骨-Sharpey纤维-肌腱复合体对腱-骨愈合的作用及机制研究

Tendon insertion injuries are commonly encountered in orthopaedic and sports medicine. These injuries are repaired by the tendon-bone interface, as we know, such repair between a soft and hard tissue (or tendon and bone) could be challenging due to inherent differences in their structure, composition and biology. Suboptimal repair of these injuries leads to recurrence and limits limb movement in affected people and lower their quality of life. Recently, it was reported that Sharpey’s fibers were responsive to mechanical loading such as running, bone related disorders such as osteoarthritis and other factors such as hormone. Because of such regulation, the Sharpey’s fibers may govern the status of tendon-bone junctions particularly during healing of injuries at these junctions. In our experience performing clinical studies and laboratory-based research, we have observed Sharpey’s fibers between tendons and bones, and the tendon-Sharpey’s fibers-bone complex was present in all phases of tendon-bone junction healing. Therefore, in this study we try to determine the effects of two essential proteins, fibronectin and integrin, which interact with the primary tendon-bone proteins, collagen, fibrin, proteoglycans, etc. on the characteristics of Sharpey’s fibers in the tendon-Sharpey’s fibers-bone complex during the healing of tendon insertion injuries. A series of in vivo and in vitro experiments will be performed to determine i) how fibronectin and integrin affect the morphological characteristics and molecular biology of Sharpey’s fibers; ii) the role of Sox9+/Scx+ puripotent cells in regulating the tendon-Sharpey’s fibers connection and bone-Sharpey’s fibers connection in the tendon-Sharpey’s fibers-bone complex; and iii) how different mechanical stresses affect the development of bone-Sharpey’s fibers-tendon complex and therefore affect the healing of tendon insertion injuries. The findings of this study will reveal the molecular mechanisms regulating the healing of tendon-bone junctions and will add new insights to effectively repair tendon insertion injuries.

腱止点损伤是创伤与修复领域常见伤病，软-硬组织结构差异大，修复困难。目前认为腱-骨愈合过程为基于Sharpey纤维的间接愈合向直接愈合逐渐转化的过程，Sharpey纤维的形成与转归对腱-骨愈合至关重要。我们发现：纤连粘蛋白和整合素可能促进骨-Sharpey纤维-肌腱”复合体形成，同时表达SOX9+/SCX+的多能干细胞可在不同牵伸应力作用下引起复合体重塑，形成腱止点典型四层结构。本课题拟明确腱-骨愈合早期纤连粘蛋白和整合素对Sharpey纤维形成的调控及对其成分、结构与功能的影响；分析SOX9+/SCX+的多能干细胞对“骨-Sharpey纤维连接”和肌腱干细胞对“肌腱-Sharpey纤维连接”形成及转化的调控；探讨不同应力对腱-骨界面干细胞分化的作用及其对“骨-Sharpey纤维-肌腱复合体”转归的影响，为促进腱-骨愈合提供新的思路和方法。

本项目围绕肌腱骨愈合过程中骨-Sharpey纤维-肌腱复合体（B-S-T）形成及其转归对愈合的影响这一科学问题，促进肌腱-骨愈合过程中纤维软骨形成和提高生物力学性能。从Sox9+/Scx+细胞、巨噬细胞、纤粘连蛋白和力学刺激多角度研究对腱骨愈合的影响，为促进腱骨愈合、提高生物力学性能提供实验证据。.按照项目计划，课题组从骨-Sharpey纤维-肌腱复合体的转归对愈合影响、愈合界面炎症细胞和干细胞转归规律和作用、纤粘连蛋白和力学刺激对腱骨愈合影响三个方面，取得了以下结果：1）发现了肌腱-骨愈合过程中的纤维软骨形成规律，明确了肌腱-骨愈合过程中骨-Sharpey 纤维-肌腱复合体（B-S-T）的形成及其转归特点；2）研究了不同时期腱-骨愈合界面 B-S-T 组成、细胞类型、细胞因子表达规律及其对愈合的影响，揭示了IL-10、TGF-β、Arg1等因子对腱骨愈合的影响；3）探讨了巨噬细胞在腱骨愈合过程中的作用和机制，发现不同类型巨噬细胞对愈合产生不同的影响，其中M2型巨噬细胞是促进愈合的重要细胞；巨噬细胞促进腱骨愈合的机制涉及到I型巨噬细胞向II型巨噬细胞的转化过程，II型巨噬细胞通过分泌IL-10、TGF-β1和TGF-β3促进了腱骨界面纤维软骨生成的影响，提高生物力学性能；4）明确了Sox9+/Scx+细胞对腱骨愈合的影响，发现了腱骨愈合过程中纤粘连蛋白促进干细胞迁移定植在损伤愈合部位，导致界面纤维软骨生成增多，生物力学性能提高；阐明愈合过程中不同应力通过改变愈合界面炎症细胞从而影响愈合效果。5）此外，还通过第四代微流控芯片技术在体外建立了腱骨愈合研究的梯度模型，同时在药物治疗、组织工程学等方面进行了探索。.综上所述，本课题在腱-骨愈合机制研究中，从腱-骨愈合的必经之路-Sharpey纤维的产生和转归入手，根据课题假设和课题技术路线，围绕 B-S-T 复合体形成的细胞及分子调控机制，以及在应力刺激作用下其组织结构重塑的特点，探讨和揭示了促进腱-骨愈合的最佳策略，较好地完成了课题任务，为以后的基础研究和临床治疗的应用提供了新的思路。