The effect of hyaluronan on CD44-mediated survival of normal and hydroxyl radical- damaged chondrocytes P. Brun, Ph.D., S. Panfilo, B.S., D. Daga Gordini,

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The effect of hyaluronan on CD44-mediated survival of normal and hydroxyl radical- damaged chondrocytes P. Brun, Ph.D., S. Panfilo, B.S., D. Daga Gordini, Ph.D., R. Cortivo, Ph.D., G. Abatangelo, M.D. Osteoarthritis and Cartilage Volume 11, Issue 3, Pages 208-216 (March 2003) DOI: 10.1016/S1063-4584(02)00352-7

Fig. 1 Effect of HA on chondrocyte viability in culture. Cells were cultured with and without HA (four concentrations tested: 0, 0.25, 0.5, 0.75, and 1.0mg/ml). Each column depicts the mean MTT value of three experiments. HA significantly enhanced cell proliferation at the 0.5mg/ml dose and at 9 days of culture. Values are expressed as optical density (OD)±standard deviation (SD). Each column depicts the mean value of three experiments. Statistical analyses: Mann–Whitney U test. Osteoarthritis and Cartilage 2003 11, 208-216DOI: (10.1016/S1063-4584(02)00352-7)

Fig. 2 Immunolocalization of CD44 on chondrocyte cell surface at 7 days of culture. The presence of CD44 was determined immunohistochemically using a monoclonal antibody against CD44. Cells were counterstained with hematoxylin. (a) Cells show a positive staining for the molecule (red color) (magnification, 400×); (b) negative control. Osteoarthritis and Cartilage 2003 11, 208-216DOI: (10.1016/S1063-4584(02)00352-7)

Fig. 3 Inhibition of HA binding to chondrocyte CD44 receptor. The addition of monoclonal anti-CD44 antibodies to chondrocyte culture medium in the presence of 0.5mg/ml HA produced a maximum inhibition of HA binding. Anti-CD44 antibodies also inhibited normal baseline proliferation of control cell cultures at 9 days. Each column depicts the mean value of three experiments. Values are expressed as optical density (OD)±standard deviation (SD). Statistical analyses; Mann–Whitney U test. Osteoarthritis and Cartilage 2003 11, 208-216DOI: (10.1016/S1063-4584(02)00352-7)

Fig. 4 Effect of HA on morphology of cells treated with oxygen-free radicals. Human chondrocytes exposed to oxygen radical flux generated by xanthine oxidase for 30min: (a) normal human chondrocytes; (b) human chondrocytes exposed to oxygen radical flux with no HA treatment; and (c) with HA treatment (0.5mg/ml). In (b) and (c), chondrocytes demonstrated cytoplasmic vacuolization induced by free-radical damage, whereas nuclear condensation was observed primarily in untreated samples (panel b). Toluidine Blue staining. Original magnification: 400×. Osteoarthritis and Cartilage 2003 11, 208-216DOI: (10.1016/S1063-4584(02)00352-7)

Fig. 5 Ultrastructure of cells treated with oxygen free radicals. Human chondrocytes exposed to oxygen radical flux generated by xanthine oxidase for 30min: (a) normal human chondrocytes; (b) human chondrocytes exposed to oxygen radical flux with no HA treatment (10000×); and (c) with HA treatment (0.5mg/ml; 8000×). Changes in the cytoplasmic were mainly vacuolization and marked enlargement of the endoplasmic reticulum, whereas in the nucleus, extensive chromatin condensation was observed. Vacuolization and nuclear damage was more pronounced in chondrocytes exposed to xanthine oxidase with no HA treatment (panel b). Osteoarthritis and Cartilage 2003 11, 208-216DOI: (10.1016/S1063-4584(02)00352-7)

Fig. 6 HA-mediated recovery of chondrocyte viability when added to cell culture both at the moment of oxidative injury and after damage. HA was added to some samples both at the moment of injury and at every media change. (a) Cells damaged at 37°C for 30min with hypoxanthine (2mM) and xanthine oxidase (20–60mU); and (b) cells damaged at 37°C for 30min with PMNs. Each column depicts the mean value of three experiments. Values are expressed as optical density (OD)±standard deviation (SD). Osteoarthritis and Cartilage 2003 11, 208-216DOI: (10.1016/S1063-4584(02)00352-7)

Fig. 7 HA-mediated recovery of chondrocyte viability when added to cell culture only after oxidative damage. HA was absent from the media culture at the moment of injury with oxygen-free radicals (hypoxanthine and xanthine oxidase), and added to media culture only after 30min of chemical injury exposure. Each column depicts the mean value of three experiments. Values are expressed as optical density (OD)±standard deviation (SD). Osteoarthritis and Cartilage 2003 11, 208-216DOI: (10.1016/S1063-4584(02)00352-7)