PROJECT SUMMARY / ABSTRACT 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 decellularized TETG (dcTETG) in our mouse model of orthotopic tracheal replacement. We identified that decellularized TETG can regenerate, restoring a functional surface airway epithelium (SAE), however outcomes are limited due to graft collapse. Using resorbable biomaterials to stabilize dcTETG, we created a Composite TETG (CTETG). We hypothesize the CTETG 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 CTETG promotes ECM regeneration in the tracheal cartilage. In our first aim, we will implant dcTETG and CTETG 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 CTETG. 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 dcTETG and CTETG 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 TETG and correlate these values with quantitative immunofluorescence of neo-epithelium. Finally, we will validate CTETG 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.