Calcification Induction and Inhibitors in VICs

SAVIC and RAVIC cells were seeded in growth media in multi-well plates at a density of 1.11×10⁴ cells/cm². Calcification was induced as described previously (22 (link),25 (link)). Cells were grown to 80% confluence (Day 0), before treating with control (1.05 mM Ca/0.95 mM Pi) or test media: 1.5 to 3.6 mM calcium (Ca) and/or 1.5 to 2.5 mM phosphate (Pi). CaCl₂ and Na₂HPO₄/NaH₂PO₄ (Sigma-Aldrich, Dorset, UK) were used to supplement ionic calcium and phosphate in the media. To study the effect of calcification inhibitors and bisphosphonates on VIC calcification, SAVICs were exposed to inorganic pyrophosphate (PPi) and etidronate (both 0.1 mM; Sigma-Aldrich). Cells were incubated for up to 7 days in a humidified atmosphere of 95% air/5% CO₂, and the medium was changed every second/third day.