such as gly- cosaminoglycan polysulphate ester (Arteparon@) and pentosan polysulphate (Cartrophen@) have been demonstrated to exhibit a chondroprotec-.
Chondroprotection,
Myth or Reality: An Experimental
By Peter Ghosh, Christine Wells,
Margaret
INDEX WORDS: Osteoarthritis; chondroprotection: degenerative joint disease; cartilage; cartilage repair.
A
CHARACTERISTIC feature of osteoarthritis (OA) and chronic rheumatoid arthritis (RA) is the progressive decline in the structural integrity of joint articular cartilage. The ability of a therapeutic substance to prevent this process has led to the concept of chondroprotection but the manner by which chondroprotection can be achieved in practise has not been adequately defined. The mechanisms responsible for failure of cartilage in osteoarthritic joints are still the subject of debate, particularly with regard to whether the primary lesion occurs within cartilage-a chondrocyte/matrix defect, in the subchondral bone-a decrease in compliance, or in the synovium-production of inflammatory mediators and/or proteinases. Irrespective of the initiating event, it is clear that once joint dysfunction is established all these tissues (cartilage, synovial fluid, synovium and subchondral bone) can contribute to progression of the disorder. The situation is summarised in Figure 1, and on these grounds, therapeutic intervention in OA should not be exclusively directed to articular cartilage but must also address the synovial inflammation”* and inadequacies which exist in the synovial fluid3 and subchondral blood s~pply.‘*~ Today, steroidal and nonsteroidal antiinflammatory drugs (NSAIDs) represent the mainstay of nonsurgical treatment of the arthritic patient. Evidence is accumulating to suggest that some of these agents may provide chondroprotection by attenuating the release from activated synovial cells of cytokines, oxygen derived free radicals, and proteinases that are capable of directly or indirectly degrading components of the cartilage matrix.‘-’ However, the effects that NSAIDs may have on the biosynthesis of hyaluronic acid (HA) by synovial fibroblasts has not been previously investigated. Recent studies’,’ with prednisone and triamcinolone hexacetonide have suggested that corticosteroids may provide a chondroprotective effect in OA, since in animal models cartilage lesions and osteophytes were reduced. Corticosteroids act by thninars in Arthritis andRheumatism,
Smith,
and Nongporn
Approach
Hutadilok
down regulating cell metabolism” and although this mode of action may be beneficial in the shortterm, by suppressing cell proliferation and the synthesis of proteinases and inflammatory mediators, the long-term consequences, particularly in relation to the inhibition of proteoglycan and HA synthesis by chondrocytes and synovial cells, has limited their application.” Polysulphated polysaccharides, such as glycosaminoglycan polysulphate ester (Arteparon@) and pentosan polysulphate (Cartrophen@) have been demonstrated to exhibit a chondroprotective effect in a variety of in vitro and in vivo systems ‘*‘l-13.however, the action of this class of drugs on synoviocyte proliferation and biosynthesis of HA has received only limited attention. In the present study, the effects of pentosan polysulphate (PPS) and three NSAIDs (tiaprofenic acid, diclofenac sodium, and piroxicam) on DNA and HA biosynthesis, by synovial fibroblast cultures obtained from OA and RA knee joints, were investigated. OVERVIEW
The preservation of articular cartilage in RA and OA by the administration of therapeutic agents is a concept that has received limited attention. Such agents are known as chondroprotective drugs, and they can preserve cartilage in several ways, directly, by effecting cartilage metabolism or indirectly, by stimulating synovial cell synthesis of hyaluronic acid or by improving subchondral-bone blood flow. Whereas the effects of antiarthritic agents on the metabolism of From the Raymond Purves Research Laboratories (University of Sydney) at the Royal North Shore Hospital of Sydney St Leonards, NS W, Australia. Peter Ghosh, PhD, FRSC, FRACI: Director, Raymond Putves Research Laboratories at the Royal North Shore Hospital of Sydney, and Associate Professor, Department of Surgery, University of Sydney, NSW, Australia: Christine Wells, BS: Research Assistant; Margaret Smith, PhD: Postdoctoral Research Fellow: Nongporn Hutadilok, MS: Research Student. Address reprint requests to Peter Ghosh, PhD. FRSC, FRAU, Raymond Purves Research Laboratories at the Royal North Shore of Sydney, St Leonards, NSW, 2065. Australia. Q 1990 by W.B. Saunders Company. 0049-0172/90/1904-1002$5.00/O
Vol 19, No 4, Suppl 1 (February), 1990: pp 3-9
3
GHOSH ET AL
PERIARTICULAR
EPIPHYSEAL VESSELS
VASCULARENGORGFMFNT
cartilage proteoglycans have been investigated in some detail, little is known of the effects such agents may have on synovial fibroblast proliferation and biosynthesis of HA. The present investigation was undertaken to address this deficiency. Synovial fibroblast cultures, derived from synovium obtained from RA and OA patients, were used for these studies. DNA synthesis was monitored by [3H] thymidine uptake and HA synthesis by incorporation of [3H] acetate into Streptomyces hyaluronidase susceptible macromolecules. Pentosan polysulphate over the concentration range of 0.002 to 2.0 pg/mL stimulated HA synthesis in cells from both RA and OA cell lines, 100% stimulation occurred at 0.25 pg/ml in the OA synoviocytes. Tiaprofenic acid at concentrations up to 2.0 pg/mL stimulated HA synthesis in cell lines obtained from OA or RA joints, but at 5.0 and 10.0 pg/mL inhibition was observed. Interestingly, at these high concentrations pentosan polysulphate and tiaprofenic acid increased [H3] thymidine incorporation into DNA. In the OA synoviocyte cell lines, diclofenac stimulated HA synthesis over the concentration range 0.25 to 10 pg/mL but inhibited synthesis in the corresponding RA cell lines at 0.1 and 0.25 pg/mL. Piroxicam exhibited negligible effect on HA synthesis in OA or RA synoviocytes at concentrations up to 1.0 pg/mL; however, a 100% stimulation of synthesis was observed
Fig 1. A schematic representation of a human joint illustrating the major pathological features, particularly in relation to abnormalities in the synovium and subchondral blood supply. The figure was modified from Kiaer’s original drawing.’
in the RA cell line at 5.0 and 10.0 pg/mL this drug.
with
MATERIALS AND METHODS
Materials Dulbecco’s modified Eagles medium (DMEM), bovine pancreatic trypsin (EC 3.4.21.4) and fetal calf serum (FCS) were obtained from Gibco-BRL (Grand Island, New York). Collagenase (from Clostridium histolyticum EC 3.4.24.3) was obtained from Sigma Chemical Co. (St Louis, MO); gentamicin from Cytosystems Pty Ltd, (NSW, Australia); streptomyces hyaluronidase ((Streptomyces hyalurobyticus) nov sp (EC 4.2.99.1)) from Miles Laboratories (Australia) Pty Ltd (Epping, NSW, Australia). Sodium [jH] acetate (specific activity 18.5 GBq/L) and [methyl-‘H] thymidine (specific activity 37 GBq/L) were from Amersham Australia Pty Ltd (North Ryde, NSW, Australia). Ninety-six well plates, 24 well plates and tissue culture flasks (75 cm*) were obtained from Greiner and Sons (Niirtingen, West Germany). The drugs studied were kindly provided by the companies indicated: tiaprofenic acid (Roussel Pharmaceuticals Pty Ltd, Castle Hill, NSW, Australia); diclofenac sodium (Ciba-Geigy Australia Pty Ltd, Pendle Hill, NSW, Australia); piroxicam (Pfizer Pharmaceutical Division, West Ryde, NSW, Australia); pentosan polysulphate (Arthropharm Pty Ltd, Bondi Junction, NSW, Australia).
Tissues Synovial specimens were obtained at the time of total joint replacement surgery or arthroscopy by Dr Nigel Hope and Dr David Sonnabend, Department of Orthopaedics, Royal North Shore Hospital of Sydney. The classification of the tissue as being derived from OA or RA joints was made by Dr David Sonnabend.
HYALURONIC
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ACID AND CHONDROPROTECTION
Culture Methods Fibroblasts were prepared from freshly collected synovial samples by a modification of the method originally described by Dayer et alI4 and described previously by Smith and Ghosh.” Briefly, the synovial membrane was finely diced, in Ca++ and and Mg’ +- free phosphate buffered saline (CMF-PBS) digested with 4 mg/mL collagenase (in DMEM containing 50 rg/mL gentamicin) for 3 hours at 37°C. An equal volume of 0.05% trypsin and 0.02% disodium ethylenediamine tetracetate (EDTA) in CMF-PBS was immediately added to the collagenase digested tissue and incubated for another hour at 37OC. The digested tissue was centrifuged at 500 g for 3 minutes and the cell pellet was then washed three times with DMEM containing 10% FCS. The cells were seeded at 1 x lo6 cells/75 cm2 flask. Cells were maintained at 37’C in a humidified (98%) CO, incubator (5% CO,, 95% air). Fibroblasts used for these experiments were collected at the fourth passage. Cells were seeded into 96 and 24 well plates at 10,000 and 30,000 cells per well respectively and used in the late log phase (70% confluence). Altogether 11 cell lines (7 RA and 5 OA) were used in this study.
Hyaluronic Acid Biosynthesis Cells seeded into the 24 well plates were pulsed for 24 hours (under standard culture conditions) with 5 PCi-[‘HIacetate per well in 400 rL of DMEM and 10% FCS, in the presence and absence of various concentrations of drugs. The media was removed from the wells and combined with washings (twice in 0.1 mol/L sodium acetate, pH 5.6). These solutions were assayed in triplicate for [‘HI-HA, as described previously.” Briefly, samples of media and washings were dialysed against 0.1 mol/L sodium acetate (pH 5.6) at 4“C over 2 days with four changes. The aliquots were then placed into two tubes. Five turbidity reducing units (TRU, as defined by Miles Laboratories) of streptomyces hyaluronidase/0.8 ml sample were added to one tube. Both tubes were incubated at 60°C for 3 hours. The samples were redialysed for 2 days against 2 changes per day of 0.1 mol/L sodium acetate pH 5.6 at 4°C. The radioactivity of aliquots was determined by scintillation spectrophotometery. The radioactivity of newly synthesized HA was calculated as the DPM of the blank sample less the DPM of the enzyme-treated sample. This value was corrected for cell number to eliminate a potential source of variation between the wells. Results were expressed as a percentage increase or decrease in HA production, when compared to the control (no drug) samples.
DNA Synthesis /(methyl-3H)-thymidine Incorporation] The cells seeded into the 96 well plates were pulsed for 24 hours (under standard culture conditions) with 1 pCi [methyl‘H] thymidine per well in DMEM and 10% FCS, and in the presence or absence of the drugs tested. The radioactive media was discarded and the cells were washed three times with CMF-PBS, 100 ILL of the trypsin/EDTA was added to each well and the cells were incubated for 2 minutes at 37°C. The cells were lysed with distilled water and lysates filtered through glass fiber filters. The filters were dried overnight at
room temperature. Radioactive DNA on the filter paper was measured by scintillation spectrophotometry. Results were expressed in terms of percentage increase or decrease in [methyl ‘H-1 thymidine uptake when compared to the control (no drug) samples.
Statistical Methods The data generated for each drug on HA and DNA synthesis in the various cell lines, for each concentration, was analysed using an unpaired Student’s f-test. The null hypothesis was that drugs exerted no effects on HA or DNA synthesis using the control population for comparison. A drug-effect was considered to occur when the difference between the two populations corresponded to P c .05.
RESULTS
Pentosan Polysulphate (PPS) Pentosan polysulphate at concentrations of up to 2.0 pg/mL stimulated HA synthesis in both RA and OA cell lines in a concentration dependent manner. In both cell lines, optimum stimulation occurred at 0.20 to 0.25 pg/mL; however, synthesis in the OA cells was approximately 25% higher (Fig 2). It should be noted, that at 20 gg/mL HA synthesis was depressed in another RA cell line (Fig 2). DNA synthesis, as determined by [3H]-thymidine uptake, was also elevated in the presence of low concentrations of this drug. The response was again concentration dependent, the greatest stimulation being observed in the RA cell line 34-5 at 25 Kg/ml (Fig 3). These results were statistically significant (P < .05). Tiaprofenic Acid The effects of tiaprofenic acid over the concentration range 0.25 to 10 pg/mL on HA production in the various OA and RA cell lines, as typified by cell lines 39-4 and 33-4, is shown in Figure 2. The effects produced by the drug were statistically significant at P 5 .O1 when analysed by an unpaired Student t-test at all concentrations used. Tiaprofenic acid significantly stimulated HA biosynthesis at concentrations between 0.25 to 2.0 pg/mL on both cell lines. Maximal stimulatory effect was observed at 0.5 hg/mL when biosynthesis was increased to between 60% and 80% above control levels. At higher concentrations (5.0 and 10.0 pg/mL tiaprofenic acid) significant inhibition of HA synthesis was produced in both cell lines. However, in the OA line HA production was inhibited to less than 40% of the control at 10.0 pg/mL, whereas in the RA
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The effects of PPS and tiaprofenic acid on HA synthesis by human synovial fibroblasts derived from RA or OA joints. Fig 2. Values ara expressed as means f standard error of the mean of percentage HA synthesis compared to nondrug treated control level. (A) The effect of PPS on RA cell line 9-9. Levels of HA synthesis et 0.2 and 2.0 pg PPS/ml were significantly different from control levels (P < .Ol, n = 3). (B) The effect of PPS on OA cell line 29-9. The levels of HA synthesis at all concentrations except 2.0 pg PPS/ml were significantly different from control values (P < .Ol. n = 3). (Cl The effect of tiaprofenic acid on RA cell line 33-4. All values were significantly different from control levels (P < .Ol, n = 4). (Dj The effect of tieprofenic acid on en OA cell line 39-4. All values were significantly different from control levels (P -G .Ol, n = 4).
line synthesis was depressed by more than 90% at 5.0 pg/mL, and 25% at 10.0 pg/mL. At higher concentrations, HA synthesis was suppressed (Fig 2). In the OA cell line 43-5 (Fig 3), [3H]thymidine incorporation was determined over the concentration range of tiaprofenic acid of 0.25 5.0 pg/mL, and statistically significant stimulation was observed at concentrations of 2.0 and 5.0 pg/mL. Diclofenac Sodium Using OA cell line 39-4 it was found that diclofenac sodium significantly stimulated HA production over the concentration range of 0.25 to 10 pg/mL. Maximal stimulation occurred at 0.5 pg/mL when a 50% enhancement of synthesis was observed (Fig 4). In contrast, HA synthesis in RA cell line 33-4 was inhibited at low concentrations of diclofenac (0.1 and 0.25 rg/ mL). However, these latter data were not statistically significant. Piroxicam Over the concentration range 0.25 to 10 Kg/ ml, piroxicam showed no significant affect on HA
production in the OA cell lines used in these experiments (Fig 4). Similarly HA synthesis in the RA cell lines as shown by cell line 33-4 (Fig 4) was not influenced by the drug at concentrations of up to 1.0 pg/mL but stimulation was dramatically increased at concentrations of 5.0 and 10.0 pg/mL (Fig 4). DISCUSSION
Hyaluronic acid is the most abundant nonaqueous, nonproteinaceous component of synovial fluid, and is responsible for the unique rheological properties of this medium.i6 Apart from the ability of HA to stabilize joint mechanics because of its high viscosity,” there is now evidence that in combination with phospholipids it may be responsible for synovial lubrication.‘8V19This view is contrary to earlier proposals which identified a glycoprotein as the lubricating entity of synovial fluid.*’ The molecular size of HA declines on average from 7.0 x lo6 in joints of normal patients to 4.8 x lo6 in synovial fluid from RA patients.*’ In both RA and OA the synovial concentration of HA may decrease because of dilution by infiltration of plasma fluid but also possibly because of the effects of free radicals
HYALURONIC
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ACID AND CHONDROPROTECTION
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Fig 3. The effects of PPS or tiaprofanic acid on DNA synthesis by human synovial fibroblasts derived from RA or OA joints. Values ara expressed es means + SEM of percentage tritiated mathylthymidine uptake compared to nondrug treated controls. (A) Effect of PPS on RA call line 34-5. Uptake at 25 pg PPS/mL was significantly different from controls (P < .05, n = 6). (B) The affect of PPS on an OA call line 274. Statistically significant values ware 0.25 gg PPSlmL (PC .OOl. n = 3): 0.5 pg PPS/mL (PC .Ol. n = 3) and 1.O pg PPS/mL W < X801,n = 3). (Cl The affect of tiaprofanic acid on an OA cell line 43-5. The values obtained at 2.0 and 5.0 gg/mL ware significantly different from controls (P < .05, n = 11).
that cause its degradation** and decreased its biosynthesis. 23 This decline in synovial fluid HA concentration and molecular size imposes abnormally high contact and shear stresses on articular cartilage and therefore contributes to its failure. From this stand point, drugs that stimulate HA biosynthesis by synovial fibroblasts could be considered to be providing chondroprotective effect. Of the drugs examined in the present study, only tiaprofenic acid and pentosan polysulphate showed a stimulatory effect on HA synthesis in both the RA and OA cell lines at concentrations that could be readily achieved in synovial fluid during normal human application (Fig 2). Piroxicam was found to have no statistical significant effect on HA biosynthesis in the OA cell lines studied and only stimulate synthesis in the RA fibroblasts at high, nontherapeutic, concentrations (Fig 4). The concentration of piroxicam has been determined to lie between 0.2 to 0.6 pg/mL in synovial fluid for a single oral dose of 20 mg of the drug. 24 Diclofenac sodium, while showing inhibitory activity on HA synthesis at low concentrations (0.1, 0.25 pg/mL) in the RA cell lines, was stimulatory to the synovial cells derived from OA patients over the entire concentration range examined (Fig 4). Assuming that these data are extrapolatable to the in vivo situation, diclofenac and more particularly, tiaprofenic acid and PPS may be valuable for normalising synovial fluid HA levels in arthritic joints. In addition, the mitogenic activity of PPS and tiaprofenic acid on synovial fibroblasts observed at high concentrations of these drugs could augment regeneration of synovial tissue that may become necrotic in the chronically inflamed joint.’ For both PPS and tiaprofenic acid, their effects on HA biosynthesis and thymidine uptake into synovial cells would appear to be inversely related (compare Figs 2 and 3). This is perhaps not so surprising since the high intracellular energy required for DNA synthesis and cell division would reduce that available for HA biosynthesis. Although the mechanism(s) by which these drugs stimulate or inhibit HA synthesis by synovial fibroblasts is presently unknown, we propose as a working hypothesis, that this action occurs at sites within the plasma membrane. Studies by Prehm*’ have shown that HA synthesis occurs at
8
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Fig 4. The effects of diclofenac and piroxicam on HA synthesis by human synovial fibroblasts derived from RA or OA joints. Values are expressed as means f SEM of percentage change in HA synthesis compared to non-drug treated control levels. (A) The effect of diclofenac on RA cell line 33-4. The values at 0.1 and 0.25 pg diclofenac/mL were significantly different from controls (P < .05, n = 4). (Bj The effect of diclofenac on OA cell line 39-4. Values at 0.25, 0.5, 1 .O and 2.0 Ag diclofenac/mL were significantly different from controls (P < .Ol, n = 41. fCj The effect of piroxicam on RA cell line 33-4. Values et 5.0 and 10 c(g piroxicam/mL were significantly different from controls (P < .Ol, n = 4). (Dj The effect of piroxicam on OA cell line 39-4. Values at 1 .O and 10 pg piroxicam/mL were significantly different from controls (P < .05. n = 4).
the inner surface of the plasmalemma, for the hyaluronate synthetase systems are located here. Hyaluronate chains are progressively elongated by the alternate addition of the substrates, uridine diphosphate (UDP)-N-acetylglucosamine and UDP-glucuronic acid, to the UDP-HA chain at the cell membrane, then moved into the extracellular space as a continuous filament. Tiaprofenic acid by virtue of its hydrophobic flat aromatic rings could be expected to readily intercalate between the phospholipid bilayer of the synoviocyte membrane and influence those proteins involved in the binding and processing of the UDP substrates required for HA synthesis. On the other hand, the highly negatively charged hydrophilic PPS could bind to cell-surface glycoproteins and, via a feedback mechanism, influence the HA synthesis indirectly. Why piroxicam, and to a lesser degree diclofenac, display differential effects to tiaprofenic acid on synoviocyte biosynthesis of HA is unclear, but could be related to the reduced ability of these drugs to intercalate with cell membrane proteins. This speculative explanation for NSAID activity on HA synthesis by fibroblasts is compatible with the recent studies of Abramson and
Weissmann. They believe that the antiinflammatory activity of most NSAIDs can be rationalized by the drug’s ability to uncouple the stimulusresponse process within secretory cells, such as neutrophils. This occurs within the cell plasmalemma where regulatory proteins, such as guanoside triphosphate binding protein (G-protein) modulate the levels of intracellular 1,4,5-triphosphate (IP,) and 1,2-diacylglycerol that in turn can elevate Ca + + and protein kinase levels within the cytoplasm. These messengers then provoke the synthesis and release of inflammatory mediators including superoxide radicals. The synoviocytes obtained from the RA and OA synovium are abnormal fibroblasts and it is possible that the pattern of HA synthesis observed in vitro arises from the irreversible binding of ligands at external cell receptors such as those proposed for the stimulation of neutrophils.26 The present studies, as well as those of Abramson and Weissmann, suggest that the biological activity of NSAIDs may be largely determined by their ability to interact with specific receptors present within the cell plasmalemma. The structural requirements of these drugs that would facilitate transport to and binding with these
HYALURONIC
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ACID AND CHONDROPROTECTION
membrane proteins would be expected to be quite different to those required for interaction with the cyclooxygenase receptor. Our studies, like those of Abramson and Weissmann,‘” therefore question the merits of searching for new antiinflammatory drugs by screening for cyclooxygenase inhibitory activity.27
In this paper, studies are described which illustrate the point that chondroprotection can be achieved in a variety of ways. Drug stimulation of HA synthesis by synovial fibroblasts is but one approach to a multifaceted problem, but clearly offers some assessment of potential chondroprotective activity.
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2. Soren A, Cooper NS, Waugh TR: The nature and designation of osteoarthritis determined by its histopathology. Clin Exp Rheum 6:41-46, 1988 3. Balazs EA, Watson D, Duff IF, et al: Hyaluronic acid in synovial fluid. I. Molecular parameters of hyaluronic acid in normal and arthritic human fluids. Arthritis Rheum 10:357376,1967 4. Hulth A, Hernborg J: Blood circulation joints. J Bone Jt Surg 50B:227, 1968
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7. Shinmei M, Kikuchi T, Masuda K, et al: Effects of interleukin-1 and anti-inflammatory drugs on the degradation of human articular cartilage. Drugs 35:33-41, 1988 (suppl 1) 8. Williams JM, Brandt KD: Triamcinolone hexacetonide protects against fibrillation and osteophyte formation following chemically induced articular cartilage damage. Arthritis Rheum 28:1267-1274, 1985 9. Pelletier J-P, Martell-Pelletier J: Protective corticosteroids on cartilage lesions and osteophyte in the Pond-Nuki dog model of osteoarthritis. Rheum 32:181-193, 1989
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10. Claman NH: Anti-inflammatory effects of corticosteroids. Clin Immunol Allergy 4:3 17-329, 1984 11. Ghosh P: Anti-rheumatic drugs and cartilage, in Brooks P (ed): Bailhere’s Clinical Rheumatology: International Practice and Research. Philadelphia, PA, Saunders, 1988, pp 309-338 12. Francis DJ, Forrest MJ, Brooks P, et al: Retardation of articular cartilage degradation by glycosaminoglycan polysulphate ester (Arteparone) pentosan polysulphate (SP54e) and DH40J in the rat air-pouch model. Arthritis Rheum 32:608-616, 1989 13. Kongtawelert P, Brooks P, Ghosh P: Pentosan polysulphate (Cartrophen@) prevents the hydrocortisone induced loss of hyaluronic acid and proteoglycans from cartilage of rabbit joints, as well as normalises the keratan sulphate levels in their serum. J Rheumatol, November, 1989
14. Dayer JM, Krane SM, Russell RGG, et al: Production of collagenase and prostaglandins by isolated adherent rheumatoid synovial cells. Proc Nat Acad Sci (USA) 73:945-949, 1976 15. Smith MM, Ghosh P: The synthesis of hyaluronic acid by human synovial fibroblasts is influenced by the nature of the hyaluronate in the extracellular environment. Rheumatol Int 7:113-122, 1987 16. Bothner H, Wik 0: Rheology’of hyaluronate. Acta Oto-Laryngologica 442:25-30, 1986 (suppl) 17. Cullis-Hill D, Ghosh P: The role of hyaluronic acid in joint stability-A hypothesis for hip displasia and allied disorders. Med Hypoth 23:171-185, 1986 18. Hills BA: The Biology of Surfactant. Cambridge, Cambridge Press, 1988, pp 279 19. Langer HE, Altmann S, Luhrs W, et al: The lubrication of the natural joint: Viscosity of hyaluronic acid and friction in the human hip, in Bergmann G, Kolbel R, Rohlmann A (eds): Biomechanics, Dordrecht, The Netherlands, Nijhoff, 1988 20. Swarm DA, Sotman S, Dixon M, et al: The isolation partial characterization of the major glycoprotein (L-G-P-I) from the articular lubricating fraction from bovine synovial fluid. Biochem J 161:473-485, 1977 21. Dahl LB, Dahl IMS, Engstrom-Laurent A, et al: Concentration and molecular-weight of sodium hyaluronate in synovial-fluid from patients with rheumatoid-arthritis and other arthropathies. Ann Rheum Dis 44:817-822, 1985 22. McNeil JD, Wiebkin OW, Betts WH, et al: Depolymerization products of hyaluronic-acid after exposure to oxygen-derived free-radicals. Ann Rheum Dis 44:780-789, 1985 23. Bates EJ, Lowther DA, Johnson CC: Hyaluronic-acid synthesis in articular-cartilage-An inhibition by hydrogenperoxide. Biochem Biophys Res Commun 132:714-720, 1985 24. Bontoux D, Phely AGX, Fourtilla J-B: Pharmacokinetits of piroxicam in synovial fluid, synovial membranes and cartilage. Royal Sot Med Intern Congr Symp 67:83-90, 1985 25. Prehm P: Hyaluronate is synthesised at plasma membranes. Biochem J 220:597-600, 1984 26. Abramson SB, Weissmann G: The mechanism of action of nonsteroidal antiinflammatory drugs. Arthritis Rheum 32:1-9, 1989 27. Vane JK: Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature 23 1:232235,1971
