A METHOD OF QUANTITATIVE AUTORADIOGRAPHY FOR THE SPATIAL LOCALIZATIONOF PROTEOGLYCAN SYNTHESIS RATES IN CARTILAGE

incorporation of [S-35]-sulfate into cartilage tissue indicates the sy nthesis of aggrecan, the large aggregating proteoglycan (PG) that endo ws cartilage with resistance to compression, Scintillation counting of tissue digests provides a quantitative measure of incorporated sulfat e but does not provide information on the spatial location of synthesi s within the tissue, Such spatially specific information is necessary to determine which cell populatons respond to diffusible factors and t o correlate local mechanical events (e.g., deformation, interstitial f luid stress) to cellular biosynthetic responses, The aim of this study was to develop and characterize a liquid emulsion autoradiography tec hnique, including an automated grain counting procedure, to derive spa tial profiles of aggrecan synthesis rates in cartilage. We chose matur e 10-14-month-old bovine humeral head articular cartilage as a model s ystem and applied a liquid emulsion autoradiography technique to [S-35 ]-sulfate-labeled, resin-embedded, and semithin-sectioned tissue expla nts, High-magnification light microscopy color images were captured on a computer, Automated image analysis for grain number determination i ncluded 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, whet her originating from single grains or grain clusters, was performed by dividing the total grain area in the image by the average area of a s ingle grain in the same image, This procedure largely eliminated the e ffects of variations of microscope light intensity, camera performance , image focus, section stain intensity, and thresholding on the result ing grain numbers. By altering the specific activity of the medium rad iolabel and the emulsion exposure times, we demonstrated a linear dose dependence, without saturation, of grain number on radioactive conten t in the underlying section, By cutting specimens in half and performi ng liquid scintillation counting on one half and autoradiography on th e other half, we found that each disintegration occurring in the secti on during exposure resulted in 0.67 +/- 0.21 grains (mean +/- SD; n = 58), Therefore, counted grain numbers can be directly converted to inc orporated sulfate, largely reflecting the synthesis of the PG aggrecan , As an example of calculated intratissue profiles of aggrecan synthes is rates, we found for the mature bovine tissue in serum-free medium t hat aggrecan synthesis increases monotonically from the articular surf ace to the radial zone by as much as tenfold.