Adult articular cartilage culture system and effects of IL-10

Previous research in this lab has established a calf epiphyseal cartilage culture system and looked at effects of a particular cytokine, interleukin 10 on cartilage. In addition to the findings of increased tissue degradation and decreased tissue regeneration, evidenced by accelerated release of matrix components and reduced uptake of essentials, it is also observed that as IL-1/3 is allowed to diffuse into the extracellular matrix from the periphery, observed degradation of tissue nevertheless centers around the blood vessels embedded inside the matrix. Although vasculature is regularly present in young cartilage tissues, mature cartilage has no blood vessels. Therefore an older culture system without vasculature will help elucidate effects of IL-11 on cartilage specific chondrocytes, with no complication from other cell types. The first purpose of this research, therefore, was to define such a culture system. This task involves modifying several experimental protocols, and conducting control experiments to define parameters for such a system. It was found that both chondrocyte and GAG concentrations are much lower in mature cartilage than in younger tissues, in addition to the much smaller overall ECM volume. Although less populous, chondrocytes in adult bovine articular cartilage have comparable synthetic capability as calf of new glycosaminoglycan (GAG) molecules. Furthermore, in culture with 20% FCS, adult cartilage loses proportionally much more GAG every day than calf, averaging about 5% of overall GAG content. Given the average parameter values and their variances, the statistical paired-t test was adopted. As sample size is directly related to the width of confidence intervals, minimum sizes were calculated for an experiment to be able to detect any possible significant changes due to treatment. From one of the control experiments, it was found that at least 40 cylindrical cartilage disks have to be included in analysis to see a deviation in bulk GAG content from control on the order of 10% due to treatment. Such numbers were obtained for other parameters as well. Treatment protocols of sufficient sample sizes were then designed to ensure conclusive results can be drawn. The second purpose, after the control experiments, was to study the effects of IL-10 on the metabolism of cartilage proteoglycans. It was found that at the concentration of 500 ng/ml, IL-10 has significant impact on mature cartilage matrix, both severely repressing new generation and promoting increased loss of tissue GAG molecules. Accelerated degradation was detectable less than 20 hours after the first introduction of IL-13. GAG synthesis is only 13% of control after 3 days of IL-10 treatment. The third purpose was to understand the correspondence of spatial distribution between introduced IL-1f and GAG loss, through histological staining. It was found that although the spatial pattern of GAG loss was as expected, no clear difference was observable between control and treatment. Unlike younger cartilage with localized GAG loss following IL-10 treatment, mature ECM releases GAG over the entire volume, making it harder to discern from histological stains. Thesis Supervisor: Martha L. Gray Title: Associate Professor