Injury to the spinal cord results in paralysis below the level of the injury, which results from neuron and oligodendrocyte cell death, axonal loss, demyelination, and critically, the limited capacity of the spinal cord neurons to regenerate. Although spinal cord neurons have the innate capacity to regenerate, they are limited by an insufficient supply of factors to promote regeneration, and an abundant supply of factors that inhibit regeneration. Our long-term goal is to develop a combination therapy based on biomaterials that bridge the injury site to control the microenvironment, which is achieved through the bridge microstructure and localized gene delivery to provide factors that stimulate and direct regeneration. This proposal develops multiple channel bridges for spinal cord regeneration that are capable of spatially controlled delivery of one or more neurotrophin encoding plasmids from each channel. DNA will be immobilized within the channels of the bridge to transfect accessory cells infiltrating the bridge, and induce the expression of neurotrophic factors that will initiate axonal elongation into and across the channel. Additionally, each channel can be loaded with a different neurotrophin encoding plasmid, or combination of plasmids, to tailor the channel for specific neural tracts. This hypothesis is based on the observations that i) DNA delivery can induce sustained, localized transgene expression in vivo, ii) neurotrophin delivery to the injury site can promote axonal elongation into a synthetic bridge, iii) cell transplantation or osmotic pump implantation does not provide a controllable concentration of neurotrophins for promoting regeneration, and iv) the spinal cord contains multiple tracts that are located at specific regions and contain different neuronal types Based on these observations, the experimental focus is on designing bridges for efficient gene transfer. These studies are subdivided into 3 specific Aims: 1) Investigate substrate-mediated DNA delivery to the channels of the bridge and characterize transgene expression (quantity, duration) and cell transfection (number). 2) Test the hypotheses that plasmids inducing neurotrophin secretion within the channels promotes localized survival of specific neuronal populations within the channel in vitro. 3) Test the hypothesis that differential expression of neurotrophins within the channels in vivo will promote axonal extension into the channels for specific neural tracts. The development of spatially regulated gene delivery will be important for regenerating complex tissue architectures, such as that observed within the spinal cord. These results will broadly impact tissue engineering, as the regeneration of complex tissue architectures is a fundamental issue. ? ? ?
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