Localized and sustained release of plasmid DNA or siRNA from biomaterial scaffolds to promote osteogenesis

There is a critical need for bone replacement tissue. Each year in the United States, roughly 0.5-1 million patients experience problematic healing of a bone fracture. Tissue engineering and regenerative medicine seek to engineer lost or damaged tissue, often with a combination of biomaterial scaffolds, bioactive factors, and transplanted cells. This thesis aimed to develop gene delivery systems that promote osteogenesis, by investigating the hypothesis that the sustained and localized delivery from a biomaterial scaffold of DNA encoding for osteogenic factors or siRNA for knockdown of osteogenic-inhibitory genes will lead to improved osteogenesis. The release of plasmid DNA and DNA complexed with poly(ethyleneimine) (PEI-DNA) from gas-foamed poly(lactic-co-glycolic acid) (PLGA) scaffolds was tailored by the incorporation of varying amounts of alginate polysaccharide, and prolonged release of PEI-DNA nanoparticles from the PLGA scaffolds was attained for at least 35 days. The use of injectable biomaterial scaffolds was also examined, as these can be placed into a defect in a minimally invasive manner. PLGA can be dissolved in an FDA-approved solvent, tetraglycol, and when injected into an aqueous environment, it will solidify. Porous injectable PLGA scaffolds were fabricated by the addition of porogens and water into the PLGA solution. These porous scaffolds were thoroughly characterized and were shown to support the growth of cells throughout the bulk of the scaffold. Furthermore, DNA was incorporated into the scaffolds, and the release rate tailored by incorporation of the DNA into microspheres. Additionally, alginate hydrogels were examined for the release of DNA and DNA incorporated into calcium phosphate nanoparticles. These injectable hydrogels containing DNA-calcium phosphate nanoparticles and preosteoblastic cells were shown to promote heterotopic osteogenesis in vivo. In addition to the delivery of DNA for upregulation of osteogenic factors, another powerful option for the promotion of osteogenesis through genetic regulation could be to deliver siRNA to downregulate factors that inhibit bone formation. siRNA incorporated into calcium-crosslinked or photocrosslinked alginate hydrogels or collagen hydrogels was released in a sustained manner for at least 1-2 weeks. The siRNA remained bioactive, capable of silencing gene expression in cells. The treatment of human mesenchymal stem cells with siRNA against osteogenic inhibitory genes was examined to determine if they could be promoted to differentiate down the osteogenic lineage via this approach. However, the silencing of five different genes identified as potential targets from the literature (GNAS, P2RY11, adenosine kinase, noggin, and chordin) failed to have a significant effect on their differentiation, indicating the complexity of stem cell fate decisions. Overall, this thesis demonstrates the utility of biomaterial scaffolds for providing sustained and localized gene delivery. The released genetic material can influence cellular behavior, and these systems offer great promise for tissue regeneration.