Development of Gene Delivery Coating for Orthopedic Implants: Enhanced Gap Junction Communication in MSCs Accelerates Ischemic Bone Fracture Healing

Abstract

Delayed fracture healing is present in 22% of the 15 million fractures that occur every year. Patients experiencing delayed healing require additional interventions and experience pain for 6 months or longer. Current treatments require invasive surgeries, or non-invasive treatments that do not address both most common causes of delayed fracture healing, compromised osteogenesis and angiogenesis. The work in this dissertation demonstrates a local gene therapy approach to accelerate fracture healing by exogenously upregulating gap junction intercellular communication (GJIC) which improves osteogenic differentiation in mesenchymal stem cells (MSCs) and angiogenesis in endothelial cells. The design includes a polymeric coating for co-immobilizing a lentiviral vector for gene delivery of GJA1, the gene for Connexin 43, and an MSC-honing peptide for enhanced cell binding. This approach minimizes off-target transduction which is a risk of systemic gene delivery in-vivo. Risk reduction is achieved by immobilizing the viral particles to the surface of an orthopedic implant, as opposed to injection in vivo gene delivery, and by co-immobilizing with a cell-binding peptide, enhances the transduction efficiency of the system. Co-immobilization of lentiviral particles and a cell-binding peptide DPI (DPIYALSWSGMA) on titanium was achieved using chemical vapor deposition of a [2,2]paracyclophane-based polymer and μContact printing of DPI, resulting in a system more efficient than gene delivery alone. In vivo tests were done to observe osteogenic differentiation in MSCs an angiogenic tubule formation, as poor vascularity and bone formation are common causes of delayed fracture healing. The test concluded that transduction with viral particles in supernatant resulted in a 1.8 fold increase in GJIC which ultimately led to upregulation of early markers of osteogenic differentiation, but did not affect MSC migration. Upregulated GJIC resulted in the doubling of length and number of tubules formed both in mono- and co-culture in 2D tubule formation assays. Once the lentiviral particles were immobilized with the MSC-binding peptide, the combined therapy increased the number of cells exposed to the virus 2.7 fold, as well as the transduction efficiency of the gene therapy 33%. This design overcomes the common in vivo gene therapy limitations which include low transduction efficiency and off target effects. To observe the effects of upregulated GJIC on bone fracture healing, a mouse model with an ischemic fracture of the tibia was used with the tibial fracture being stabilized by an intramedullary rod coated with the polymeric gene delivery coating. The therapy resulted in increased anabolic activity at the callus, presenting early cartilage formation and a 46% larger callus size and revascularization of the fracture callus on day 7. By day 14, the gene therapy resulted in 40% more bone volume in the callus. Upregulation of Cx43 improved the vascularity in an ischemic tibial fracture and osteogenic differentiation resulting in increased bone volume at the fracture site. Together these results indicate that upregulated cell-cell communication in MSCs accelerated bone fracture healing, by inducing osteogenesis and angiogenesis at the fracture site. This work is a framework for an adaptable localized lentiviral gene delivery used to mitigate limitations of viral gene delivery, such as systemic viral delivery, low transduction efficiency and difficulties delivering to a target cell type in a heterogeneous cell population.