Oxidative stress induces expression of osteoarthritis markers procollagen IIA and 3B3(−) in adult bovine articular cartilage I.M. Khan, Ph.D., S.J. Gilbert,

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Oxidative stress induces expression of osteoarthritis markers procollagen IIA and 3B3(−) in adult bovine articular cartilage I.M. Khan, Ph.D., S.J. Gilbert, Ph.D., B. Caterson, Ph.D., L.J. Sandell, Ph.D., C.W. Archer, Ph.D. Osteoarthritis and Cartilage Volume 16, Issue 6, Pages 698-707 (June 2008) DOI: 10.1016/j.joca.2007.10.004 Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Cell death in bovine articular cartilage explants following exposure to increasing concentrations of H2O2. (A) Ethidium homodimer-1 was used to identify dead cells in sections of explants following 7 days exposure to H2O2 in the presence of standard medium plus ITS. All nuclei were counterstained using DAPI (bar: 100μm). (B) Quantification of percentage cell death following 7-day culture in increasing concentrations of H2O2 either in standard medium or standard medium plus ITS (n=5; P<0.05). Osteoarthritis and Cartilage 2008 16, 698-707DOI: (10.1016/j.joca.2007.10.004) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 (A) immunofluorescent detection of 3B3(−) reactivity in 14-day cartilage explants following a single treatment with increasing concentrations of H2O2. Most of the labeling was confined to the pericellular and interritorial extracellular matrix of surface and upper middle zones of the cartilage, labeling was highest in explants exposed to 0.5mM H2O2. (B) 3B3(−) epitope expression over 21 days, 0.5mM H2O2 treated explants were removed at 7-day intervals and probed with antibody. (C) Quantification of the sum of sGAG released into the culture medium from 0 to 14 days normalized to the wet weight of explants (n=3; P<0.05). Osteoarthritis and Cartilage 2008 16, 698-707DOI: (10.1016/j.joca.2007.10.004) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 (A) QPCR gene expression analysis of iNOS mRNA levels in explants exposed to a single dose of 0.1–1.0mM H2O2 and cultured for 14 days in standard medium plus ITS (n=3; P<0.05). (B) immunofluorescent detection of nitrotyrosine immunoreactivity in cartilage explants treated with a single dose of 1mM hydrogen peroxide and cultured for 14 days. Treated explants were labeled along the surface and had more pronounced cellular labeling than controls, especially in the deep zones. Representative images from experiments carried out on explants from three different donors (bar: upper panel 100μm and lower panel 50μm). Osteoarthritis and Cartilage 2008 16, 698-707DOI: (10.1016/j.joca.2007.10.004) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 (A) Immunohistochemical detection of procollagen type IIA in control, 0.1mM, 0.5mM and 1.0mM H2O2 treated cartilage explants cultured for 14 days (bar: 100μm). Note the intense pericellular labeling for procollagen type IIA in deep zone chondrocytes of explants subjected to a single dose exposure of 0.5mM H2O2. Representative images are shown from experiments using explants from three different donors. (B) Graph of relative quantity of collagen type II mRNA expression in explants treated with increasing concentrations of hydrogen peroxide compared to untreated explants as determined by QPCR (n=3; P<0.05). Osteoarthritis and Cartilage 2008 16, 698-707DOI: (10.1016/j.joca.2007.10.004) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Immunolocalisation of 3B3(−) epitopes and procollagen type IIA propeptide in osteoarthritic bovine cartilage. Mild and severely osteoarthritic cartilage, modified Mankin scores 4 and 9–12, respectively, was obtained from aged matched animals to other bovine material used in this study. Serial sections were probed with polyclonal rabbit anti-collagen type IIA, mouse anti-3B3(−) and detected using either fluorscein-conjugated or peroxidase-conjugated secondary antibodies. The white arrows denote the surface of the cartilage; block arrows highlight clusters of cells at the surface. Nuclei were counterstained with propidium iodide (bar: 100μm). Osteoarthritis and Cartilage 2008 16, 698-707DOI: (10.1016/j.joca.2007.10.004) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 QPCR gene expression analysis of collagen type X and matrilin-1 in oxidatively stressed cartilage explants. (A) There was no increase in collagen type X expression between untreated and treated samples (n=4), any gene expression that was present was due to chondrocytes within the calcified layer, a small amount of which was carried over in the process of removing the explants from the joint. (B) Matrilin-1 expression increased in a dose-dependent manner following a single dose of increasing concentrations of hydrogen peroxide, we observed a 26-fold increase in expression (n=4; P<0.05) in explants exposed to 1mM hydrogen peroxide and cultured for 14 days post-treatment. Osteoarthritis and Cartilage 2008 16, 698-707DOI: (10.1016/j.joca.2007.10.004) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions