Biomechanical influence of ECM remodeling on the developing enthesis

ECM重塑对发育中的生物力学影响

基本信息

批准号：
10263389
负责人：
Sarah Calve
金额：
$ 32.86万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10263389
关键词：
3-Dimensional Address Adolescent Adult Affect Affinity Chromatography Algorithms Amino Acids Architecture Atomic Force Microscopy Biomechanics Cartilage Cells Cellular Morphology Cellularity Chemistry Cicatrix Collaborations Confocal Microscopy Cues Development Developmental Process Disease Embryo Embryonic Development Engineering Environment Extracellular Matrix Extracellular Matrix Proteins Failure Forelimb Fructose Growth Guidelines Heparan Sulfate Proteoglycan Immunofluorescence Immunologic Individual Instruction Knowledge Label Laboratories Maps Mass Spectrum Analysis Measurement Measures Mechanics Metabolic Methionine Methods Modeling Modulus Morphology Mus Muscle Musculoskeletal Natural regeneration Operative Surgical Procedures Optics Play Polymers Protein Biosynthesis Protein Dynamics Proteins Research Personnel Role Skeletal Muscle Structure Techniques Tendon force Tendon structure Testing Three-Dimensional Image Three-Dimensional Imaging Time Tissue Viability Tissues Work analog base bone cell behavior cell motility comparative design functional group high resolution imaging image processing imaging biomarker in vivo innovation interstitial knock-down laser capture microdissection mechanical load mechanical properties microscopic imaging novel perlecan postnatal progenitor protein degradation regenerative therapy repaired scaffold success three-dimensional visualization tissue repair

项目摘要

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.

项目摘要尽管工作了数十年，但工程脚手架的成功很少恢复源头，组织将肌肉生成的力平滑地从肌腱转移到骨头。这个区域是容易因机械加载过多而失败，在许多情况下，界面不能通过外科手术由于埃文的复杂性和低细胞性，重新建立了。设计脚手架的原因缺乏恢复受损分节的能力是设计主要模仿体系结构和成熟组织的组成。脚手架设计中很少考虑的是组织在开发过程中进行大量ECM重塑，在指导细胞中起着重要作用成熟组织形成的行为。研究人员无法利用这些启发性脚手架设计的提示由于有关组成，营业额，组织和发展肌肉骨骼组织的机械性能。我们的长期目标是创建可以生物力学的脚手架来指导细胞重建损坏组织；因此，确定如何在体内完成这一点至关重要。为了实现我们的目标，我们需要第一个解决以下问题：1）ECM表达在整个过程中的动态是什么编队？ 2）这些组件如何在3D中组织？ 3）这个组织如何影响机械环境？ 4）机械载荷如何调节源组件？直接将ECM蛋白掺入到发育中的肌腱，灌肠和软骨的基质中，我们将用非典型氨基酸（NCAA）在鼠发育的各个阶段将组织标记。这 NCAA上的生物正交手柄可实现新合成蛋白的识别和定位使用点击化学。查看单个ECM组件如何相对于细胞的空间分布发育中的ENTHENES，我们将使用光学清除方法可视化含有的鼠组织荧光标记为肌腱和软骨祖细胞。使用共聚焦显微镜和3D图像处理算法，我们将表征由于细胞内，细胞和组织量表的形态如何变化开发和胚胎运动。为了测试以下假设在胚胎和产后运动开始时，我们将利用我们的新型原子力显微镜方法，可以测量细胞和ECM在可行组织中的刚度。这个假设将会通过采用肌肉发育不全的MDG模型，可以直接测试骨骼肌的小鼠系胚胎发生过程中抑制收缩力。通过将机械性能与组成相关联和结构表征，我们希望确定一组脚手架参数，以促进细胞再生所必需的行为。