Purpose. This study has researched the affect of different methodologies of harvesting and analysing the samples in determining the mediators emerging after the rat articular cartilage injury. Materials and Methods. One hundred and forty-four male wistar rats were divided into 2 groups. Synovial fluid samples were taken from all of the rats. We entered into the right knees of the rats in group I under anaesthesia and took cartilage tissue samples from their distal femur. Samples were taken as reference values for enzyme linked immunosorbent assay (ELISA) and histopathological evaluations. We entered into the right knees of the rats in group II and formed complete layer of cartilage injury in their medial femoral condyles. At the end of the 15th day, the rats were sacrificed after taking synovial fluid samples from their right knees creating defect in the rats in group II. The molecular markers in the synovial fluid and cartilage tissue samples which were taken from the experimental and control groups (MMP-9, MMP-13, TIMP-1, TNF-α, and NO) were analysed by direct or indirect methodologies. SPSS 18.0 Package program was used in the statistical evaluation. Students t-test where the measurement variables between the experimental and control groups were compared was applied. Receiver Operating Characteristics (ROC) curves were used in the determination of the diagnostic sufficiency from the tissue. Results. No difference was found between TIMP-1 and MMP-9 levels in synovial fluid and cartilage tissue. From the molecular markers, when MMP-9, MMP-13, NO, TIMP-1, TNF-α′, the area under ROC curve, and P values were examined, MMP-13 ( , 95% CI: 0.70–0.85), NO ( , 95% CI: 0.72–0.86), and TNF-α ( , 95% CI: 0.91–0.98) results were found to be statistically significant. Inferences. The indirect ELISA protocol which we apply for the cartilage tissue as an alternative to synovial lavage fluid is a reliable method which can be used in the determination of articular cartilage injury markers. 1. Introduction The molecular markers which emerge in the process after the articular cartilage injury are used in the monitoring of degenerative diseases, prognosis determination, monitoring of response to the treatment, and identification of the disease mechanism in molecular level [1, 2]. One important purpose of the measurement of molecular determinants is to examine the disease quantitatively in the early stages when the cartilage injury has not been radiologically determined yet. Chondrocytes have a directing role in the metabolism including the construction and destruction of
References
[1]
G. Nagyeri, M. Radacs, S. Ghassemi-Nejad et al., “TSG-6 protein, a negative regulator of inflammatory arthritis, forms a ternary complex with murine mast cell tryptases and heparin,” The Journal of Biological Chemistry, vol. 286, no. 26, pp. 23559–23569, 2011.
[2]
K. Tsuritani, J. Takeda, J. Sakagami et al., “Cytokine receptor-like factor 1 is highly expressed in damaged human knee osteoarthritic cartilage and involved in osteoarthritis downstream of TGF-β,” Calcified Tissue International, vol. 86, no. 1, pp. 47–57, 2010.
[3]
T. A. Stupina, V. D. Makushin, and M. A. Stepanov, “Experimental morphological study of the effects of subchondral tunnelization and bone marrow stimulation on articular cartilage regeneration,” Bulletin Experimental Biology and Medicine, vol. 153, no. 2, pp. 289–293, 2012.
[4]
M. A. Jordan, G. S. Van Thiel, J. Chahal, and S. J. Nho, “Operative treatment of chondral defects in the hip joint: a systematic review,” Current Reviews in Musculoskeletal Medicine, vol. 5, no. 3, pp. 244–253, 2012.
[5]
O. S. Ertas and A. Kayali, “An overview on analytical method validation,” Journal of Faculty Pharmacy, vol. 34, no. 1, pp. 41–57, 2005.
[6]
G. Szepesi, M. Gazdag, and K. Mihalyfi, “Selection of high-performance liquid chromatographic methods in pharmaceutical analysis. III. Method validation,” Journal of Chromatography A, vol. 464, no. 2, pp. 265–278, 1989.
[7]
A. Kayal, O. Sogut, and B. Tuncel, “Some considerations on analytical methods validations, bioavailability, bioequivalence and pharmacokinetic studies,” European Journal of Drug Metabolism and Pharmacokinetics, vol. 20, no. 4, pp. 271–274, 1995.
[8]
A. Marin, A. López-Gonzálvez, and C. Barbas, “Development and validation of extraction methods for determination of zinc and arsenic speciation in soils using focused ultrasound: application to heavy metal study in mud and soils,” Analytica Chimica Acta, vol. 442, no. 2, pp. 305–318, 2001.
[9]
J. G. Morrison, P. White, S. McDougall et al., “Validation of a highly sensitive ICP-MS method for the determination of platinum in biofluids: application to clinical pharmacokinetic studies with oxaliplatin,” Journal of Pharmaceutical and Biomedical Analysis, vol. 24, no. 1, pp. 1–10, 2000.
[10]
V. Grdinic and J. Vukovic, “Prevalidation in pharmaceutical analysis, part 1, fundamentals and critical discussion,” Journal of Pharmaceutical and Biomedical Analysis, vol. 35, no. 1, pp. 489–512, 2004.
[11]
P. Garnero, J. C. Rousseau, and P. D. Delmas, “Molecular basis and clinical use of biochemical markers of bone, cartilage, and synovium in joint diseases,” Arthritis & Rheumatism, vol. 43, no. 1, pp. 953–968, 2000.
[12]
D. A. Walsh, P. Verghese, G. J. Cook, et al., “Lymphatic vessels in osteoarthritic human knees,” Osteoarthritis Cartilage, vol. 20, no. 5, pp. 405–412, 2012.
[13]
Y. Henrotin, B. Kurz, and T. Aigner, “Oxygen and reactive oxygen species in cartilage degradation: friends or foes?” Osteoarthritis and Cartilage, vol. 13, no. 8, pp. 643–654, 2005.
[14]
A. Ostalowska, E. Birkner, M. Wiecha et al., “Lipid peroxidation and antioxidant enzymes in synovial fluid of patients with primary and secondary osteoarthritis of the knee joint,” Osteoarthritis and Cartilage, vol. 14, no. 2, pp. 139–145, 2006.
[15]
K. Haugbro, J. C. Nossent, T. Winkler, Y. Figenschau, and O. P. Rekvig, “Anti-dsDNA antibodies and disease classification in antinuclear antibody positive patients: the role of analytical diversity,” Annals of the Rheumatic Diseases, vol. 63, no. 4, pp. 386–394, 2004.
[16]
K. J. Jepsen, F. Wu, J. H. Peragallo et al., “A syndrome of joint laxity and impaired tendon integrity in lumican- and fibromodulin-deficient mice,” The Journal of Biological Chemistry, vol. 277, no. 38, pp. 35532–35540, 2002.
[17]
B. E. Gencosmanoglu, M. Eryavuz, and S. Dervisoglu, “Effects of some nonsteroidal anti-inflammatory drugs on articular cartilage of rats in an experimental model of osteoarthritis,” Research in Experimental Medicine, vol. 200, no. 3, pp. 215–226, 2001.
[18]
A. J. Hough, “Pathology of osteoarthritis,” in Arthritis and Allied Conditions, W. J. Koopman, Ed., pp. 1945–1968, Williams & Wilkins, Baltimore, Md, USA, 1997.
[19]
J. D. Sandy, C. R. Flannery, P. J. Neame, and L. S. Lohmander, “The structure of aggrecan fragments in human synovial fluid. Evidence for the involvement in osteoarthritis of a novel proteinase which cleaves the Glu 373-Ala 374 bond of the interglobular domain,” The Journal of Clinical Investigation, vol. 89, no. 5, pp. 1512–1516, 1992.
[20]
S. Tseng, A. H. Reddi, and P. E. Di Cesare, “Cartilage oligomeric matrix protein (COMP): a biomarker of arthritis,” Biomarker Insights, vol. 4, pp. 33–44, 2009.
[21]
L. S. Lohmander, L. A. Hoerrner, and M. W. Lark, “Metalloproteinases, tissue inhibitor, and proteoglycan fragments in knee synovial fluid in human osteoarthritis,” Arthritis & Rheumatism, vol. 36, no. 2, pp. 181–189, 1993.
[22]
L. S. Lohmander, H. Roos, L. Dahlberg, L. A. Hoerrner, and M. W. Lark, “Temporal patterns of stromelysin-1, tissue inhibitor, and proteoglycan fragments in human knee joint fluid after injury to the cruciate ligament or meniscus,” Journal of Orthopaedic Research, vol. 12, no. 1, pp. 21–28, 1994.
[23]
M. Feldmann, F. M. Brennan, and R. N. Maini, “Role of cytokines in rheumatoid arthritis,” Annual Review of Immunology, vol. 14, pp. 397–440, 1996.
[24]
L. Connell and I. B. McInnes, “New cytokine targets in inflammatory rheumatic diseases,” Best Practice & Research, vol. 20, no. 5, pp. 865–878, 2006.
[25]
M. Sharif, E. George, L. Shepstone et al., “Serum hyaluronic acid level as a predictor of disease progression in osteoarthritis of the knee,” Arthritis & Rheumatism, vol. 38, no. 6, pp. 760–767, 1995.
[26]
C. Melchiorri, R. Meliconi, L. Frizziero, et al., “Enhanced and coordinated in vivo expression of inflammatory cytokines and nitric oxide synthase by chondrocytes from patients with osteoarthritis,” Arthritis & Rheumatism, vol. 41, no. 12, pp. 2165–2174, 1998.
[27]
R. Hilf, J. L. Wittliff, W. D. Rector, E. D. Savlov, T. C. Hall, and R. A. Orlando, “Studies on certain cytoplasmic enzymes and specific estrogen receptors in human breast cancer and in nonmalignant diseases of the breast,” Cancer Research, vol. 33, no. 9, pp. 2054–2062, 1973.
[28]
F. A. Van de Loo, O. J. Arntz, F. H. van Enckevort, P. L. van Lent, and W. B. van den Berg, “Reduced cartilage proteoglycan loss during zymosan-induced gonarthritis in NOS2-deficient mice and in anti-interleukin-1-treated wild-type mice with unabated joint inflammation,” Arthritis & Rheumatism, vol. 41, no. 4, pp. 634–646, 1998.
[29]
R. F. Loeser, “Integrins and cell signaling in chondrocytes,” Biorheology, vol. 39, no. 1-2, pp. 119–124, 2002.
[30]
A. R. Poole, F. Guilak, and S. B. Abramson, “Etiopathogenesis of osteoarthritis,” in Osteoarthritis, Diagnosis and MEdical/Surgical Management, R. W. Moskowitz, Ed., pp. 27–49, Lippincott, Philadelphia, Pa, USA, 4th edition, 2007.
[31]
S. R. Goldring and M. B. Goldring, “The role of cytokines in cartilage matrix degeneration in osteoarthritis,” Clinical Orthopaedics and Related Research, no. 427, supplement, pp. S27–S36, 2004.
[32]
S. Kamekura, K. Hoshi, T. Shimoaka et al., “Osteoarthritis development in novel experimental mouse models induced by knee joint instability,” Osteoarthritis and Cartilage, vol. 13, no. 7, pp. 632–641, 2005.
[33]
A. R. Poole, M. Kobayashi, T. Yasuda, et al., “Type II collagen degradation and its regulation in articular cartilage in osteoarthritis,” Annals of the Rheumatic Diseases, vol. 61, supplement 2, pp. 78–81, 2002.
