Biomechanical influence of ECM remodeling on the developing enthesis

项目总结 尽管经过了几十年的努力，但在工程支架方面几乎没有成功的成果 修复末端后，组织将肌肉产生的力量顺利地从肌腱传递到骨骼。这一地区是 容易因机械负荷过大而失败，在许多情况下，界面不能进行手术 由于末端的复杂性和低细胞密度而重建。工程脚手架缺乏 修复受损牙齿的能力是设计主要模仿建筑和 成熟组织的组成。在支架设计中很少考虑的是组织 在发育过程中经历广泛的细胞外基质重塑，这在指导细胞 在成熟组织形成过程中的行为。研究人员一直无法利用这些有益的东西 脚手架设计提示由于对脚手架的组成、周转、组织和 发育中的肌肉骨骼组织的力学特性。 我们的长期目标是创造能够通过生物力学引导细胞重建受损的支架 因此，确定这是如何在体内完成的是至关重要的。为了实现我们的目标，我们需要 首先回答以下问题：1)细胞外基质在诱导过程中的表达动态是什么 编队？2)这些组件如何在3D中组织？3)该组织如何影响 机械环境？4)机械负荷是如何调节牙齿装配的？ 为了直接定量ECM蛋白掺入到发育中的腱、末端和软骨的基质中， 我们将用非典型氨基酸(NCAA)标记小鼠发育不同阶段的组织。这个 NCAA上的生物正交柄能够识别和定位新合成的蛋白质 使用点击化学。查看单个ECM组件相对于中的单元格的空间分布情况 在正在开发的末端，我们将使用光学透明方法来可视化包含 荧光标记的肌腱和软骨祖细胞。使用共焦显微镜和3D图像处理 算法，我们将描述细胞内、细胞和组织尺度上的形态如何由于 发育和胚胎能动性。为了检验这样一种假设，即末端的僵硬程度将发展为 更陡峭的梯度在胚胎和出生后的运动开始时，我们将利用我们的新原子力 一种显微镜方法，可以测量活组织内细胞和细胞外基质的硬度。这一假说将 通过使用肌肉发育不全的MDG模型直接进行测试，在该模型中，骨骼肌 在胚胎发育过程中，收缩能力受到抑制。通过将机械性能与成分相关联 和结构特征，我们希望确定一组支架参数，这将促进细胞 齿槽再生所必需的行为。

PROJECT SUMMARY Despite decades of work, there has been little success in engineering scaffolds that can successfully restore the enthesis, the tissue smoothly transfers muscle-generated force from tendon to bone. This region is prone to failure from excessive mechanical loading and in many cases the interface cannot be surgically reestablished due to the complexity and low cellularity of the enthesis. A reason engineered scaffolds lack the ability to restore the damaged enthesis is that the design predominantly mimics the architecture and composition of the mature tissue. What is rarely taken into consideration in scaffold design is that tissues undergo extensive ECM remodeling during development, which plays a significant role in directing cellular behavior in the formation of the mature tissue. Researchers have been unable to capitalize on these instructive cues for scaffold design due to the limited knowledge regarding the composition, turnover, organization and mechanical properties of developing musculoskeletal tissues. Our long-term objective is to create scaffolds that can biomechanically direct cells to rebuild damaged tissues; therefore, it is critical to identify how this is accomplished in vivo. To achieve our objective, we need to first address the following questions: 1) What are the dynamics of ECM expression over the course of enthesis formation? 2) How are these components organized in 3D? 3) How does this organization influence the mechanical environment? 4) How does mechanical loading regulate enthesis assembly? To directly quantify ECM protein incorporation into the matrix of developing tendon, enthesis and cartilage, we will label tissues at various stages of murine development with non-canonical amino acids (ncAAs). The bioorthogonal handles on the ncAAs enable the identification and localization of newly synthesized proteins using click chemistry. To see how individual ECM components are spatially distributed with respect to cells in the developing enthesis, we will use optical clearing methods to visualize murine tissues containing fluorescently labeled tendon and cartilage progenitors. Using confocal microscopy and 3D image processing algorithms, we will characterize how morphology at the intracellular, cellular and tissue scale change due to development and embryonic motility. To test the hypothesis that the stiffness across the enthesis will develop a steeper gradient upon the onset of embryonic and postnatal motility, we will utilize our novel atomic force microscopy method that can measure the stiffness of cells and ECM within viable tissues. This hypothesis will be directly tested by employing the mdg model of muscular dysgenesis, a mouse line in which skeletal muscle contractility is inhibited during embryogenesis. By correlating the mechanical properties with the compositional and structural characterization, we expect to identify a set of scaffold parameters that will promote cellular behaviors necessary for enthesis regeneration.