A biochemical histochemical analysis of the efficiency of a low ionic strength solution in extracting cartilage extracellular matrix components

RESUMO

The extracellular matrix of cartilage is composed of collagen, proteoglycans and non-collagenous proteins. These components may interact sufficiently strongly with each other, so that they can only be extracted using chaotropic agents such as 4 M quanidine chloride. Extraction in this normally preceded by homogenization in phosphate buffered saline (PBS) solution (0.15 NaCl, 5mM phosphate buffer, pH 7.4) followed by centrifugation to obtain a precipitate which is then extracted with guanidine chloride. In this work, the suitability of PBS alone for extracting the components of chicken xiphoid cartilage was compared with that of guanidine chloride. The stability of the matrix components obtained was also analyzed. Proteins with molecular masses of 30, 40, 52, 58 and 110 kDa were identified in the PBS extract. Small proteoglycans were detected in guanidine chloride extracts, but not in PBS extracts, suggesting that these molecules were strongly bound to other components of the matrix. The examination of cartilage sections stained with toluidine blue and Ponceau S after exposure to saline and guanidine chloride, showed that although saline did not remove substantial amounts of glycosaminoglycans it caused structural changes in the organization of the extracellular matrix. However, this effect was much less than with guanidine chloride.