Ascorbic acid

To stimulate calcification, human VICs were cultured with inorganic phosphate-osteogenic medium (IP-OM) (complete media supplemented with 2 mM sodium dihydrogen phosphate (Beyotime Biotech, Jiangsu, China), 75 mM ascorbic acid (Beyotime Biotech), 100 nM dexamethasone (Beyotime Biotech) and 10⁻⁷ mM insulin (Beyotime Biotech)) for 7–9 days, modified from previously described approaches [4 (link), 37 (link), 38 (link)]. Calcific nodules could be observed by day 3. The osteogenic differentiation medium was changed every 2 days.

C3H10T1/2 cells (Stem Cell Bank, Chinese Academy of Sciences) were cultured at 37˚C with 5% CO₂ in DMEM (Gibco, Thermo Fisher Scientific) supplemented with 10% FBS (Gibco, Thermo Fisher Scientific) The culture medium was changed every 2 days and the cells were passaged at ~85% confluence. At ~85% confluence, osteogenic differentiation was induced by osteogenic medium (DMEM supplemented with 10% FBS, 10 nM dexamethasone, 10 mM β-glycerophosphate and 50 µg/ml ascorbic acid (Beyotime)) for 7 days at 37˚C with 5% CO₂. Lipogenic differentiation was induced by lipogenic medium (DMEM supplemented with 10% FBS, 0.5 mM isobutylmethylxanthine (Merck, USA), 125 nM indomethacin, 1 μM dexamethosone, 20 nM insulin, and 1 nM Liothyronine (MCE, USA)). After 2 days, cells were switched to maintenance medium (DMEM supplemented with 10% FBS, 1 nM Liothyronine, 20 nM insulin, 0.5 μM rosiglitazone (MCE, USA)) for 5 d at 37 ˚C with 5% CO₂.