Figure 1.

Osteogenic stimuli induce extracellular matrix mineralization and Ca deposition of valve interstitial cells (VICs). (a–f) Confluent VICs (passage number 5–8) were maintained in growth medium (GM) or in osteogenic medium (OM) obtained by supplementing growth medium (GM) with phosphate (P) (0–2.5 mmol/L) and Ca (0–1.2 mmol/L). (a) Bright-field microscope image of human VICs in passage number 5, 100× magnification. (b,c) Ca deposition in the extracellular matrix (ECM) was evaluated by Alizarin Red (AR) staining after 7 days of treatment. Representative images of stained plates from three independent experiments and quantification are shown. (d) Ca content of the HCl-solubilized ECM is presented. (e) Cell viability was determined by MTT assay after 7 days of treatment. (f) Protein expression of RUNX2 was determined from whole cell lysates (24 h). Membranes were reprobed for β-actin. Representative Western blots and relative expression of RUNX2 normalized to β-actin from three independent experiments are shown. Results are presented as mean ± SD of three independent experiments performed in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.