Measuring and Mapping Functional Properties of Extracellular Matrix (ECM)

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

The unique properties of cartilage originate from the architecture and organization of its ECM. Articular cartilage is a load-bearing tissue that protects the bone, distributes stress and provides a smooth surface for joint movement. The ECM of cartilage consists of a fibrous collagen network, which is prestressed by the osmotic swelling pressure exerted by large negatively charged proteoglycan (PG) assemblies embedded within it. The PG macromolecular assemblies absorb fluid and inflate the collagen matrix. The load-bearing ability of cartilage is governed by its swelling pressure, which depends on the concentration of the main macromolecular components and ions in the ECM, and their mutual interactions. In unloaded equilibrium the osmotic swelling pressure of the PGs is balanced by the elastic stress developed within the collagen network. The load bearing behavior of cartilage is sensitive to both biochemical and microstructural changes occurring in development, disease, degeneration, and aging. We are developing noninvasive in vitro methods to determine structure/function relationships of ECM components using novel MR imaging methods, which have the potential for early diagnosis of cartilage disorders and diseases. We have also developed a magnetization transfer (MT) MRI method, which is capable to detect immobile protons (e.g., protons on the collagen backbone), which are not detectable by conventional water proton MRI methods owing to their short transverse relaxation time, T2. To visualize these invisible protons the magnetization of these molecules is transferred to the free water, which is visible by MRI. In a pilot study we have compared the results obtained for the concentrations of the main cartilage constituents by our MT MRI method and high definition infrared spectroscopic (HDIR) imaging measurements made on the same samples. Our novel approach has the potential to map tissue structure and functional properties in vivo and non-invasively. The hydration of cartilage defines its swelling and load bearing ability. To study cartilage hydration, an array of complementary techniques is required that probe not only a wide range of length and time scales, but are also statistically representative of the heterogeneous sample. Controlled hydration or swelling using the osmotic stress technique provides a direct means of determining functional properties of cartilage and of other ECMs. Our earlier measurements revealed the role of the collagen network in limiting the hydration of normal (healthy) cartilage and ensuring a high PG concentration in the matrix, which is essential for effective load bearing. We also demonstrated that the loss of collagen network stiffness is consistent with the degradation of cartilage observed in osteoarthritis (OA). To quantify the effect of hydration on cartilage properties we developed a tissue micro-osmometer to perform experiments in a practical and rapid manner. This instrument is capable to measure very small changes in the amount of water absorbed by small tissue samples (less than 1 microgram tissue) as a function of the equilibrium activity (vapor pressure) of the surrounding tissue water. We use osmotic pressure measurements to determine the contributions of individual components of ECM to the total tissue swelling pressure. We have also developed a method for mapping the local elastic and osmotic properties of cartilage using the Atomic Force Microscope (AFM) together with the tissue micro-osmometer. Many of the impediments that previously hindered the use of AFM to probe soft inhomogeneous samples, particularly biological tissues, were addressed by our new approach that utilizes the precise scanning capabilities of the AFM to generate large volumes of compliance data from which the relevant elastic properties can be extracted. In conjunction with results obtained from high-resolution scattering measurements, tissue micro-osmometry, and biochemical analysis, this technique allows us to map the spatial variations in the osmotic modulus within tissue specimens. Knowledge of the local osmotic properties of cartilage is particularly important, given that the osmotic modulus determines the compressive resistance of the tissue to external load. We also made rheological measurements to determine the dynamic properties of cartilage PGs (chondroitin sulfate, hyaluronic acid, aggrecan and aggrecan-HA complex) at the macroscopic level. In the context of cartilage function in the joints, the dynamic response of the constituents is particularly important because the timescale of slow joint movement is significantly different from that of rapid joint movement. In the case of relatively slow motion of joints, the dynamics of joint movement is governed by the viscoelastic complex fluid nature of cartilage, while in the rapid motion of joints, the elastic (gel-like) nature of cartilage becomes prevalent. A unique feature of cartilage is that its proteoglycans exhibit a hierarchical bottlebrush structure at multiple length scales, which emerges from molecular and supramolecular self-assembly. Collagen fibers constitute a fine fibrous network that constrains these proteoglycan aggregates. Our systematic measurements of aggrecan/HA systems revealed that the osmotic modulus of the aggrecan-HA complex is enhanced with respect to that of the random assemblies of aggrecan bottlebrushes, providing direct evidence that complex formation among aggrecan and HA molecules significantly improves the load-bearing ability of cartilage. We also demonstrated that aggrecan-HA assemblies exhibit microgel-like behavior and they are remarkable insensitive to changes in the ionic environment, particularly to calcium (Ca+2) ion concentration. These results are consistent with the role of aggrecan as an ion reservoir buffering calcium content in cartilage and bone. Based on the results of structural studies, we developed a biomimetic hydrogel model of cartilage consisting of a stiff polymer matrix, made of polyvinyl alcohol (PVA) with embedded microgel particles consisting of crosslinked polyacrylic acid (PAA) microparticles. In our model the PVA network corresponds to the collagen matrix, while the charged PAA microgel particles represent the PG assemblies. The high swelling ability of the PAA gel causes the composite gel to be prestressed by osmotic pressure in water, which contributes to the improvement of the gel's mechanical properties. This type of gel is conceptually different from conventional filler-reinforced systems. In the PVA/PAA system the swollen PAA particles inflate the PVA network. The swelling of the PAA is limited by the tensile stress developing in the PVA matrix, which increases as the gel absorbs more liquid. This behavior is opposite to the decrease of the elastic pressure observed in regular gels. We studied the effects of different factors corresponding to changes occurring in age- and disease-related alterations of cartilage. Similar information cannot be obtained from measurements made on biological tissues because their composition and physical properties (e.g,. stiffness, charge density) cannot be independently varied. We have demonstrated that the osmotic and mechanical behaviors of our biomimetic model systems reproduce the properties of healthy and osteoarthritic human cartilage remarkably well. To better understand the role of the two primary components, the network polymer, and the embedded particles, we developed molecular dynamics (MD) simulation methods to generate a wide range of gel-like structures from compact to open gels. We use MD to better understand the origin of the physical and chemical interactions among ECM components to enable out development of larger length scale continuum descriptions of ECM tissue behavior and functional properties.