LOCALIZED GENE THERAPY TO STIMULATE BONE REPAIR

Participants: D. Scott, E.P. Frankenburg, K.A. Sweet, B. Nolan, R. Taylor, D.C. Kayner, M.W. Stock, S. A. Goldstein

Keywords: localized gene therapy, fracture healing, tissue engineering

Introduction

While most fractures heal predictably and robustly, a class of injuries characterized as "fractures at risk" have been demonstrated to heal poorly and require surgical intervention and often augmentation with bone graft or bone graft substitutes. Many factors have been demonstrated to be associated with the lack of healing, ranging from the degree of trauma, mechanical stability, amount of soft tissue trauma, anatomical site, and potential underlying metabolic or physiologic disorders. Autologous bone grafts have been established as the gold standard of treatment, however, donor site morbidity, as well as limitations in available volume of material continues to stimulate a search for alternative sources or therapies. Substitutes have included allografts, calcium phosphate cements, and many other osteoconductive or space occupying materials.

Since the discovery of specific growth factors and cytokines that stimulate bone repair, many investigators have devised ways for delivering recombinant proteins to fracture sites or bone defects in an effort to induce formation. The use of these recombinant proteins has been met with mixed success due to rapid degradation of the delivered proteins, dosing difficulties and other unknown factors. Our laboratory, in collaboration with several colleagues, has developed an alternative strategy utilizing localized transient gene therapy to promote bone repair. During the past several years we have demonstrated that plasmid DNA encoding for osteotropic factors can be delivered by means of a three-dimensional structural matrix surgically implanted into a bone defect. These studies demonstrated in situ transfection of wound repair fibroblasts, resulting in expression and synthesis of the encoded protein and subsequent formation of bone. The purpose of this current study is to further investigate the potential for utilizing localized gene therapy to promote bone formation and, in particular, evaluate specific genes or combinations of genes, and matrices on the repair process.

Materials and Methods

The study utilized our previously developed femoral defect model in 400-500 gram Sprague-Dawley rats. External fixators are secured on the femurs bilaterally after percutaneous placement of four pins using specialized instrumentation and guides. After fixation, 5 mm defects are created in the mid diaphyses of both limbs, creating a critical sized defect which we have shown leads to a non-union in 100% of animals. The gene matrix combinations are then surgically placed in the defects and the wounds are closed. This study involves the use of nine groups of animals with at least five animals per group. Combinations of genes, including PTH _1-34, as well as several angiogenic and mitogenic factors are being utilized. Variations in the delivery matrix are also being evaluated. Weekly radiographs are being taken post surgically until eight weeks when all animals are euthanized. Following euthanasia, repair sites are evaluated using microradiographic, histologic and biomechanical assays.

Progress

Approximately 50 animals have been entered into the study and most have already been euthanized. Early results suggest that the model effectively serves as an assay for promotion of bone formation. Studies during the next several months will finalize the quantification of bone formation as a function of gene and matrix combinations.