IN SITU BMSC SEEDING OF 3D PRINTED SCAFFOLDS USING CELL-RELEASING HYDROGELS In the current proposal, we will investigate the effect of in situ BMSC seeding of 3D printed polyHIPE grafts on bone formation. The combination of the cell-releasing hydrogel carrier with advanced 3D manufacturing technologies has the potential to generate a graft with patient-specific geometries and enhanced bone regeneration. To this end, we recently developed a multi-modal printing system to generate tissue engineered scaffolds that mimic the native structure of bone. In this system, fumarate-based emulsion inks with hierarchical porosity (polyHIPE) were reinforced with a poly(lactic acid) shell to achieve simultaneous improvements in permeability and compressive properties. In addition to the design of scaffold properties, success as a bone graft depends on the delivery or recruitment of bone marrow stromal cells (BMSC) that aid regeneration through a variety of mechanisms including serving as new centers of bone formation and secretion of trophic factors that modulate inflammation, stimulate angiogenesis, and limit fibrosis. We have developed a biodegradable cell- releasing hydrogel carrier that cures in situ to seed our 3D printed bone graft with BMSC at the time of surgery with subsequent cell release onto the scaffold after the initial inflammatory period. In situ BMSC seeding of scaffolds has the potential to minimize the costs, treatment delays, and regulatory hurdles of extended pre- culture periods. At the end of the grant period, we will have identified the target cell-release profile that improves cell retention and BMSC-initiated osteogenesis in an ectopic bone model. This will provide strong evidence of the osteoinductive character of the cell-seeded bone graft and support future investigation in a large animal orthotropic model (R01). In addition to improving bone grafting procedure, these studies will validate a method of BMSC delivery that can be used in a broad range of applications.