Neuronal growth cones are highly dynamic structures at the end of neurites, which explore the environment and establish new connections during nervous system development and regeneration following injury and disease. It is well known that growth cones respond to diverse types of environmental cues such as a molecular cues, stiffness, topography, and electrical fields. However, the biophysics and molecular mechanisms behind the growth cone’s response to extracellular stiffness remain unclear. Previous computational modeling of substrate-cytoskeleton coupling from our lab revealed that soft to intermediate substrate stiffness results in the fastest growth cone responses. To experimentally validate these findings, we fabricated polyacrylamide (PAA) hydrogels with either uniform or gradient stiffness. On gels with uniform stiffness, we found that neurite outgrowth by Aplysia bag cell neurons exhibits a biphasic behavior between 0.3 and 30 kPa, with 3 kPa being the optimal stiffness. These results were confirmed with PAA gels with a stiffness gradient between 0.3 and 3 kPa or between 3 and 10 kPa. Additionally, traction force microscopy revealed a biphasic dependence of the growth cone’s traction stress on substrate stiffness, exhibiting the highest average stress of 16 Pa on gels with a stiffness of 3 kPa. These findings are in agreement with the substrate-cytoskeletal coupling model predicting the fastest neurite growth and the highest traction forces on soft to intermediate substrate stiffness.
Laura Pulido (Sun,) studied this question.