36 Local non-viral gene delivery to immunomodulate and enhance fracture healing
Although 80 percent of fractures typically heal without complications, there is a small proportion (≤ 20%) that experience delayed healing or non-union. In patients with complications, there is a typical involvement of excessive pro-inflammatory cytokines and/or failure to resolve inflammation due to defective regulatory mechanisms. Thus, immunomodulation of the local fracture microenvironment, such as by enhancing anti-inflammatory cytokine production, could be an effective way to enhance fracture healing. Accordingly, the overall objective of this study is to develop an innovative gene-based therapy that mitigates the negative effects of inflammation while providing a structural template for new bone formation.
In this study, a collagen-hydroxyapatite scaffold is used as a platform for the delivery of pDNA, encoding for interleukin-1 receptor antagonist (IL-1Ra), complexed to the robust non-viral gene delivery vector, polyethyleneimine (PEI). We utilize pDNA encoding for GFP and Gaussia luciferase as reporter genes to determine the transfection efficiency and gene expression profile of PEI-pDNA nanoparticles in rat bone marrow-derived mesenchymal stem cells (MSCs). The effect of PEI-pDNA nanoparticles on cell viability was evaluated using cell titer blue assay. The conditioned medium of PEI-pIL-1Ra transfected cells was checked for IL-1Ra bioactivity using HEK-Blue-IL1b reporter cell line. The capacity of IL-1Ra gene activated scaffolds to mitigate IL-1b induced osteogenesis was determined by micro-CT analysis.
We have determined that PEI-pDNA nanoparticles can achieve a transient gene expression profile in rat bone marrow-derived mesenchymal stem cells (MSCs), with a transfection efficiency of 14.8±1.8%. The PEI-pDNA nanoparticles had a limited effect on cell viability after 10 days in culture, in terms of their metabolic activity. Cells transfected with PEI-pIL-1Ra were found to produce functional IL-1Ra, with the capacity to antagonize IL-1b-mediated alkaline phosphatase activity. Nanoparticles carrying pDNA encoding for IL-1Ra have been successfully incorporated into collagen-hydroxyapatite scaffolds, as verified by reporter assays. Osteogenic mineralization within scaffolds can be inhibited by exposure to IL-1b, and that this negative effect could then remarkably be mitigated in scaffolds incorporating PEI-pIL-1Ra nanoparticles.
The transient nature of therapeutic gene expression in our approach offers a key advantage as it potentially enables the preservation of the initial pro-inflammatory response to fracture, which is crucial to the healing cascade. Utilizing our therapy is also advantageous over other approaches including recombinant protein delivery and viral-based gene delivery methodologies. Studies are currently on-going to evaluate the therapeutic efficacy of our approach in a femoral osteotomy model in rats.