Intact triple helical collagen molecules are highly resistant to proteolytic enzymes, whereas degraded (unwound) collagen is easily digested. This fact was exploited to develop a simpli fied method for the quantification of the amount of degraded collagen in the collagen net work of connective tissues. Essentially, the method involves extraction of proteoglycans with 4 M guanidinium chloride, selective digestion of degraded collagen by a-chymotrypsin, hydrolysis in 6 M HC1 of the released fragments as well as the residual tissue, and then mea surement of the amount of hydroxyproline in both pools* Since the digestion of degraded collagen by a-chymotrypsin and measurement of hydroxyproline is not restricted to a sper cific collagen type, this technique can be applied to a wide variety of connective tissues. The method was validated with articular cartilage. Levels of in situ degraded collagen were about four-fold higher in degenerated (fibrillated) cartilage than in its healthy counterpart derived from the same donor. More detailed investi gations revealed that the collagen damage in degenerated cartilage is more extensive at the cartilage surface than in the region adjacent to bone. This was not the case in healthy carti lage; identical low values were obtained at the surface and close to the bone. An impaired collagen network has been hypothesized to be the reason for the swelling of cartilage in os-4 ■* teoarthritis (OA). The present paper presents the first experimental evidence to support this hypothesis: more damage to the collagen network (i.e., more degraded collagen molecules within fibrils) is linearly related to more extensive swelling of the OA tissue in hypotonic saline.
This pilot study represents the first evidence of differential susceptibility to OA in guinea-pigs. Comparison of these two strains of guinea-pig has revealed that increased metabolism within the affected tissues, cartilage and bone, is associated with the development and progression of OA. This work demonstrates that the Strain 13 is a viable age-matched control to the Hartley strain and merits a more in depth evaluation of the contribution of bone and bone metabolism to OA.
Matrix metalloproteinases (MMPs) are involved in physiological tissue remodeling and pathological conditions like tumour metastasis and joint destruction. Until now, no convenient and sensitive MMP-activity assay in crude media like synodal fluid has been available. Therefore, the highly soluble fluorogenic substrate TNO211 (Dabcyl-Gaba-Pro-Gin-Gly-LeuGIu(EDANS)-AIa-Lys-NH2), containing the MMP cleavable Gly-Leu bond and EDANS/Dabcyl as fluorophore/quencer combination, was synthesized and characterized as an MMP specific substrate. We show that the fluorogenic assay using ] NO211 is sensitive and can detect MMP activity in culture medium from endothelial cells and untreated synovial fluid (SF) from RA and OA patients, and control subjects. MMP activity in SF significantly increased in the order C < OA < RA, thus the frequent use of OA samples as control in studies on RA is debatable.A ey words: Matrix metalloproteinase; Synovial fluid; I luorescence quenching
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