Tissue engineering strategies that enable nerve regeneration will require methods that can promote and direct neurite extension across the lesion. In this report, we investigate an in vitro combinatorial approach to directed neurite outgrowth using gene delivery from topographically patterned substrates, which can induce expression of neurotrophic factors to promote neurite extension and direct the extending neurites. Poly(lactide-co-glycolide) (PLG), which has been used to fabricate conduits or bridges for regeneration, was compression molded to create channels with 100, 150, and 250 microm widths. DNA complexes were immobilized to the PLG, and cells cultured on the substrate were transfected with efficiencies dependent on channel width and DNA amount. A co-culture model consisting of primary neurons and accessory cells was employed to investigate neurite outgrowth within the channels. Localized secretion of nerve growth factor (NGF) by the accessory cells promoted neuron survival and neurite extension. Neurons cultured in channels with NGF expression exhibited longer primary neurites than in the absence of channels. Neurons cultured in smaller width PLG microchannels exhibited a greater degree of directionality and less secondary sprouting than larger channels. Finally, surface immobilization allowed for the delivery of distinct plasmids from each channel, which may enable channels to be tailored for specific nerve tracts. This approach demonstrates the ability to combine gene delivery with physical guidance, and can be tailored to target specific axonal populations with varying neurotrophic factor requirements.
Animals Cell Line