Cartilage hydration (water uptake) is essential for load bearing and lubrication since it is the osmotic pressure that causes it to resist applied loads. Cartilage swelling is also exquisitely sensitive to changes occurring in development, disease, degeneration, and aging. Moreover, controlled hydration or swelling of cartilage provides a means of determining functional properties of cartilage and of other extracellular matrices. Specifically, we have used controlled hydration of cartilage to measure important physical/chemical properties of the collagen network and 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 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 the 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 was that it required many person-days to study a single cartilage specimen, so it was not suitable to routine pathological analysis or for use in tissue engineering applications. Secondly, a significant amount of tissue was required to obtain the osmotic titration curves, contributing to long equilibration times. We recently developed a new 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 a 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. To illustrate the applicability of the new apparatus, we measured the swelling pressure of tissue-engineered cartilage specimen. The micro-osmometer will eventually permit us to obtain a profile of the osmotic compressibility or stiffness as a function of depth from the articular surface to the bone interface. It will also allow us to quantify the contributions of individual components of ECM (such as aggrecan and hyaluronic acid) to the total osmotic pressure. Moreover, it should allow us to assess the osmotic compatibility and mechanical integrity of developing tissues and of tissue-engineered cartilage (or ECM) in regenerative medical applications.
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