Effects of mechanical vibration on DNA and proteoglycan syntheses in cultured articular chondrocytes

The objective of this study was to determine the effects of mechanical vibration loading on DNA and proteoglycan syntheses in cultured rabbit articular chondrocytes. Chondrocyte culture plates were placed in a vibratory apparatus and subjected to a mechanical vibratory load at various frequencies and periods during culture. Mechanical vibration was applied at a sinusoidal waveform of 1.4 G-acceleration with frequencies of 200, 300, 400, 800, and 1600 Hz. ³H-thymidine and H₂ ³⁵SO₄ incorporation were used to detect radiolabeled DNA and proteoglycan syntheses, respectively. A frequency of 300 Hz showed a time-dependent augmentation of DNA synthesis and gave a maximal increase on day 3 with periodic vibration (8 h per day), and at 72 h or longer with continuous vibration. It also promoted proteoglycan synthesis in long-term cul-ture (from 3 to 15 days) by periodic vibration. However, frequencies above 400 Hz suppressed biosynthesis. These results suggest that mechanical vibration at certain frequencies may modulate the biosynthetic response of articular chondrocytes. Received: May 30, 2000 / Accepted: October 4, 2000     

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.     

The effect of rheumatoid synovial fluid macrophages on DNA,glycosaminoglycan and collagen synthesis by synovial fibroblasts

Summary The effects of soluble factors secreted by peripheral blood monocytes and rheumatoid synovial fluid macrophages were tested on human synovial fibroblast cultures. Both monocytes and macrophages liberated factors which reduced DNA synthesis (³H-thymidine incorporation) by synovial fibroblasts. Monocyte and macrophage factors stimulated hyaluronic acid synthesis. The activation obtained with rheumatoid synovial macrophages was considerably greater than that with monocytes. Foetal bovine serum was found to have a clear stimulatory effect on the synthesis of collagen and other proteins by fibroblasts. The effects of monocyte and macrophage factors on protein synthesis in synovial fibroblasts were small: collagen synthesis was slightly increased relative to other extracellular proteins.     

Effect of human growth hormone on proteoglycan synthesis in cultured rat chondrocytes

We have previously demonstrated that hGH stimulates DNA synthesis in cultured chondrocytes in the absence of serum. The present study is concerned with the effects of hGH on proteoglycan synthesis by cultured chondrocytes. Chondrocytes were isolated from rat rib growth cartilage by collagenase digestion, plated in plastic dishes, transferred to serum-free MCDB 104 medium, and incubated for 24 h to establish growth arrest. The cultures were then preincubated for 0-24 h with various concentrations of hGH and ovine prolactin (oPrl) and finally pulse-labelled for 30 min with radioactive sulphate in the presence of hormone. hGH, but not oPrl, stimulated sulphate incorporation with an apparent maximum at 50 ng/ml (approximately 170%). The stimulatory effect was apparent after 2 h and maximal after 3h preincubation. After 12 h the stimulatory effect had decreased to insignificant levels. Qualitative analysis of isolated proteoglycans indicated that the stimulation of sulphate incorporation by hGH is exerted at the level of protein synthesis with little effect on glycosylation and sulphation. Further experiments are required to demonstrate whether the stimulatory effect on proteoglycan synthesis is a specific phenomenon or represents one aspect of a general stimulation on cell metabolism in preparation for DNA synthesis.     

Expression of CD44 in normal and rheumatoid synovium and cultured synovial fibroblasts.

OBJECTIVE--To determine if expression of CD44, the principal receptor for hyaluronan, was altered in rheumatoid (RA) synovium and cultured rheumatoid synovial fibroblasts. METHODS--Synovium was obtained from normal adult human joints (n = 4) and from joints of patients with RA (n = 5). Specific monoclonal antibodies to CD44 were used in immunofluorescence of whole synovium and cultured synovial fibroblasts and in quantitative Western blotting and ELISA of CD44 in cultured synovial fibroblasts. RESULTS--CD44 was restricted to the lining layer in normal synovium but present, in reduced concentrations, throughout rheumatoid synovium. Cultured rheumatoid cells were 19% larger in area and showed far fewer and less extensive CD44-positive cytoplasmic extensions, together with reduced staining intensity compared with normal. Quantitative Western blotting normalised for cell protein showed a 75% reduction (normal = 1754 (835), rheumatoid = 409 (84) mean (SD) arbitrary units) in the amount of CD44 in rheumatoid cells compared with normal, and enzyme linked immunosorbent assay (ELISA) of cultured cell monolayers normalised for cell number indicated a 29% reduction (normal = 0.707 (0.110), rheumatoid = 0.504 (0.103), mean (SD) optical density at 405 nm). CONCLUSIONS--Rheumatoid synovial cells showed altered morphology and reduced CD44 expression compared with normal cells. CD44, by means of modulated associations with the cytoskeleton, may be involved in cell shape change.     

Interactions between human osteoarthritic chondrocytes and synovial fibroblasts in co-culture

OBJECTIVE: To imitate the in vivo joint situation and to allow cell interactions, a co-culture system of human osteoarthritic chondrocytes and synovial fibroblasts from a single joint was established and characterized with or without stimulation by IL-1 beta. METHODS: Culture settings included chondrocytes in alginate alone, synovial fibroblasts in monolayer alone and a co-culture of both. Proteoglycan (PG) synthesis was measured by 35S-incorporation, PG content by a dimethylmethylene blue assay, DNA content by a fluorometric assay, and prostaglandin-E2 and IL-1 beta release by ELISA. RESULTS: In co-culture PG synthesis by chondrocytes was significantly reduced in the presence of IL-1 beta (1 ng/ml) compared to controls. PG content of chondrocyte cultures was reduced for controls and IL-1 beta treated co-cultures. Synovial fibroblasts in co-culture did not show significant change of PG synthesis or content when compared to cells in mono-cell culture. PG release into the medium was relatively high in co-cultures. IL-1 beta significantly decreased the proliferation rate of chondrocytes in co-cultures and slightly increased prostaglandin-E2 release. CONCLUSIONS: Co-culturing of osteoarthritic chondrocytes and synovial fibroblasts from a single human joint allows interactions between both entities and may offer a useful tool to study the effects of mediators or new drugs under more in vivo like conditions compared to mono-cell cultures.     

Copper modulation of extracellular matrix synthesis by human articular chondrocytes

OBJECTIVE: The effects of Cu2+ on human articular chondrocytes, arising from both N (normal) and OA (osteoarthritic) cartilage, were investigated "in vitro". METHODS: Chondrocytes, cultured in high density, were incubated with copper chloride (0.01-0.25 microM/mL). Proteoglycan and collagen were assessed by incorporation of [35S]-Sulfate and [3H]-Proline. SDS-PAGE analysis was performed to quantify the ratio of type II to type I collagen. RESULTS: Cu2+ neither increased proteoglycan synthesis by chondrocytes. of origin N or OA, nor influenced their proliferation rate. Collagen synthesis was increased. This effect is time and concentration dependant: in cultures treated for 12 days, collagen synthesis stimulation was +20% and +26% (P < 0.02) in N and OA cultures respectively, the ratio of type II to type I collagen was slightly increased. This effect was more obvious in OA cell lines than in N ones. CONCLUSION: The observations suggest that Cu2+ upregulates collagen anabolism in human articular chondrocytes.     

Human milk stimulates DNA synthesis and cellular proliferation in cultured fibroblasts.

Human milk contains a mitogenic factor that stimulates DNA synthesis and cell division in mouse and human fibroblasts in vitro. Milk at a concentration of 1% (vol/vol) is as active in stimulating DNA synthesis as is 5% (vol/vol) human serum and 10% (vol/vol) calf serum. The mitogenic activity of human milk is destroyed by incubation with trypsin and chymotrypsin. However, neither urea, guanidine hydrochloride-dithiothreitol, nor exposure to pH 1 will inactivate the milk-derived growth factor. Gel filtration and isoelectric focusing indicate that the mitogenic activity of human milk has a molecular weight between 14,000 and 18,000 and an isoelectric point between 4.4 and 4.7.     

Histamine stimulates prostaglandin E production by rheumatoid synovial cells and human articular chondrocytes in culture

Histamine stimulates prostaglandin E (PGE) production by cultures of adherent rheumatoid synovial cells and human articular chondrocytes. When subcultured synovial fibroblasts or human articular chondrocytes were "primed" by preincubation with conditioned media from primary adherent rheumatoid synovial cell cultures (synovial factor), each produced even higher PGE levels upon histamine exposure. This histamine stimulation was prevented by histamine H1, but not H2, antagonists and was more marked if serum was absent from the culture media. Thus, histamine-induced PGE production by these cells is mediated via H1 receptor activation and subsequent arachidonic acid liberation.     

Metabolism of collagen in aspartylglycosaminuria: Decreased synthesis by cultured fibroblasts

Decreased collagen synthesis may explain the connective tissue symptoms (e.g. skeletal deformations and susceptibility to hernias) frequently present in AGU patients. Products from the incomplete intracellular degradation of glycoproteins can interfere with collagen synthesis in AGU. Aspartylglycosaminuria might thus provide a model for studying the regulation of collagen synthesis.     

Acetaldehyde and lactate stimulate collagen synthesis of cultured baboon liver myofibroblasts

Cells with electron-microscopic characteristics of myofibroblasts were isolated from baboon liver biopsy specimens by collagenase digestion and Percoll density gradient centrifugation and then cultured. The cultures consisted of only one cell type. By immunofluorescence, these cells synthesized collagen types I, III, and IV and laminin. Typical features of myofibroblasts were maintained throughout many passages in the culture. To study the effects of ethanol (and its oxidation product acetaldehyde and associated metabolite lactate) on myofibroblast collagen synthesis, the cell cultures were incubated for 24 h in a medium containing either 50 mM ethanol, 200 microM acetaldehyde, or 5 mM lactate. The cells did not contain significant alcohol dehydrogenase activity. Acetaldehyde stimulated significantly (p less than 0.05) myofibroblast collagen synthesis without changing noncollagen protein synthesis or proline pools. Lactate caused a significant (p less than 0.02) increase in intracellular proline pool and collagen synthesis. Ethanol itself did not have any effect on collagen synthesis of myofibroblasts. The stimulation of collagen synthesis of hepatic myofibroblasts by acetaldehyde and lactate may contribute to the development of alcoholic liver fibrosis, as alcohol intake is known to elevate acetaldehyde and lactate in tissues and blood.     

Insulin-like growth factor stimulation of chondrocyte proteoglycan synthesis by human synovial fluid

We investigated the role of insulin-like growth factors (IGFs) as regulating factors of cartilage metabolism in human synovial fluid (SF), using a bovine explant culture system that was shown to respond to recombinant IGF-1 in vitro. SF from rheumatoid arthritis (RA) patients and from control patients was found to stimulate chondrocyte proteoglycan synthesis in bovine articular cartilage. A monoclonal antibody directed primarily against IGF-1 (and to some extent, IGF-2) partially blocked the stimulatory action of serum and totally blocked the stimulation by SF. These findings indicate that IGFs are major regulating factors of cartilage proteoglycan synthesis in human SF. In addition, we measured serum and SF levels of IGF-1 in RA patients and control patients, using a radioimmunoassay. No difference in immunoreactive serum IGF-1 was detected between patients and controls. The IGF-1 levels in SF were consistently lower than in serum, for both patient groups. No differences in IGF-1 concentration were found between RA and non-RA SF. The relevance of these data with respect to joint inflammation is discussed.     

Connective tissue synthesis by cultured scleroderma fibroblasts. I. In vitro collagen synthesis by normal and scleroderma dermal fibroblasts

The authors have been unable to demonstrate an increase in collagen synthesis by fibroblasts isolated from sclerodermatous skin. In order to elucidate this problem further, scleroderma fibroblasts were biopsied from upper dermis, from lower (including subcutaneous) dermis, and from adjacent clinically noninvolved skin. All cell lines failed to show a significant increase in collagen synthesis when they were compared to control fibroblast lines. One difference among them was that fibroblasts from involved areas showed a rate of collagen synthesis equal to or less than cells isolated from adjacent clinically noninvolved sites.     

The effect of synovial tissue on the synthesis of proteoglycan by the articular cartilage of young pigs

Explants of pig articular cartilage were grown in organ culture in the presence of synovial tissue; controls consisted of paired explants that were cultivated in isolation. To find whether the synovial tissue affected synthesis of sulfated proteoglycan by the cartilage, ³⁵SO₄ was added to the medium and its incorporation into the cartilage examined by both biochemical assay and autoradiography. The synovial tissue severely inhibited the uptake of ³⁵SO₄, but if the synovium was removed after 8 days cultivation and the cartilage was grown in isolation for a further 4 days, incorporation of ³⁵SO₄ equalled and sometimes surpassed that of controls which had been grown without synovium continuously for 12 days. Synovium did not prevent the formation of new cartilage on the cut surfaces of the explants, but it reduced the incidence of newly formed cartilage.     

Elevated levels of insulin-like growth factor (IGF) binding protein-3 in rheumatoid arthritis synovial fluid inhibit stimulation by IGF-I of articular chondrocyte proteoglycan synthesis

The objective of this study was to quantify insulin-like growth factor (IGF) binding proteins (IGFBPs) in the synovial fluid (SF) and plasma of patients with rheumatic diseases and to study the role of these proteins in the regulation of cartilage proteoglycan (PG) synthesis. Immunological determination of IGFBP-2, IGFBP-3, IGF-I, IGF-II, interleukin-1β (IL-1β) and tumour necrosis fac-tor α (TNFα) was undertaken in the SF and plasma of 115 patients with rheumatoid arthritis (RA; n = 53), osteoarthritis (OA; n = 44) and other rheumatic disorders. We also determined the effects of SF on bovine cartilage PG synthesis in culture. IGFBP-2 and IGFBP-3 were elevated in the plasma (by 38% and 28%, respectively) and SF (by 56% and 59%, respectively) of patients with RA compared to age- and sex-matched OA controls (determined by RIA and confirmed by Western ligand blot). IGF-I and IGF-II did not differ significantly between the two groups. OA SF, and, to a lesser extent, RA SF stimulated cartilage PG synthesis in culture, and more than 60% of this activity was neutralised by a specific monoclonal anti-IGF-I antibody. Human IGFBP-3 dose-dependently inhibited the stimulation of cartilage PG synthesis effected by SF or human IGF-I. In RA patients, the SF concentration of IGFBP-3 was positively correlated with SF levels of IL-1β and TNFα, with the serum level of C-reactive protein and with the erythrocyte sedimentation rate. We concluded that IGF-I is, under the conditions studied, the most important anabolic factor in human SF with respect to articular cartilage PG synthesis. The bioactivity of IGF-I in joints is modulated by IGFBP-3, which is elevated in RA SF compared to OA SF. Elevated IGFBP-3 in RA SF may reduce the availability of IGF-I to articular chondrocytes, thus interfering with cartilage PG synthesis in RA. Received: 30 November 1996 / Accepted: 19 February 1997