Spatial gradients of chemotropic factors from immobilized patterns to guide axonal growth and regeneration

In this study, we investigated the effect of varying localized concentration gradients of NGF and Sema3A on the axonal outgrowth of embryonic chick DRG explants and primary neurons in vitro. Immobilized 2D NGF or Sema3A micropatterns were produced using photolithography on tissue culture cover slips. Two distinct regions were identified: slow, with little or no change in concentration of chemotropic factor; and steep, with a transition from low to high. The direction of axonal outgrowth was defined as proximal or distal, with proximal growing towards the higher concentration of immobilized NGF/Sema3A and vice versa for distal. Axons grew preferentially in the proximal direction when explants were seeded onto steep NGF, and distally in response to steep Sema3A. On slow NGF, or on slow Sema3A there was no difference in the directional specificity of axonal outgrowth. DRG primary neurons seeded onto steep NGF migrated proximally, whereas neurons seeded onto slow NGF migrated in all directions. Conversely, neurons seeded onto steep or slow Sema3A did not extend any axons. Our 2D immobilized micropatterns of chemotropic factors show promise for further development of in vitro nerve tissue engineering studies.