C. Bassleer

Effects of peptidic glycosaminoglycans complex on human chondrocytes cultivated in three dimensions

Human chondrocytes from the pelvic joint were cultivated in suspension; under these conditions, after a few days, cells aggregated. These chondrocytes were morphologically differentiated (round shape, situated inside cavities and surrounded by a matrix synthesized during cultivation) and biosynthetically differentiated (synthesis of type II collagen and cartilage proteoglycans (PG) (Bassleer et al. In vitro 22, 115-120, 1986). In this work, we present the metabolic and cellular effects of a peptidic-glycosaminoglycan (P-GAG) complex isolated from calf cartilage and bone marrow. We analyzed the effects of P-GAG on DNA synthesis (appreciated by 3H-thymidine incorporation into DNA), on type II collagen and on PG synthesis analyzed by specific radioimmunoassays. According to its final concentration in culture medium, P-GAG was able to stimulate proliferation or to favor the production of specific components of cartilage matrix, type II collagen and PG.

Effects of Meloxicam Compared to Acetylsalicylic Acid in Human Articular Chondrocytes

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.

Effects of ipriflavone and its metabolites on human articular chondrocytes cultivated in clusters

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.

Effects of sodium naproxen on differentiated human chondrocytes cultivated in clusters

SummaryThe effects of different pharmacological concentrations of the nonsteroidal 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.

Radioimmunoassay for Human Type Ii Collagen

Human articular cartilage type II collagen (h coll.II) was purified and used to develop a radioimmunoassay. The sequential saturation procedure allowed a sensitivity of 3 ng/tube. The intra and between assay coefficients of variation were less than 10 and 20% respectively in the linear part of the curve. The assay was highly specific for native human articular type II collagen. There was no cross-reactivity with other constituents of cartilage: human proteoglycans, fibronectin, laminin and hyaluronic acid did not interfere with the assay. No cross-reactivity existed with bovine collagen types I, III, IV. However, native collagens from human placenta (I, III, IV, V, VI), rat and calf skin type I collagens and bovine type II collagen produced a weak cross-reaction only at high doses. Concerning the latter, inhibition curves were not parallel. Parallelism of inhibition curves were observed for dilution of type II collagen, produced by human chondrocytes in three-dimensional culture. All of these characteristics indicate that radioimmunoassy of type II collagen is a very sensitive and specific method available for the study and quantification of type II collagen in in vitro experimental conditions.

Proteoglycans synthesized by human chondrocytes cultivated in clusters

Proteoglycan metabolism was studied by a specific human cartilage proteoglycan radioimmunoassay in human chondrocytes cultivated in clusters. In this culture system, after a few days, previously dissociated chondrocytes were aggregated. They then synthesized a new cartilage matrix and were morphologically differentiated; they had a round shape and were situated inside small individual cavities (lacunae). The amounts of proteoglycan released into culture medium and present in chondrocyte clusters were maximal on the third to fifth day of culture; production decreased and stabilized from the 10th day to the end of culture. During the first days of culture, monomeric proteoglycans were present in large proportion; they gradually decreased between the sixth and 11th day of culture. These results suggest a modified synthesis of proteoglycan and hyaluronic acid during cultivation.

Effects of Ximoprofen and Acetylsalicylic Acid on Human Articular Chondrocytes in 3-Dimensional Culture

SummaryDifferent pharmacological concentrations of 2 nonsteroidal anti-inflammatory drugs (NSAIDs), acetylsalicylic acid (ASA) and ximoprofen (XP), were tested on human chondrocytes cultivated in clusters. DNA synthesis, measured by [3H]thymidine uptake into DNA, was depressed by ASA at concentrations typical of those found in synovial fluid and plasma after the administration of a single oral dose (500mg). Similarly, proteoglycans, assayed by a specific radioimmunoassay, were depressed in culture medium, whereas type II collagen was not modified. In contrast, XP did not affect these chondroformative processes in chondrocytes. Both NSAIDs were potent inhibitors of prostaglandin E2 (PGE2) synthesis, XP being more efficient than ASA. These experiments demonstrated that although XP inhibits PGE2 synthesis, it does not depress chondroformative parameters in human cartilage in vitro.Different pharmacological concentrations of 2 nonsteroidal anti-inflammatory drugs (NSAIDs), acetylsalicylic acid (ASA) and ximoprofen (XP), were tested on human chondrocytes cultivated in clusters. DNA synthesis, measured by [3H]thymidine uptake into DNA, was depressed by ASA at concentrations typical of those found in synovial fluid and plasma after the administration of a single oral dose (500mg). Similarly, proteoglycans, assayed by a specific radioimmunoassay, were depressed in culture medium, whereas type II collagen was not modified. In contrast, XP did not affect these chondroformative processes in chondrocytes. Both NSAIDs were potent inhibitors of prostaglandin E2 (PGE2) synthesis, XP being more efficient than ASA. These experiments demonstrated that although XP inhibits PGE2 synthesis, it does not depress chondroformative parameters in human cartilage in vitro.

Characterization of proteoglycans produced by human chondrocytes cultivated in clusters

After enzymatic dissociation from their matrix, human chondrocytes from hip joint were cultivated in suspension, in flasks containing 2 ml of Dulbeccos Modification of Eagles Medium (Flow) supplemented with 10% foetal calf serum (Gibco). Flasks were disposed on a gyrotory shaker (100 rpm) at 37~ [1]. After a few days, cells aggregated and secreted a new intercellular matrix composed of type II collagen and cartilage proteoglycans (PG) [1]. Using a specific human cartilage PG radioimmunoassay [2], we analyzed PG produced by human chondrocytes in dusters.