When soldiers and Veterans injure their extremities, tendons are likely to be disrupted. Without intact tendon-bone units, extremities cannot function properly. These injuries may be mid-substance tendon ruptures, or tears at the tendon-bone interface (TBI). The most difficult aspect of tendon reconstruction is adequate healing of this interface with bone. To date, the healing process of the TBI is poorly understood. The goals of this project are to determine the cellular and molecular mechanisms of TBI healing, and to develop tissue engineered TBI constructs that can be used for extremity reconstruction. It is hypothesized that tissue-engineered composite constructs consisting of tendon and bone would allow reconstruction with tendon-to-tendon and bone-to-bone healing. This would be faster and stronger, thus offering earlier mobilization and improved outcomes. Because there is a paucity of research on the process of TBI healing and tissue engineered TBI composite constructs, the following research objectives are proposed: The initial objective is to use an animal model of TBI healing based on the rat Achilles tendon to develop tendon-bone constructs. Decellularization of rat Achilles tendon-calcaneal bone segments and reseeding with adipoderived stem cells (ASCs) will be performed to create tissue engineered composite constructs. This process will be optimized with platelet-rich plasma (PRP) supplementation and the use of a novel tendon hydrogel as a carrier. The hydrogel is produced from decellularized tendon powder, is reconstituted into liquid form, and becomes a solid, yet porous gel at body temperature. It is hypothesized that the strength of bone-bone healing and tendon-tendon healing will exceed that of tendon-bone healing. Furthermore, repairs made with reseeded TBI grafts would be stronger than repairs performed with decellularized TBI grafts. In order to test this, tissue engineered rat Achilles TBI constructs will be re-implanted to assess fo short-term and long-term biocompatibility and repair strength. Techniques to assess healing will include immunohistochemistry, in situ hybridization, MTS measurement of tendon and bone-tendon junction biomechanical strength, high resolution Xray, and micro CT. Lastly, the ultimate goal would be translation to the production of human TBI composite grafts for clinical use in the reconstruction of extremity injuries. Human tissue engineered TBI constructs will be created based on optimized decellularization and reseeding techniques from the previous objectives. The specific anatomic structure to be produced initially is the distal phalanx-flexor tendon graft. The strength of native versus decellularized composite grafts will be tested. This proposal will ultimately allow the development of human composite TBI grafts for use in the reconstruction of traumatic and degenerative conditions of the extremities in soldiers and Veterans.