Alizarin red dye solution

After osteogenic induction for 7 or 21 days, cell line was fixed with 4% paraformaldehyde (Sigma) for 30 min at a room temperature, and then washed 3–5 times with 1 × PBS for 3–5 min; Secondly, BCIP/NBT staining solution (Cyagen, Santa Clara, CA) or Alizarin red (Solarbio) were added to the cells and incubated at a room temperature for 5–30 min in the dark. Until the expected color appeared, BCIP/NBT or Alizarin red dye solution (Cyagen) were removed and washed 2–3 times with distilled water and taken pictures.

Dulbecco’s modified Eagle medium with low glucose (LG-DMEM), F-12 medium, trypsin, GlutaMAX, non-essential amino acids, fetal bovine serum (FBS), and adipocyte/osteocyte/chondrocyte differentiation basal medium and their corresponding supplements were purchased from Gibco (NY, USA). Antibiotics (penicillin and streptomycin) were purchased from Sino American Biotechnology (Shanghai, China). HA was obtained from Seebio Biotech, Inc. (Shanghai, China). Bovine serum albumin (BSA), hematoxylin, saturated oil red O dye solution, Marker I DNA Ladder, and Gold View Type I nucleic acid staining were obtained from Solarbio (Beijing, China). Alizarin red dye solution and Toluidine blue were purchased from Cyagen (Shanghai, China) and Sangon Biotech (Shanghai, China), respectively. SB431542 was obtained from Selleck (Shanghai, China). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Beijing, China). Human IL-10 ELISA Kit and human TGF-β1 ELISA Kit were purchased from Biosamite (Shanghai, China). All other chemicals were of analytical grade.