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：
9552708
负责人：
Sarah Calve
金额：
$ 36.82万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9552708
关键词：
Address Adolescent Adult Affect Affinity Chromatography Algorithms Amino Acids Architecture Atomic Force Microscopy Behavior 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 Imagery 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 overexpression perlecan postnatal progenitor protein degradation regenerative therapy repaired scaffold success 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.

项目概要尽管经过了几十年的努力，在能够成功地实现这一目标的工程脚手架方面却几乎没有取得成功。恢复附着点后，组织顺利地将肌肉产生的力从肌腱转移到骨骼。该地区是容易因过度的机械负载而发生故障，并且在许多情况下，该接口无法通过手术进行由于附着点的复杂性和低细胞结构而重新建立。工程支架缺乏的原因恢复受损端点的能力在于该设计主要模仿了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 ECM 重塑，这在指导细胞中发挥着重要作用成熟组织形成中的行为。研究人员无法利用这些指导性的成果由于对组成、周转、组织和结构的了解有限，因此对脚手架设计的提示发育中的肌肉骨骼组织的机械特性。我们的长期目标是创建能够通过生物力学指导细胞重建受损的支架纸巾；因此，确定这是如何在体内实现的至关重要。为了实现我们的目标，我们需要首先解决以下问题：1）在插入过程中 ECM 表达的动态是什么形成？ 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）机械负载如何调节附着点组装？为了直接量化 ECM 蛋白掺入发育中的肌腱、附着点和软骨的基质，我们将用非规范氨基酸（ncAA）标记小鼠发育各个阶段的组织。这 ncAA 上的生物正交处理能够识别和定位新合成的蛋白质使用点击化学。了解各个 ECM 成分相对于细胞的空间分布在发育中，我们将使用光学透明方法来可视化含有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、细胞和组织尺度的形态学如何因发育和胚胎运动。为了检验这样的假设：附着点的刚度会产生在胚胎和出生后运动开始时梯度更陡，我们将利用我们新的原子力显微镜方法可以测量活组织内细胞和 ECM 的硬度。这个假设将通过采用肌肉发育不全的 mdg 模型直接进行测试，该模型是一种小鼠品系，其中骨骼肌胚胎发生过程中收缩力受到抑制。通过将机械性能与成分相关联和结构表征，我们期望确定一组支架参数，以促进细胞附着点再生所必需的行为。