SUMMARY Approximately 30% of U.S. adults suffer from tendon and ligament injuries. These injuries often require repair (e.g., rotator cuff) or reconstruction (e.g., anterior cruciate ligament (ACL)) to re-integrate the midsubstance with the adjacent bone. Recreating the zonal tendon-to-bone insertion site (i.e., enthesis) is critical to restoring normal function. Zonal enthesis formation involves anchoring collagen fibers, synthesizing proteoglycan-rich fibrocartilage, and mineralizing this fibrocartilage. The hedgehog (Hh) signaling pathway is critical to the formation of a zonal insertion during development by promoting the formation of unmineralized and mineralized fibrocartilage zones of the enthesis. 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, we utilize innovative transgenic murine ACL reconstruction models to elucidate the mechanisms of zonal tendon-to-bone repair in order to develop novel therapies to improve the repair outcome. Our preliminary data demonstrate that Hh signaling promotes zonal tendon-to-bone integration following ACL reconstruction. The objective of this proposal is to locally stimulate hedgehog signaling via scaffold delivery of Hh agonist to increase the formation of zonal tendon-to-bone attachments. Our hypothesis is that delivery of the agonist will stimulate the local progenitor cells to proliferate and then differentiate into fibrochondrocytes in the attachments, leading to improved tunnel integration. We will use multiplexed, mineralized cryohistology to monitor the zonal attachments and novel mechanical testing to assess tunnel integration strength. If the Hh pathway could be harnessed therapeutically, it would result in a paradigm shift in the treatment of these debilitating injuries.