Functional Properties Of Extracellular Matrix

细胞外基质的功能特性

基本信息

批准号：
8736808
负责人：
PETER J. BASSER
金额：
$ 37.07万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8736808
关键词：
Address Affect Aging Atomic Force Microscopy Behavior Biochemical Biological Biological Models Biomechanics Bovine Cartilage Cartilage Cell Adhesion Charge Chemical Structure Chemicals Childhood Collaborations Collagen Collagen Fiber Complex Data Development Disease Disease Resistance Electrostatics Engineering Ensure Environment Equilibrium Exhibits Extracellular Matrix Fluorescence Fluorides Foundations Gel Goals Growth Human Hyaluronic Acid Hydration status Hydrogels Image Individual Joints Knowledge Lead Length Lubrication Maps Measurement Measures Mechanics Microscopic Modeling Molecular Neutrons Osmotic Pressure Osteoarthrosis Deformans Persons Phase Physics Physiological Pilot Projects Polymers Procedures Property Protective Agents Proteoglycan Public Health Quartz Regenerative Medicine Relaxation Research Personnel Resistance Resolution Roentgen Rays Role Sampling Scanning Scanning Probe Microscopes Severities Shapes Slice Solutions Specimen Spectrum Analysis Structure Structure-Activity Relationship Surface Swelling System Techniques Time Tissue Engineering Tissue Sample Tissues Titrations Tooth structure Trypsin Universities Variant Water Weight-Bearing state aggrecan analog aqueous base bone chemical property density design high throughput analysis implantation improved instrument light scattering loss of function nanostructured novel physical property pressure repaired research study response scaffold self assembly success tissue regeneration uptake vapor water vapor

项目摘要

Understanding the physical and chemical mechanisms affecting cartilage behavior is essential to predict its biomechanical properties, particularly its load-bearing and lubricating abilities, which are governed by osmotic and electrostatic forces that strongly depend on tissue hydration. Such understanding is also a prerequisite for the success of tissue engineering and regenerative medicine strategies to grow, repair, and reintegrate cartilage. The biomechanical behavior of cartilage is sensitive to both biochemical and microstructural changes occurring in development, disease, degeneration, and aging. To study cartilage physical properties (e.g., osmotic swelling properties and hydration) an array of techniques is required that probe not only a wide range of length scales but also statistically representative volumes of the sample. Controlled hydration provides a direct means of determining functional properties of cartilage and of other tissues. Specifically, we have used controlled hydration of cartilage to measure physical/chemical properties of the collagen network and of the proteoglycans (PG) independently within the extracellular matrix. This approach entailed modeling the cartilage tissue matrix as a composite material consisting of two distinct phases: a collagen network and a concentrated PG solution trapped within it. In pilot studies, we used this approach to determine pressure-volume curves for the collagen network and PG phases in native and in trypsin-treated normal human cartilage specimen, as well as in cartilage specimen from osteoarthritic (OA) joints. In both normal and trypsin-treated specimens, collagen network stiffness appeared unchanged, whereas in the OA specimen, collagen network stiffness decreased. Our findings highlighted the role of the collagen network in limiting normal cartilage hydration, and in ensuring a high PG concentration, and thus, swelling pressure within the matrix, both of which are essential for effective load bearing in cartilage and joint lubrication, but are lost in OA. A shortcoming of this approach was that it required tissue slices to obtain these osmotic titration curves. This lead to long equilibration times requiring several person-days to study a single cartilage specimen, making this approach unsuitable for routine pathological analysis or for use in tissue engineering applications. More recently, we designed and built a new tissue micro-osmometer to perform these experiments practically and rapidly. This instrument can measure minute amounts of water absorbed by small tissue samples (< 1 microgram) as a function of the equilibrium activity (pressure) of the surrounding water vapor. A quartz crystal sensitively and precisely detects the water uptake of the tissue specimen attached to its surface. Varying the equilibrium vapor pressure surrounding the specimen induces controlled changes in the osmotic pressure of the tissue layer. To demonstrate the applicability of the new apparatus, we measured the swelling pressure of tissue-engineered cartilage specimen. We use the micro-osmometer to obtain a profile of the osmotic compressibility or stiffness of multiple cartilage specimens simultaneously as a function of depth from the articular surface to the bone interface. It also allows us to assess the mechanical integrity of developing tissues and osmotic compatibility of tissue-engineered cartilage (or ECM) with the hope of improving integration and viability following implantation in regenerative medicine applications. Moreover, osmotic pressure measurements allow us to quantify the contributions of individual components of ECM (e.g., aggrecan, hyaluronic acid (HA) and collagen) to the total swelling pressure. Our recent osmotic pressure measurements on aggrecan/HA systems showed evidence of self-assembly of the bottlebrush shaped aggrecan subunits into microgel-like assemblies. We found that aggrecan microgels of several microns in size coexist with smaller aggregates, as well as individual aggrecan molecules. The results also indicate that in the presence of HA, the formation of the aggrecan/HA complex at low aggrecan concentrations reduces the osmotic pressure. However, in the physiological concentration range the osmotic modulus of the aggrecan-HA complex is enhanced with respect to that of the random assemblies of aggrecan bottlebrushes, confirming that the aggrecan/HA complex increases the load bearing ability of cartilage. We have also developed a new experimental procedure for mapping the local elastic properties of cartilage using the atomic force microscope (AFM). Many of the impediments that have previously hindered the use of the AFM in high-throughput analysis of inhomogeneous samples, particularly biological tissues, have been addressed. The technique utilizes the precise scanning capabilities of AFM to generate large volumes of compliance data from which we extract the relevant elastic properties. In conjunction with scattering measurements, micro-osmometry and biochemical analysis, this technique allows us to map the spatial variations in the osmotic modulus of tissues and gels. We mapped the osmotic modulus of bovine cartilage samples by combining tissue micro-osmometry with force-deformation measurements made by the AFM. Knowledge of the local osmotic properties of cartilage is particularly important since the osmotic modulus defines the compressive resistance to external load. We found that the water retention is stronger in the upper and deep zones of cartilage, where collagen fibers are ordered, than in the middle zone where they are randomly arranged. We have constructed the elastic and osmotic modulus maps for the different layers. The latter that is a combination of the elastic and swelling properties, exhibits much stronger spatial variation reflecting the highly heterogeneous character of the tissue. A major objective of tissue engineering is to mimic the ECM environment. However, the complexity of interactions between ECM and cells makes it difficult to design materials for regenerative medicine applications. Previous studies have indicated that the chemical structure of the scaffold is critical. Molecular factors (e.g., hydrophilic or hydrophobic character of the polymer, stiffness, charge density) significantly influence cell adhesion, spreading and growth. In collaboration with researchers at the Carnegie Mellon University we developed novel nanostructured hydrogels, which have potential as an artificial ECM, and can act as a macroscopic scaffold for tissue regeneration. Our complementary microscopic and macroscopic measurements made on aggrecan, HA, and aggrecanHA solutions indicate that the osmotic pressure, molecular organization, and dynamic response of PG assemblies are governed by the bottlebrush-shaped aggrecan molecule. Aggrecan subunits spontaneously self-assemble into microgel-like assemblies in aqueous solution. Complexation with HA reinforces the aggrecan assemblies. The relaxation rate of the aggrecanHA system is slightly slower than that of the pure aggrecan solution, indicating that the connectivity of the two components only weakly influences the dynamics of the aggrecanHA complex. A comparison between the osmotic pressure of engineered cartilage and that of model systems reveals that the load-bearing properties of cartilage are primarily governed by the PG assemblies. Collectively, these approaches are helping us get closer to a physical/chemical understanding of extracellular matrix and the basis of functional properties of normal cartilage, and its changes in development, as well as possible explanations for loss of function in disease, degeneration and in abnormal development.

了解影响软骨行为的物理和化学机制对于预测其生物力学特性至关重要，特别是其承载和润滑能力，这些能力受到强烈依赖于组织水合作用的渗透力和静电力的控制。这种理解也是组织工程和再生医学策略成功生长、修复和重新整合软骨的先决条件。软骨的生物力学行为对发育、疾病、退化和衰老过程中发生的生化和微观结构变化敏感。为了研究软骨的物理特性（例如，渗透膨胀特性和水合），需要一系列技术，不仅可以探测各种长度尺度，还可以探测具有统计代表性的样本体积。控制水合作用提供了确定软骨和其他组织功能特性的直接方法。具体来说，我们使用软骨的受控水合来独立测量细胞外基质内胶原网络和蛋白聚糖 (PG) 的物理/化学特性。这种方法需要将软骨组织基质建模为由两个不同相组成的复合材料：胶原蛋白网络和其中捕获的浓缩 PG 溶液。在初步研究中，我们使用这种方法来确定天然和胰蛋白酶处理的正常人类软骨样本以及骨关节炎 (OA) 关节软骨样本中胶原蛋白网络和 PG 相的压力-体积曲线。在正常和胰蛋白酶处理的标本中，胶原网络刚度似乎没有变化，而在 OA 标本中，胶原网络刚度下降。我们的研究结果强调了胶原蛋白网络在限制正常软骨水合、确保高 PG 浓度以及基质内膨胀压力方面的作用，这两者对于软骨的有效承载和关节润滑至关重要，但都丢失了在办公自动化中。这种方法的缺点是它需要组织切片才能获得这些渗透滴定曲线。这导致平衡时间较长，需要几个人天的时间来研究单个软骨样本，使得该方法不适合常规病理分析或组织工程应用。最近，我们设计并制造了一种新型组织微渗透压计，可以实际、快速地进行这些实验。该仪器可以测量小组织样本（< 1 微克）吸收的微量水，作为周围水蒸气平衡活动（压力）的函数。石英晶体灵敏而精确地检测附着在其表面的组织样本的吸水量。改变样本周围的平衡蒸气压会引起组织层渗透压的受控变化。为了证明新装置的适用性，我们测量了组织工程软骨样本的膨胀压力。我们使用微渗透压计同时获得多个软骨样本的渗透压缩性或刚度曲线，作为从关节表面到骨界面的深度的函数。它还使我们能够评估发育中组织的机械完整性和组织工程软骨（或 ECM）的渗透相容性，以期提高再生医学应用中植入后的整合性和生存能力。此外，渗透压测量使我们能够量化 ECM 各个成分（例如聚集蛋白聚糖、透明质酸 (HA) 和胶原蛋白）对总膨胀压的贡献。我们最近对聚集蛋白聚糖/HA 系统进行的渗透压测量表明，刷状聚集蛋白聚糖亚基自组装成微凝胶状组件的证据。我们发现几微米大小的聚集蛋白聚糖微凝胶与较小的聚集体以及单个聚集蛋白聚糖分子共存。结果还表明，在HA存在的情况下，在低聚集蛋白聚糖浓度下形成聚集蛋白聚糖/HA复合物会降低渗透压。然而，在生理浓度范围内，聚集蛋白聚糖-HA复合物的渗透模量相对于聚集蛋白聚糖瓶刷的随机组装的渗透模量有所增强，证实聚集蛋白聚糖/HA复合物增加了软骨的承载能力。我们还开发了一种新的实验程序，使用原子力显微镜（AFM）绘制软骨的局部弹性特性。以前阻碍 AFM 在非均质样品（特别是生物组织）的高通量分析中使用的许多障碍都已得到解决。该技术利用 AFM 的精确扫描功能生成大量合规数据，我们从中提取相关的弹性属性。结合散射测量、微渗透压测定和生化分析，该技术使我们能够绘制组织和凝胶渗透模量的空间变化图。我们通过将组织微渗透压测定法与 AFM 进行的力变形测量相结合，绘制了牛软骨样品的渗透模量。了解软骨的局部渗透特性尤其重要，因为渗透模量定义了对外部负载的压缩阻力。我们发现，胶原纤维有序排列的软骨上层和深层区域的保水性比随机排列的中间区域更强。我们构建了不同层的弹性和渗透模量图。后者是弹性和膨胀特性的组合，表现出更强的空间变化，反映了组织的高度异质性特征。组织工程的一个主要目标是模拟 ECM 环境。然而，ECM 和细胞之间相互作用的复杂性使得设计用于再生医学应用的材料变得困难。先前的研究表明支架的化学结构至关重要。分子因素（例如聚合物的亲水或疏水特性、硬度、电荷密度）显着影响细胞粘附、扩散和生长。我们与卡内基梅隆大学的研究人员合作开发了新型纳米结构水凝胶，它具有作为人工 ECM 的潜力，并且可以作为组织再生的宏观支架。我们对聚集蛋白聚糖、HA 和聚集蛋白聚糖HA 溶液进行的互补微观和宏观测量表明，PG 组装体的渗透压、分子组织和动态响应是由瓶刷状聚集蛋白聚糖分子控制的。聚集蛋白聚糖亚基在水溶液中自发地自组装成微凝胶状组件。与 HA 络合可增强聚集蛋白聚糖的组装。 aggrecanHA 系统的弛豫速率比纯 aggrecan 溶液稍慢，表明两个组件的连接性仅对 aggrecanHA 复合物的动力学产生微弱影响。工程软骨的渗透压与模型系统的渗透压之间的比较表明，软骨的承载特性主要由 PG 组件控制。总的来说，这些方法正在帮助我们更接近对细胞外基质和正常软骨功能特性的基础及其发育变化的物理/化学理解，以及对疾病、退化和异常中功能丧失的可能解释。发展。