Quantitative Analysis of Cartilage Components

Collagen: To remove the extractable collagen pool, 1 ml of 1 mg/ml α-chymotrypsin (TLCK Treated, type VII: from bovine pancreas, Sigma) in 50 mM Tris, pH 7.6, containing proteinase inhibitors (1 mM EDTA, 1 mM iodoacetamide and 10 μg/ml pepstatin-A) was added to 50 mg of diced cartilage sample. After incubation at 37°C overnight the supernatant was removed. The supernatant and the saline, used for equilibration, were diluted quantitatively 1:1 with 12 N HCl and the residue was immersed in 6 N HCl to be hydrolyzed at 110°C overnight. The hydrolysates were then dried at 90°C, reconstituted with 0.5 ml distilled water and dried again to remove traces of HCl. Finally, samples were dissolved in 0.5 ml distilled water and clarified by adding charcoal resin decolorizer (prepared from equal amounts of activated charcoal and AG-1 X8 anion exchange resin). The amount of hydroxyproline (μg/mg tissue) was measured by colorimetric methodology at 550 nm using L-4- hydroxyproline (Fluka) as a standard
[22 (link)].
Results are reported as μg hydroxyproline, while in the discussion we refer to collagen content. The extractable collagen pool is expressed as percentage of the total collagen amount, as previously reported
[23 (link)].
GAG: To another 10 mg of diced cartilage sample 1 μl of 19 mg/ml papain (papaya latex, EC 3.4.22.2, Sigma) in 0.1 M Tris–HCl, pH 7.2, containing 10 mM disodium EDTA and 5 mM cysteine-HCl, was added. After incubation at 60°C for 18 hours the digest was removed
[24 (link)]. The amount of GAG was determined by a commercially available colorimetric kit using dye-precipitation of sulphated GAGs with Alcian blue
[25 (link)].