A study will be conducted on the hyaluronate (HA) and link glycoprotein (GPL) molecules extractable, under associative conditions, from rabbit articular cartilage. The main purpose of this project is to study the earliest changes occurring in induced osteoarthritic samples and to compare it with normal ones. Such changes are characterized by a disaggregation of the proteoglycans and an increase in the tissue water content. At the early stages the disease is assumed to be reversible. It is hypothesized that these stages reflect a change in the HA-binding capacity in the middle and lower zones of the cartilage matrix. This property of the HA seems to be mediated by its interaction with one or more glycoproteins. The absence of some of the glycoproteins could, consequently, provoke a disruption of the matrix molecular organization. This phenomenon could result from the specific action of a biological factor (lysozyme or collagenase) and a special interest will be given to the possible role of lysozyme as a triggering factor in osteoarthritis. Two HA fractions, the """"""""free"""""""" HA and the HA bound in proteoglycan aggregates, will be isolated and purified by a sequence of equilibrium and rate zonal centrifugations. Their interaction with the glycoproteins, similarly isolated, and with exogenous cartilage lysozyme, will be studied by means of reaggregation experiments in vitro. These experiments will be conducted in both dissociative and associative conditions. The purified components, as well as the resulting products of aggregation, will be characterized by an array of methodologies. These include: light-scattering (conventional and quasielastic), microviscometry (with electronic detection), SDS gel electrophoresis, immunological identifications and ultracentrifugal characterization. The latter includes: polydispersity determination in Cs2SO4 isovolumetric density gradients, molecular weight by equilibrium centrifugation in capillary microcells, and analytical rate zonal centrifugation, also in isovolumetric gradients. Some innovations introduced are: new sectorial cells for swinging buckets, the use of non-solvating electrolytic media (Cs2SO4), and a pretreatment of normal cartilage with highly purified collagenase. The latter is used to optimize the yield of proteoglycans extractable under associative conditions. Dissociations would blur, completely, the difference between the free and bound HA fractions being studied. It is expected that a comparison of these fractions will help to attain the long term objective of this project.
