Ars 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).

2 × 10⁴/well cells were seeded in the lower wells of a 24-well plate. After reaching 60–70% confluence, USCs were induced using osteogenic medium (Cyagen, China). Col I, CSM/Col I and BMP2-CSM/Col I hydrogels were added into the upper inserts, respectively. At Days 4, 7, 14 and 21, cells were stained with a BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China), and osteogenic USCs were stained with blue. The level of alkaline phosphatase (ALP) was quantified using an ALP assay kit (Nanjing Jiancheng Bioengineering Institute, China) according to the user manual.
Calcium deposits were visualized using ARS staining (Cyagen, China) at Days 7, 14 and 21. To quantify the calcium content, orange-red deposition was dissolved by 10% (w/v) cetylpyridine (TCI, China), and OD values were measured at 540 nm.