Directing neurite growth in 3d collagen scaffolds with gradients of mechanical properties

Biomaterial scaffolds for nerve and spinal cord regeneration must riot only promote neurite re-growth but also direct it. Several cell types, including neurons, respond to the mechanical properties of the Substrate on which they are grown. We believe that gradients of mechanical properties can be used to direct neurons. To spatially control the mechanical properties, gradients of genipin - a naturally occurring, cell-tolerated crosslinking agent - are created in 3D through a compliant collagen gel using microfluidics. Gradients of mechanical properties are evaluated by measuring genipin-induced fluorescence, which we have previously correlated to mechanical properties. Growth of neurites was evaluated in gels Of uniform stiffness and a gradient generated by incubation in 0 to 1 mM genipin for 12hrs to produce approximately an order of magnitude change in the shear modulus. Neurite growth was evaluated 5 days after gradient formation. Neurites demonstrated a directional bias against the gradient of stiffness. These results demonstrate that neurites can respond to subtle gradients of mechanical properties within a 3D scaffold and point to opportunities to manipulate properties for directed nerve and spinal cord regeneration.