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Functional Properties Of Extracellular Matrix

细胞外基质的功能特性

基本信息

批准号：
9550271
负责人：
PETER J. BASSER
金额：
$ 33.15万
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/9550271
关键词：
5 year old Affect Aging Atomic Force Microscopy Award Biochemical Biological Models Biomechanics Bovine Cartilage Cartilage Cell Adhesion Cells Charge Chemical Structure Chemicals Chemistry Childhood Collaborations Collagen Collagen Fiber Complex Crystallization Data Degenerative polyarthritis Development Disease Disease Resistance Ensure Environment Equilibrium Exhibits Extracellular Matrix Extracellular Structure Fiber Fluorescence Fluorides Foundations Gel Goals Growth Human Hyaluronic Acid Hydration status Hydrogels Hydrophobicity Image Individual Joints Knowledge Laws Lead Legal patent Length Lubrication Magnetic Resonance Imaging Maps Measurement Measures Mechanics Methods Microscopic Modeling Modulus Molecular Monitor Nanostructures National Institute of Child Health and Human Development Neutrons Osmotic Pressure Pathologic Persons Phase Physics Pilot Projects Polymers Procedures Property Protective Agents Proteoglycan Public Health Quartz Regenerative Medicine Research Research Activity Research Personnel Resistance Resolution Roentgen Rays Role Sampling Scanning Scanning Probe Microscopes Severities Slice Specimen Spectrum Analysis Structure Structure-Activity Relationship Surface Swelling System Techniques Time Tissue Engineering Tissue Sample Tissues Tooth structure Trypsin Universities Variant Water Weight-Bearing state aggrecan analog base cartilage degradation chemical property clinical application density design experimental study high resolution imaging hydrophilicity imaging biomarker imaging modality improved in vivo insight instrument light scattering mechanical properties molecular scale nanostructured novel optical imaging pre-clinical pressure repaired response scaffold self assembly tissue regeneration tissue support frame uptake vapor water vapor

项目摘要

Understanding cartilage's functional and biomechanical properties, particularly its load-bearing and lubricating abilities, requires deep knowledge of the properties of and interactions among cartilage's primary constituents. This knowledge is also a prerequisite for successful tissue engineering and regenerative medicine strategies to grow, repair, and reintegrate cartilage. Cartilage functional properties are influenced by biochemical and microstructural changes occurring in development, degeneration, aging and diseases. At the macroscopic length scale, controlled hydration provides a direct means of determining tissue stiffness as well as electromechanochemical properties of its collagen and proteoglycan (PG) components. We have used controlled hydration of cartilage to measure physical/chemical properties of the collagen network and of the proteoglycans (PG) independently within the ECM. This approach entailed modeling the cartilage tissue matrix as a composite medium consisting of two distinct phases: a collagen network and a concentrated PG solution trapped within it. In these classical studies, we determined "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 restraining 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 which are lost in OA. A shortcoming of this approach, however, was that it required excised tissue slices to perform this analysis. This lead to long equilibration times requiring several person-days to study a single cartilage specimen, making this approach unsuitable for routine pathological analysis, tissue engineering, preclinical or clinical applications. Subsequently, we designed and built a novel tissue micro-osmometer to perform these experiments practically and rapidly (US Patent No. 7,380,477). 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 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. We have also developed an experimental procedure for mapping the local elastic properties of cartilage using the atomic force microscope (AFM). This technique utilizes the precise scanning capabilities of AFM to generate large volumes of compressibility data from which we extract the relevant elastic properties. We mapped the osmotic modulus of bovine cartilage samples by combining tissue micro-osmometry with AFM force-deformation measurements. Knowledge of the local osmotic properties of cartilage is particularly important since the osmotic modulus defines the tissue's compressive resistance to external loads. We found that the water retention is stronger in the upper and deep zones of cartilage than in the middle zone. 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. We have begun correlating mechanical measurements with observations made by MRI imaging on the same cartilage specimens. This approach has the potential to develop novel imaging biomarkers to aid in MSK MRI applications. A major objective of tissue engineering is to mimic the ECM environment to enable the regrowth of functional tissue. 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 scaffold, stiffness, charge density of the polymer) 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, which can act as a macroscopic scaffold for tissue regeneration. Experimental results obtained by macroscopic (osmotic swelling pressure measurements) and microscopic techniques (SANS, SAXS, DLS) aimed a quantifying the interactions between the main macromolecular components of the ECM, yield insights both into the properties of aggrecan assemblies at a supramolecular level and into the mechanism of load bearing of cartilage. Measurements made on aggrecan, hyaluronic acid (HA), and aggrecan/HA solutions indicate that the osmotic pressure, molecular organization, and dynamic response of PG assemblies are governed by the bottlebrush-shaped aggrecan molecule. Osmotic pressure measurements allow us to quantify the contributions of individual components of ECM (e.g., aggrecan, HA, and collagen) to the total swelling pressure. Such measurements on aggrecan/HA systems showed evidence of self-assembly of the bottlebrush shaped aggrecan subunits into microgel-like aggregates. We showed the complexation with HA enhances aggrecan assembly. The osmotic pressure of aggrecan-HA complexes decreases when decreasing the ratio of aggrecan to HA. SANS reveals that there is no significant interpenetration between neighboring aggrecan molecules as they are squeezed together. Neither osmotic pressure measurements nor SANS indicate significant interactions between the collagen network and the aggrecan bottlebrushes. We have also developed a multiscale experimental approaches to study ECM in which quantitative MR imaging is combined with chemical composition mapping via High-Definition Infrared (optical) Imaging (HDIR) and high resolution mechanical maps made by AFM. Our pilot study on 2.5 year-old bovine cartilage yields consistent results between the NMR parameters, the chemical composition and structure of the ECM, and its mechanical properties. HDIR is being used to validate MR imaging methods to measure tissue composition. Our hope is to identify and quantify molecular and microstructural changes that allow us to monitor early changes in the tissue that may later lead to cartilage degeneration. This research activity was previously supported by the NICHD DIR Director's Award. We are now developing hydrogels that mimic the mechanical and osmotic properties of cartilage. Studies on well-defined model systems may open new avenues to understand the biomechanical properties of cartilage such as its load bearing ability, and may explain how molecular scale properties like matrix stiffness, charge density, affect macroscopic mechanical and swelling properties.

了解软骨的功能和生物力学特性，特别是其承重和润滑能力，需要深入了解软骨主要成分的特性和相互作用。这些知识也是成功的组织工程和再生医学策略来生长、修复和重新整合软骨的先决条件。