Impact of substrate stiffness on vascular smooth muscle cell behavior: A comparison of uniform and gradient substrate stiffness

The mechanical compliance of underlying substrata has been emerging as an important environmental cue that influences various cell behavior such as cell morphology and motility. Local variations in tissue mechanical properties have been reported in vivo, for example, at sites of angiogenesis or atherosclerosis; therefore variation in extracellular matrix (ECM) stiffness may be an important signal that guides cell behavior in vivo. Recently, in vitro studies have shown that fibroblasts and vascular smooth muscle cells (VSMCs) prefer to migrate from relatively compliant to adjacent relatively stiffer substrate. However, this phenomenon, termed durotaxis, has not yet been rigorously demonstrated. This requires a systematic comparison of cell behavior on substrata with uniform and varying gradient stiffness. The determination of quantitative relationships between scaffold properties and cell response would not only provide insight to normal and pathologic processes but may also aid in the design of scaffolds for tissue engineering applications.; To quantify the dependence of VSMC morphology and motility on substrates with uniform and gradient stiffness, we used polyacrylamide (PAAM) hydrogels as our model system. We developed a novel system to generate well-defined stiffness gradients by integrating microfluidics technology and photopolymerization of acrylamide and measured the stiffness of the gradient gels using atomic force microscopy (AFM).; We investigated VSMC morphology and motility on substrates with uniform stiffness ranging from 5 to 250 kPa and found that cell spreading and elongation (polarization) increases with stiffness. Using a random cell walk model, we found that cell motility also increased with substrate stiffness. We investigated stiffness gradients ranging from 0 to 6 kPa/100 mum and found that VSMC morphology was not significantly different from that on uniform stiffness substrates. However, significant durotaxis was observed on the gradient substrata. We quantified durotaxis by determining the McClutcheon-Othmer tactic index (used previously for chemotaxis) and found that durotactic index increases with the magnitude of the gradient. Additionally, we also assessed VSMC durotaxis morphologically by examining cell orientation with respect to the gradient direction and further evaluated the dependence of durotaxis on the gradient. The knowledge gained from this study, combined with further investigations into molecular mechanisms and dynamics underlying durotaxis, may aid in the design of new therapeutic strategies and vascular graft design to control VSMC migration.