Studying Mechanotransduction in Late Embryonic Development to Inform Tendon Tissue Engineering

项目总结/摘要 肌腱和韧带损伤是残疾和疼痛的常见原因。在许多情况下，受伤的组织不能 直接修复，必须用移植材料替换。理想情况下，组织工程生物材料可以 用于此目的;然而，没有组织工程构建体已成功地用于重建人类， 肌腱或韧带断裂。肌腱置换失败的主要原因是， 大多数组织工程方法不能复制正常的肌腱发育。无支架技术 基于细胞自组装的细胞能够产生与早期细胞的结构和力学相匹配的组织。 胚胎腱然而，它们不能经历肌腱发育后期的关键阶段， 胶原原纤维伸长并融合在一起，产生基本上更硬和更强的材料。一个原因 这是因为该结构缺乏通常由肌肉在体内提供的机械刺激。事实上， 鸡胚肌肉活动在肌腱发育后期达到峰值，这种肌肉活动负责 增加胚胎肌腱的模量。尽管如此，尽管结构的机械载荷 尽管改善了组织力学，但它们仍然无法匹配机械性能的数量级增加， 在鸡胚肌腱中观察到。一个关键的障碍是缺乏关于 机械转导机制决定细胞对机械刺激的反应， 肌腱发育了解肌腱细胞如何对机械刺激做出反应，不仅因为肌肉 但是在发育过程中发生的组织结构和力学的局部变化也是必要的 开发出可以成功复制肌腱功能的生物材料。因此，本项目的目标是 确定介导组织结构多尺度变化的机械传导机制， 在后期肌腱发育过程中观察到的力学。具体而言，该项目将（1）确定多尺度 结构和力学变化发生在后期肌腱发育和（2）决定 驱动这些变化的机械转导机制。总的假设是，细胞感觉机械 通过细胞-细胞和细胞-基质相互作用的组合刺激，并且这些机械转导事件 是驱动肌腱正常发育的关键这将通过抑制胚胎肌肉来评估 通过位点特异性基因敲除在胚胎肌腱中的活性和扰动机械传导信号 在鸡胚的卵外培养期间。这些手法对肌腱结构和力学的影响 将通过多尺度力学测试、计算建模和 超微结构成像这项工作是第一次调查的机械转导机制驱动的 结构和机械变化观察后期肌腱发展。调查结果将提供 增强组织工程结构的基础，以开发可成功替代 病变的肌腱和韧带

Project Summary/Abstract Tendon and ligament injuries are a common cause of disability and pain. In many cases, the injured tissue cannot be repaired directly and must be replaced with a graft material. Ideally, a tissue engineered biomaterial could be used for this purpose; however, no tissue engineered construct has been successfully used to reconstruct human tendon or ligament ruptures. A primary reason for the failure in producing successful tendon replacements is that most tissue engineering approaches do not replicate normal tendon development. Scaffold-free techniques based on cellular self-assembly are able to generate tissues that that match the structure and mechanics of early embryonic tendon. However, they are unable to undergo a critical phase in late tendon development where the collagen fibrils elongate and fuse together generating a substantially stiffer and stronger material. One reason for this is that the constructs lacked the mechanical stimulation normally provided in vivo by muscles. In fact, chick embryo muscle activity peaks during late tendon development and this muscle activity is responsible for increasing the modulus of embryonic tendons. Nevertheless, while mechanical loading of constructs does improve tissue mechanics, they still fail to match the order-of-magnitude increase in mechanical properties observed in embryonic chick tendons. A critical barrier is the lack of knowledge regarding the mechanotransduction mechanisms that determine the cellular response to mechanical stimulation and drive late tendon development. Understanding how tendon cells respond to mechanical stimuli due to not only muscle loading but also the local changes in tissue structure and mechanics that occur during development is necessary to develop biomaterials that can successfully replicate tendon function. Therefore, the objective of this project is to identify the mechanotransduction mechanisms that mediate the multiscale changes in tissue structure and mechanics observed during late tendon development. Specifically, this project will (1) identify the multiscale structural and mechanical changes that occur during late tendon development and (2) determine the mechanotransduction mechanisms driving these changes. The overall hypothesis is that cells sense mechanical stimuli through a combination of cell-cell and cell-matrix interactions and that these mechanotransduction events are essential for driving proper tendon development. This will be evaluated by inhibiting embryonic muscle activity and perturbing mechanotransduction signaling in embryonic tendons via site-specific gene knockout during ex ovo culture of chicken embryos. The effects of these manipulations on tendon structure and mechanics will be determined by a novel combination of multiscale mechanical testing, computational modeling, and ultrastructural imaging. This work is the first investigation of the mechanotransduction mechanisms driving the structural and mechanical changes observed during late tendon development. The findings will provide the foundation for enhancing tissue engineered constructs to develop biomaterials that can successfully replace diseased tendons and ligaments.