Fig. 2.

Collagen and elastin production by osteoblasts and VSMCs. (A) Osteoblasts and VSMCs were cultured for 7 or 14 days and then stained to visualise collagen (red) and elastin (black) fibres. Representative light microscopy images showing widespread deposition of collagen (highlighted by the arrows) in early and mature, bone-forming osteoblasts. Mineralised matrix in bone-forming cultures stained dark brown/black and was associated with collagen staining. No obvious collagen or elastin staining in control VSMCs or calcifying VSMCs via light microscopy. Regions of calcification in VSMC cultures also stained dark brown/black (highlighted by the arrows). Immunofluorescent staining for Col1α1 showed extensive collagen expression and deposition in osteoblast cultures. Control and calcifying VSMCs also expressed Col1α1 protein but immunostaining was primarily cell associated rather than extracellular. Staining for SM22α showed uniform expression across osteoblast and VSMC cell layers. Scale bars, light microscopy = 50 μm, immunofluorescence = 50 μm. (B)Col1α1 mRNA expression was up to 11-fold lower in control and calcifying VSMCs, relative to bone-forming osteoblasts. (C) Soluble collagen levels increased with osteoblast differentiation and bone formation (black bar). No soluble collagen was detected in control and calcifying VSMC cultures [ND = not detected]. (D)Elastin mRNA expression was ∼2-fold higher in control VSMCs than bone-forming osteoblasts; calcifying VSMCs and osteoblasts displayed similar levels of elastin expression. (E) Cell-associated elastin was up to 50% higher in control VSMCs compare to calcifying VSMCs and osteoblasts. Values are mean ± SEM (n = 6 replicate wells, n = 4 RNA sets), * = p < 0.05, *** = p < 0.001.