These results confirm a significant increase in cell death after injurious compression and suggest that most cell death observed here was by an apoptotic process.

Incorporation of [35S]-sulfate into cartilage tissue indicates the synthesis of aggrecan, the large aggregating proteoglycan (PG) that endows cartilage with resistance to compression. Scintillation counting of tissue digests provides a quantitative measure of incorporated sulfate but does not provide information on the spatial location of synthesis within the tissue. Such spatially specific information is necessary to determine which cell populations respond to diffusible factors and to correlate local mechanical events (e.g., deformation, interstitial fluid stress) to cellular biosynthetic responses. The aim of this study was to develop and characterize a liquid emulsion autoradiography technique, including an automated grain counting procedure, to derive spatial profiles of aggrecan synthesis rates in cartilage. We chose mature 10-14-month-old bovine humeral head articular cartilage as a model system and applied a liquid emulsion autoradiography technique to [35S]-sulfate-labeled, resin-embedded, and semithin-sectioned tissue explants. High-magnification light microscopy color images were captured on a computer. Automated image analysis for grain number determination included a color thresholding procedure to discriminate grains from the lightly stained structural image and computation of the average area of a single grain from each image. Determination of grain number, whether originating from single grains or grain clusters, was performed by dividing the total grain area in the image by the average area of a single grain in the same image. This procedure largely eliminated the effects of variations of microscope light intensity, camera performance, image focus, section stain intensity, and thresholding on the resulting grain numbers. By altering the specific activity of the medium radiolabel and the emulsion exposure times, we demonstrated a linear dose dependence, without saturation, of grain number on radioactive content in the underlying section. By cutting specimens in half and performing liquid scintillation counting on one half and autoradiography on the other half, we found that each disintegration occurring in the section during exposure resulted in 0.67 +/- 0.21 grains (mean +/- SD; n = 58). Therefore, counted grain numbers can be directly converted to incorporated sulfate, largely reflecting the synthesis of the PG aggrecan. As an example of calculated intratissue profiles of aggrecan synthesis rates, we found for the mature bovine tissue in serum-free medium that aggrecan synthesis increases monotonically from the articular surface to the radial zone by as much as tenfold.

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We developed a new quantitative histochemical method for mapping aggrecan content in articular cartilage and applied it to models of cartilage degradation. Ruthenium hexaammine trichloride (RHT) forms co-precipitates with aggrecan, the main proteoglycan component of cartilage, and was previously found to be a good fixative in aiding the maintenance of chondrocyte morphology. We show that these RHT-aggrecan precipitates generate a positive chemographic signal on autoradiographic emulsions, in the absence of any radioactivity in the tissue section, via a process similar to the autometallographic process used previously for localization of trace metals ions in tissues. By exploiting the inherent depth-dependence of aggrecan concentration in adult articular cartilage, we demonstrated that the density of silver grains produced by RHT-derived chemography on autoradiographic emulsions correlated with locally measured aggrecan concentration as determined by the dimethylmethylene blue assay of microdissected tissue from these different depths of cartilage. To explore the benefits of this new method in monitoring tissue degradation, cartilage explants were degraded during culture using interleukin-1 (IL-1) or digested after culture using chondroitinase and keratinase. The RHT chemographic signal derived from these samples, compared to controls, showed sensitivity to loss of aggrecan and distinguished cell-mediated loss (IL-1) from degradation due to addition of exogenous enzymes. The RHT-derived chemographic grain density represents an interesting new quantitative tool for histological analysis of cartilage in physiology and in arthritis.