Functional Properties Of Extracellular Matrix

细胞外基质的功能特性

• 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
• 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

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，尤其是其承载和润滑能力至关重要，其载荷和润滑能力受渗透性和静电力的控制，这些力极大地依赖于组织水合。这种理解也是组织工程成功和再生医学策略的成功的先决条件。 软骨的生物力学行为对发育，疾病，变性和衰老中发生的生化和微结构变化敏感。为了研究软骨的物理特性（例如，渗透肿胀特性和水合），需要一系列技术，不仅要探测样品的长度范围范围很大，而且还需要统计上代表性的量。受控的水合提供了确定软骨和其他组织功能性能的直接方法。 具体而言，我们已经使用软骨的控制水合来测量胶原蛋白网络的物理/化学特性和蛋白聚糖（PG）在细胞外基质中独立的。这种方法需要将软骨组织基质建模为一个由两个不同阶段组成的复合材料：胶原蛋白网络和被困在其中的浓缩PG溶液。在试点研究中，我们使用这种方法来确定天然和胰蛋白酶处理的正常人软骨标本中的胶原蛋白网络和PG相的压力量曲线，以及来自骨关节炎（OA）关节的软骨标本。在正常和胰蛋白酶处理的标本中，胶原网络的刚度似乎没有变化，而在OA标本中，胶原网络刚度降低。我们的发现强调了胶原蛋白网络在限制正常软骨水合的作用，并确保较高的PG浓度，从而在基质内进行膨胀压力，这两种压力对于在软骨和关节润滑中有效负载至关重要，但在OA中丢失。 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。这导致了长时间的平衡时间，需要几个人的时间来研究单个软骨标本，从而使该方法不适合常规病理分析或用于组织工程应用。最近，我们设计并构建了一个新的组织微量射击器，以实际和快速地执行这些实验。 该仪器可以测量由小组织样品（<1微克）吸收的微量水作为周围水蒸气的平衡活性（压力）的函数。石英晶体敏感，精确地检测到附着在其表面的组织样品的吸水。改变样品周围的平衡蒸气压力会引起组织层渗透压的受控变化。 为了证明新设备的适用性，我们测量了组织工程软骨标本的肿胀压力。我们使用微敏度计来获得同时从关节表面到骨头界面的深度的渗透压缩性或刚度的刚度。 它还使我们能够评估发展组织的机械完整性以及组织工程软骨（或ECM）的渗透兼容性，希望在再生医学应用中植入后提高整合和生存能力。 此外，渗透压测量值使我们能够量化ECM（例如Aggrecan，透明质酸（HA）和胶原蛋白）对总肿胀压力的各个组件的贡献。我们最近对AggreCan/HA系统进行的渗透压测量显示了瓶装刷状的脂肪脂肪亚基的自组装的证据。我们发现，几微米的几微米的Aggrecan微凝胶与较小的聚集体以及单个脂肪分子共存。结果还表明，在HA存在的情况下，在低脂肪浓度下的脂肪组/HA复合物的形成降低了渗透压。然而，在生理浓度范围内，相对于脂肪奶油瓶刷的随机组装，脂肪素HA络合物的渗透模量得到了增强，这证实了脂肪蛋白/HA复合物可以提高软骨的负载能力。 我们还开发了一种新的实验程序，用于使用原子力显微镜（AFM）绘制软骨的局部弹性特性。 许多以前妨碍了AFM在对不均匀样本（尤其是生物组织的无外观样本）中使用的障碍。该技术利用AFM的精确扫描功能来生成大量合规性数据，我们从中提取相关的弹性属性。结合散射测量，微观测量和生化分析，该技术使我们能够绘制组织和凝胶渗透模量的空间变化。我们通过将组织微观测定法与AFM进行的力变形测量相结合，从而绘制了牛软骨样品的渗透模量。由于渗透模量定义了对外部负载的抗压强度，因此对软骨的局部渗透特性的了解尤为重要。我们发现，与随机排列的中部区域相比，在软骨的上部和深区中，水的retention留更强。我们已经为不同层构建了弹性和渗透模量图。后者是弹性和肿胀特性的组合，表现出更强的空间变异，反映了组织的高度异质性。 组织工程的主要目标是模仿ECM环境。但是，ECM与细胞之间相互作用的复杂性使得为再生医学应用设计材料很难。先前的研究表明，支架的化学结构至关重要。分子因子（例如，聚合物的亲水性或疏水特征，刚度，电荷密度）显着影响细胞粘附，扩散和生长。与卡内基梅隆大学的研究人员合作，我们开发了新颖的纳米结构水凝胶，这些水凝胶具有人为的ECM潜力，并且可以充当组织再生的宏观脚手架。 我们对Aggrecan，HA和Aggrecanha溶液进行的互补微观测量和宏观测量表明，PG组件的渗透压，分子组织以及动态响应受瓶洗奶瓶形的Aggrecan分子的控制。 Aggrecan亚基自发地自组装成水溶液中的微凝胶样组件。 与HA的络合增强了AggreCan组件。 Aggrecanha系统的松弛速率比纯Aggrecan溶液的松弛速率稍慢，这表明这两个组件的连通性仅对Aggrecanha复合物的动力学产生弱影响。 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受PG组件的控制。 总的来说，这些方法有助于我们更接近对细胞外基质的物理/化学理解，以及正常软骨的功能特性及其发育变化的基础，以及可能对疾病，变性和异常发育功能丧失的可能解释。