Fig. 1.

Comparison of in vitro bone formation and VSMC calcification. Representative phase contrast microscopy images (alizarin red stained) and whole well scans (unstained) of osteoblast and VSMCs after 14 days in culture. (A) Abundant formation of mineralised bone nodules by primary osteoblasts cultured with 2 mM β-glycerophosphate; unmineralised deposited collagen is highlighted by the arrow. (B) Widespread, non-specific mineral deposition was observed in osteoblasts cultured with 10 mM β-glycerophosphate (highlighted by the arrow). (C) No calcification in control VSMC cultures. VSMCs cultured with (D) 2 mM and (E) 10 mM β-glycerophosphate for 14 days do not calcify. (F) VSMCs grown in 2 mM sodium orthophosphate form small, discrete regions of calcification. These structures are significantly smaller than osteoblast bone nodules and are less visible in the whole well scans. (G) Osteoblasts cultured with sodium orthophosphate also display non-specific mineral deposition (white arrow). Scale bars: phase contrast microscopy images = 250 μm, tissue culture wells = 0.5 cm. (H) The overall level of deposited calcium was similar in cultures of bone-forming osteoblasts and calcifying VSMCs (2 mM phosphate) but ∼90% lower in control VSMCs. Values are mean ± SEM (n = 6 replicate wells), *** = p < 0.001.