Contribution of calcium-containing crystals to cartilage degradation and synovial inflammation in osteoarthritis Y.Z. Liu, A.P. Jackson, S.D. Cosgrove

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General Information Osteoarthritis and Cartilage

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Contribution of calcium-containing crystals to cartilage degradation and synovial inflammation in osteoarthritis Y.Z. Liu, A.P. Jackson, S.D. Cosgrove Osteoarthritis and Cartilage Volume 17, Issue 10, Pages 1333-1340 (October 2009) DOI: 10.1016/j.joca.2009.04.022 Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 X-ray diffractograms of (a) t-CPPD, (b) m-CPPD, (c) m-CPPTβ and (d) DCPD. Osteoarthritis and Cartilage 2009 17, 1333-1340DOI: (10.1016/j.joca.2009.04.022) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Micrographs of the calcium-containing crystals that have been generated using the methods described in the text: (a), m-CPPD; (b), t-CPPD; (c), DCPD; (d), m-CPPTβ; (e), crushed m-CPPTβ and (f), extract from OA cartilage. The scale bar represents 25μm. Osteoarthritis and Cartilage 2009 17, 1333-1340DOI: (10.1016/j.joca.2009.04.022) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 An image of human femoral condyles that has been used in this study highlighting the cartilage and the exposed bone. The arrow indicates visible crystal deposition on the surface of the cartilage. Osteoarthritis and Cartilage 2009 17, 1333-1340DOI: (10.1016/j.joca.2009.04.022) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 An image of primary human OA synovoial cells derived from the OA synovium. The inset is a view of the cell cytoplasm that contains crystals. Osteoarthritis and Cartilage 2009 17, 1333-1340DOI: (10.1016/j.joca.2009.04.022) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 X-ray diffractograms of material extracted from the OA knee cartilage from four donors. Osteoarthritis and Cartilage 2009 17, 1333-1340DOI: (10.1016/j.joca.2009.04.022) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Crushing of synthetic m-CPPTβ enhanced the catabolic effects in primary OA chondrocytes. Primary OA chondrocytes from three different donors were treated with crushed or uncrushed m-CPPTβ crystals for 24h. The production of NO (n=3) over the 24h treatment period was determined. Bars represent mean±s.e.m. Osteoarthritis and Cartilage 2009 17, 1333-1340DOI: (10.1016/j.joca.2009.04.022) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 Induction of NO (a) and MMP-13 (b) in primary OA chondrocytes and PGE2 (c) release in primary OA synoviocytes by synthetic crushed m-CPPTβ, t-CPPD, m-CPPD and DCPD crystals. The cells from three to five different donors were treated with crushed m-CPPTβ, t-CPPD, m-CPPD or DCPD crystals for 24h. The production of NO (n=5), MMP-13 (n=3) and PGE2 (n=5) over the 24h treatment period was determined. Bars represent mean±s.e.m. Osteoarthritis and Cartilage 2009 17, 1333-1340DOI: (10.1016/j.joca.2009.04.022) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 8 Induction of NO (a), MMP-13 (b) and PGE2 (c) release in primary OA chondrocytes and PGE2 (d) release in primary OA synoviocytes by purified endogenous OA knee joint crystals. The crystals were prepared from five different OA crystalline cartilage samples and tested. The cells from four to eight different donors were treated with the purified crystals or IL-1β (0.1ng/ml) for 24h. The production of NO (n=8), MMP-13 (n=5) and PGE2 (n=4) from chondrocytes and PGE2 (n=6) from synoviocytes over the 24h treatment period was determined. Bars represent mean±s.e.m. Osteoarthritis and Cartilage 2009 17, 1333-1340DOI: (10.1016/j.joca.2009.04.022) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 9 Calcium phosphate and pyrophosphate crystal-induced NO release is not TLR2 dependent. Primary OA chondrocytes from three different donors were pre-treated with a TLR2 blocking antibody T2.5 at 100μg/ml or IgG1 control for an hour before addition of synthetic and endogenous crystals (0.3mg/ml) or the TLR2 ligand Pam3CSK (1μg/ml). The endogenous crystals were prepared from five different OA crystalline cartilage samples and named as D1, D2, D3, D4 and D5. Following 24h treatment the production of NO (n=3) over the treatment period was determined. Bars represent mean±s.e.m. *P<0.05, significantly different from corresponding IgG1 value. Osteoarthritis and Cartilage 2009 17, 1333-1340DOI: (10.1016/j.joca.2009.04.022) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions