Durotaxis, a phenomenon that cells move according to changes in stiffness of the extra cellular matrix, has emerged as a crucial parameter controlling cell migration behavior. The current study provides a simple method to generate three-dimensional continuous stiffness variations without changing other physical characteristics of the extra cellular environment. Using Finite Element simulations, the stiffness and the stiffness gradient variations are evaluated quantitatively, leading to an analysis of the dependence of cell migration behavior on the substrate stiffness parameters. We tested various cell lines on several 3-D environments. The durotaxis results show that the cell migration velocity does not have any consistency with the stiffness of the substrate, rather it is more related to the stiffness gradient of the substrate. This finding suggests a new mechanism underlying the durotaxis phenomenon, highlighting the importance of the substrate stiffness gradient, rather than the stiffness itself.

In this study, we investigate the effects of micron-scale surface patterns on the alignment of individual cells and groups of cells. Using a simple replication molding process we produce a number of micron-scale periodic wavy patterns with different pitch and depth. We observe C2C12 cells as they grow to confluence on these patterns and find that, for some geometries, cell-cell interaction leads to global alignment in a confluent culture when individual cells would not align on the same pattern. Three types of alignment behavior are thus defined: no alignment, immediate alignment, and alignment upon confluence. To further characterize this response, we introduce a non-dimensional parameter that describes the aligning power of a periodic pattern based on its geometry. The three types of alignment behavior can be distinguished by the value of the alignment parameter, and we identify values at which the transitions in alignment behavior occur. Applying this parameter to data from the current and several earlier studies reveals that the parameter successfully describes substrate aligning power over a wide range of length scales for both wavy and grooved features.