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

XII 型胶原蛋白在细胞和基质介导机制中的关键作用，调节肌腱结构和功能在发育和损伤反应中的获得

基本信息

批准号：
10629438
负责人：
Nathaniel A. Dyment
金额：
$ 34.85万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-20 至 2026-05-31
项目状态：
未结题

项目摘要

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.

肌腱分层结构的建立对于机械功能至关重要。在胚胎发生过程中，肌腱祖细胞需要协调的细胞-细胞和细胞-基质通讯。在组装 ECM 之前组织成线性阵列并建立细胞间通信，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 ECM 的组织。众所周知，XII 胶原蛋白可通过形成桥来调节胶原纤维的组装。纤维之间，我们最近的数据表明，缺乏胶原蛋白 XII 的肌腱表现出纤维堆积减少和明显的纤维结构域的丧失，我们还发现这些肌腱的肌腱细胞紊乱。排列和间隙连接组织，表明胶原蛋白 XII 在细胞组织、细胞中的新作用沟通，并建立一个有组织的肌腱细胞网络，但是破坏的程度。由于胶原蛋白 XII 缺乏而导致的肌腱分层结构是由无序的细胞排列驱动的通信或无组织的 ECM 的沉积仍然不清楚，因此，我们的首要任务仍然不清楚。目标是确定胶原蛋白 XII 在调节肌腱细胞组织中的时间作用，分层我们的总体假设是，在肌腱发育和愈合过程中，结构和机械功能。除了 ECM 原纤维组装之外，胶原蛋白 XII 在 ECM 之前调节细胞排列和通讯在发育和愈合过程中沉积，这对于建立正常的肌腱结构功能至关重要。我们将使用新型组织靶向和可诱导的 Col12a1 敲除小鼠模型来专门针对肌腱在发育和愈合过程中，这些小鼠模型将与创新药物结合使用。多尺度方法来评估组织水平力学、细胞组织和通讯、纤维排列、目标 1 将定义胶原蛋白 XII 在调节细胞排列中的时间作用。肌腱生长和发育期间的 ECM 组装将被诱导 Col12a1 的靶向敲除。整个肌腱发育过程（Scx-Cre 驱动器；目标 1a）或细胞组织建立后（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进行评估。目标 2 将定义胶原蛋白 XII 的时间作用。使用 SMA-CreERT2 驱动器调节肌腱愈合过程中的细胞排列和 ECM 组装， Col12a1 敲低将针对腹膜衍生的祖细胞，这是愈合的主要贡献者在目标 2a 中，表达 αSMA 的细胞将在增殖期成为目标，而在目标 2b 中，将在增殖期结束时针对表达 SMA 的细胞来分离贡献我们将利用复杂且严格的分层结构/功能措施来通过建立时间序列来定义肌腱形成过程中细胞和 ECM 组件之间的相互作用这些创新研究将为未来的治疗提供指导。