Figure 6.

Comparison of pro-degenerative and pro-calcifying conditions. (A) Alizarin red S staining and quantification of 2D VIC cultures (n = 16) after 7 d under pro-degenerative (pd, β-GP + CaCl2; blue line) and pro-calcifying (pc, NaH₂PO₄, red line) conditions compared to control conditions. (B) Images and transmitted light images of AV leaflets after 28 d cultivation under pd and pc conditions and analysis of optical density (OD). Data (n = 6) are mean ± SEM. Alizarin red S (C), Movat’s pentachrome (D), and AP staining (E, purple areas) of AV leaflets under pd, pc, and control conditions after 28 d cultivation. Scale bar indicates 100 µm. Representative images of five different experiments are shown. Analysis of AP (F) and phosphate (G) in supernatants of 2D VIC cultures (d2–d7) and AV leaflet cultures (d3–d27) under pd (blue line) and pc (red line) conditions compared to control conditions (gray line). Data (n = 5) are mean ± SEM. p-values (*: pd vs. control, #: pc vs. control) are calculated by using Kruskal–Wallis test with Dunn’s multiple comparison post hoc test. * and #: p < 0.05; ** and ##: p < 0.01; *** and ###: p < 0.001.