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.

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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.

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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

We describe a new principle for assessment of the activity of proteolytic enzymes of all classes and show the application of this principle for the quantitative assay of bacterial collagenase and human matrix metalloproteinases (MMPs). Central to this new principle is the presence of a proenzyme that can be activated into an active enzyme by a single proteolytic event. The regular activation sequence in the proenzyme is replaced using protein engineering by an artificial sequence recognized by the proteinase to be determined. The latter can act as an activator for the newly engineered proenzyme. In the present paper a simple colorimetric assay for the determination for MMPs is described based on this principle. With the aid of protein engineering, a modified pro-urokinase has been prepared in which the activation sequence normally recognized by plasmin (Pro-Arg-Phe-Lys upward arrowIle-Ile-Gly-Gly) has been replaced by a sequence expected to be recognized and hydrolysed by many MMPs (Arg-Pro-Leu-Gly upward arrowIle-Ile-Gly-Gly). The active urokinase resulting from activation of the modified pro-urokinase by a MMP could be measured either directly, using a specific chromogenic peptide substrate for urokinase, or indirectly via urokinase-catalysed plasminogen activation. The response of the assay to equal molar quantities of active MMPs decreases in the order MMP-2>MMP-9>MMP-1>MMP-3>MMP-7. The detection limit for MMP-9 was below 15 pM, corresponding to 3. 75x10(-15) mol per assay. Using the assay, increased MMP activity was detected in synovial tissue extracts from rheumatoid arthritis patients compared with those from osteoarthritis patients, and in stomach tumour extracts as compared with normal stomach tissue extracts.

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Objective-To investigate the influence of age, osteoarthritis (OA), and osteochondrosis (OC) on the matrix metalloproteinase (MMP) activity in the synovial fluid (SF) of equine joints. Methods-SF was collected from normal and osteoarthritic metacarpophalangeal joints (normal: 14 adult, 28 juvenile; OA: 22 adult). And from normal and osteochondrotic tarsocrural joints (5 months: 11 normal, 8 OC; 11 months: 7 normal, 6 OC). Subsequently, overall MMP activity was measured. Results-The level of active MMPs was almost twofold higher in SF from juvenile horses (age up to 11 months) than in SF from mature animals (4-30 years; p<0.001). In juvenile horses MMP activity was higher in 5 month old foals than in 11 month old foals (p<0.01). In adult horses MMP activity was independent of age. In OA joints the activity was nearly twice as high as in normal joints (p<0.001). In OC joints MMP activity was not significantly diVerent from normal, age matched, control joints. Conclusions-MMP activity in SF from normal adult joints is not related to age. In juvenile joints MMP activity is significantly higher than activity in joints from adult animals. It is hypothesised that the gradual decrease in MMP activity with increasing age reflects the declining metabolic activity resulting from ceasing growth and the accompanying decrease in cartilage remodelling. The increased MMP activity in osteoarthritic joints most likely reflects matrix destruction. In osteochondrosis MMP mediated matrix degradation appears not to be diVerent from normal joints. (Ann Rheum Dis 1998;57:697-699) Matrix metalloproteinases (MMPs) are essential for normal matrix turnover and play a key part in pathological conditions like osteoarthritis (OA). Matrix turnover in adult articular cartilage is extremely slow. In young individuals metabolism will have to be maintained at a substantially higher level to allow for growth and remodelling.