Anterior cruciate ligament (ACL) injuries are one of the leading causes of training and sports related injuries and contribute to post-traumatic osteoarthritis. Torn ACLs are commonly reconstructed, instead of repaired, utilizing a tendon graft that is passed through bone tunnels created in the femur and tibia positioned at the native ACL footprints. Even with advancements in surgical technique and rehabilitation strategies, graft failure and recurrent knee instability are unfortunate complications. Additionally, rehabilitation following surgery is critical to a successful outcome, with premature return to activity resulting in an increased risk of graft failure and reinjury. Consequently, there is an unmet clinical need to improve and expedite treatment of these debilitating injuries, to get patients back to their active lifestyles while minimizing the risk of graft failure. Recreating the zonal tendon- to-bone insertion site (i.e., enthesis) is critical to restoring normal function following these injuries. 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 this zonal insertion during growth and development by promoting the formation of unmineralized and mineralized fibrocartilage zones of the enthesis. Recent studies by our group demonstrate that this pathway has a similar role in producing fibrocartilage within zonal attachments in the bone tunnels following ACL reconstruction. Therefore, our long- term goal is to develop therapies that leverage this pathway to improve repair outcomes and expedite recovery. The objective of this proposal is to locally deliver a small molecule Hh signaling agonist to increase the formation of zonal tendon-to-bone attachments. We will conduct an extensive in vitro release study to test the duration and bioactivity of SAG released from our innovative BiLDS scaffolds in Aim 1. We will then translate this novel delivery system to treat NZW rabbits following ACL reconstruction in Aim 2 via localized delivery of the agonist in the bone tunnels. Our hypothesis is that delivery of the agonist will stimulate the local progenitor cells to proliferate and then differentiate into fibrochondrocytes in the attachments. To test this hypothesis, in Aim 2, we will assess the extent of tunnel integration by measuring proliferation of the progenitor pool, zonal attachment formation via mineralized cryo-histomorphometry, and integration strength via anterior-posterior drawer and uniaxial mechanical testing. Additionally, we will determine the long-term effects of agonist release on joint health by measuring changes to the synovium and articular cartilage. Finally, we will use activity monitoring post-surgery and longitudinal in vivo MRI imaging to track the animal rehabilitation and healing response. Successfully harnessing the Hh pathway therapeutically could result in...