Critical role of collagen XII in cell- and matrix-mediated mechanisms regulating acquisition of tendon structure and function in development and the injury response

肌腱层次结构的建立是肌腱功能发挥的关键。这一严密控制的过程 需要协调的细胞-细胞和细胞-矩阵通信。在胚胎发育过程中，肌腱前体细胞 在组装ECM之前，组织成线性阵列并建立细胞-细胞通信，这表明 细胞决定ECM的组织结构。细胞也在线性阵列内克隆扩张，这表明细胞外基质也 指示单元格组织。已知的是，XII胶原蛋白通过形成桥来调节胶原纤维的组装 在纤维之间，我们最近的数据显示，缺乏XII胶原蛋白的肌腱显示出纤维堆积减少和 丢失不同的纤维域。有趣的是，我们还发现这些肌腱有无序的腱细胞。 排列和缝隙连接组织，表明胶原XII在细胞组织、细胞 沟通，建立有组织的腱细胞网络。然而，扰乱的程度 XII胶原蛋白缺乏所致的肌腱分级结构是由细胞排列紊乱和 沟通或由杂乱无章的细胞外基质沉积仍未阐明。因此，我们最重要的是 目的是建立XII胶原蛋白在调节肌腱细胞组织中的时间作用，分层 肌腱发育和愈合过程中的结构和机械功能。我们的全球假设是，在 除了ECM纤维组装外，XII胶原蛋白还调节ECM之前的细胞排列和通讯 在发育和愈合过程中的沉积，这是建立正常的肌腱结构功能的关键。 我们将使用新的组织靶向和可诱导的Col12a1基因敲除小鼠模型来特定靶向肌腱 在发育和愈合过程中。这些鼠标型号将与创新的 多尺度方法评估组织水平力学、细胞组织和通信、纤维排列 和原纤维大小/组织。目标1将确定XII胶原蛋白在调节细胞排列中的时间作用 肌腱生长发育过程中的细胞外基质组装。将诱导靶向击倒Col12a1 在肌腱发育过程中(SCX-CRE驱动程序；目标1a)或在细胞组织建立之后(SCX- CreERT2驱动程序；目标1b)。还将使用3D细胞凝胶结构进行时间研究，以评估 体内没有混淆变量的组织形成。目标2将定义XII胶原蛋白的时间角色 在肌腱愈合过程中调节细胞排列和细胞外基质组装。使用SMA-CRERT2驱动程序， Col12a1基因敲除将针对周膜衍生的祖细胞，这些祖细胞是愈合的主要贡献者 受伤后的肌腱。在Aim 2a中，表达sma的细胞将在增殖期成为靶点，而 在目标2b中，sma表达细胞将在增殖期结束时作为靶点以分离贡献。 到ECM组件。我们将利用复杂和严格的层级结构/功能措施来 通过建立TIMA确定肌腱形成过程中细胞和ECM组装之间的相互作用 XII胶原蛋白在这些过程中的作用。这些创新的研究将为未来的治疗提供指导。

Establishment of tendon hierarchical structure is critical to mechanical function. This tightly controlled process requires coordinated cell-cell and cell-matrix communication. During embryogenesis, tendon progenitors organize into linear arrays and establish cell-cell communication prior to assembling ECM, suggesting that cells dictate ECM organization. Cells also clonally expand within linear arrays, suggesting that the ECM also dictates cell organization. Collagen XII is known to regulate collagen fibril assembly by forming bridges between fibrils, and our recent data show that collagen XII-deficient tendons exhibit reduced fibril packing and loss of distinct fiber domains. Interestingly, we also found that these tendons have disordered tenocyte arrangement and gap junction organization, indicating a novel role for collagen XII in cell organization, cell communication, and establishing an organized tenocyte network. However, the extent to which disrupted tendon hierarchical structure due to collagen XII deficiency is driven by disordered cellular arrangement and communication or by the deposition of disorganized ECM remains unelucidated. Therefore, our overarching goal is to establish the temporal roles of collagen XII in regulating tendon cell organization, hierarchical structure, and mechanical function during tendon development and healing. Our global hypothesis is that, in addition to ECM fibril assembly, collagen XII regulates cellular arrangement and communication prior to ECM deposition during development and healing, which is pivotal to establishing normal tendon structure-function. We will use novel tissue-targeted and inducible Col12a1 knockout mouse models to specifically target tendons during development and healing. These mouse models will be used in conjunction with an innovative multiscale approach to assess tissue level mechanics, cell organization and communication, fiber alignment, and fibril size/organization. Aim 1 will define the temporal roles of collagen XII in regulating cell arrangement and ECM assembly during tendon growth and development. Targeted knockdown of Col12a1 will be induced throughout tendon development (Scx-Cre driver; Aim 1a) or following establishment of cell organization (Scx- CreERT2 driver; Aim 1b). Temporal studies will also be conducted using 3D cell-gel constructs to evaluate tissue formation without confounding variables found in vivo. Aim 2 will define the temporal roles of collagen XII in regulating cell arrangement and ECM assembly during tendon healing. Using the SMA-CreERT2 driver, Col12a1 knockdown will be targeted to peritenon-derived progenitors, the primary contributors to healing tendon following injury. In Aim 2a, SMA-expressing cells will be targeted during the proliferative phase, while in Aim 2b, SMA-expressing cells will be targeted at the end of the proliferative phase to isolate contributions to ECM assembly. We will utilize sophisticated and rigorous measures of hierarchical structure/function to define the interplay between cell and ECM assembly in tendon formation through establishment of temporal roles for collagen XII in these processes. These innovative studies will provide guidance for future therapies.