Investigating the role of substrate stiffness in the persistence of valvular interstitial cell activation.

During heart valve remodeling and in many disease states, valvular interstitial cells (VICs) shift to an activated myofibroblast phenotype characterized by enhanced synthetic and contractile activity. Pronounced alpha smooth muscle actin (αSMA)-positive stress fibers, the hallmark of activated myofibroblasts, are also observed in VICs cultured on stiff substrates especially in the presence of transforming growth factor-beta1 (TGF-β1), however, the detailed relationship between stiffness and VIC phenotype has not been explored. The goal of this study was to characterize VIC activation as a function of substrate stiffness over a wide range of stiffness levels including that of diseased valves (stiff), normal valves (compliant), and hydrogels for heart valve tissue engineering (very soft). VICs obtained from porcine aortic valves were cultured on stiff tissue culture plastic to activate them, then, cultured on collagen-coated polyacrylamide substrates of predefined stiffness in a high-throughput culture system to assess the persistence of activation. Metrics extracted from regression analysis demonstrate that relative to a compliant substrate, stiff substrates result in higher cell numbers, more pronounced expression of αSMA-positive stress fibers, and larger spread area which is in qualitative agreement with previous studies. Our data also indicate that VICs require a much lower substrate stiffness level to "deactivate" them than previously thought. The high sensitivity of VICs to substrate stiffness demonstrates the importance of the mechanical properties of materials used for valve repair or for engineering valve tissue.