Glucosamine sulfate did not affect DNA synthesis nor coll II production but caused a statistically significant stimulation of PG production by chondrocytes from human osteoarthritic cartilage cultured for up to 12 days in 3-dimensional cultures.

These in vitro studies suggest that ACS is able to increase matrix component production by human chondrocytes and to inhibit the negative effects of IL-1 beta.

Articular diseases, such as osteoarthritis, is the clinical expression of the loss of cartilage function. COX inhibitors are widely used in the treatment of such pathologies for their beneficial effects on inflammation but often produce a negative activity on cartilage synthesis. In this study, we determined the effect of different prodelphinidins, the major compounds isolated from Ribes nigrum leaves, on the proteoglycans (PGs), type II collagen (coll. II) and prostaglandin E(2) (PGE(2)) production by differentiated human chondrocytes cultivated in long term (12 days) and in clusters as well as their inhibition potential on COX-1 and COX-2 in vitro. Gallocatechin trimer (GC-GC-GC) showed the higher stimulation of PGs and coll. II production (1 microg ml(-1)) and the synthesis of PGE(2) was significantly reduced by gallocatechin dimer (GC-GC), gallocatechin-epigallocatechin (GC-EGC) and GC-GC-GC at 10 and 100 microg ml(-1). The inhibition of PGE(2) synthesis was confirmed by the in vitro test on purified COX enzymes, showing the selectivity of prodelphinidins on COX-2. However, the prodelphinidins had no effects on COX activity in the whole blood assay. Our studies suggest that the prodelphinidins fractions from R. nigrum may be useful as an additive agent in the prevention of osteoarthritis.

SUMMARYCartilage was taken from the macroscopically normal part of human femoral heads immediately after orthopedic surgical operations for total prothesis consecutive to hip arthrosis. Alter clostridial collagenase digestion and repeated washings, chondrocytes (10 s cells) were cultivated in a gyrotory shaker (100 rpm}. Under these conditions, cells were kept in suspension and after 3 to 5 d formed a flaky aggregate which, on Day 10, became dense. These chondrocytes were morphologically differentiated: they had a round shape, were situated inside cavities, and were surrounded by a new matrix. Histochemical methods showed the presence of collagen and polysaccharides in cell cytoplasm and in intercellular matrix, and the immunofluorescence method using specific antisera (anticartilage proteoglycans and anti-type II collagen) showed that these two constituents were in intercellular matrix. The measurement of the amounts of proteoglycans (PG) released into culture medium and those present in chondrocyte aggregate (by a specific PG radioimmunoassay} showed a maximum production on Days 3 to 5 of culture, then the production decreased and stabilized (from Day 10 to the end of culture). The observed difference between the amounts of PG in aggregates after 20 d and those after 2 h of culture demonstrated that PG neosynthesis did occur during cultivation. This conclusion was supported by other results obtained by [l'C]glucosamine incorporation in chondrocyte aggregates. Moreover, the aggregate fresh weight related to cell number (appreciated by DNA assay} increased significantly with culture duration. Three-dimensional chondrocyte culture represents an interesting model: chondrocytes were differentiated morphologically as well as biosynthetically and synthesized a new cartilage matrix.

The effects of different pharmacological concentrations (0, 5, 10, 100, and 1000 ng/mL) of synthetic human calcitonin (hCT) and salmon calcitonin (sCT) on the incorporation of [3H]thymidine and production of proteoglycans (PG) and type II collagen (coll II) by human articular chondrocytes during a 20-day period were studied in a tridimensional chondrocyte culture model. [3H]Thymidine uptake was measured in chondrocyte clusters, and specific PG and coll II RIAs were performed every 4 days on the culture medium and cell aggregates; total PG and coll II production were also assessed at different culture durations by adding the amounts found in culture media and their corresponding clusters. Incubation with hCT or sCT did not affect [3H]thymidine uptake regardless of the dose. For each culture period, PG and coll II release into culture medium, cluster content, and total production increased significantly in a dose-dependent manner. Cumulative curves for these parameters showed a progressive significant increase with culture duration at hCT and sCT doses of 0, 5, and 10 ng/mL. Cumulative curves obtained with 10, 100, and 1000 ng/mL were seldom significantly different from one another. No differences emerged between the use of hCT or sCT. Thus, CT exerted no proliferative effect on human articular chondrocytes in tridimensional culture, but displayed a dose-dependent and prolonged stimulatory effect on PG and coll II production. CT may possess chondroprotective properties in addition to its other known effects.

Meloxicam is a new nonsteroidal anti-inflammatory drug (NSAID) derived from enolic acid, which has displayed potent anti-inflammatory properties in animal studies combined with low gastrointestinal toxicity. Other NSAIDs have been shown, in vitro, to have a variety of effects on cartilage repair processes in diseased articular cartilage. The aim of this study was to ascertain the effects of meloxicam on some of these processes using in vitro models. Acetylsalicylic acid, a NSAID whose characteristics have been previously elucidated in the models, was used as an active comparator. The effects of meloxicam were different from those of acetylsalicylic acid on chondrocyte clusters. At pharmacologically active concentrations, meloxicam was a potent inhibitor of prostaglandin-E₂ production. However, all chondroformative processes were unaffected by meloxicam as indicated by a lack of effect on DNA synthesis and on type-II collagen and proteoglycan levels in chondrocyte culture medium and clusters, while acetylsalicylic acid decreased proteoglycan production and cell proliferation. Consequently, these in vitro findings suggest that meloxicam does not adversely affect the reparative processes active within the cartilage matrix of a diseased joint. This study represents a sound basis for future studies to establish the effects of meloxicam on osteoarthritis disease progression.

Ipriflavone (IP) is an isoflavone derivative that was suggested to have bone-sparing effects in post-menopausal and senile osteoporosis. A moderate stimulatory effect of IP and its metabolites on proliferation of osteoblastic cells was reported in rat osteoblastic osteosarcoma cell line. We investigated the effects of different concentrations (0, 1, 10 and 100 micrograms/ml) of IP and its metabolites (MET I, II, III and V) on the incorporation of [3H] thymidine and production of proteoglycans (PG) and type II collagen (COL II) by human articular chondrocytes during a 12-day period, in a three-dimensional chondrocyte culture model. [3H]thymidine uptake was measured in chondrocyte clusters, and specific PG and COL II radioimmunoassays were performed every 4 days on the culture medium and cell clusters. Incubation with IP or its metabolites did not affect [3H]thymidine uptake regardless of the dose. PG released into the culture medium and PG cluster content rose significantly (P < 0.025) in presence of IP (1, 10 and 100 micrograms/ml). MET I increased PG release in culture medium (10 and 100 micrograms/ml) and PG cluster content (100 micrograms/ml). MET II has no effect on PG production. MET III increased PG in culture medium (100 microgram/ml) but did not influence PG cluster content while MET V (100 micrograms/ml) increased both PG release in culture medium and PG cluster content. COL II release in culture medium and COL II cluster content were significantly (P < 0.025) increased in presence of IP (10 and 100 micrograms/ml), MET III (1, 10 and 100 micrograms/ml) or MET V (100 micrograms/ml). MET I and II did not significantly affect COL II production.

The effects of different pharmacological concentrations of the non-steroidal anti-inflammatory drug (NSAID) sodium naproxen (NAP) were tested on several metabolic functions of differentiated human chondrocytes cultivated in clusters and compared with the action of acetylsalicylic acid (ASA). DNA synthesis was significantly inhibited by ASA but not by NAP. Proteoglycan production was also markedly decreased by ASA, while synthesis of type II collagen was not modified. By contrast, NAP did not affect these chondroformative processes. Both NSAIDs were potent inhibitors of prostaglandin E2 production. These results indicate that in terms of the parameters tested NAP does not lead to deleterious effects on human articular chondrocytes cultured in vitro.