(93c) Three-Dimensionally Directed DRG Neurite Outgrowth within Hydrogels in a Co-Culture Model Using Gene Delivery

Injury to the spinal cord results in loss of function below the level of injury, for which current strategies are unable to restore function. Spinal cord neurons have the potential to regenerate, yet regeneration is limited by the resulting inhibitory environment. A permissive environment that can promote and direct neurite growth through a combination of biochemical signals and physical guidance has the potential to enhance neurite extension across the injury site. We are developing bridges from biomaterials to create an environment that will support regeneration in the injured spinal cord. Hydrogels are an attractive biomaterial for spinal cord regeneration as their mechanics are similar to native soft tissue and can support cell adhesion and migration. Gene delivery from the hydrogels is being investigated to modulate the local microenvironment to promote and guide tissue formation. The efficiency of non-viral strategies must be increased for efficacy; thus, we are investigating the design of hydrogels for maximizing gene delivery, and must balance these design properties for gene delivery with the design to support neurite outgrowth. Poly(ethylene glycol) (PEG)-based enzymatically degradable hydrogels formed with RGD peptides support cell adhesion and matrix degradation. Encapsulation of HT-1080 cells and lipoplexes within the hydrogel resulted in maximal transfection levels that were sustained for up to 16 days in hydrogels formed with 10% PEG and 5 mM RGD. Release studies using radiolabeled DNA indicated that 40%, 75%, and 80% of the vector is release by Days 1, 5, and 10 of the culture, respectively. The entrapped HT-1080 cells were then used in a co-culture system with dorsal root ganglia (DRG) explants to investigate neurite outgrowth in three-dimensions. The HT-1080 cells serve as targets for transfection, and they did not support neurite outgrowth in the absence of transfection with an NGF-encoding plasmid. Hydrogels that supported neurite outgrowth within the co-culture ranged from 7.5% and to 10% PEG and 2 to 5 mM RGD. Lower PEG contents and higher RGD concentrations were insufficient to support neurite outgrowth. Transfection with pRK5-NGF lipoplexes promoted neuron survival and neurite extension within the hydrogels. Our results demonstrate the ability to modulate hydrogel parameters to transfect accessory cells, which can subsequently promote neurite extension in vitro, providing a foundation for the ultimate translation to spinal cord regeneration in vivo.