L ascorbic acid phosphate

Passage 2 ADSCs were harvested and seeded in 12 well plates (2 × 10⁴ cells/cm²) and incubated to 90% confluence. Then, the conventional growth medium was replaced with osteogenic differentiation medium; 100 mL osteogenic differentiation medium contains 10 mL fetal bovine serum, 1 mL 100 U/mL penicillin and streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 10 μL 10⁻⁸ mol/L dexamethasone, 1 mL 0.1 mmol/L L-ascorbic acid phosphate (Beyotime, Shanghai, China), 1 mL 2 mmol/L Glutamine (Beyotime, China) and Sodium β-glycerophosphate. Alpha MEM was applied (Hyclone, Logan, UT, USA) to up 100 mL. The medium was renewed every 3 days till day 21. ADSCs were fixed with 4% formaldehyde for 60 min and thereafter stained with 2% alizarin red to observe the effect of osteogenic staining.

When the cells reached to 90% confluence, the conventional growth medium was replaced with osteogenic differentiation medium, which was prepared by 15 mL 15% foetal bovine serum [FBS (Gibco, USA)], 10 μL 10⁻⁸ mol/L dexamethasone sodium phosphate (Beyotime, China), 1 mL 1.8 mmol/L KH₂PO₄ (Beyotime, China), 1 mL 100 U/mL penicillin (Gibco), 1 mL 100 U/mL streptomycin (Gibco), 1 mL 0.1 mmol/L L‐ascorbic acid phosphate (Beyotime, China), 1 mL 2 mmol/L Glutamine (Beyotime, China) and Alpha MEM (Hyclone, USA) to up 100 mL. The medium was renewed every 3 days till Day 7 or Day 14. Mineral deposit was then detected by ARS Staining (Cyagen, USA). According to the manufacturers’ instructions, the cells were fixed with 2 mL of 4% neutral formaldehyde solution for 30 minutes. After removing formaldehyde and washing the plate with PBS, ARS solution was added for 5 minutes. The staining cells were observed by a light microscope (OLYMPUS, Japan).