these SFs for 4 days and chondrocyte activity was determined by glycosaminoglycan (GAG) turnover. In explants cultured in post-exercise SF, GAG synthesis ...
British Journal of Rheumatology 1998;37:671–676
LOADING-INDUCED CHANGES IN SYNOVIAL FLUID AFFECT CARTILAGE METABOLISM B. M. VAN DEN HOOGEN,*† C. H. A. VAN DE LEST,*† P. R. VAN WEEREN,* F. P. J. G. LAFEBER,‡ M. LOPES-CARDOZO,† L. M. G. VAN GOLDE† and A. BARNEVELD* *Department of General and Large Animal Surgery, Utrecht University, †Department of Veterinary Basic Sciences, Biochemistry Section, Utrecht University and ‡Department of Rheumatology & Clinical Immunology, University Hospital Utrecht, The Netherlands SUMMARY The purpose of this study was to determine whether changes in the synovial fluid (SF ) induced by in vivo loading can induce an alteration in the metabolic activity of chondrocytes in vitro. Therefore, SF was collected from ponies after a period of box rest and after they had exercise for a week. Normal, unloaded articular cartilage explants were cultured in 20% solutions of these SFs for 4 days and chondrocyte activity was determined by glycosaminoglycan (GAG) turnover. In explants cultured in post-exercise SF, GAG synthesis was enhanced and GAG release was diminished when compared to cultures in pre-exercise SF. SF analysis showed that levels of insulin-like growth factors (IGF-I and IGF-II ) tended to be higher in post-exercise SF, while no differences were found in metalloproteinase activity, hyaluronic acid and protein concentrations. This study showed that anabolic effects of joint loading on cartilage are, at least partially, mediated by alterations in the SF. K : Exercise, Synovial fluid, Cartilage, Glycosaminoglycans, Equine, IGF-I, IGF-II, Matrix metalloproteinase, Explant culture.
M reports have demonstrated the influence of mechanical stress on cartilage metabolism, involving the turnover of proteoglycans (PG), one of the main matrix components and of importance to the physical properties of cartilage. Animal studies in vivo have shown that immobilization of joints leads to a decrease in PG synthesis and total PG content in cartilage [1–3], whereas the PG content is enhanced in cartilage of animals that were exercised regularly [4–6 ]. Static compression of articular cartilage in vitro decreases PG synthesis and increases the loss of PG [7, 8]. In contrast, dynamic stress increases PG synthesis and promotes the retention of PG in cartilage explants and in chondrocyte cultures [7–10]. Both the frequency and severity of joint loading are also important determinants in the development of osteoarthritis, a degenerative joint disease characterized by damage to the articular cartilage [11]. This is particularly evident with horses since a considerable number of horses end their athletic career prematurely because of osteoarthritis. The general idea is that the underlying cause of this disease is an imbalance between the synthesis and breakdown of matrix macromolecules. This is reflected in an early stage of disease by loss of PG molecules [5, 12], which have a relatively rapid turnover [13]. Mechanical loading of the joint can induce a variety of physiological changes that may contribute to the signalling of the load to the chondrocyte. Deformation of the cartilage induces changes in fluid flow, fluid
pressure, osmotic pressure, electric potential gradients, interstitial pH, cell–matrix interactions, and the shape of the chondrocyte and its nucleus. Each of these factors has been shown to influence chondrocyte activity in vitro [7, 14–18], but whether these factors act directly on intracellular pathways or indirectly by means of humoral signals, autocrine or paracrine, is not known. Another open question is whether chondrocytes are the only cells that signal loading or whether other intra-articular structures can also respond to exercise and therefore contribute to the observed effects of loading on chondrocytes in vivo. For example, cells of synovial tissue may well respond to loading by producing humoral factors such as growth hormones, cytokines and enzymes which are released into the synovial fluid (SF ). Furthermore, as a filtrate of the blood, SF can also contain systemic factors induced by exercise that influence chondrocyte metabolism. Since SF is in direct contact with cartilage and is the important source of nutrition of cartilage, all these humoral factors found in the SF may contribute to maintaining cartilage integrity. The aim of the present study was to determine whether the influence of joint loading on chondrocyte activity is only a direct effect due to cartilage deformation, or is also mediated by humoral signals. Therefore, we investigated whether SF, taken from ponies postexercise when compared to pre-exercise, alters PG metabolism of autologous unloaded cartilage in explant culture. In addition, differences in composition of pre- and post-exercise SF in levels of total protein, hyaluronic acid, active matrix metalloproteinases (MMPs), and insulin-like growth factor I and II (IGF-I and IGF-II ), which could mediate these effects, were determined.
Submitted 1 October 1997; revised version accepted 23 January 1998. Correspondence to: B. M. Van den Hoogen, Laboratory of Veterinary Biochemistry, Utrecht University, PO Box 80.176, 3508 TD Utrecht, The Netherlands.
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MATERIALS AND METHODS Collecting synovial fluid samples Eight Shetland ponies (male, 2 yr old, ~250 kg) had box rest for at least 1 month before an SF sample (~1 ml ) from the carpal joints was taken aseptically (pre-exercise SF ). Then the ponies were submitted to a mild to moderate exercise programme, consisting of 0.5 h trotting in a horse walker every day during 1 week. At the eighth day, the ponies were galloped for 10 min on a treadmill and 30 min thereafter another SF sample was taken from the contralateral carpal joints (post-exercise SF ). No additional agents such as anti-coagulants were added to the SF. The SF samples were centrifuged (3000 g for 15 min) immediately after sampling and the supernatants were stored at −20°C. Cartilage explant culture Articular cartilage tissue culture was performed as described by Lafeber et al. [19]. Cartilage was obtained post mortem from the femoral condyles of the same Shetland ponies that had been used to collect the SF samples. The ponies had had box rest for at least 1 month after the exercise before they were killed for educational purposes. Full-thickness cartilage was cut into square pieces (~2 × 2 mm) and the wet weight (ww) of the explants (5–20 mg) was determined aseptically. The explants were cultured individually in 200 ml of medium in round-bottomed 96-well polystyrene microtitre plates (Nunc, Roskilde, Denmark) at 37°C in a humidified atmosphere of 5% CO in air. 2 The standard medium, consisting of Dulbecco’s modified Eagle’s medium (DMEM ) (Gibco, Breda, The Netherlands), 0.85 m ascorbic acid, 2 m glutamine, 100 IU/ml penicillin and 100 IU/ml streptomycin sulphate, was enriched with 10% heat-inactivated pooled pony serum. Pilot study for culture of cartilage explants in synovial fluid To determine whether it was possible to use cell-free SF without anti-coagulation additives, we compared equine cartilage explants cultured in several concentrations of equine SF with culture in medium with 10% horse serum. After explants had been pre-cultured for 1 day, medium was replaced by 20% SF in DMEM, 50% SF in DMEM, 100% SF, or 10% horse serum in DMEM, all with the vitamin, amino acid and antibiotic additives as indicated above which were added in a negligible volume (
