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

项目摘要 此F31奖学金申请提供了McKay矫形外科协作团队的培训计划 宾夕法尼亚大学的研究实验室和一项创新的研究计划，为铁木尔先生做好准备 卡马利蒂诺夫成为肌肉骨骼领域的独立科学家。大约30%的成年人在 美国人患有肌肉骨骼疾病，其中近一半是肌腱和 韧带受伤。这些损伤通常需要修复(例如，肩袖肌腱)或重建(例如，前 十字韧带(十字韧带)，以使肌腱中间物质与底层矿化骨重新结合。 重建带状肌腱到骨的附着点(即末端)是恢复正常组织功能的关键。 带状突起的形成包括锚定胶原纤维，合成富含蛋白多糖的纤维软骨，以及 矿化这个纤维软骨。Hedgehog(HH)信号通路是一种有效的带状肌腱到肌腱转导通路的调节器。 在生长和发育过程中的骨植入形成，因为它促进未矿化和 矿化的纤维软骨带。不幸的是，在传统的肌腱到骨骼修复中研究这一途径 一直具有挑战性，因为这些修复模型不能充分将胶原纤维固定在骨骼上，更不用说 产生纤维软骨区。相反，当肌腱移植物穿过时，韧带重建 骨隧道可以在隧道内产生带状附着物。因此，我们利用创新的转基因小鼠 前交叉韧带重建模型依次阐明带状肌腱-骨附着的形成机制 开发新的治疗方法以改善传统的肌腱修复效果。我们的初步数据表明 HH信号促进前交叉韧带重建后的带状肌腱-骨整合。这样做的目的是 建议定义Sonic Hedgehog(Shh)和India Hedgehog(IHH)在促进 成人肌腱到骨的修复。我们的假设是，HH通路在带状肌腱到肌腱的转归过程中起着双相作用。 前交叉韧带重建后的骨附着形成，Shh在修复的早期阶段起促进作用 祖细胞库的扩大和IHH促进纤维软骨的分化和成熟。我们将使用 复合矿化冷冻组织学和激光捕获显微切割结合微流控qPCR阵列进行监测 用于评估隧道综合强度的带状附着体形成和新的力学测试。瞄准 HH途径的特定配体将提供关键的机械洞察力，以了解某些配体是否比 在隧道整合过程的特定阶段具有重要意义，为小说研究开辟了新的途径 改善成人肌腱到骨的修复的治疗目标。

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.