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 probe 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, and thus, swelling pressure within the matrix, both of which are essential for effective load bearing in cartilage and joint 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. A shortcoming of this approach was that it required tissue slices to obtain these osmotic titration curves. This lead to long equilibration times requiring several person-days to study a single cartilage specimen, making this approach unsuitable for routine pathological analysis or for use in tissue engineering applications. More recently, we designed and built a new tissue micro-osmometer to perform these experiments practically and rapidly. 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 sensitively and precisely detects the water uptake of the tissue specimen attached to its surface. 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 mechanical integrity of developing tissues and osmotic compatibility of tissue-engineered cartilage (or ECM) with the hope of improving integration and viability following implantation in regenerative medicine applications. 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 (HA) 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 aggregates, as well as individual aggrecan molecules. The results also indicate that in the presence of HA, 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 increases the load bearing ability of cartilage. Our combined static and dynamic scattering measurements (small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS), static light scattering (SLS), dynamic light scattering (DLS), and neutron spin-echo) demonstrate that aggrecan assemblies exhibit remarkable insensitivity to changes in ionic environment, particularly to calcium ion (Ca+2) concentration. This result is consistent with the role of aggrecan as an ion reservoir mediating calcium metabolism in cartilage and bone. We have also developed a new experimental procedure for mapping the local elastic 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 analysis 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 from which we extract the relevant elastic properties. In conjunction with scattering measurements, micro-osmometry and biochemical analysis, this technique allows us to map the spatial variations in the osmotic modulus of tissues and gels. We mapped the osmotic modulus of bovine cartilage samples by combining tissue micro-osmometry with force-deformation measurements made by the AFM. Knowledge of the local osmotic properties of cartilage is particularly important since the osmotic modulus defines the compressive resistance to external load. We found that the water retention is stronger in the upper and deep zones of cartilage, where collagen fibers are ordered, than in the middle zone where they are randomly arranged. We have constructed the elastic and osmotic modulus maps for the different layers. The latter that is a combination of the elastic and swelling properties, exhibits much stronger spatial variation reflecting the highly heterogeneous character of the tissue. A major objective of tissue engineering is to mimic the ECM environment. However, the complexity of interactions between ECM and cells makes it difficult to design materials for regenerative medicine applications. Previous studies have indicated that the chemical structure of the scaffold is critical. Molecular factors (e.g., hydrophilic or hydrophobic character of the polymer, stiffness, charge density) significantly influence cell adhesion, spreading and growth. In collaboration with researchers at the Carnegie Mellon University we developed novel nanostructured hydrogels, which have potential as an artificial ECM, and can act as a macroscopic scaffold for tissue regeneration. These nanostructured hybrid hydrogels with cleavable nanogels enable controlled-release of sequential encapsulated biomolecules, such as multiple growth factors required at distinct stages during the regenerative process. Collectively, these approaches are helping us get closer to a physical/chemical understanding of the basis of functional properties of normal cartilage, and its changes in development, as well as possible explanations for loss of function in disease, degeneration and in abnormal development.
