Engineering Gene Delivery from Hydrogels for Regenerative Medicine Applications

Hydrogels are promising for regenerative medicine applications because they can be readily tailored and their physical properties closely resemble native tissues. Gene delivery from hydrogels can increase their bioactivity as tissue engineering scaffolds through the delivery of tissue inductive factors that promote tissue morphogenesis. However, insufficient levels and duration of expression limit their use as a therapy for tissue regeneration. Thus, the work in this thesis investigated the mechanisms to achieve effective gene delivery within hydrogels. Hydrogels crosslinked with cell degradable enzymes physically retain DNA based on their small mesh size, which requires cell migration to access the entrapped vector. Cell migration increased levels of transgene expression by enabling cells to contact greater amounts of DNA, but also facilitated the release of DNA from the hydrogel. Enhancing vector retention using cationically charged affinity peptides that reversibly bind DNA significantly enhanced the levels of transgene expression. However, excessive binding limited cellular internalization and gene transfer. Intermediate binding maximally enhanced transgene expression, which significantly enhanced neurite outgrowth in vitro when delivering vector encoding for neurotrophic factor. Incorporating hydrogel macroporosity was subsequently investigated to maintain structural integrity and facilitate the ingrowth of cells in vivo. Macroporosity supported the growth of transduced cells to prolong the localized elevated transgene expression levels. Prolonging the expression of gene therapy vectors encoding for VEGF demonstrated the capability of these hydrogels to direct the formation of tissue by inducing observable angiogenesis within hydrogel macropores. Hydrogels capable of gene delivery provide a combinatorial approach for nerve regeneration, with the hydrogel supporting neurite outgrowth and gene delivery inducing the expression of tissue inductive factors. Hydrogel design maximized neurite outgrowth in a neuronal co-culture model when the requirements for promoting gene transfer were effectively balanced with the requirements for supporting neurite outgrowth. These mechanistic studies identify the components hydrogel design parameters that promote gene transfer to facilitate tissue growth. Building complexities into this platform has the potential to provide the presentation of multiple physical and biochemical cues required to engineer functional tissue replacements.