The vital wound healing process is critically dependent on sequential and overlapping stages that can be hindered by factors including diseases/conditions, systemic variables, or aging. Thus, there exists a need to aid the body when it is unable to repair tissue on its own. Currently, there is limited success in bioengineered matrices for tissue repair. Vascularization of the scaffold remains the primary cause of construct and host-integration failure. Vascularization ensures a route for nutrient, oxygen, and waste transport to facilitate cell survival deep within the construct for new tissue formation. Thus, we proposed to develop a porous hyaluronic acid (HA) hydrogel system to deliver non-viral genes to promote angiogenesis for improved wound healing.

Our lab had previously shown that porous HA hydrogels facilitated more cellular infiltration in vivo in murine subcutaneous implant and wound healing models when compared to non-porous gels of the same composition. With newfound validation that porous hydrogel architecture was necessary, new techniques were developed and reduced the microsphere template processing time from 17-22 h to 1-2 h with introduction of liquid handling to minimize human error and inconsistencies with comparable results. The investigation of natural scaffolds in cutaneous wound healing further demonstrated that porous hydrogel architecture results in more rapid wound closure with more stable, mural-covered blood vessels, even when compared to fibrin—a scaffold commonly chosen due to its inherent role in natural wound healing. Incorporation of non-viral DNA aimed to enhance the therapeutic capacity of the scaffolds and was tested by loading DNA via caged nanoparticle encapsulation (CnE) and comparing it to methods utilized in literature. DNA loading was paired with hyaluronidase (HAase) treatment of the gel to loosen the polymer network for cell-mediated degradation and resultant transfection. In vitro and in vivo transfection was significantly improved with CnE loaded gels and transgene expression was a function of HAase concentration. HAase treatment was then applied to efforts in developing a sequential gene delivery system. Two hydrogel systems (surface coated and two phase) were tested in vitro that led to an in vivo application of a hybrid system. Bioluminescence imaging showed that in vivo transgene expression profiles suggested dual gene delivery rather than sequential. Investigation of the effect of spatio-temporal presentation of pro-angiogenic plasmids showed that a homogenous presentation of pVEGF and pPDGF polyplexes resulted in more rapid wound closure and mural-covered blood vessels than hydrogels with spatially separated polyplexes by day 7.

With further optimization and modification, we believe the proposed hydrogel system(s) are capable of controlled delivery of non-viral genes for cutaneous tissue repair. Although the focus of this dissertation was focused on repairing skin wounds, a well developed hydrogel system can deliver any type or combination of genes to yield numerous therapeutic effects.