This proposal combines the efforts of two independent research groups in a project to develop an advanced 3D bioprinted trachea toward repair of defects due to disease or trauma in pediatric patients. While there have been groundbreaking advances in tracheal repair that have leveraged 3D printing to fabricate patient-specific stents, the next stage in tracheal repair and pediatric-stage regenerative medicine as a whole, is to create tissues that not only restore function near term, but also achieve long term patency and will grow with the child. 3D bioprinting is uniquely poised to address this challenge, however, advances are needed in both the fabrication technologies and the biomaterials used. Here we propose to develop a novel 3D bioprinted trachea that can serve as scaffold for direct implantation and regeneration. By combining advanced 3D bioprinting of extracellular matrix (ECM) protein hydrogels with trachea decellularization and established animal models, we are uniquely positioned to integrate these capabilities and create a graft that can remodel into functional tissue. We have specifically chosen the trachea because of the critical need to develop technologies to match the unique anatomical structure of pediatric patients across a diverse range of ages. Our exciting preliminary data and detailed experimental plan supports this innovative approach. The R21 phase is designed to develop and demonstrate in vitro fabrication, mechanical integrity and biological function. Aim 1 will develop and characterize tracheal ECM bioink for 3D printing. Aim 2 will engineer the composition, structure and mechanics of the 3D printed tracheas to recapitulate that of native and decellularized tracheas. Aim 3 will compare the bioactivity of 3D printed tracheas to decellularized tracheas. If the R21 milestones are met, the R33 phase will evaluate the in vivo performance of the 3D bioprinted trachea in a porcine model to demonstrate formation of functional tissue (Aim 4) and the ability to grow over a 9 month period while maintaining patency (Aim 5). By the end of the project, we will have developed and validated proof-of-concept that a 3D bioprinted ECM protein scaffold can reform functional tissue in vivo and potentially eliminate the need for future surgeries.