Articular cartilage is a load-bearing tissue consisting of a fibrous collagen network that encapsulates large proteoglycan (PG) assemblies, which absorb fluid and inflate the collagen matrix. The load-bearing ability of cartilage is governed by the swelling pressure, which depends on the concentration of the main macromolecular components of the extracellular matrix (ECM) and their mutual interactions. At 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 applications to determine structure/function relationships of ECM components using novel MR imaging methods, which has the potential for early diagnosis of cartilage diseases. In collaboration with Uzi Eliav (Tel Aviv University), we 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 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. Our novel approach has the potential to map tissue structure and functional properties in vivo and non-invasively. Simultaneously, we determine the microscopic mechanical properties of cartilage (making elastic and osmotic modulus maps) by AFM nano-indentation. Cartilage hydration is a key determinant of its 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. 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. 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. 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. We demonstrated that aggrecan-HA assemblies exhibit microgel-like behavior and they are remarkable insensitive to changes in the ionic environment, particularly to Ca+2 ion concentration. The results are consistent with the role of aggrecan as an ion reservoir mediating calcium metabolism in cartilage and bone. We found that the viscoelastic response of aggrecan solutions is consistent with previous osmotic compressibility and neutron scattering studies indicating supramolecular molecular assemblies in solution. We gained insights into the evolution of the complex formation between aggrecan and HA molecules by rheological measurements. These measurements indicated that the aggrecan molecules are gradually organized onto the HA backbone, rigidifying the backbone of the bottlebrush and a gel is formed. Novel models and methods to measure ion transport through polyelectrolyte gels, serving as cartilage models, and cartilage slices are being developed.
| Horkay, Ferenc; Basser, Peter J; Hecht, Anne-Marie et al. (2017) Structure and Properties of Cartilage Proteoglycans. Macromol Symp 372:43-50 |
| Puwal, Steffan; Roth, Bradley J; Basser, Peter J (2017) Heterogeneous anisotropic magnetic susceptibility of the myelin-water layers causes local magnetic field perturbations in axons. NMR Biomed 30: |
