PROJECT SUMMARY Pediatric long-segment airway defects are caused by congenital malformations or result from trauma, infection, or malignancy. Although rare, these defects are often fatal. There is currently no established surgical technique to repair long-segment tracheal defects and the reconstructive options remain heroic. Tissue engineering has the potential to replace failed tissue with a normal, living organ. Despite its potential, clinical outcomes of tissue engineered tracheal grafts (TETG) have been poor. The main barriers to translation of tracheal replacement are graft collapse and delayed epithelialization. We assessed the performance of partially decellularized tracheal grafts (PDTG) in our mouse model of orthotopic tracheal replacement. Using resorbable biomaterials to stabilize PDTG, we created a Composite Tracheal Graft (CTG). We hypothesize the CTG can improve overall survival in long-segment tracheal replacement, attenuate graft collapse, promote extracellular matrix (ECM) production and SAE differentiation. To test this hypothesis, we will first assess how CTG promotes ECM regeneration in the tracheal cartilage. In our first aim, we will implant PDTG and CTG in a mouse model of tracheal replacement and quantify ECM production and mechanical properties. Using a conditional knock-out of chondrocyte-mediated ECM production, we will then assess the impact on graft chondrocytes on ECM production. In our second aim, we will define how SAE differentiation is promoted by CTG. We hypothesize that modification of graft dimensions with splinting reduces wall shear stress (WSS) resulting in improved epithelial differentiation. To test the effect of WSS on SAE differentiation, we will implant PDTG and CTG of normal and small diameter, thus increasing WSS by reducing graft radius. To quantify WSS, we will use computational fluid dynamics (CFD) to topographically map WSS through the grafts and correlate these values with quantitative immunofluorescenceof neo-epithelium. Finally, we will validate CTG performance in an ovine model of tracheal replacement in our third aim. Using routine radiographic and endoscopic surveillance, we will quantify animal survival, clinical manifestations, graft dimensions, and graft regeneration. This proposal advances the field of airway tissue engineering through the development of a composite tissue engineered tracheal graft and defining the mechanical factors contributing to graft regeneration.