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

肌腱分级结构的建立对力学功能至关重要。这个严格控制的过程 需要协调的小区-小区和小区-矩阵通信。在胚胎发育过程中，肌腱祖细胞 组织成线性阵列，并在组装ECM之前建立细胞间通信，这表明 细胞决定ECM组织。细胞也在线性阵列中克隆扩增，这表明ECM也 决定了细胞的结构。已知胶原蛋白XII通过形成桥来调节胶原原纤维组装 我们最近的数据表明，胶原蛋白XII缺乏的肌腱表现出减少的原纤维包装， 不同纤维域的损失。有趣的是，我们还发现这些肌腱的肌腱细胞排列紊乱 排列和间隙连接组织，表明胶原蛋白XII在细胞组织，细胞 沟通，并建立一个有组织的tenocyte网络。然而，在多大程度上扰乱了 由于胶原蛋白XII缺乏导致的肌腱分级结构由无序的细胞排列驱动， 通信或由无序ECM的沉积造成的，仍然没有阐明。因此，我们的总体 目的是建立胶原XII在调节肌腱细胞组织中的时间作用， 结构和肌腱发育和愈合过程中的机械功能。我们的全球假设是， 除了ECM原纤维组装外，胶原XII在ECM形成之前调节细胞排列和通讯。 在发育和愈合过程中沉积，这是建立正常肌腱结构-功能的关键。 我们将使用新的组织靶向和诱导型Col 12 a1敲除小鼠模型来特异性靶向肌腱 在发育和愈合过程中。这些小鼠模型将与一种创新的 多尺度方法来评估组织水平力学，细胞组织和通信，纤维排列， 和原纤维尺寸/组织。目的1将明确胶原XII在调节细胞排列中的时间作用 和ECM组装。将诱导Col 12 a1的靶向敲低 在整个肌腱发育过程中（Scx-Cre驱动程序;目标1a）或在细胞组织建立后（Scx- CreERT 2驱动程序; Aim 1b）。还将使用3D细胞凝胶构建体进行时间研究，以评价 组织形成，无体内发现的混杂变量。目的2将定义胶原蛋白XII的时间作用 调节肌腱愈合过程中细胞排列和ECM组装。使用SMA-CreERT 2驱动程序， Col 12 a1敲除将靶向腹膜衍生的祖细胞，这是愈合的主要贡献者 肌腱损伤后。在Aim 2a中，在增殖期将靶向表达hSMA的细胞，而 在目标2b中，将在增殖期结束时靶向表达hSMA的细胞以分离贡献 至ECM组件。我们将利用复杂而严格的层次结构/功能措施， 通过建立时间序列来确定肌腱形成中细胞和ECM组装之间的相互作用， 胶原蛋白XII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.