(173a) DNA Loaded Multiple Channel Bridges for Spinal Cord Regeneration

Spinal cord injury results in paralysis below the level of injury, for which therapeutic strategies are ineffective at promoting axonal regrowth across the injury site. Since 1980, bridges have been developed in which polymer scaffolds are implanted to span the injury site and support and direct axonal elongation. We propose to develop bridges capable of localized DNA delivery that will promote protein production by endogenous cells at the injury site to create a permissive environment for axon regeneration. Poly(lactide-co-glycolide) (PLG) multiple channel bridges were fabricated by a gas foaming/particulate leaching method. Plasmid DNA was mixed with PLG microspheres and NaCl (porogen) before foaming. A DNA incorporation efficiency of greater than 50% was obtained, and a sustained release was observed in vitro over a timespan of 5 days. Plasmid encoding for luciferase or enhanced green fluorescence protein (EGFP) were loaded into the bridge to quantify transgene expression with a luciferase assay or assess the location and identity of transfected cells by immunohistochemistry, respectively. Bridges releasing luciferase plasmid were initially investigated in a subcutaneous mouse model to non-invasively monitor transgene expression using the Xenogen imaging system. Transgene expression was observed for up to 90 days, but the expression profile was dependent upon the DNA loading and pore size. Higher doses led to longer transgene expression, while larger pore sizes led to better cell infiltration and transgene expression. For the spinal cord injury model, bridges were implanted into a rat spinal cord lateral hemisection to determine the extent of transgene expression and the distribution and identity of transfected cells in the spinal cord. Initial studies with bridges with small pore sizes (38-63 ìm) and seven 250 ìm channels demonstrated luciferase expression at high levels during the initial 3 days which decreased thereafter. EGFP staining however, showed a high number of transfected cells up to 4 weeks. A double fluorescent staining revealed that most of the transfected cells were fibroblasts, followed by astrocytes and Schwann cells. Macrophages, however, were present in high numbers but were not transfected. In conclusion, a biodegradable multiple channel PLG bridge was engineered and implanted into a rat spinal cord hemisection model. Bridges loaded with DNA resulted in transgene expression at the injury site for over 4 weeks, with fibroblasts as the main cell type transfected. The subcutaneous mouse model has shown that transgene expression has the potential to be manipulated by varying the bridge fabrication parameters. Multiple channel bridges capable of DNA delivery provide a powerful approach to promote axonal regrowth by providing a physical support for axonal elongation and inducing the expression of stimulating neurotrophic factors.