The biomechanical behavior of cartilage is sensitive to both biochemical and microstructural changes occurring in development, disease, degeneration, and aging. To study cartilage physical properties (e.g., osmotic swelling properties and hydration) an array of techniques is required that probes not only a wide range of length scales but also statistically representative volumes of the sample. Controlled hydration provides a direct means of determining functional properties of cartilage and of other extracellular matrices. Specifically, we have used controlled hydration of cartilage to measure physical/chemical properties of the collagen network and of the proteoglycans (PG) independently within the extracellular matrix. This approach entailed modeling the cartilage tissue matrix as a composite material consisting of two distinct phases: a collagen network and a concentrated proteoglycan solution trapped within it;applying various known levels of equilibrium osmotic stress;and using physical-chemical principles and additional experiments to determine a """"""""pressure-volume"""""""" relationship for both the PG and collagen phases independently. In pilot studies, we used this approach to determine 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 specimen, collagen network stiffness appeared unchanged, whereas in the OA specimen, collagen network stiffness decreased. Our findings highlighted the role of the collagen network in limiting normal cartilage hydration, and in ensuring a high PG concentration in the matrix, both of which are essential for effective load bearing in cartilage and lubrication, but are lost in OA. These data also suggest that the loss of collagen network stiffness, and not the loss or modification of PGs may be the incipient event leading to the subsequent disintegration of cartilage observed in OA. One shortcoming of this approach, however, was that it required a significant amount of tissue to obtain the osmotic titration curves. This lead to long equilibration times requiring person-days to study a single cartilage specimen, making this approach unsuitable for routine pathological analysis or for use in tissue engineering applications. Recently, we designed and built a new tissue micro-osmometer to perform these experiments in a practical and rapid manner. 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 detects the water uptake of the tissue specimen attached to its surface. The high sensitivity of its resonance frequency to small changes in the amount of adsorbed water allows us to measure the mass uptake of the tissue specimen precisely. Varying the equilibrium vapor pressure surrounding the specimen induces controlled changes in the osmotic pressure of the tissue layer. To validate the methodology, we used synthetic polymer gels with known osmotic properties. The micro-osmometer permits us to obtain a profile of the osmotic compressibility or stiffness of multiple cartilage specimens simultaneously as a function of depth from the articular surface to the bone interface. It also allows us to assess the osmotic compatibility and mechanical integrity of developing tissues and of tissue-engineered cartilage (or ECM) with the hope of improving integration and viability following implantation. To demonstrate the applicability of the new apparatus, we measured the swelling pressure of tissue-engineered cartilage specimen. Moreover, osmotic pressure measurements allow us to quantify the contributions of individual components of ECM (e.g., aggrecan, hyaluronic acid and collagen) to the total swelling pressure. Our recent osmotic pressure measurements on aggrecan/hyaluronic acid systems showed evidence of self-assembly of the bottlebrush shaped aggrecan subunits into microgel-like assemblies. We found that aggrecan microgels of several microns in size coexist with smaller associations, as well as individual aggrecan molecules. The results also indicate that in the presence of hyaluronic acid, the formation of the aggrecan-HA complex at low aggrecan concentrations reduces the osmotic pressure. However, in the physiological concentration range the osmotic modulus of the aggrecan-HA complex is enhanced with respect to that of the random assemblies of aggrecan bottlebrushes, confirming that the aggrecan/HA complex is improves load bearing function in cartilage. Our combined static and dynamic scattering measurements (small-angle X-ray scattering, small-angle neutron scattering, static light scattering, dynamic light scattering, neutron spin-echo) demonstrate that aggrecan assemblies exhibit remarkable insensitivity to changes in ionic environment, particularly to calcium ion concentration. This result is consistent with the role of aggrecan as an ion reservoir mediating calcium metabolism in cartilage and bone. We developed a method for mapping the local elastic and viscoelastic properties of cartilage using the atomic force microscope (AFM). Many of the impediments that have previously hindered the use of the AFM in high-throughput probing of inhomogeneous samples, particularly biological tissues, have been addressed. The technique utilizes the precise scanning capabilities of a commercial AFM to generate large volumes of compliance data and extracts the relevant elastic properties from these data. In conjunction with results obtained from scattering measurements, micro-osmometry and biochemical analysis, this technique will allow us to map spatial variations in the osmotic modulus of various tissue samples. Knowledge of the local osmotic properties of cartilage is particularly important since the osmotic modulus defines the compressive resistance to external load. At the molecular level we investigated the effect of ionic environment on the structure of aggrecan molecules adsorbed onto well-controlled surfaces. The conformation of the aggrecan were visualized using the AFM. Understanding aggrecan adsorption onto a charged surface provides insight into its interactions with bone and implant surfaces in the biological milieu.
