Integration of gene therapy and biomaterials can be used to stimulate tissue regeneration by directing the actions of progenitor cells, either endogenous or transplanted. The scaffold, typically a biodegradable and biocompatible polymeric structure, must function to provide the cells with the mechanical support required for growth and with the biochemical signals to guide tissue regeneration. Delivering DNA encoding for biologically active proteins within the scaffold is a versatile approach to guide tissue regeneration that can target most cellular processes. However, systems for efficient and controlled DNA delivery and expression by cells, a process termed transfection, remain to be developed. A novel DNA delivery strategy, termed substrate-mediated delivery, that enhances and localizes transfection from a biomaterial was developed. First, a strategy to deliver DNA by immobilization to a cell adhesive substrate was developed. In this approach, DNA is complexed with a cationic polymer to alter the surface charge density, reduce the effective hydrodynamic radius, and protect against degradation. The resulting DNA/cationic polymer complex is then immobilized to the substrate by two mechanisms: (i) tethering using the neutravidin-biotin interaction and (ii) electrostatic using an ionic bond between the negatively charged surface and the positively charged complex. Using neutravidin modified polystyrene substrates, where immobilization by electrostatic interactions is limited, efficient transfection represented a balance between binding and release of the complex. Transfection by the DNA modified polystyrene substrates produced 100-fold higher expression than through bolus delivery of DNA, the traditional approach. Second, hydrogels composed of hyaluronic acid and collagen were developed as the cell adhesive substrate to translate the system to in vivo applications. These hydrogels are biodegradable and can be topographically patterned to guide cell growth. In vivo implantation of the hydrogels in a rabbit ear wound healing model enhanced healing. Finally, transfections mediated by complexes immobilized to the hydrogel resulted in up to 48% of NIH/3T3 cells expressing the transgene, and with expression spatially regulated along the topographical pattern. The delivery of DNA from a biocompatible and biodegradable hydrogel can enhance the applicability of non-viral gene delivery to tissue engineering applications.