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OBJECTIVES: Matrix metalloproteinases (MMPs) produced by chondrocytes play a role in the development of cartilage degradation in joint diseases. Moreover, inhibition of MMP secretion by macrophages accumulating in arteriosclerotic plaques would account for the plaque stabilising activity of statins in cardiovascular patients. Recently, simvastatin has been shown to inhibit both developing and established collagen induced arthritis in a murine model. We thus decided to investigate the effect of simvastatin on the production of MMP-3 from cultured interleukin (IL)1 stimulated human chondrocytes. METHODS: Cells from human cartilage, obtained from eight subjects with osteoarthritis undergoing surgery for total hip prostheses, were cultured in the presence of different concentrations of simvastatin (5, 10, and 50 micromol/l) with and without IL1beta (5 ng/ml). MMP-3 level was measured in the culture medium after 48 h of incubation. RESULTS: IL1beta stimulation of chondrocytes increased MMP-3 concentration in the cultures (from 0.69 (0.09) to 1.94 (0.12) ng/microg protein). Incubation with simvastatin was associated with a dose dependent reduction in MMP-3 increase, both in the presence (-15%, -17%, and -26% with 5, 10, and 50 micromol/l, respectively) and in the absence (-32% with 50 micromol/l) of IL1beta. The inhibiting effect of simvastatin was completely reversed by the addition of mevalonate (100 micromol/l) or farnesol (10 micromol/l). CONCLUSIONS: Our data show that simvastatin, by blocking HMGCoA-reductase and interfering in the prenylation processes, is able to inhibit MMP-3 production from cultured human chondrocytes that have been either unstimulated or stimulated with IL1beta, thus suggesting a possible additional mechanism for statins in counteracting chronic joint disease related cartilage damage.

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OBJECTIVE: This study investigated the effect of hydrostatic cyclical pressure on the cell ultrastructure and cytoskeleton of normal and osteoarthritis (OA) human cultivated chondrocytes in vitro. METHODS: The different effects of pressurization with sinusoidal waves at a minimum pressure of 1 MPa, a maximum pressure of 5 MPa and a frequency of 0.25 Hz for 3 hrs on normal and OA chondrocytes were assessed by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and immunoflurescence microscopy (IF). RESULTS: Structural differences exist between normal and OA chondrocytes at the nuclear, cytoplasmic and cytoskeletal level. Pressurization did not alter the normal chondrocytes, but had a beneficial effect on OA chondrocytes, by increasing the number of cell organelles responsible for synthesis activities. IF examination has shown that the distribution of actin protein in normal chondrocytes is polarized on the apical sides of the cellular cytoplasm. However, in OA chondrocytes the signal of the actin protein is not as well defined. Similarly, the localization of the tubulin protein in normal and OA cells also appears to be different. Hydrostatic pressure did not cause any modification in the cytoskeletal organization of the OA chondrocytes. CONCLUSION: This study confirms the different morphology, structure and cytoskeletal aspect of normal and OA chondrocytes and the important role played by pressure on cell morphology. The recovery of OA chondrocytes observed by an increase of cytoplasmic organelles does not seem to involve the cytoskeleton.

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OBJECTIVE: To determine the dissolving ability (DA) of linear pentasodium tripolyphosphate (PSTP), cyclic trisodium metaphosphate (TSMP), polymeric sodium metaphosphate (SMP) on synthetic crystals of calcium pyrophosphate dihydrate (CPPD) and on crystalline aggregates of menisci from patients with chondrocalcinosis (CC). METHODS: Synthetic CPPD crystals were mixed with phosphate buffered saline (PBS), which contained the different polyphosphates, for one hour at 37 degrees C. The calcified menisci were obtained from the knees of four female patients with CPPD disease who underwent total arthroscopic meniscectomy for degenerative meniscal lesions. Meniscal cryosections and fragments were incubated in SMP (15 mg/ml PBS) at 37 degrees C for one hour and 24 hours, respectively. Histological evaluation on meniscal samples after polyphosphate incubation was carried out by ordinary transmitted light microscopy and polarised light microscopy. The dissolution of CPPD crystals by polyphosphates was assessed by atomic absorption spectroscopy, which determined the amount of calcium liberated from synthetic crystals and meniscal fragments. Cytotoxicity of SMP was evaluated by tetrazolium salt assay and by an ultrastructural study on cultured chondrocytes. RESULTS: SMP and PSTP showed higher DA on CPPD crystals than TSMP. Analysis of the DA values at increasing concentrations of SMP showed that a concentration of 15 mg/ml completely dissolved 2.0 mg CPPD crystals. The solution of meniscal CPPD crystals showed a significant increase of calcium concentration after three hours and 24 hours of SMP incubation (p=0.0001; Kruskal-Wallis analysis of variance) compared with fragments incubated in PBS control solution. Macroscopic and microscopic evaluation of meniscal specimens showed a notable reduction of CPPD deposits. A 50% inhibitory dose on cultured chondrocytes was reached at the maximum concentration of SMP used in this work (15 mg/ml); ultrastructural analysis did not show morphological alterations in the treated cells. CONCLUSION: The results of this study indicate that linear polyphosphates are effective in dissolving both synthetic and ex vivo CPPD crystal aggregates. This suggests a potential therapeutic use for these molecules in the treatment of symptomatic CC.

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Objective This study investigated the in vitro effects of chondroitin sulfate (CS) on human articular chondrocytes cultivated in the presence or in the absence of interleukin-1beta (IL-1beta) during 10 days of culture with and without pressurization cycles. Design The effects of CS (10 and 100 microg/ml) with and without IL-1beta were assessed in the culture medium of cells exposed to pressurization cycles in the form of synusoidal waves (minimum pressure 1 Mpa, maximum pressure 5 Mpa) and a frequency of 0.25 Hz for 3 h by immunoenzymatic method on microplates for the quantitative measurement of human proteoglycans (PG). On the 4th and 10th day of culture the cells were used for morphological analysis by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Results The presence of IL-1beta determines a significant decrease in PG concentration measured in the culture medium. When the cells are cultured in the presence of IL-1beta and CS, a statistically significant restoration of PG levels is observed. Under pressurization conditions, we observed that PG concentration in the medium of cells presents a significant increase at baseline conditions, in the presence of IL-1beta+CS10 and IL-1beta+CS100, but not with IL-1beta alone. The results concerning metabolic evaluation are confirmed by the morphologic findings obtained by TEM and SEM. Conclusions These in vitro studies confirm the protective role of CS, which counteracts the IL-1beta induced effects and they confirm the importance of pressure on chondrocyte metabolism and morphology.

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BACKGROUND: Spa therapy is frequently used in daily rheumatological practice, but its benefit remains to be evaluated. The purpose of this paper is to evaluate the effectiveness and tolerability of mud-packs and mineral baths with fluorurate radioactivity water from the thermal resort of Merano (Bolzano) versus short wave therapy in patients with osteoarthritis of the knees. METHODS: Forty-eight patients were treated for a period of two weeks with mineral water baths and mineral mud-packs and twenty-four patients were treated with short-wave therapy for the same period. Assessment criteria were spontaneous pain ratings on a visual analogue scale (VAS), functional impairment (Lequesne's index), quality of life index (AIMS1), analgesic and/or non-steroidal anti-inflammatory drugs consumption and patient and physician assessment of effectiveness and tolerability of treatments. These criteria were recorded at the beginning and at the end of the treatments and after 3 months. RESULTS: A significant improvement (p < 0.0001) in the Lequesne's index, in the VAS and a significant decrease in analgesic consumption was achieved in both groups up to 15 days. The improvement remains to the end of the follow-up period only in the group treated with spa therapy. CONCLUSIONS: This study suggests that spa therapy has a prolonged, beneficial, symptomatic effect in osteoarthritis of the knees.

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To evaluate the efficacy and tolerability of the new diclofenac-N-(2-hydroxyethyl)-pyrrolidine lecithin gel (DHEP lecithin gel, with 1.3% DHEP and 2.4% lecithin) compared with the efficacy and tolerability of diclofenac-N-(2-hydroxyethyl)-pyrrolidine gel (DHEP gel) in peri and extraarticular inflammatory states, a controlled, randomized, double-blind clinical study was conducted. Two groups of 50 patients each were enrolled and were given one of the two different formulations with a slight massage on the painful area three times a day for 10 consecutive days. Patients received a self-evaluation notebook in which to record daily assessment of spontaneous pain (Huskisson's visual analogue scale). On days 0, 3 and 10, the patients were visited by the investigator. All patients completed the study. The assessment of spontaneous pain showed that although pain decreased in both groups, the decrease was more marked in patients taking DEHP lecithin gel and that it reached a statistically significant difference at days 5, 6, 7 and 8. This decrease was also confirmed by assessments on the ordinal scale. Although periarticular swelling disappeared in both groups, swelling severity decreased sooner in patients taking DHEP lecithin gel. The efficacy and safety of both treatments was judged to be good or excellent by 70% of the patients in each group. The efficacy of the active principle, pyrrolidine salt, is confirmed. Moreover, the formulation containing lecithin passes through the skin lipid barrier more easily than the formulation without lecithin and is as valid as the other in the therapy of rheumatic disorders. Finally, DHEP lecithin gel preparation has a quicker therapeutic action on symptoms, such as spontaneous pain and local swelling, than DHEP gel.

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In this work we studied the morphological and ultrastructural aspects of normal and osteoarthritic (OA) human articular chondrocytes cultivated in alginate gel for 48 hours. After this period the chondrocytes in Petri dishes were exposed to cyclic pressurization (minimum pressure 1 MPa and maximum pressure 5 MPa) at 0.25 Hz frequency for three hours. In other loading procedures the cells were exposed to continuous pressure (24 MPa) for three hours. Some dishes were not pressurised and these served as controls. The cells were then fixed for transmission electron microscopy (T.E.M.) and for scanning electron microscopy (S.E.M.). No ultrastructural changes were observed in normal chondrocytes exposed at physiological pressure. OA cells placed under physiological pressure showed a partial recovery on morphological and ultrastructural aspects. Normal and OA samples exposed to continuous pressure (24 MPa) showed a morphological worsening in both T.E.M. and S.E.M. studies.

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Recent studies have outlined the role of bisphosphonates, and particularly clodronate, as potential therapeutic agents for inflammatory and degenerative joint diseases. On this basis, we carried out an open, non comparative pilot trial to evaluate the effects of clodronate on synovial fluid concentration of some inflammatory mediators (prostaglandin E2, leukotriene B4 and tromboxane B2) after intra-articular, repeated administrations in 20 patients (7 males and 13 females) with synovitis secondary to knee osteoarthritis. At the end of the treatment period, statistically significant reductions (p < 0.05) of spontaneous pain and pain on active movement, evaluated by means of a 100 mm visual analogue scale (VAS), were reported. Linear regression analysis showed that the decrease of pain was correlated with the bisphosphonate induced reduction of prostaglandin E2 levels. These results, in spite of the limitation due to the open design of the trial suggest a possible role of bisphosphonates in the treatment of knee osteoarthritis.

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