36 Local non-viral gene delivery to immunomodulate and enhance fracture healing

Lackington William A.1, Gomez Maria A.1, Vazquez Arlyng2/3, O’Brien Fergal J. 2/3, Thompson Keith1

  1. AO Research Institute Davos, Davos, Switzerland
  2. Tissue Engineering Research Group, Dept. of Anatomy & Regenerative Medicine, Royal College of Surgeons in Ireland, University of Medicine and Health Sciences, Dublin, Ireland
  3. AMBER Centre, Royal College of Surgeons in Ireland, Dublin, Ireland

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.