Measuring and Mapping Functional Properties of Extracellular Matrix (ECM)

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Articular cartilage is a load-bearing connective tissue that covers the ends of bones and provides a smooth, low-friction, and wear-resistant lubricated surface to facilitate joint movement. The unique properties of cartilage originate from the architecture and organization of its ECM. Cartilage ECM consists of a fibrous collagen network, which is prestressed by the osmotic swelling pressure exerted by large negatively charged proteoglycan (PG) assemblies embedded in the collagen network. 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). We used 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 our tissue micro-osmometer. 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. To date, no satisfactory theoretical framework embodying the relationship between molecular/supramolecular structure, macroscopic properties and biological function of either synthetic or natural polyelectrolyte brush molecules has been developed, making experimental studies of these molecules important for future progress in understanding the arthritic and other medical conditions related to the disfunction of bottlebrush molecules. We compared the behavior of aggrecan with that of model bottlebrush polymers sodium poly(acrylate) bottlebrushes. Previous studies have shown that aggrecan, the major cartilage proteoglycan, possesses exceptional properties that make this molecule well suited for its multiple biological roles in cartilage and other connective tissues. We demonstrated that aggrecan exhibits a significantly different solution organization from common linear polyelctrolytes. Our molecular dynamics simulations indicated that the increased chain backbone solvation in aggrecan changes the nature of the inter-polyelectrolyte interaction from long-ranged to short-ranged associative interaction. For polyelectrolytes having a strong degree of chain solvation such as aggrecan, this relatively short-range and multi-functional sticky interaction gives rise to the formation of fractal-like aggregates. This behavior can be expected to have implications for understanding biological function and disease. The extreme insensitivity of the structure of aggrecan solutions to changes in salt concentrations is another striking difference of aggrecan from most synthetic polyelectrolytes that directly impacts the stability of these materials under a wide range of solutions encountered in living systems. 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 biophysical model the PVA network corresponds to the collagen matrix, while the charged PAA microgel particles represent the PG assemblies. The PAA microgel particles inflate the PVA polymer matrix and generate prestress in the PVA network. 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. Our molecular dynamics simulations to determine the effects of molecular interactions on cartilage structure and functional properties are in progress.