Articular chondrocytes and synoviocytes in culture: influence of antioxidants on lipid peroxidation and proliferation

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Articular chondrocytes and synoviocytes in culture: influence of antioxidants on lipid peroxidation and proliferation Bodo Kurz and Michael SchUnke Anatomisches Institut der Universitat Kiel, Olshausenstrasse 40, D-24098 Kiel, Germany

Summary. Chondrocytes and synoviocytes are the main cell types in articular joints. Articular cartilage is fed by synoviocytes via synovial fluid and has a low partial oxygen pressure. Thus, chondrocytes show oxygen radical protective mechanisms in vivo and are unproteeted against these factors under common culture conditions. We investigated the influence of ascorbic acid, Fe2+, glutathione and alpha-tocopherol on lipid peroxidation and proliferation of rat articular chondrocytes and rabbit synoviocytes (HIG-82) in vitro. A combination of ascorbic acid and Fe2+ induced the production of thiobarbituric acid-reactive material as a marker of radical-mediated lipid peroxidation in homogenates and/or supernatants of cultured chondrocytes and synoviocytes. The amount of lipid peroxidation of chondrocytes was about 3-fold higher than that of synoviocytes. Ascorbic acid or Fe2+ alone had no significant influence on the production of thiobarbituric acid-reactive material. Lipid peroxidation could be abolished by addition of the radical scavenger alphatocopherol, whereas glutathione had no effect. 25-50 j.lM alpha-tocopherol decreased the ascorbic acid - (100 j.lg/ ml) and Fe2 + - (3 j.lM) induced lipid peroxidation to a basal level. Moreover, ascorbic acid inhibited the proliferation of rat chondrocytes and rabbit synoviocytes measured by eH]-thymidine incorporation. Alpha-tocopherol and glutathione had no influence on the proliferation of chondrocytes but alpha-tocopherol decreased the growth of synoviocytes and increased the anti-proliferative effect of ascorbic acid on these cells. The importance of these findings for the use of ascorbic acid, glutathione and alphatocopherol in chondrocyte and synoviocyte cultures, or the influence of these molecules on the etiology and treatment of articular diseases will be discussed. Correspondence to: B. Kurz

Ann Anat (1997) 179: 439-446 © Gustav Fischer Verlag

Key words: Chondrocyte - Culture - Ascorbic acid - Proliferation - Lipid peroxidation - Alpha-tocopherol - Synoviocyte - Glutathione

Introduction Numerous studies have reported on the cultivation and investigation of chondrocytes and synoviocytes, because these cells are the main articular cell types and targets or origin of lots of widespread diseases such as rheumatoid arthritis, osteoarthritis or osteochondrosis dissecans. Since chondrocytes dedifferentiate quickly under common culture conditions, several cultivation techniques were employed, including 3-dimensional perfusion cultures (Bujia et al. 1995), high cell density cultures (Farquharson and Whitehead 1995) and nonadhering or adherent monolayer cultures (Balmain et al. 1995, Yan et al. 1994, Aulthouse et al. 1994). In nearly all cases chondrocytes are cultivated in the presence of ascorbic acid, since collagen synthesis is an ascorbic acid-dependent mechanism (Ryan et al. 1991; Murad et al. 1981). Ascorbic acid is known as an antioxidant, but in some cases it can be involved in hydroxyl radical-induced lipid peroxidation. Ascorbic acid reduces Fe3 + to the hydroxyl radical inductor Fe2+, and hydroxyl radicals cause lipid peroxidation (Halliwell and Gutteridge 1984; Rowley and Halliwell 1983; Winterbourn 1981). Free Fe2+ is added to nearly all culture media and also included in pathogenically changed synovial fluid (Ahmadzadeh et al. 1989; Halliwell and Gutteridge 1984). It has the power to induce lipid peroxidation in vitro or in vivo, if radical scavengers are missing. Tschan et al. (1990) demonstrated a better survival of avian chondrocytes in vitro by the addition of catalase. We therefore investigated the influence of ascorbic acid, free Fe2+ and

the radical scavengers alpha-tocopherol and glutathione on lipid peroxidation and proliferation of rat articular chondrocytes and rabbit synoviocytes (cell line· HIG-82) in vitro, because cell proliferation is also inhibited by oxygen radicals (Vincent et al. 1991). Our study shows that rat chondrocytes undergo a strong and synoviocytes a weaker lipid peroxidation in the presence of free Fe2+ and ascorbic acid, and that this effect can be abolished by the addition of alpha-tocopherol. Additional data showing the inhibition of rat chondrocyte proliferation by ascorbic acid and of synoviocyte proliferation by ascorbic acid and alpha-tocopherol will be presented, and it will be suggested that ascorbic acid-induced inhibition of proliferation is a non-radical-mediated mechanism.

or without stimulants) and homogenized. Samples were divided and every 500 III volume was used for the lipid peroxidation assay or for a protein assay. Lipid peroxidation in the samples was estimated by measuring the formation of thiobarbituric-reactive material (TBAR), i. e., malondialdehyde (MDA). The assay was performed as described by Villacara et al. (1989) and Buege and Aust (1978), with some modifications. The 500 III samples were mixed with 1 ml thiobarbituric acid solution (0.375%, containing 20% trichloracetic acid and 0.25 M HCl) and incubated at 100°C for 15 min. Thereafter the samples were cooled and centrifugated at 1500 x g for 10 min. The absorbance of the supernatant was measured in a spectrophotometer at 533 nm using MDA as standard (thiobarbituric acid, MDA, ascorbic acid and alpha-tocopherol from Sigma, Deisenhofen, Germany; Fe2S04 from Merck, Darmstadt, Germany).

Proliferation assay

Materials and methods The proliferation was measured by a modified method described elsewhere for thymic epithelial cells (Kurz et al. 1996). Chondrocytes and synoviocytes were isolated and/or cultivated as described above. 100,000 chondrocytes or 25,000 synoviocytes/cm2 were seeded into 4 cm2 wells and cultured in a normal medium. After 24 h agonists were added as 1000- or 100-fold stock solutions in ethanol (alpha-tocopherol) or water (ascorbic acid, glutathione). After an additional 48 h the cells were incubated in 1 ml medium containing the agonists and 11lCi [3H]-thymidine (Amersham, Buckinghamshire, UK; code TRK 565) for 5 h. The supernatant was then discarded and cells successivly washed with PBS, twice with methanol, water, 10% trichloracetic acid and water (5 min each). Cells were dissolved in 1 ml 0.3 M NaOH (15 min), neutralized with 1 ml 0.3 M HCl and, after addition of 10 ml scintillation liquid (Hydroluma, Baker Chemicals, GrossGerau, Germany; code 8584), measured in a beta-counter.

Isolation and cultivation of chondrocytes Rat articular cartilage was obtained from knees of 4 day old Wistar rats that had been removed aseptically and washed twice in Hank's buffered salt solution (HBSS, containing 100 U/ml penicillin/streptomycin and amphotericin; from Seromed). The knees were incubated in 0.1 % trypsin/phosphate-buffered saline (PBS) for 2 x 30 min at 37°C and transferred into 50% fetal calf serum (FCS)/PBS. Under a binocular the articular cartilage was freed from connective tissue, muscles and bone material, and the dissected cartilage was transferred for 6 h into a collagenase solution (collagenase from Clostridium histolyticum, Sigma No. C1889; 1 mg/ml in Ham F12 containing 100 Vlml penicillin/streptomycin and amphotericin and 1% FCS) at 37°C. After centrifugation at 1000 g for 5 min, the supernatant was discarded and the pellet resuspended in 10 ml medium (Ham F12, 10 % FCS, 100 U/ml penicillin/streptomycin and amphotericin) and filtered through a 20 Ilm nylon mesh. The vital cells in the final cell suspension were counted by the trypan blue exclusion method with a hemacytometer and seeded in a density of 100,000 chondrocytes/cm2 (cultured at 37°C; 5 % CO 2 atmosphere). Cells were characterized by measurement of collagen type II synthesis and immunocytochem~stry against collagen type II.

Cultivation of HIG-82 synoviocyte cell line HIG-82 (from the American Type Culture Collection) is a synoviocyte cell line predominantly of type B cells, isolated after a spontaneous establishment of an aging, late passage culture of rabbit synovium primary cultures (Georgescu et al. 1988). The cells were seeded at a density of 25,000 cells/cm2 and cultured in Ham F12 (10% FCS, 100 U/ml penicillin, streptomycin and amphotericin). The cells were subcultured weekly.

Electron microscopy Rat chondrocytes and synoviocytes were cultured on coverslips as described above. The coverslips were rinsed twice with PBS, fixed with 5% glutaraldehyde in PBS for 1 hat 4°C, rinsed again twice with PBS and treated with 2% aqueous OS04 for 1 h. The samples were dehydrated with ascending concentrations of ethanol, and embedded in araldite. The coverslips were discarded and ultra-thin sections prepared. The sections were incubated for 15 min with saturated uranyl acetate in 70% methanol, hydrated in descending concentrations of methanol and incubated for 5 min in a plumbic citrate solution in a COz-poor atmosphere.

Results Cell cultures

Lipid peroxidation Confluent monolayers of or synoviocytes (culture washed twice with HBSS. bation buffer by scraping

cultured chondrocytes (culture day 6) day 4) in 10 cm2 Petri dishes were The cells were harvested in 1 ml incuwith a rubber police man (HBSS with

Dissociated cells from cartilage of knees of 4 day-old Wistar rats were cultured as mono layers. The cells were characterized by the predominant production of collagen type II (data not shown) and grew in polygonal patterns without cell contacts. The cells showed the ultrastructural

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Fig. 1. Articular chondrocytes of a rat knee after 7 days in culture. a) Difference interference contrast microscopy. The cells grow in a monolayer and contain vesicles (arrow). Bar = 60 J.lm. b) Phase contrast microscopy. The cells have a polygonal shape and some vesicles in the cytoplasm. Many cells have two nucleoli (arrow). Bar =60 J.lm. c) Electron microscopy. In the cells large amounts of rough endoplasmic reticulum (arrow), many free ribosomes and some vesicles in the cytoplasm (small arrows) are detected. The nucleus consists predominantly of euchromatin. Bar = 2.5 J.lm.

Fig. 2. Synoviocytes (HIG-82) after 4 days in culture. a) Light microscopy of a semi-thin section of HIG-82 cells counterstained with hemalum. The spindle-shaped cells are in tight contact and have many nucleoli (arrow). The cells are aligned in one direction. Bar =60 J.lm. b) Electron microscopy of HIG-82 cells in culture. The cells contain euchromatin-rich nuclei and many vesicles, ribosomes and mitochondria. Bar = 2.5 J.lm.

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features of active, protein-producing cells (Fig. 1). Spindle-shaped cells, as a morphological marker for dedifferentiation, appeared after one to two weeks in small spots at the edge of the layer. HIG-82 synoviocytes were cultured as a monolayer and formed lines of spindle-shaped cells. They had many nucleoli and showed a large number of organelles like metabolically active cells on the ultrastructural level. Cell contacts were not visible (Fig. 2).

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Lipid peroxidation was measured by quantifying the amount of thiobarbituric acid-reactive material (TBAR). Experiments with homogenates of cultured rat chondrocytes showed that ·Fe2+ alone had no influence on the TBAR production (Fig. 3). In the presence of ascorbic

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Fig. 6. Influence of alpha-tocopherol (black circles) and glutathione (squares) on the Fe2+ (3 j.lM)- and ascorbic acid (100 11g/ ml)-induced thiobarbituric acid-reactive material (TBAR) production from rat chondrocyte homogenate in vitro. Values ± standard deviation (n = 4).

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acid, there was a dose-dependent induction of lipid peroxidation. Ascorbic acid also induced TBAR production in homogenates of chondrocytes and synoviocytes in a dose-dependent manner in the presence of free Fe2+ (Figs. 4 and 5). The amount of TBAR/j.!g membrane protein was much higher for chondrocytes than for synoviocytes (about 3-fold). This hydroxyl radical-mediated damage to the membranes could be abolished by the ad-

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Fig. 9. Influence of alpha-tocopherol without (black circles) and with (white circles) ascorbic acid (100 ~glml) and gluathione (black squares) with ascorbic acid on the proliferation of cultured synoviocytes measured by [3H]-thymidine incorporation. Values ± standard deviation (n =3).

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Fig. 10. Influence of alpha-tocopherol and glutathione on the proliferation of cultured rat chondrocytes measured by [3H]-thymidine incorporation. Alpha-Tocopherol (squares) and glutathione (black circles) without, and alpha-tocopherol (circles) and glutathione (black squares) with ascorbic acid (100 llg/ml). Values ± standard deviation (n = 3).

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Table 1. Influence of ascorbic acid and alpha-tocopherol on thiobarbituric acid-reactive material (TBAR) production in the supernatants of rat chondrocytes cultivated for 12 h in HBSS Stimulus

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Cell proliferation

Proliferation of the cells was measured by incorporation of [3H]-thymidine. The experiments showed a dose-dependent inhibition of rat chondrocyte and rabbit synoviocyte proliferation by ascorbic aCid (Figs. 8 and 9). Alphatocopherol had no influence on the proliferation of chondrocytes, either alone or in combination with ascorbic aCid (Figs. 8 and lO). However, alpha-tocopherol inhibited proliferation of synoviocytes dose-dependently, and increased the anti-proliferative effect of ascorbic acid (Fig. 9). Glutathione had no influence on the proliferation of these cells (Fig. 10).

Discussion Most cell types protect themselves against toxic oxygen radicals by decomposing them with antioxidants or special enzymes (i. e. catalase, superoxide dismutases, glutathione peroxidases; Cadenas 1989). Articular chondrocytes normally do not need such kinds of protective mechanisms, because cartilage is a tissue with a low partial oxygen pressure (Stockwell 1983). Under normal culture conditions however, partial oxygen pressure is about 21 'Yo, so that the chondrocytes here undergo a stronger oxygen radical attack and need additional antioxidants. Ascorbic aCid is an antioxidant and a widespread medium supplement for cultures of articular chondrocytes, since it is also commonly known as a stimulator of procollagen mRNA synthesis, hydroxylation and collagen secretion, e. g. for chondrocytes of chickens, swine and cattle (Ryan et al. 1991; Pacifici and Iozzo 1988; Sandell and Daniel 1988). Thus, ascorbic acid is an important medium component for a rich extracellular matrix production in vitro.

However, ascorbic acid also catalyses Fez+-dependent oxidative radical reactions (Halliwell and Gutteridge 1984; Rowley and Halliwell 1983; Winterbourn 1981). These reactions can induce lipid peroxidation and extensive disruption of cellular and organelle membranes via hydroxyl radicals, and are thought to be one of the major components in the etiology of inflammatory joint diseases such as rheumatoid arthritis (Minotti and Aust 1987; Rowley et al. 1984; Blake et al. 1981). In our studies we observed a strong dose-dependent induction of thiobarbituric acid-reactive material by a combination of Fez+ and ascorbic aCid in homogenates and supernatants of rat articular chondrocytes and rabbit synoviocytes (HIG-82). Most culture media contain free Fez+ (Ham F12 for example contains 3 J.lM). Micromolar concenttations of free Fez+ also exist in the synovia of patients with rheumatoid diseases (Abmadzadeh et al. 1989; Halliwell and Gutteridge 1984). Thus, the addition of ascorbic acid to cultivated chondrocytes initiates a kind of "in vitro rheumatoid disease", if no radical scavengers are present. The cells undergo an oxidative stress, which promotes cell death and dedifferentiation. One scavenger that influences ascorbic acid reactions is alpha-tocopherol. We have shown here that 25-50 J.lM of alpha-tocopherol decrease the Fez+- and ascorbic acid-induced lipid peroxidation of chondrocyte and synoviocyte homogenates to a basal level. Therefore, we suggest the addition of alphatocopherol to ascorbic aCid-containing chondrocyte cultures. Comparable results reported by Tschan et al. (1990) indicated a better survival of avian chondrocytes in culture after the addition of HzOz-reducing catalase, and Watkins et al. (1996) demonstrated the beneficial effect of alpha-tocopherol on avian growth cartilage. Alpha-tocopherol is discussed in connection with the treatment of inflammatory joint diseases. Heliovaara et al. (1994) described a higher risk of rheumatoid arthritis in patients with low serum levels of alpha-tocopherol. Fairburn et al. (1992) detected lower alpha-tocopherol levels in the synovia of patients with inflammatory joint diseases than in that of control persons, while serum levels were normal. One reason for the protective effect of alpha-tocopherol must be its ability to terminate the process of lipid peroxidation. In our experiments we demonstrated this radical scavenger effect for articular chondrocytes and synoviocytes in vitro. The amount of lipid peroxidation, measured by TBAR production, was 3-fold lower for synoviocytes than for chondrocytes. The reason might be the less efficient radical scavenger mechanisms in chondrocytes as compared to synoviocytes. Enzymes like superoxide dismutase and catalase that protect cells from oxygen radical attack are not present in chondrocytes, apart from those in premineralized cartilage where they have low activity (Matsumoto et al. 1991). Synoviocytes express these enzymes copiously and protect themselves. In addition to the termination of lipid peroxidation, we could demonstrate an inhibition of rabbit synoviocyte proliferation by alpha-tocopherol. The lining layer of syn-

444.

ovium develops a hyperplasia in cases of rheumatoid arthritis (Veale et al. 1993; Mohr et al. 1975). This hyperplasia might be inhibited by the influence of alphatocopherol on synoviocyte proliferation, and thus demonstrate another healing and/or protective mechanism of this vitamin in rheumatoid arthritis. This has, however, to be confirmed for human tissue. Oxygen-free radicals are also known to stop chondrocytes in their proliferation by making a G2-arrest (Vincent et al. 1991). We have demonstrated here a strong inhibition of cellular growth in rat chondrocyte cultures (and in human chondrocytes of infants, data not shown) by ascorbic acid. The same was reported for chicken chondrocytes by Rosselot et al. (1992), but the radical scavenger alpha-tocopherol failed to abolish this inhibition in our experiments. Other authors have also demonstrated the positive influence of ascorbic acid on the proliferation of rabbit chondrocytes (McDevitt et al. 1988; Wright et al. 1988; Benya et al. 1984). In view of the different species-dependent effects of ascorbic acid on chondrocyte proliferation, and the failure of alpha-tocopherol to minimize the ascorbic acid-induced inhibition of rat chondrocyte proliferation in our studies, we suggest that the regulation of chondrocyte growth by ascorbic acid is not mediated via oxygen radicals. Thus, the addition of ascorbic acid negatively influences multiplication of rat, chicken or human chondrocytes in vitro. Moreover, Monsonego et al. (1993) demonstrated a repression of collagen type II gene expression in avian chondrocytes by this vitamin. Ascorbic acid is necessary for a rich chondrocyte collagen production, but it also damages the cells by interaction with free Fe2+, if no scavengers such as alpha-tocopherol are available. Ascorbic acid must therefore be used with caution as a bivalent molecule in the culture of articular chondrocytes, and, unlike alpha-tocopherol, it is probably not useful in the treatment of arthritic diseases. Acknowledgements. We would like to thank Rita Kirsch and Elisabeth Schongarth for their excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft. Sonderforschungsbereich 367 "Molekulare Mechanismen entzUndlicher und degenerativer Prozesse".

References Ahmadzadeh N, Shingu M, Nobunaga M (1989) Iron-binding proteins and free iron in synovial fluids of rheumatoid arthritis patients. Clin Rheumatol 8: 345-351 Aulthouse AL, Becker K, Beck M (1994) Proteoglycan synthesis by cultured human chondrocytes. Microsc Res Tech 28: 520526 Balmain N, von Eichel B, Toury R, Belquasmi F, Hauchecorne M, Klaus G, Mehls 0, Ritz E (1995) Calbindin-D28K and D9K and 1,25(OH)2 vitamin D3 receptor immunolocalization and mineralization induction in long-term primary cultures of rat epiphyseal chondrocytes. Bone 17: 37-45

Benya PD, laffe S, Raffo A (1984) The capacity of chondrocytes to respond to serum is enhanced by organ culture in the absence of serum, stimulated by serum, and modified by ascorbate. Arch Biochem Biophys 232: 323-336 Blake DR, Hall ND, Bacon PA, Dieppe PA, Halliwell B, Gutteridge Ml (1981) The importance of iron in rheumatoid diseases. Lancet 2: 1142-1144 Buege lA, Aust SD (1978) Microsomal lipid peroxidation. In Methods Enzymol 52: 302-310 Bujia 1, Rotter N, Minuth W, Burmester G, Hammer C, Sittinger M (1995) Cultivation of human cartilage tissue in a 3-dimensional perfusion culture chamber: characterization of collagen synthesis. Laryngorhinootologie 74: 559-563 Cadenas E (1989) Biochemistry of oxygen toxicity. Annu Rev Biochem 58: 79-110 Fairburn K, Grootveld M, Ward Rl, Abiuka C, Kus M, Williams RB, Winyard PG, Blake DR (1992) Alpha-tocopherol, lipids and lipoproteins in knee-joint synovial fluid and serum from patients with inflammatory joint disease. Cli Sci Coleh 83: 657-664 Farquharson C, Whitehead CC (1995) Differentiation and mineralization in chick chondrocytes maintained in a high cell density culture: a model for enchondral ossification. In-vitro Cell Dev BioI Anim 31: 288-294 Georgescu HI, Mendelow D, Evans CH (1988) HlG-82: an established cell line from rabbit periarticular soft tissue, which retains the "activatable" phenotype. In vitro Cell Develop BioI 24: 1015-1022 Halliwell B, Gutteridge MC (1984) Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem 1 219: 1-14 Heliovaara M, Knekt P, Aho K, Aaran RK, Alfthan G, Aromaa A (1994) Serum antioxidants and risk of rheumatoid arthritis. Ann Rheum Diss 53: 51-53 Kurz B, von Gaudecker B, Krisch B, Mentlein R (1996) Rat thymic epithelial cells in vitro and in situ: characterization by immunocytochemistry and morphology. Cell Tissue Res 283: 221-229 Matsumoto H, Silverton SF, Debolt K, Shapiro 1M (1991) Superoxide dismutase and catalase activities in the growth cartilage: relationship between oxidoreductase activity and chondrocyte maturation. 1 Bone Miner Res 6: 569-574 McDevitt CA, Lipman 1M, Ruemer RJ, Sokoloff L (1988) Stimulation of matrix formation in rabbit chondrocyte cultures by ascorbate: Characterization of proteoglycans. 1 Orthop Res 6: 518-524 Minotti G, Aust SD (1987) The role of iron in the initiation of lipid peroxidation. Chern Phys Lipids 44: 191-208 Mohr W, Wild A, Paulini K (1975) Proliferation of synovial tissue in rats with adjuvant disease. Path Microbiol43: 1-9 Monsonego E, Halevy 0, Gertler A, Volokita M, Schickler M, Hurvitz S, Pines M (1993) Growth hormone receptors in avian epiphyseal growth-plate chondrocytes. Gen Comp Endocrinol 92: 179-188 Murad S, Grove D, Lindberg KA, Reynolds G, Sivarajah A, Pinnel SR (1981) Regulation of collagen synthesis by ascorbic acid. Proc Natl Acad Sci USA 78: 2879-2882 Pacifici M, lozzo RV (1988) Remodeling of the rough endoplasmic reticulum during stimulation of procollagen secretion by ascorbic acid in cultured chondrocytes. A biochemical and morphological study. 1 BioI Chern 263: 2483-2492 Rosselot G, Reginato AM, Leach RM (1992) Development of a serum-free system to study the effect of growth hormone and insulinlike growth factor-Ion cultured postembryonic growth plate chondrocytes. In vitro Cell Dev Bioi 28 A: 235-244

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Rowley D, Gutteridge JM, Blake D, Farr M, Halliwell B (1984) Lipid peroxidation in rheumatoid arthritis: thiobarbituric acidreactive material and catalytic iron salts in synovial fluid from rheumatoid patients. Clin Sci 6: 691-695 Rowley DA, Halliwell B (1983) Formation of hydroxyl radicals from hydrogen peroxide and iron salts by superoxide- and ascorbate-dependent mechanisms: relevance to the pathology of rheumatoid disease. Clin Sci 64: 649-653 Ryan LM, Kurup I, Cheung HS (1991) Stimulation of cartilage inorganic pyrophosphate elaboration by ascorbate. Matrix 11: 27fr281 Sandell U, Daniel JC (1988) Effects of ascorbic acid on collagen mRNA levels in short term chondrocyte cultures. C(Jnnect Tissue Res 17: 11-22 Stockwell RA (1983) Metabolism of Cartilage. In Cartilage Vol 1, Hall BK (ed) Academic Press Inc, Orlando, Fl. 253-273 Tschan T, Hoerler I, Houze Y, Winterhalter KH, Richter C, Bruckner P (1990) Resting chondrocytes in culture survive without growth factors, but are sensitive to toxic oxygen met" abolites. J Cell BioI 111: 257-260 Veale D, Yanni G, Rogers S, Barnes L, Bresnihan B, Fitzgerald 0 (1993) Reduced synovial membrane macrophage numbers, ELAM-1 expression, and lining layer hyperplasia in psoriatic arthritis as compared with rheumatoid arthritis. Arthritis and Rheumatism 36: 893-900

Villacara A, Spatz M, Dodson RF, Com C, Bembry J (1989) Effect of arachidonic acid on cultured cerebromicrovascular endothelium: permeability, lipid peroxidation and membrane "fluidity". In Acta Neuropathol 78: 310-316 Vincent F, Corral M, Defer N, Adolphe M (1991) Effect of oxygen free radicals on articular chondrocytes in culture: c-myc and c-Ha-ras messenger RNAs and proliferation kinetics. Exp Cell Res 192: 333-339 Watkins BA, Xu H, Threk JJ (1996) Linoleate impairs collagen synthesis in primary cultures of avian chondrocytes. PSEBM 212: 153-159 Winterbourn CC (1981) Hydroxyl radical production in body fluids. Roles of metal ions, ascorbate and superoxide. Biochern J 198: 125-131 Wright GC Jr, Wei XQ, McDevitt CA, Lane BP, Sokoloff L (1988) Stimulation of matrix formation in rabbit chondrocyte cultures by ascorbate. 1. Effect of ascorbate analogs and betaaminopropionitrile. J Orthop Res 6: 397-407 Yan WQ, Yang TS, Hou LZ, Suzuki F, Kato Y (1994) Effect of concanavalin A on morphology and DNA synthesis of resting chondrocyte cultures. Shih-Yen-Sheng-Wu-Hsueh-Pao 27: 300-305

Accepted May 15, 1997

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