VEGF CHEMICALLY MODIFIED MRNA-LOADED MATRIX FOR VASCULARIZED BONE ENGINEERING

Spontaneous vascularization of critical-size bone grafts is not sufficiently rapid to ensure efficient bone formation. Angiogenic gene therapy suffers from safety issues and limitations. Delivery of chemically modified mRNAs (cmRNA) are a promising approach for the safe transient expression of transgenes. Here we investigated the efficacy of VEGF cmRNA to promote coupling of vascularization and bone formation in engineered grafts. Osteogenic grafts were prepared with human bone marrow mesenchymal cells (BMSC) and calcium phosphate microparticles in fibrin, containing 0.3 (Low), 3 (Med) or 30 (High) μg/ml of a VEGF-encoding cmRNA. Vascularization, osteogenic commitment and bone formation were assessed 1, 4 and 8 weeks after ectopic implantation in nude mice. The translational potential of VEGF cmRNA was tested in a critical femoral defect in rats. After 1 week in vivo , the Low dose stimulated vascular invasion and density compared to controls. Higher cmRNA doses (Med and High) significantly increased endothelial cell density on the outer portion of the constructs, but actually impaired blood vessel in-growth. Quantification of Osterix+ human cells showed that Low VEGF cmRNA promoted BMSC osteogenic commitment, but higher VEGF doses failed to do so. After 4 weeks, the Low VEGF condition stimulated deposition of dense mineralized matrix, while grafts with the Med and High doses mostly contained fibrous tissue. After 8 weeks, grafts with Low VEGF developed a full bone organ with mature bone tissue and bone marrow. In contrast, higher VEGF cmRNA doses caused significant bone loss. Preliminary data show that VEGF cmRNA delivery could also improve vascular density within the defect area in the critical model. Quantification of bone tissue is currently ongoing. These data show that a VEGF cmRNA-loaded matrix effectively promotes rapid vascularization and bone formation in engineered grafts, with a clear dose-dependency. The combination of safety and efficacy by cmRNA delivery within a biomaterial holds promise for translation in regenerative medicine.