Effects of extracellular matrix type in modulating cell migration on highly tunable mechanical gradient hydrogels
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Mechanical compliance has been demonstrated to be a key determinant of cell behavior, directing processes such as spreading, migration, and differentiation. Durotaxis, directional migration from softer to more stiff regions of a substrate, has been observed for a variety of cell types on mechanical gradients with absolute stiffnesses and gradient rates spanning multiple orders of magnitude. Recent stiffness mapping experiments have shown that local changes in tissue stiffness in disease are often accompanied by an altered extracellular matrix composition in vivo. However, the importance of extracellular matrix composition in cellular responses to mechanical gradients has not yet been thoroughly explored. To address this problem, we have developed a method to produce polyacrylamide hydrogels featuring highly tunable gradients in mechanical stiffness that allow for independent control of the absolute substrate stiffness and gradient rate. Maskless lithography is used to micropattern glass slides with hydrophobic and hydrophilic silanes to constrain the geometry of pre-gel solutions and establish a predictable cross-linker diffusion gradient, resulting in consistent linear mechanical gradients upon polymerization. This feature, together with the ability to control ECM composition independent of substrate stiffness, allows us to isolate the effects of mechanical and biological signals on cell migratory behavior. Using this system, we have tracked the migration of vascular smooth muscle cells and NIH 3T3 fibroblasts in vitro on mechanical gradient and uniform stiffness hydrogels and quantitatively analyzed differences in cell migration as a function of extracellular matrix composition. Our results show that both vascular smooth muscle cells and NIH 3T3 fibroblasts will exhibit durotaxis on mechanical gradients coated with fibronectin but not on those coated with laminin, demonstrating that extracellular matrix type can act as a regulator of a cell’s response to mechanical gradients. Interestingly, NIH 3T3 fibroblasts were also observed to migrate randomly on gradients coated with a mixture of both fibronectin and laminin, suggesting that there may be a complex interplay in the cellular response to mechanical gradients in the presence of multiple extracellular matrix signals. These findings indicate that the composition of the adhesion ligand is a critical determinant of a cell’s migratory response to mechanical gradients.