Ascorbate

For osteogenic differentiation cells were firstly grown to 100% confluence, and then cultured with medium containing α-MEM with 10% FBS, ascorbate, β-glycerophos-phate and dexamethasone (Cyagen Biosciences, Jiangsu, China) for nine days. Medium was changed every 3 days. Alizarin red staining was performed and recorded using an inverted microscope (Olympus, Tokyo, Japan). In addition, mRNA expression of Runx2 and osteocalcin (OCN) was analyzed with qPCR. To assess adipogenic differentiation cells were cultured at 100% confluence for 2∼3 weeks in α-MEM containing 10% FBS, L-glutamine, insulin, isobutylmethylxanthine, rosiglitazone and dexame-thasone (Cyagen Biosciences, Jiangsu, China). Medium was changed every 2∼3 days and then Oil red staining was performed. For chondrogenic differentiation, 3−4×10⁵ suture mesenchyme cells were cultured as a pellet at the bottom of a 15 ml aseptic tube in 0.5 ml α-MEM medium supplemented TGF-β3, dexamethasone, ascorbate, sodium pyruvate and proline (Cyagen Biosciences, Jiangsu, China) and changed every 3 days. After 21 days culture, the cell microsphere was formalin-fixed and paraffin-em-bedded and then sectioned at 4 mm, stained with Alcian blue.

For osteogenic differentiation, rADSCs at passage 3 were inoculated at 4×10³ cells/cm² and cultured for 2 weeks in osteogenic induction medium [MEM containing 10% FBS, 100 µg/ml ascorbate, 0.1 µM dexamethasone and 10 mM β-glycerophosphate (All from Cyagen Biosciences, Suzhou, Jiangsu, China)]. rADSCs were then fixed with 4% paraformaldehyde (PFA, Boster Biosciences, Beijing, China) for 20 min, washed with PBS for 3 times, afterward, incubated with 1 mg/ml Alizarin Red (Sigma-Aldrich, St. Louis, MO) solution for 30 min to stain for calcium deposition. For adipogenic differentiation, rADSCs at passage 3 were cultured for 2 weeks in adipogenic induction medium [high glucose-DMEM containing 10% FBS, 1 µM dexamethasone, 0.5 mM methyl-isobutylxanthine, 10 µg/ml insulin, and 100 µM indomethacin (All from Cyagen Biosciences, Suzhou, Jiangsu, China)]. Then, cells were fixed with 4% PFA for 20 min, washed with PBS for 3 times, afterward, immersed in 0.3% Oil Red O solution (Sigma-Aldrich, St. Louis, MO) in 60% isopropanol for 30 min to assess lipid droplet formation.