Summary Approximately 30% of U.S. adults suffer from tendon and ligament injuries, which frequently occur near insertion sites into bone (i.e., entheses) and do not spontaneously heal. Growth and development studies have demonstrated a critical role for hedgehog (Hh) signaling in driving zonal enthesis formation but it's role in adult enthesis repair is largely unknown. Zonal enthesis formation involves anchoring collagen fibers, synthesizing proteoglycan-rich fibrocartilage, and mineralizing this fibrocartilage. Hh promotes this fibrocartilage formation. Unfortunately, studying this pathway in traditional tendon-to-bone repair has been a challenge since these repair models do not sufficiently anchor collagen fibers to bone, much less produce zones of fibrocartilage. Conversely, ligament reconstructions, where a tendon graft is placed through bone tunnels, can produce zonal attachments. Therefore, ligament reconstruction models, such as the anterior cruciate reconstruction model proposed in this application, can be employed to study the mechanisms that regulate zonal tendon-to-bone repair in the adult. This proposal will address this gap in knowledge by targeting the hedgehog pathway genetically and pharmacologically during tendon-to-bone repair following ACL reconstruction in novel transgenic mouse models. We will define the roles of the hedgehog pathway in specific stages of the repair response from the expansion of the progenitor pool to production of fibrocartilage and bone within zonal tendon-to-bone attachments during the tunnel integration process. By modulating the pathway pharmacologically, we will determine the potential for this pathway to be targeted in a translational fashion that could lead to novel therapies in the future. Our central hypothesis is that the Hh pathway is a critical positive regulator of zonal enthesis formation in the adult and therefore stimulation of the pathway will improve tendon- to-bone repair.