title: Ruthenium hexaammine trichloride chemography for aggrecan mapping in cartilage is a sensitive indicator of matrix degradation.
abstract: 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.
authors.affiliation: Chemical and Biomedical Engineering, Ecole Polytechnique, Montreal, Quebec, Canada.
authors.forename: M D
authors.initials: MD
authors.lastname: Buschmann
authors.forename: A M
authors.initials: AM
authors.lastname: Maurer
authors.forename: E
authors.initials: E
authors.lastname: Berger
authors.forename: P
authors.initials: P
authors.lastname: Perumbuli
authors.forename: E B
authors.initials: EB
authors.lastname: Hunziker
mesh_terms: D053674:Aggrecans; D000818:Animals; D002358:Cartilage, Articular / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects; D002417:Cattle; D002810:Chondroitinases and Chondroitin Lyases / Q000378:metabolism; D046508:Culture Techniques; D005109:Extracellular Matrix / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure; D016326:Extracellular Matrix Proteins; D006651:Histocytochemistry / Q000379:methods*; D006811:Humerus / Q000473:pathology; D007091:Image Processing, Computer-Assisted; D007375:Interleukin-1 / Q000494:pharmacology; D037181:Lectins, C-Type; D010447:Peptide Hydrolases / Q000378:metabolism; D010781:Photography; D011509:Proteoglycans / Q000302:isolation & purification*; D017975:Ruthenium Compounds
chemical_list: D053674:Aggrecans; D016326:Extracellular Matrix Proteins; D007375:Interleukin-1; D037181:Lectins, C-Type; D011509:Proteoglycans; D017975:Ruthenium Compounds; C031584:hexammineruthenium; D010447:Peptide Hydrolases; C035855:keratinase; D002810:Chondroitinases and Chondroitin Lyases