G1452

For chondrogenesis, 1 × 10⁶ CSPCs at passage 2 were centrifuged in 15 mL polypropylene tubes to obtain cell pellets and cultured in MesenCult™-ACF chondrogenic differentiation medium (Catalogue #05455, Stem Cell) for 4 weeks. After differentiation, pellets were fixed and sectioned. Chondrogenesis was analysed by staining with toluidine blue (G3668, Solarbio). For osteogenesis, CSPCs at passage 2 were harvested and cultured in 24-well plates (20,000 cells/well). After 90% confluence, the osteogenic differentiation medium was replaced [low glucose DMEM (10567-014, Gibco) with 10% FBS, 10 mM β-glycerophosphate (50020, Sigma), 50 mM L-ascorbate-2-monophosphate (A7631, Sigma), and 1 μM dexamethasone (D1756, Sigma)] for 3 weeks. The osteogenic cells were fixed and stained with alizarin red (G1452, Solarbio). To induce adipogenesis, CSPCs at passage 2 were harvested and cultured in 24-well plates (20,000 cells/well). After 90% confluence, the osteogenic differentiation medium high glucose DMEM (11965-092, Gibco) was replaced with 10% FBS, 100 μM indomethacin (I7378, Sigma), 0.5 mM IBMX (I5879, Sigma), 10 μg/mL insulin (91077C, Sigma), and 1 μM dexamethasone (D1756, Sigma) for 2 weeks. Oil Red O (G1260, Solarbio) staining was performed to assess the adipogenicity potency.

After approximately 7 days post seeding, the primary BMSCs were switched into osteogenic medium (OGI, containing 10 nM dexamethasone, 10 mM β-glycerophosphate, and 50 μM ascorbic acid in α-MEM containing 10% FBS), or recombinant bone morphogenic protein 2 (BMP2, 300 ng/mL) to induce osteoblast differentiation. After osteogenic induction for 14 days, cells were fixed and subjected to ALP staining (C3206, Beyotime, Shanghai, China). After osteogenic induction for 21 days, the cells were subjected to alizarin red staining (ARS) (G1452, Solarbio, China).