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Effects of low molecular weight hyaluronan combined with carprofen on canine osteoarthritis articular chondrocytes and cartilage explants in vitro

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Abstract

Intra-articular injection with non-steroidal anti-inflammatory drugs (NSAIDs) is used to treat inflammatory joint disease, but the side effects of NSAIDs include chondrotoxicity. Hyaluronan has shown positive effects on chondrocytes by reducing apoptosis and increasing proteoglycan synthesis. The purposes of this study were to evaluate the effects of low molecular weight hyaluronan (low MW HA), carprofen 25 mg/ml, carprofen 12.5 mg/ml, and a combination of HA and carprofen on canine osteoarthritis (OA) articular chondrocytes and a cartilage explant model in terms of cell viability, extracellular matrix remaining, and gene expression after exposure. In chondrocyte culture, MTT assay was used to evaluate the chondrotoxicity of IC50 and IC80 of carprofen with HA. In cartilage explant culture, two kinds of extracellular matrix (uronic acid and collagen) remaining in cartilage were used to evaluate cartilage damage for 14 d after treatment. Expression of COL2A1, AGG, and MMP3 was used to evaluate the synthesis and degradation of the matrix for 7 d after treatment. In chondrocyte culture, low MW HA could preserve OA chondrocyte viability but could not reduce the chondrotoxicity level of carprofen (P < 0.05). In explant culture, low MW HA combined with 12.5 mg/ml carprofen caused less destruction of uronic acid and collagen structure when compared with the control (P < 0.05). Low MW HA caused high expression levels of COL2A1 and AGG in OA cartilage (P < 0.05); HA combined with carprofen resulted in higher COL2A1 and AGG expression levels than carprofen alone.

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References

  • Aigner T, Zien A, Gehrsitz A, Gebhard PM, McKenna L (2001) Anabolic and catabolic gene expression pattern analysis in normal versus osteoarthritic cartilage using complementary DNA-array technology. Arthritis Rheum 44:2777–2789. doi:10.1002/1529-0131(200112)44:12<2777::AID-ART465>3.0.CO;2-H

    Article  CAS  PubMed  Google Scholar 

  • Archer CW, Francis-West P (2003) The chondrocyte. Int J Biochem Cell Biol 35:401–404

    Article  CAS  PubMed  Google Scholar 

  • Armstrong S, Lees P (1999) Effects of R and S enantiomers and a racemic mixture of carprofen on the production and release of proteoglycan and prostaglandin E2 from equine chondrocytes and cartilage explants. Am J Vet Res 60:98–104

    CAS  PubMed  Google Scholar 

  • Benton HP, Vasseur PB, Broderick-Villa GA, Koolpe M (1997) Effect of carprofen on sulfated glycosaminoglycan metabolism, protein synthesis, and prostaglandin release by cultured osteoarthritic canine chondrocytes. Am J Vet Res 58:286–292

    CAS  PubMed  Google Scholar 

  • Blumenkrantz N, Asboe-Hansen G (1973) New method for quantitative determination of uronic acids. Anal Biochem 54:484–489

    Article  CAS  PubMed  Google Scholar 

  • Brew CJ, Clegg PD, Boot-Handford RP, Andrew JG, Hardingham T (2010) Gene expression in human chondrocytes in late osteoarthritis is changed in both fibrillated and intact cartilage without evidence of generalised chondrocyte hypertrophy. Ann Rheum Dis 69:234–240. doi:10.1136/ard.2008.097139

    Article  CAS  PubMed  Google Scholar 

  • Chang JK, Wu SC, Wang GJ, Cho MH, Ho ML (2006) Effects of non-steroidal anti-inflammatory drugs on cell proliferation and death in cultured epiphyseal-articular chondrocytes of fetal rats. Toxicology 228:111–123. doi:10.1016/j.tox.2006.08.028

    Article  CAS  PubMed  Google Scholar 

  • Coleman PJ, Scott D, Mason RM, Levick JR (2000) Role of hyaluronan chain length in buffering interstitial flow across synovium in rabbits. J Physiol 526:425–434

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Cook JL, Kuroki K, Visco D, Pelletier JP, Schulz L, Lafeber FP (2010) The OARSI histopathology initiative—recommendations for histological assessments of osteoarthritis in the dog. Osteoarthr Cartil 18:S66–S79. doi:10.1016/j.joca.2010.04.017

    Article  PubMed  Google Scholar 

  • Denizot F, Lang R (1986) Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 89:271–277

    Article  CAS  PubMed  Google Scholar 

  • Ding C (2002) Do NSAIDs affect the progression of osteoarthritis? Inflammation 26:139–142. doi:10.1023/A:1015504632021

    Article  CAS  PubMed  Google Scholar 

  • Esko JD, Kimata K, Lindahl U (2009) Proteoglycans and sulfated glycosaminoglycans. In: Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME (eds) Essentials of glycobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 229–248

    Google Scholar 

  • Fukuda K, Takayama M, Ueno M, Oh M, Asada S, Kumano F, Tanaka S (1997) Hyaluronic acid inhibits interleukin-1-induced superoxide anion in bovine chondrocytes. Inflamm Res 46:114–117

    Article  CAS  PubMed  Google Scholar 

  • Fukui N, Ikeda Y, Ohnuki T, Tanaka N, Hikita A, Mitomi H, Ochi T (2008) Regional differences in chondrocyte metabolism in osteoarthritis: a detailed analysis by laser capture microdissection. Arthritis Rheum 58:154–163. doi:10.1002/art.23175

    Article  CAS  PubMed  Google Scholar 

  • Garnero P, Ayral X, Rousseau JC, Christgau S, Sandell LJ, Dougados M, Delmas PD (2002) Uncoupling of type II collagen synthesis and degradation predicts progression of joint damage in patients with knee osteoarthritis. Arthritis Rheum 46:2613–2624. doi:10.1002/art.10576

    Article  CAS  PubMed  Google Scholar 

  • Ghosh P, Guidolin D (2002) Potential mechanism of action of intra-articular hyaluronan therapy in osteoarthritis: are the effects molecular weight dependent. Semin Arthritis Rheum 32:10–37

    Article  CAS  PubMed  Google Scholar 

  • Goldberg VM, Buckwalter JA (2005) Hyaluronans in the treatment of osteoarthritis of the knee: evidence for disease-modifying activity. Osteoarthr Cartil 13:216–224. doi:10.1016/j.joca.2004.11.010

    Article  CAS  PubMed  Google Scholar 

  • Hashizume M, Mihara M (2009) Desirable effect of combination therapy with high molecular weight hyaluronate and NSAIDs on MMP production. Osteoarthr Cartil 17:1513–1518. doi:10.1016/j.joca.2009.04.018

    Article  CAS  PubMed  Google Scholar 

  • Hazewinkel HAW, Van den Brom WE, Theyse LFH, Pollmeier M, Hanson PD (2008) Comparison of the effects of firocoxib, carprofen and vedaprofen in a sodium urate crystal induced synovitis model of arthritis in dogs. Res Vet Sci 84:74–79. doi:10.1016/j.rvsc.2007.02.005

    Article  CAS  PubMed  Google Scholar 

  • Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res 6(10):986–94

  • Henrotin Y, Chevalier X, Deberg M, Balblanc JC, Richette P, Mulleman D, Osteoarthritis Group of French Society of Rheumatology (2013) Early decrease of serum biomarkers of type II collagen degradation (Coll2-1) and joint inflammation (Coll2-1 NO2) by hyaluronic acid intra-articular injections in patients with knee osteoarthritis: a research study part of the Biovisco study. J Orthop Res 31:901–907. doi:10.1002/jor.22297

    Article  CAS  PubMed  Google Scholar 

  • Jalbă BA, Jalbă CS, Vlădoi AD, Gherghina F, Stefan E, Cruce M (2011) Alterations in expression of cartilage-specific genes for aggrecan and collagen type II in osteoarthritis. Rom J Morphol Embryol 52:587–591

    PubMed  Google Scholar 

  • Jayasuriya CT, Chen Q (2012) Cartilage extracellular matrix integrity and OA. InTech Open Access. Retrieved from: http://www.intechopen.com/source/pdfs/28932/InTech-Cartilage_extracellular_matrix_integrity_and_oa.pdf

  • Kawasaki K, Ochi M, Uchio Y, Adachi N, Matsusaki M (1999) Hyaluronic acid enhances proliferation and chondroitin sulfate synthesis in cultured chondrocytes embedded in collagen gels. J Cell Physiol 179:142–148. doi:10.1002/(SICI)1097-4652(199905)179:2<142::AID-JCP4>3.0.CO;2-Q

    Article  CAS  PubMed  Google Scholar 

  • Kolar K (1990) Colorimetric determination of hydroxyproline as measure of collagen content in meat and meat products: NMKL collaborative study. J Assoc Off Anal Chem 73:54–57

    CAS  PubMed  Google Scholar 

  • Laurent TC, Fraser JR (1992) Hyaluronan. FASEB J 6:2397–2404

    CAS  PubMed  Google Scholar 

  • Lisignoli G, Grassi F, Zini N, Toneguzzi S, Piacentini A, Guidolin D, Facchini A (2001) Anti-Fas-induced apoptosis in chondrocytes reduced by hyaluronan: evidence for CD44 and CD54 (intercellular adhesion molecule 1) involvement. Arthritis Rheum 44:1800–1807. doi:10.1002/1529-0131(200108)44:8<1800::AID-ART317>3.0.CO;2-1

    Article  CAS  PubMed  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402–8

  • Matyas JR, Atley L, Ionescu M, Eyre DR, Poole AR (2004) Analysis of cartilage biomarkers in the early phases of canine experimental osteoarthritis. Arthritis Rheum 50:543–552. doi:10.1002/art.20027

    Article  CAS  PubMed  Google Scholar 

  • Mihara M, Higo S, Uchiyama Y, Tanabe K, Saito K (2007) Different effects of high molecular weight sodium hyaluronate and NSAID on the progression of the cartilage degeneration in rabbit OA model. Osteoarthr Cartil 15:543–549. doi:10.1016/j.joca.2006.11.001

    Article  CAS  PubMed  Google Scholar 

  • Mu SC, Liu HC, Wu JY, Lee MTM, Chuang HP, Chen LK, Chen YT (2009) A large kindred of early-onset osteoarthritis of the knee and hip: excluding the link to COL2A1 gene. Rheumatology 48:371–374. doi:10.1093/rheumatology/kep010

    Article  CAS  PubMed  Google Scholar 

  • Okada Y, Shinmei M, Tanaka O, Naka K, Kimura A, Nakanishi I, Bayliss MT, Iwata K, Nagase H (1992) Localization of matrix metalloproteinase 3 (stromelysin) in osteoarthritic cartilage and synovium. Lab Invest 66:680–690

    CAS  PubMed  Google Scholar 

  • Peng H, Zhou J, Liu S, Hu Q, Ming J, Qiu B (2010) Hyaluronic acid inhibits nitric oxide-induced apoptosis and dedifferentiation of articular chondrocytes in vitro. Inflamm Res 59:519–530. doi:10.1007/s00011-010-0156-x

    Article  CAS  PubMed  Google Scholar 

  • Qiu B, Liu SQ, Peng H (2008) Influence of sodium hyaluronate on iNOS expression in synovium and NO content in synovial fluid of rabbits with traumatic osteoarthritis. Chin J Traumatol 11:293–296

    Article  CAS  PubMed  Google Scholar 

  • Struglics A, Larsson S, Pratta MA, Kumar S, Lark MW, Lohmander LS (2006) Human osteoarthritis synovial fluid and joint cartilage contain both aggrecanase- and matrix metalloproteinase-generated aggrecan fragments. Osteoarthr Cartil 14:101–113. doi:10.1016/j.joca.2005.07.018

    Article  CAS  PubMed  Google Scholar 

  • Takahashi K, Goomer RS, Harwood F, Kubo T, Hirasawa Y, Amiel D (1999) The effects of hyaluronan on matrix metalloproteinase-3 (MMP-3), interleukin-1beta(IL-1beta), and tissue inhibitor of metalloproteinase-1 (TIMP-1) gene expression during the development of osteoarthritis. Osteoarthr Cartil 7:182–190. doi:10.1053/joca.1998.0207

    Article  CAS  PubMed  Google Scholar 

  • Terauchi R, Takahashi KA, Arai Y, Ikeda T, Ohashi S, Imanishi J, Kubo T (2003) Hsp70 prevents nitric oxide-induced apoptosis in articular chondrocytes. Arthritis Rheum 48:1562–1568. doi:10.1002/art.11040

    Article  CAS  PubMed  Google Scholar 

  • Uthman I, Raynauld JP, Haraoui B (2003) Intra-articular therapy in osteoarthritis. Postgrad Med J 79:449–453. doi:10.1136/pmj.79.934.449

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Wang CT, Lin YT, Chiang BL, Lin YH, Hou SM (2006) High molecular weight hyaluronic acid down-regulates the gene expression of osteoarthritis-associated cytokines and enzymes in fibroblast-like synoviocytes from patients with early osteoarthritis. Osteoarthr Cartil 14:1237–1247. doi:10.1016/j.joca.2006.05.009

    Article  PubMed  Google Scholar 

  • Williams A, Smith JR, Allaway D, Harris P, Liddell S, Mobasheri A (2013) Carprofen inhibits the release of matrix metalloproteinases 1, 3, and 13 in the secretome of an explant model of articular cartilage stimulated with interleukin 1beta. Arthritis Res Ther 15:R223. doi:10.1186/ar4424

    Article  PubMed Central  PubMed  Google Scholar 

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Acknowledgments

This study was supported by the 50th CMU Anniversary Ph.D. program and the Faculty of Veterinary Medicine, Chiang Mai University. The authors are also grateful for research funding from the Chiang Mai University (CMU) through the research administration office provides budget to our Excellence Center in Osteology Research and Training Center (ORTC). We gratefully thank the Thailand Excellence Center for tissue engineering and stem cells, Department of Biochemistry, Faculty of Medicine, Chiang Mai University for their kind support.

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Correspondence to Korakot Nganvongpanit.

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Editor: T. Okamoto

Thippaporn Euppayo and Korakot Nganvongpanit contributed equally to this work.

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Euppayo, T., Siengdee, P., Buddhachat, K. et al. Effects of low molecular weight hyaluronan combined with carprofen on canine osteoarthritis articular chondrocytes and cartilage explants in vitro. In Vitro Cell.Dev.Biol.-Animal 51, 857–865 (2015). https://doi.org/10.1007/s11626-015-9908-9

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  • DOI: https://doi.org/10.1007/s11626-015-9908-9

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