Human Cartilage Explants Research Articles

Some recombinant human cytokines stimulate glycosaminoglycan synthesis in human synovial fibroblast cultures and inhibit it in human articular cartilage cultures

Recombinant human cytokines were compared for their effects on glycosaminoglycan (GAG) synthesis in human synovial fibroblast cultures and human articular cartilage explant cultures. In fibroblast cultures, recombinant human interleukin-1 alpha (rHuIL-1 alpha), rHuIL-1 beta, and recombinant human tumor necrosis factor alpha (rHuTNF alpha) stimulated hyaluronic acid (HA) production and, to a lesser extent, sulfated GAG production, while recombinant human gamma-interferon did not have a significant effect. Half-maximal stimulation of HA by rHuIL-1 beta was 0.14 pM, while stimulation for rHuIL-1 alpha and rHuTNF alpha was 1.6 pM and 32 pM, respectively. Indomethacin (10 micrograms/ml) had no influence on HA stimulation by cytokines, while hydrocortisone (2-10 micrograms/ml) caused a significant reduction. In articular cartilage cultures, the cytokines inhibited production of sulfated GAGs. The activity of rHuIL-1 beta was greater than that of rHuIL-1 alpha (half-maximal inhibition at 0.71 pM and 4.7 pM, respectively) and both were considerably more active than rHuTNF alpha; gamma-interferon again had no significant effect. Neither indomethacin nor hydrocortisone influenced cytokine-induced inhibition by either rHuIL-1 preparation. These studies indicate that cytokines released during an inflammatory process may affect GAG synthesis in human joint tissues and may have opposite effects on GAG synthesis in different types of connective tissues.

Potential influences of ketoprofen on human healthy and osteoarthritic cartilage in vitro.

Ketoprofen (Orudis, Rhône-Poulenc) is an anti-inflammatory drug with analgesic properties that is used in diseases such as rheumatoid arthritis and osteoarthritis (OA). It is therefore of interest to know whether ketoprofen has a direct influence on cartilage metabolism. We studied the effects of ketoprofen in therapeutic concentrations, on proteoglycan (PG) turnover in explants of human cartilage. The cartilage specimens were divided into three groups: healthy young (less than or equal to 3 yrs) n = 8, healthy old (mean 56 yrs) n = 13 and OA cartilage (greater than or equal to 65 yrs) n = 15. The rate of PG synthesis at day 4 of the culture was measured by the uptake of 35S sulphate. Cartilage PG content and PG release into the medium were determined over 8 days of culture. Ketoprofen stimulated the rate of PG synthesis of young cartilage, but not of old cartilage. In OA cartilage both stimulation and suppression occurred. Ketoprofen had no influence on cartilage PG content and PG release of healthy or OA cartilage during the 8 days of culture. The cartilage was examined histologically, and graded for severity of OA. There was no relation between the severity of OA and the effect of ketoprofen.

Differential responses of old human cartilage explants to synovial- and mononuclear-cell factors.

To investigate mechanisms of cartilage destruction that may apply to rheumatoid arthritis, young and old human, and young porcine, articular cartilage was cultured for 8 days and the effects on proteoglycan (PG) metabolism of normal synovium supernatant (NSS), rheumatoid synovial fluid (RFL), and blood mononuclear cell supernatant (MCF) were studied. The effects were chondrocyte-mediated. An inverse correlation was found between baseline net PG synthesis and the effect of NSS on PG synthesis. Responses of young (porcine and human) cartilage were similar. In young cartilage the three agents induced PG depletion by suppression of net PG synthesis. In old cartilage NSS and RFL induced PG depletion, whereas MCF did not. In cartilage of low baseline net PG synthesis, NSS and MCF stimulated both PG release and PG synthesis; NSS stimulated predominantly PG release, and MCF predominantly PG synthesis. In conclusion, young and old human cartilage differ in the quality of their in vitro response to potentially catabolic factors. This may be due to the difference in baseline net PG synthesis. Synovial extracts differ from mononuclear-cell supernatants in their effects on old cartilage. It is suggested that this is caused by the presence, in different relative amounts, of factors that influence either PG synthesis or PG release.

94 HUMAN PLACENTAL LACTOGEN PROMOTES CELL PRCLTFERATION, DNA SYNTHESIS, AND SOMATOMEDIN RELEASE BY ISOLATED HUMAN FETAL CONNECTIVE TISSUES

Since placental lactogens have been implicated in the control of fetal metabolism, and in the release of somatomedins (SM) by fetal tissues, we examined the actions of human placental lactogen (HPL) on the growth of isolated human fetal myoblasts, fibroblasts and cartilage explants, and on their release of immunoassayable SM-C. Myoblasts were derived from skeletal muscle and fibroblasts from the skin of abortuses delivered between 12 to 17 weeks after prostaglandin induction. Costal cartilage explants were obtained from one hemithorax. After myoblasts and fibroblasts were plated at low density (0.3 × 105 cells per well) HPL (50-1000 ng ml−1) caused a dose-dependent increase in cell number over a 7 day incubation, becoming statistically significant between 50 and 250 ng/ml with individual experiments. After sub-confluent mycblasts and fibroblasts were growth-restricted by exposure to medium containing 1% fetal calf serum, HPL caused a significant increase in [3H] thymidine uptake during a subsequent exposure at similar concentrations. Human growth hormone (HGH) failed to increase thymidine incorporation by myoblasts or fibroblasts, while neither HPL or HGH affected isotope uptake by cartilage explants. Culture medium conditioned for 44 h by exposure to myoblasts or fibroblasts was assayed for SM-C by RIA after extraction with acid-ethanol. The SM-C present in conditioned medium was significantly elevated after exposure of cells to HPL (250 ng ml−1) but not HGH. The results show that HPL is mitogenic for cultured human fetal myoblasts and fibroblasts and that this may be due partly to an increase in SM-C release by the cells.

Delayed aggregation of proteoglycans in adult human articular cartilage.

The biosynthesis and macromolecular organization of proteoglycans was studied in explants of adult human articular cartilage. In a series of pulse-chase experiments, labelling with (35S)sulphate, it was shown that the proteoglycan monomer is synthesized as a precursor that has a low affinity for hyaluronic acid. These findings suggest a possible mechanism by which the rate of incorporation of proteoglycans into the extracellular matrix may be controlled.

Characterization of proteins from human synovium and mononuclear leucocytes that induce resorption of cartilage proteoglycan in vitro.

Both human synovial tissue in culture and lectin-stimulated mononuclear leucocytes produced a protein that induced proteoglycan resorption in explants of bovine nasal cartilage and human articular cartilage. On gel filtration the protein had Mr 16000-20000 and on isoelectric focusing its pI was 5.2-5.3. The protein corresponded to catabolin, which has previously been identified as a product of cultured porcine synovial tissue and mononuclear leucocytes. The action of partially purified human catabolin was not inhibited by cortisol, although the activity of the leucocyte supernatants from which it had been isolated was inhibited. For this reason it is not possible to be sure that the active factor detected in the bioassay of the crude leucocyte culture supernatants is in fact catabolin.

Stimulation of DNA synthesis by ascorbate in cultures of articular chondrocytes.

The addition of 0.2 mM Na L-ascorbate increased the incorporation of 3H-thymidine by rabbit articular chondrocytes in cell and organ culture. The stimulatory response of explants to ascorbate was potentiated by pretreatment of the cartilage with 0.2% clostridial collagenase (type 1) or trypsin for 15-30 minutes. In explants there was a latent period of 3 to 4 days before increased labeling of the nuclei could be detected. The effect was transient and declined after 8 days of culture. It was more evident in organ cultures of immature (3-month-old) than 2- to 3-year-old rabbits. Age differences were not detected in cell cultures. Explants of adult human articular cartilage were stimulated by ascorbate when the medium was supplemented with 10% fresh human serum but not by fetal bovine serum. The findings indicated that synthesis of DNA by articular chondrocytes in situ is regulated by responsiveness of the cells proper to compounds such as vitamin C, by properties of the extracellular matrix, and by factors in the serum. Ascorbate was cytotoxic at concentrations greater than 0.2 mM in the presence of certain batches of serum.

Characterization of collagenase, other metallo-proteinases and an inhibitor (TIMP) produced by human synovium and cartilage in culture.

1. Explants of human synovium from normal, rheumatoid and osteoarthritic subjects produced proteinases in culture that could degrade connective tissue macromolecules at neutral pH. 2. The proteinases detected in the media were predominantly of the class requiring metal ions for activity, and occurred in either partially or wholly latent forms. Activation could be achieved by treatment with either trypsin or the organo-mercurial, 4-aminophenylmercuric acetate. 3. Explants of both normal and osteoarthritic synovium produced an inhibitor of collagenase in the early days of culture, similar to that previously described for rabbit tissues. In contrast, no free inhibitor could be detected in the media of rheumatoid synovial cultures. 4. Explants of normal human articular cartilage produced latent collagenase and free inhibitor in culture. 5. These findings suggest that the activity of tissue-derived metallo-proteinases may be controlled by locally synthesized inhibitors, by mechanisms analogous to those already described for rabbit model systems. Changes in the synthesis and degradation of inhibitor could be important in human diseases in which destruction of connective tissue is a prominent feature.