Combined polymeric scaffold delivery of condensed DNA, protein, and stem cells for bone tissue engineering

Bone tissue engineering has emerged to address the limitations associated with traditional therapies to promote bone regeneration. Gene therapy approaches that utilize the localized delivery of plasmid DNA encoding for osteogenic growth factors, via three dimensional biodegradable polymers, is a promising approach to facilitate bone formation. However, the low transfection efficiency of plasmid DNA is an obstacle to the success of this approach, and must be overcome to achieve clinical application. Poly(ethylenimine) (PEI) has been shown to be highly effective in increasing the transfection efficiency of plasmid DNA in various in vitro and in vivo studies. It is hypothesized that the local delivery of condensed DNA encoding for osteogenic growth factor within porous scaffolds leads to increased transfection efficiency and enhanced bone regeneration.; A scaffold delivery system incorporating condensed DNA was developed to demonstrate its capability in directing bone formation. Release of uncondensed DNA from poly(lactide-co-glycolide) scaffolds was rapid, while release was significantly slower when condensed DNA was incorporated. In vitro transfection was observed only in scaffolds incorporating condensed DNA, while no transfection was observed in scaffolds incorporating uncondensed DNA. Implantation of scaffolds incorporating condensed beta-galactosidase plasmid into the subcutaneous tissue of rats resulted in high gene expression for the entire 15-week duration of the experiment. The utility of this system for bone regeneration was tested by the delivery of a plasmid DNA encoding for bone morphogenetic protein-4 (BMP-4). Bone formation within a cranial critical sized defect was enhanced with sustained and localized delivery of the condensed DNA, as contrasted to the delivery of plasmid DNA. Further, this system can also be used for the combined delivery of condensed plasmid DNA encoding for BMP-4, vascular endothelial growth factor (VEGF), and human bone marrow stromal cells, which led to a significant increase in bone formation as compared to any combination of other conditions. The last result highlighted the importance of VEGF-mediated angiogenesis in bone tissue development. This thesis demonstrated the feasibility of attaining increased in vivo localized transfection via the delivery of condensed DNA, and this system can be used for simultaneous cell transplantation to enhance bone regeneration.