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

项目摘要 这个F31奖学金申请提供了一个培训计划，在麦凯骨科的协作团队 宾夕法尼亚大学的研究实验室和一项创新的研究计划， Kamalitdinov成为肌肉骨骼领域的独立科学家。大约30%的成年人在 美国患有肌肉骨骼疾病，其中几乎一半是肌腱和 韧带损伤这些损伤通常需要修复（例如，旋转套肌腱）或重建（例如，前 交叉韧带（ACL）），以重新整合肌腱中间物质与下面的矿化骨。 重建带状肌腱-骨插入部位（即，附着点）对于恢复正常组织功能至关重要。 带状附着点的形成包括锚定胶原纤维，合成富含蛋白多糖的纤维软骨， 矿化纤维软骨Hedgehog（Hh）信号通路是带状腱-肌间连接的有效调节因子。 在生长和发育过程中，骨插入形成，因为它促进了未矿化和 矿化纤维软骨区。不幸的是，在传统的肌腱-骨修复中研究这一途径， 因为这些修复模型不能充分地将胶原纤维锚在骨上， 产生纤维软骨区。相反，肌腱移植物穿过的韧带重建 骨隧道可以在隧道中产生区域性附着。因此，我们利用创新的转基因小鼠， ACL重建模型，以阐明带状肌腱-骨附着形成的机制， 开发新的治疗方法来改善传统的肌腱修复效果。我们的初步数据表明， Hh信号促进ACL重建后带状腱-骨整合的目的 一个建议是定义音速刺猬（Shh）和印度刺猬（Ihh）在促进 成人腱骨修复我们的假设是，Hh通路在带状肌腱-- ACL重建后的骨附着形成，Shh在修复的早期阶段起作用， 祖细胞库的扩增和Ihh促进纤维软骨分化和成熟。我们将使用 多路矿化冷冻组织学和激光捕获显微切割与微流体qPCR阵列，以监测 区域连接形成和新型机械测试，以评估隧道整合强度。靶向 Hh途径的特异性配体将提供关键的机制见解，以了解某些配体是否更容易被 重要的是在隧道集成过程的特定阶段，开辟新的研究途径， 治疗目标，以改善成人肌腱骨修复。

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.