A method of quantitative autoradiography for the spatial localization of proteoglycan synthesis rates in cartilage.

Abstract

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.

A cartilage growth mixture model for infinitesimal strains: solutions of boundary-value problems related to in vitro growth experiments

“…This tensor description of growth allows for anisotropic growth, which may be necessary to include for modeling cartilage as experimental results (Buschmann et al 1996) suggest that proteoglycans may be preferably deposited in a plane normal to the direction of 3 If X and x denote material and spatial coordinates, respectively, then H s =∂u s /∂X ~∂u s /∂x in the linear theory. 4 The superscript α will be used to designate the proteoglycans (p), collagens (c), and others (oth).…”

“…It is decomposed into two equations by assuming that the apparent density changes only because of the elastic part of the deformation. Using (A7) and (5) 3 and introducing the infinitesimal fluid density change n w , 6 the referential continuity equations linearize to…”

“…The local continuity equations (A6) become 6 This follows the linearization approach of (Green and Naghdi 1970). 7 Eqn.…”

“…The type of experiment that was conducted in (Buschmann et al 1996) that quantified the spatial location of molecular deposition in the extracellular matrix could be used to fully characterize each growth tensor. Furthermore, the effect of various biochemical regulators on the growth law may be studied.…”

The Organization of Aggrecan in Human Articular Cartilage

“…Although no analysis was made of the tissues after fixation, embedding, and sectioning, cetylpyridinium chloride was included in the fixative to prevent leaching of the proteoglycans during processing (17).…”