Biomechanical influence of ECM remodeling on the developing enthesis

项目摘要 尽管几十年的工作，有工程脚手架，可以成功地成功， 恢复附着点，组织顺利地将肌肉产生的力量从肌腱传递到骨骼。该区域 并且在许多情况下该界面不能通过外科手术 由于附着点的复杂性和低细胞性而重新建立。工程支架缺乏 修复受损附着点的能力在于设计主要模仿结构， 成熟组织的组成。在支架设计中很少考虑的是， 在发育过程中经历广泛的ECM重塑，这在指导细胞增殖中起着重要作用。 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 由于对支架的组成、周转、组织和结构的了解有限， 发育中的肌肉骨骼组织的机械性能。 我们的长期目标是创造一种支架，它可以通过生物力学的方式引导细胞重建受损的细胞。 组织;因此，关键是要确定这是如何在体内完成的。为了实现我们的目标，我们需要 首先解决以下问题：1）在附着点过程中ECM表达的动态是什么 阵型？2)这些组件在3D中是如何组织的？3)这个组织如何影响 机械环境？4)机械负载如何调节端点组装？ 为了直接定量ECM蛋白掺入发育中的肌腱、附着点和软骨的基质中， 我们将用非标准氨基酸（ncAA）标记小鼠发育不同阶段的组织。的 ncAA上的生物正交手柄能够识别和定位新合成的蛋白质 使用点击化学。为了了解单个ECM组分如何在空间上相对于细胞分布， 在发育中的附着点，我们将使用光学透明方法来可视化小鼠组织， 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，我们将表征细胞内，细胞和组织尺度的形态如何变化， 发育和胚胎运动。为了检验整个附着点的刚度将发展为 在胚胎和出生后运动开始时，我们将利用我们的新原子力 显微镜方法可以测量活组织内细胞和ECM的硬度。这一假设将 通过采用肌肉发育不全的MDG模型直接进行测试， 在胚胎发生期间收缩性被抑制。通过将机械性能与组成相关联， 和结构表征，我们希望确定一组支架参数，将促进细胞 附着点再生所需的行为。

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.