Although the function of proteoglycans within the tendon extracellular matrix are not fully understood, changes in their turnover have been associated with tendinopathies. In contrast to cartilage, aggrecanases are constitutively expressed and active in tendon, indicative of a high rate of aggrecan turnover. Clinical trials investigating the use of active site MMP inhibitors have been confounded by side-effects which involve tendonitis and “musculoskeletal syndrome”. Such side effects may relate to non-specific inhibition of tendon aggrecanases required to maintain normal metabolic homeostasis. The purpose of this study, therefore, was to compare the rate turnover of tendon and cartilage proteoglycans derived from the same joint and to determine the effect of MMP inhibitors (actinonin and marimastat) on aggrecan catabolism. Deep digital flexor tendon explants from compressed and tensional regions were dissected from young and mature bovine. Explants were precultured and then cultured for a further 4 days with or without marimastat (0–2 M) or actinonin (0–200 M). Proteoglycan and lactate quantification, Western blot analysis of degradation products and RT-PCR analyses were performed on these samples. In a separate experiment for measurement of proteoglycan turnover, explants were set up as described above then pulse chase labelled with [35S] sulphate. The rate of turnover of 35S-labelled proteoglycans from the matrix of tendon (and articular cartilage obtained from the same animal) was subsequently calculated from the amount of 35S-labelled macromolecules appearing in the medium each day and that remaining in the matrix of explants at the termination of culture. Proteoglycan turnover (presumably predominantly aggrecan) was markedly higher in tendon versus cartilage. This difference was apparent in tendons from all regions and ages. Both marimastat and actinonin inhibited aggrecanase-mediated proteoglycan catabolism in both tendon and cartilage explants. As expected mRNA expression for the aggrecanases, MMPs and TIMPs was unaffected by addition of these inhibitors to the culture medium. Aggrecan turnover in tendon is higher than that of articular cartilage, which may be attributed to distinct physiological properties of this proteoglycan in tendon. Importantly, immunohistochemical staining for aggrecan in tendon indicates its presence in between collagen fibres and fibril bundles and thus aggrecan aggregates may dissipate resultant compressive loads by resisting the flow of water in these locations. In addition, aggrecan may facilitate the sliding of fibrils during the small amount of elongation of the tendon whilst under tension. Thus, the half-life of tendon aggrecan is significantly reduced because it constantly participates in repeated resistance to compression. Our data also demonstrates that both marimastat and actinonin can inhibit aggrecanase-mediated proteoglycan catabolism in tendon cultures. This suggests that the occurrence of “musculoskeletal syndrome” in clinical trial patients may be due to the fact that these inhibitors affect the activity of aggrecanases in tendon, thus preventing them from playing their normal role in tendon aggrecan turnover and consequently perturbing normal physiological function.

Introduction: Previous studies have demonstrated that exposure of normal bovine and human osteoarthritic cartilage to n-3 polyunsaturated fatty acids (PUFAs) such as those present in fish oils can modulate the expression and activity of the degradative and inflammatory factors that are responsible for cartilage destruction [1,2]. In these studies, supplementation of cartilage explant cultures with n-3 PUFAs resulted in an abrogation of aggrecanase activity as well as mRNA expression of mediators of inflammation. To date, few studies have examined the effect of PUFAs on the metabolism of other tissues within the musculoskeletal system, therefore the present work examines the effect of n-3 PUFA supplementation on tendon metabolism.

Methods: Bovine deep digital flexor tendon explants were obtained from the compressed region of young metacarpophalangeal joints (2-week-old) and supplemented with eicosapentaenoic acid (EPA), as previously described [2]. Release of proteoglycan metabolites was analysed using Western blotting whilst RT-PCR analysis was used to examine the mRNA expression patterns of matrix proteases and inflammatory agents.

Results: Exposure to the n-3 fatty acid, EPA, markedly changed the overall lipid composition profile of the tendon with major changes occurring in the supplemented fatty acid (i.e., EPA), with a concomitant percentage reduction in other polyunsaturated fatty acids. Aggrecanase activity was present in the media from control cultures, as expected [3]. However, supplementation with EPA had no effect on this activity, in contrast to articular cartilage where aggrecanase catabolites were absent from the conditioned media following treatment with n-3 PUFAs [1,2]. mRNA expression for the inflammatory mediators (COX-2, IL-1β, TNF), ADAMTS-5, MMPs and TIMPs was also unchanged following supplementation with EPA, again contrasting with articular cartilage where mRNA expression was abolished.

Discussion: This study demonstrates that exposure of bovine tendon explant cultures to an n-3 PUFA, EPA, had no effect on the mRNA expression or activity of aggrecanases; similarly, expression of the inflammatory mediators was also unaffected. Importantly, within this musculoskeletal tissue, aggrecanases are constitutively active and appear to be involved in normal, everyday turnover of aggrecan, in contrast to non-pathological articular cartilage where aggrecanase-generated metabolites are only detected following treatment with catabolic agents. Similarly, COX-2 mRNA expression is present constitutively within tendons whereas in cartilage it is absent under basal (unstimulated) conditions. These data demonstrate that the incorporation of n-3 PUFAs have a differential effect on the regulatory mechanisms which control gene expression within articular cartilage versus tendon.