C. Pascual Garrido, A. A. Hakimiyan, L. Rappoport, T. R. Oegema, M. A

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Anti-apoptotic treatments prevent cartilage degradation after acute trauma to human ankle cartilage C. Pascual Garrido, A.A. Hakimiyan, L. Rappoport, T.R. Oegema, M.A. Wimmer, S. Chubinskaya Osteoarthritis and Cartilage Volume 17, Issue 9, Pages 1244-1251 (September 2009) DOI: 10.1016/j.joca.2009.03.007 Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Experimental set-up. (A) Fresh human tali (cartilage with bone attached) individually potted in polymethylmethacrylate with cartilage surface aligned parallel to the front end of a 4mm diameter cylindrical impactor. Areas that will be subjected to injury are outlined by Indian ink; (B) pneumatic, pressure controlled impaction device; (C) schematic depiction of tested areas (core, ring, and control) in the polymethylmethacrylate potted human tali. Osteoarthritis and Cartilage 2009 17, 1244-1251DOI: (10.1016/j.joca.2009.03.007) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Cell survival detected by live/dead assay in the control, non-treated samples. (A) Representative photomicrographs of cartilage sections at days 2 (left column) and 14 (right column) of culture. (a and b) Non-impacted cartilage; (c and d) impacted core regions; (e and f) adjacent ring regions; arrows indicate the areas of impact. Original magnification 100×. (B–D) Quantitative assessment of cell survival in the superficial layer (B), middle/deep layer (C), and in the entire cartilage section (D) corresponding to photomicrographs shown in A. Day 0 means immediately after injury. (*) Represents statistical difference between impacted and non-impacted controls (t test, P<0.05). Osteoarthritis and Cartilage 2009 17, 1244-1251DOI: (10.1016/j.joca.2009.03.007) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Comparison of cell survival detected by live/dead assay between treated and non-treated impacted samples. (A) Representative photomicrographs of core and ring cartilage sections shown at day 14 of culture. (a and b) Non-treated impacted controls; (c and d) treatment with P188; (e and f) treatment with caspase-3 inhibitor; (g and h) treatment with caspase-9 inhibitor. Original magnification 100×. (B and C) Quantitative assessment of cell viability corresponding to photomicrographs shown in A at various time points of culture in the superficial layer of the core (B) and the ring (C). (*) Represents statistical difference between treated and non-treated groups at the corresponding time points (P<0.05). Osteoarthritis and Cartilage 2009 17, 1244-1251DOI: (10.1016/j.joca.2009.03.007) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Quantitative assessment of cell death by apoptosis in the superficial (A and C) and middle/deep (B and D) cartilage layers of the impacted core (A and B) and adjacent ring (C and D). Values are shown as the percentage of Tunel-positive cells; comparison is made between treatments. (*) Represents statistical difference between treated and non-treated groups at corresponding time points when P<0.01; (^) represents statistical difference when P<0.05. Osteoarthritis and Cartilage 2009 17, 1244-1251DOI: (10.1016/j.joca.2009.03.007) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Histological assessment of cartilage explants. (A) Representative photomicrographs of cartilage explants stained with Safranin O/fast green taken at day 2 (left column) and day 14 of culture (right column). (a and b) Non-impacted control; (c and d) impacted, non-treated control; (e and f) explants treated with P188 for 48h; (g and h) explants treated with caspase-3 inhibitor; (i and j) explants treated with caspase-9 inhibitor. Original magnification 40×. Mankin score of the core (B) and ring (C) areas of the impacted, non-treated control or cartilage explants treated with P188, caspase-3 or caspase-9 inhibitors. (*) Represents statistical difference between impacted non-treated control and treated groups at corresponding time points when P<0.05. Osteoarthritis and Cartilage 2009 17, 1244-1251DOI: (10.1016/j.joca.2009.03.007) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions