L. C. Davies, B. Sc. , Ph. D. , E. J. Blain, B. Sc. , Ph. D. , B

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Chondroitin sulphate impedes the migration of a sub-population of articular cartilage chondrocytes L.C. Davies, B.Sc., Ph.D., E.J. Blain, B.Sc., Ph.D., B. Caterson, B.Sc., Ph.D., V.C. Duance, B.Sc., Ph.D. Osteoarthritis and Cartilage Volume 16, Issue 8, Pages 855-864 (August 2008) DOI: 10.1016/j.joca.2007.12.005 Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Schematic representation of the in vitro cartilage explant model for measuring chondrocyte migration and integration. The explant was excised from the bovine metacarpophalangeal joint (1) and cut using a sharp scalpel blade (2). The excised cartilage was orientated to give a full thickness exposed cut edge for chondroitinase ABC treatment (3) and embedded in agarose leaving only the cut edge exposed and flush with the surrounding agarose (4). Osteoarthritis and Cartilage 2008 16, 855-864DOI: (10.1016/j.joca.2007.12.005) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Numbers of chondrocytes isolated from (A) young and (B) mature articular cartilages migrating on native and chondroitinase ABC digested aggrecan over 24h. Values are expressed ±s.e.m. (n=3) *P<0.05. Osteoarthritis and Cartilage 2008 16, 855-864DOI: (10.1016/j.joca.2007.12.005) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Immunofluorescence photomicrographs, using monoclonal antibodies 1-B-5 and 2-B-6, demonstrating the presence of chondroitinase ABC generated CS-stubs originating from the exposed cut edge (white dotted line). CS-stub formation is present in the superficial (A–D), transitional (E–H) and deep (I–L) zones of the articular cartilage explant, with increasing depth from the cut edge with time in culture (up to 24h). Chondroitinase ABC generated CS-stubs are labelled with FITC (green) and nuclei counterstained with propidium iodide (red). Bar=50μm. Osteoarthritis and Cartilage 2008 16, 855-864DOI: (10.1016/j.joca.2007.12.005) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Fluorescence photomicrographs illustrating ethidium homodimer (dead) labelled chondrocytes within full depth articular cartilage (A) 8h chondroitinase ABC treated and (B) PBS treated explants. The cut edge is depicted by a white dotted line. SZ=superficial zone and DZ=deep zone. Bar=50μm. Osteoarthritis and Cartilage 2008 16, 855-864DOI: (10.1016/j.joca.2007.12.005) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Immunofluorescence photomicrographs demonstrating the migration of CMFDA-labelled young articular chondrocytes into (A) PBS control and (B) chondroitinase ABC digested explants over 28 days in culture. The antibodies 2-B-6 and 1-B-5 and a secondary Texas Red® conjugated antibody have been used to highlight the chondroitinase ABC generated CS-stubs (scale bar=50μm). The cut edge is depicted by a white dotted line. (C) Quantification of migrating CMFDA-labelled chondrocytes (green) visualised in (A) and (B). Data are presented as mean±s.e.m. (n=3; *P<0.05). Osteoarthritis and Cartilage 2008 16, 855-864DOI: (10.1016/j.joca.2007.12.005) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Immunofluorescence photomicrographs demonstrating the migration of CMFDA-labelled mature articular chondrocytes into (A) PBS control and (B) chondroitinase ABC digested explants over 28 days in culture. The antibodies 2-B-6 and 1-B-5 and a secondary Texas Red® conjugated antibody have been used to highlight the chondroitinase ABC generated CS-stubs (scale bar=50μm). The cut edge is depicted by a white dotted line. (C) Quantification of migrating CMFDA-labelled chondrocytes (green) visualised in (A) and (B). Data are presented as mean±s.e.m. (n=3; *P<0.05, **P<0.01, ***P<0.001). Osteoarthritis and Cartilage 2008 16, 855-864DOI: (10.1016/j.joca.2007.12.005) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 High magnification fluorescence photomicrographs of migrated CMFDA-labelled chondrocytes (green) isolated from young and mature articular cartilages into the superficial (A–B), transitional (C–D) and deep (E–F) zones of the chondroitinase ABC treated articular cartilage explants. Arrowheads indicate migrated chondrocytes displaying characteristic morphology of cells within their respective zones. Bar=10μm. Osteoarthritis and Cartilage 2008 16, 855-864DOI: (10.1016/j.joca.2007.12.005) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions