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 load bearing behavior of cartilage is sensitive to both biochemical and microstructural changes occurring in development, disease, degeneration, and aging. Cartilage hydration is a key determinant of its load bearing properties. 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 extracellular matrices (ECM). 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. A quartz crystal detects the water uptake of a specimen attached to its surface. The high sensitivity of its resonance frequency to small changes in the amount of adsorbed water makes it possible to determine the water uptake of the tissue with high precision. We used osmotic pressure measurements to determine the contributions of individual components of ECM (e.g., aggrecan, hyaluronic acid (HA), and collagen) to the total tissue swelling pressure. Our measurements on 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 improves the load-bearing ability of cartilage. Our combined static and dynamic scattering measurements (SAXS, SANS, SLS, DLS, neutron spin-echo) demonstrated that aggrecan-HA assemblies exhibit microgel-like behavior and remarkable insensitivity to changes in the ionic environment, particularly to Ca+2 concentration. The results are consistent with the role of aggrecan as an ion reservoir mediating calcium metabolism in cartilage and bone. We have 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 inhomogeneous samples, particularly biological tissues, were addressed by this new approach that utilizes the precise scanning capabilities of a commercial 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, 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 have begun noninvasive in vitro applications of critical tissue-sciences understanding of structure/function relationships of ECM components to develop and design novel MR imaging methods, which has the potential for early diagnosis of cartilage diseases. Specifically, we are developing imaging methods directed at measuring key compositional and structural features of cartilage ECM, which we can use to estimate functional properties of the tissue with the aid of a biophysical modeling framework. In collaboration with Uzi Eliav (Tel Aviv University) we developed a novel 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 MRI owing to their short 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. The results show that our novel approach has the potential to map tissue structure and functional properties in vivo and noninvasively.