Temporal roles of Sonic and Indian Hedgehog in tunnel integration following anterior cruciate ligament reconstruction

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Project Summary This F31 fellowship application provides a training plan with a collaborative team in the McKay Orthopaedic Research Labs at the University of Pennsylvania and an innovative research plan to prepare Mr. Timur Kamalitdinov to be an independent scientist in the musculoskeletal field. Approximately 30% of adults in the United States suffer from a musculoskeletal condition, with almost half of those conditions being tendon and ligament injuries. These injuries often require repair (e.g., rotator cuff tendons) or reconstruction (e.g., anterior cruciate ligament (ACL)) to re-integrate the tendon midsubstance with the underlying mineralized bone. Recreating the zonal tendon-to-bone insertion site (i.e., enthesis) is critical for restoring normal tissue function. Zonal enthesis formation involves anchoring collagen fibers, synthesizing proteoglycan-rich fibrocartilage, and mineralizing this fibrocartilage. The Hedgehog (Hh) signaling pathway is a potent regulator of zonal tendon-to- bone insertion formation during growth and development as it promotes the formation of the unmineralized and mineralized fibrocartilage zones. Unfortunately, studying this pathway in traditional tendon-to-bone repair has been challenging because 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 passed through bone tunnels can produce zonal attachments in the tunnels. Therefore, we utilize innovative transgenic murine ACL reconstruction models to elucidate the mechanisms of zonal tendon-to-bone attachment formation in order to develop novel therapies to improve traditional tendon repair outcomes. Our preliminary data demonstrate that Hh signaling promotes zonal tendon-to-bone integration following ACL reconstruction. The objective of this proposal is to define the spatiotemporal roles of Sonic hedgehog (Shh) and Indian hedgehog (Ihh) in promoting adult tendon-to-bone repair. Our hypothesis is that the Hh pathway plays a biphasic role during zonal tendon-to- bone attachment formation following ACL reconstruction, with Shh acting in the early stages of repair to promote expansion of the progenitor pool and Ihh facilitating fibrocartilage differentiation and maturation. We will use multiplexed mineralized cryohistology and laser capture microdissection with microfluidic qPCR arrays to monitor the zonal attachment formation and novel mechanical testing to assess tunnel integration strength. Targeting specific ligands of the Hh pathway will provide key mechanistic insights into whether certain ligands are more important during specific stages of the tunnel integration process, opening new avenues of research into novel therapeutic targets to improve adult tendon-to-bone repair.