}. The stained images were captured by a stereo fluorescence microscope (Zeiss).