Toluidine blue sodium borate

rASCs were induced to differentiate into adipocytes, osteoblasts, and chondrocytes. For adipogenesis, the cells were cultured in adipogenic induction medium (DMEM supplemented with 10% FBS, 10⁻⁷ M dexamethasone (Sigma, St. Louis, MO), 100 μΜ indomethacin (Sigma), 100 μM 3-isobutyl-1-methyl-xanthine (Sigma), and 10 mg/L insulin (Invitrogen)), and the medium was refreshed every 2 days. Two weeks later, the cells were fixed with 4% paraformaldehyde (PFA) and stained with Oil red O (Sigma). For osteogenesis, the cells were maintained in osteogenic induction medium (DMEM supplemented with 10% FBS, 10 mM β-glycerol phosphate (Sigma, CA), 50 μM L-ascorbate-2-phosphate (Sigma), and 5 ng/mL recombinant human bone morphogenetic protein-2 (HumanZyme, Chicago, IL)) and the medium was changed with fresh one every 2 days. One week later, the cells were examined for alkaline phosphatase (AKP) activity by vector blue alkaline phosphatase substrate kit III (Vector, Burlingame, CA). For chondrogenesis, the cells were cultured in chondrogenic induction medium which includes DMEM supplemented with 10% FBS, 10 ng/mL TGFβ1 (HumanZyme, Chicago, IL), 0.1 mol/L dexamethasone (Invitrogen), 50 mg/L L-ascorbate-2-phosphate (Sigma), and 50 g/L ITS (Invitrogen). The cells were cultured for two weeks and fixed in 4% PFA and stained with toluidine blue sodium borate (Sigma).

hUCMSCs were induced to differentiate into adipocytes, osteoblasts and chondrocytes. For adipogenesis, the cells were cultured in adipogenic induction medium (DMEM supplemented with 10% FBS, 10⁻⁷ M dexamethasone (Sigma, St. Louis, MO), 100 μΜ indomethacin (Sigma), 100 μM 3-isobutyl-1-methyl-xanthine (Sigma) and 10 mg/L insulin (Invitrogen)), and the medium was refreshed every 2 days. Two weeks later, the cells were fixed with 4% paraformaldehyde (PFA) and stained with Oil-Red O (Sigma). For osteogenesis, the cells were maintained in osteogenic induction medium (DMEM supplemented with 10% FBS, 10 mM β-glycerol phosphate (Sigma), 50 μM L-ascorbate-2-phosphate (Sigma) and 5 ng/mL recombinant human bone morphogenic protein-2 (HumanZyme, Chicago, IL)) and the medium was changed with fresh one every 2 days. One week later, the cells were examined for alkaline phosphatase (AKP) activity by vector blue alkaline phosphatase substrate kit III (Vector, Burlingame, CA). For chondrogenesis, the cells were cultured in chondrogenic induction medium includes DMEM supplemented with 10% FBS, 10 ng/mL TGF-β1 (PeproTech, Cranbury, NJ, USA), 0.1 mol/L dexamethasone (Invitrogen), 50 mg/L L-ascorbate-2-phosphate (Sigma) and 50 g/L ITS (Invitrogen). The cells were cultured for two weeks and fixed in 4% PFA, and stained with toluidine blue sodium borate (Sigma).