Development of a Polymeric Gene Delivery Vector for Application in Osteochondral Tissue Engineering
Needham, Clark James
Mikos, Antonios G.
Doctor of Philosophy
In this work, the polymeric gene delivery vector poly(ethyenimine)-hyaluronic acid (bPEI-HA)was optimized for transfection efficiency, incorporated into microparticles for controlled release, and applied directly in an oligo[poly(ethylene glycol) fumarate] (OPF) composite scaffold for osteochondral tissue generation. First, the effect of bPEI-HA synthesis parameters, specifically primary amines concentration, ligand targeting, and overall charge on the effectiveness of the vectors were investigated by altering the type and amount of hyaluronic acid (HA) oligosaccharide in the polymer. It was found that the length of the HA oligosaccharide had the most significant effect on cytotoxicity and transfection efficiency with human mesenchymal stem cells, while molar incorporation of HA, as opposed to the saccharide length and HA mass incorporation, had the greatest effect on zeta potential, but a minor effect on both cytotoxicity and transfection efficiency. Next, bPEI-HA/DNA complexes were incorporated into poly(DL-lactic-co-glycolic acid) (PLGA) microparticles and compared to microparticles containing bPEI/DNA complexes at several incorporation concentrations. It was found that the addition of HA to the bPEI vector allowed for increased loading concentration within these systems and significantly altered release kinetics without changing the morphology of the particles. Furthermore, the incorporation of HA onto the bPEI backbone significantly increased the transfection efficiency of the complexes released from the corresponding microparticle formulation. Finally, bPEI-HA was complexed with DNA encoding for the transcription factors RUNX2 and the SOX trio and incorporated into a composite hydrogel scaffold which was implanted into a rat osteochondral defect for 6 weeks. The in vitro release of this system was characterized and found to have a significant burst release over the first week of exposure to water. The in vivo analysis showed that the incorporation of DNA encoding for RUNX2 in the bone layer of these scaffolds significantly increased bone growth. The results also indicate that a spatially loaded combination of RUNX2 and SOX trio DNA loading leads to significantly better healing than empty hydrogels or either factor alone. Finally, the results of this study suggest that subchondral bone is necessary for correct cartilage healing. This research demonstrates the potential for gene delivery, and specifically bPEI-HA combined with transcription factor DNA, to be applied to in vivo osteochondral situations and result in improved tissue growth and quality.
Gene delivery; BPEI-HA; Osteochondral Regeneration