Smooth muscle cells (SMCs) play a significant role in the pathogenesis of atherosclerosis. 2D cultures elucidated valuable information about the interaction between SMCs and extracellular matrix (ECM) components. However, 3D constructs better represent the native vascular environment. Furthermore, a limited number of studies addressed the effect of ECM stiffness on SMCs phenotype. We investigated the effect of stiffness of different ECM substrates by modulating their concentrations, including

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... e effect on morphology, proliferation, expression of the contractile protein α-smooth muscle actin (α-SMA) and deposition of collagen type I (Col I) and collagen type III (Col III) proteins. At low concentrations of Col I gels and Col I gels supplemented with 10% fibronectin (Fn), SMCs exhibited non-elongated, 'hill-and-valley' shape and large mean cellular area, indicating a hypertrophic morphology, characteristic of the synthetic phenotype. However, with increasing concentration, mean cellular area and proliferation relative to cells cultured in 2D dropped. Whole protein secretion into the culture media and deposition of Col I and Col III generally decreased with increasing stiffness. Moreover, percentage of α-SMA+ SMCs decreased with increasing gel concentration, pointing to a shift towards the synthetic phenotype. Supplementing Col I with 10% Laminin (Ln) maintained higher cellular area and aspect ratio at all gel concentrations and did not change α-SMA expression significantly, compared to Col I alone or Col I + Fn. Overall, these results demonstrate that ECM components and stiffness could provide the tools to modulate the phenotype and function of SMCs in vitro, which allows for the control of properties of engineered tissues.