Effects of chondroitin sulfate and interleukin-1 beta on human articular chondrocytes cultivated in clusters.

OBJECTIVE: To test the effects of chondroitin sulfate (ACS, a glycosaminoglycan of cartilage) with and without interleukin-1 beta (IL-1 beta) on human articular chondrocytes cultivated in clusters and in long-term (0-16 days or 16-32 days). DESIGN: Chondrocyte productions of proteoglycans (PGs), type II collagen (coll-II) and prostaglandin E2 (PGE2) were assayed by specific radioimmunoassays applied to conditioned culture media and to clusters. RESULTS: During the two culture periods (0-16 days or 16-32 days), ACS (100-1000 micrograms/ml) increased total PG production and had no effect on the production of coll-II by chondrocytes. During the first 16 days, ACS (500-1000 micrograms/ml) decreased total PGE2 synthesis. IL-1 beta decreased PG and coll-II productions and increased PGE2 synthesis. During the first period (0-16 days), while the cluster is forming, ACS counteracted the IL-1 beta-induced effects on PG (500-1000 micrograms ACS/ml), coll-II (100-1000 micrograms ACS/ml) and PGE2 (500-1000 micrograms ACS/ml) productions. During the second period (16-32 days), when the cluster is already formed, ACS counteracted the IL-1 beta-induced effects on total PG (100-1000 micrograms ACS/ml), coll-II (1000 micrograms ACS/ml) and PGE2 (1000 micrograms ACS/ml) productions. CONCLUSION: 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.

Stimulation of proteoglycan production by glucosamine sulfate in chondrocytes isolated from human osteoarthritic articular cartilage in vitro.

OBJECTIVE: This study investigated the in-vitro effects of a crystalline glucosamine sulfate (GS) preparation on DNA synthesis and on proteoglycan (PG) and type II collagen (coll II) production by human articular chondrocytes isolated from human osteoarthritic articular cartilage in a 3-dimensional culture system for 4, 8, and 12 days. MATERIALS AND METHODS: Human articular chondrocytes from osteoarthritic femoral heads were isolated from their matrix by collagenase digestion and then cultured in suspension. Under constant agitation, cells aggregated and formed a cluster within a few days. The effects of GS (1-100 micrograms/ml) on chondrocytes were determined by quantifying DNA synthesis (by measurement of [3H]-thymidine uptake) as well as PG and coll II production using radiommunoassays (RIAs) specific for coll II and to human human cartilage PG. Cross-reaction with GS in the RIAs was not detected. Moreover, PG size distribution was determined by exclusion chromatography under associative conditions to determine the association of PG monomers with hyaluronic acid (HA) to form large molecular weight PG aggregates. RESULTS: Under the above conditions, PG production in culture media and chondrocyte clusters was increased by GS (10-100 micrograms/ml). DNA synthesis and coll II production were not modified by GS. In addition, GS did not modify the physico-chemical form of PG produced by cells during culture. CONCLUSIONS: 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.

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 tiaprofenic acid and acetylsalicylic acid on human articular chondrocytes in 3-dimensional culture.

Two nonsteroidal antiinflammatory drugs (NSAID: acetylsalicylic acid, ASA, and tiaprofenic acid, TA) were tested on differentiated human chondrocytes cultivated in clusters. DNA synthesis was depressed by ASA at therapeutic concentrations. The amount of proteoglycans in culture medium was decreased by ASA, whereas type II collagen was not modified. By contrast, TA did not affect chondroformative processes in chondrocytes. Both NSAID were potent inhibitors of prostaglandin E2 (PGE2) synthesis, TA being more efficient than ASA. From these experiments, we conclude that TA and ASA inhibit PGE2 synthesis; TA did not depress chondroformative variables in human cartilage in vitro, while ASA induced a decrease of DNA and proteoglycan syntheses.

In vitro effects of Etodolac and acetylsalicylic acid on human chondrocyte metabolism.

The effects of two nonsteroidal anti-inflammatory drugs (NSAIDs), Etodolac (ETO) and acetylsalicylic acid (ASA), used at pharmacological concentrations, were tested on several metabolic parameters of human chondrocytes cultivated in three-dimensional culture. The results indicated that proteoglycan synthesis was significantly decreased by ASA treatment, whereas Etodolac did not affect this parameter. Neither ASA nor Etodolac modified type-II collagen production. Both NSAIDs were potent inhibitors of PGE2 production, but Etodolac was more efficient at equimolar concentrations. In contrast, collagenolytic activity was unaffected by Etodolac.

Effects of sodium naproxen on differentiated human chondrocytes cultivated in clusters.

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.

In-vitro evaluation of drugs proposed as chondroprotective agents.

Three proposed chondroprotective agents (CP), namely glucosamine sulfate (GAS), chondroitin sulfate (CS) and glycosaminoglycan-peptide complex (GP-C), were tested on differentiated human articular chondrocytes cultured in clusters. Chondrocyte productions of proteoglycans (PG), type II collagen (coll. II) and prostaglandin E2 (PGE2) were established by specific radioimmunoassays applied to the culture medium (CM) and in chondrocyte clusters (CC). Collagenolytic activity was assayed in CM. DNA synthesis, studied by measuring 3H-thymidine incorporation, was unaffected by CS and GAS. GP-C, at low concentration, stimulated DNA synthesis. GP-C, at higher doses, induced a high increase in PG and coll. II productions. GAS and CS induced a stimulatory effect limited to PG production. None of the CP tested here affected the basal PGE2 production by human chondrocytes.

Effects of hormones and local growth factors on articular chondrocyte metabolism.

Human chondrocyte proliferation and production of matrix components such as proteoglycans and type II collagen (coll. II) were studied in an in vitro model of differentiated chondrocytes. It clearly appears that several hormones such as growth hormone, calcitonin, androgens and parahormones such as insulin like growth factor I and epidermal growth factor stimulate chondrocyte proliferation and coll. II and proteoglycan synthesis. These hormones and parahormones have no effect on either prostaglandin production or release and activation of collagenase. From our investigations in vitro, articular chondrocytes are target cells for hormones and local factors mainly responsible of chondroformation.

In vitro assays of chondrocyte functions: the influence of drugs and hormones.

Human articular chondrocytes may be cultured in three dimensions, according to a method already validated. This model allows us to study the repair processes of the cartilage, by measuring the proliferative activity of chondrocytes and the synthesis of two major constituents of matrix: proteoglycans and type II collagen. Some substances are characterised by stimulatory effect on DNA synthesis and no effect or a defective effect on matrix components: this is the case for Epidermal Growth Factor. Others are able to stimulate (hGH) or to depress (acetyl salicylic acid) both chondrocyte proliferation and matrix components synthesis. Finally, some substances called "chondroprotective", such as the glycosaminoglycan-peptide complex, GP-C (Rumalon) stimulate either the proliferative response or the synthesis of proteoglycans and type II collagen, according to the dose.

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.

Effects of human and salmon calcitonin on human articular chondrocytes cultivated in clusters.

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.

Effects of hormones and drugs on cartilage repair.

When cartilage is attacked, either by an excess of charges or by biochemical agents, its morphological and functional impairment is associated with a local homeostatic reaction; this includes a proliferative response (assessed by 3H-thymidine incorporation) and a stimulation of proteoglycan and type II collagen (II coll) synthesis. This homeostatic reaction may be studied in vitro using tridimensional culture of human chondrocytes. Cartilage clusters are formed after 4 days' culture and chondrocytes multiply for the first 15 days of culture. Furthermore, human cartilage proteoglycans and II coll, assayed by specific radioimmunoassays, are released into the culture medium and constitute the new matrix of the clusters. Moreover, it appears that these human chondrocytes are targets for several hormones capable of stimulating a proliferative response without affecting proteoglycan and II coll synthesis.

Effects of etodolac on human chondrocytes cultivated in three dimensional culture.

Human chondrocytes in three-dimensional culture were incubated for up to 20 days in the presence of etodolac, a nonsteroidal anti-inflammatory drug which penetrates readily into the synovial fluid. Even at an etodolac concentration of 80 micrograms/ml, DNA synthesis, proteoglycan synthesis, and type-II collagen synthesis were unchanged. Collagenase production was also unaffected by etodolac (60 micrograms/ml). In contrast, prostaglandin E2 production was reduced by 84% in the presence of 60 micrograms/ml of etodolac. The 80 micrograms/ml concentration is 5 times that found in the serum of subjects treated with 200 mg of etodolac twice a day for 6 days, and 33 times the concentration in synovial fluid of arthritic patients treated with etodolac 200 mg twice a day for 7 days. These in vitro results indicate that anti-inflammatory levels of etodolac may not damage articular cartilage in vivo.

New diagnostic tools and methodological approaches: an outlook to the future.

In spite of recent progress, the aetio-pathogenesis of a large number of osteoarticular diseases is as yet not well known. This is apparent for commonly occurring diseases such as osteoarthritis (OA), rheumatoid arthritis (RA), type I osteoporosis (post-menopausal) and type II osteoporosis (senile). As a result of the lack of knowledge about these pathogenic factors, therapeutics often remain symptomatic and not completely satisfactory. Nowadays there is a need for methodological tools to explore the causes or pathogenic factors of these conditions. Moreover, in OA, there is at present no way to predict the development of this disease and to follow its evolution, hence, there is a need for specific markers. These biological and methodological developments are useful for searching for new molecules able to compensate for the pathogenic anomalies demonstrated in RA and OA. In this paper, we will discuss some of the methodological developments now in progress which may provide solutions to some of the aetiopathogenic, diagnostic and therapeutic problems related to RA and OA.

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.

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.