The effect of hyaluronan on CD44-mediated survival of normal and hydroxyl radical-damaged chondrocytes.
OBJECTIVE
To identify the CD44-receptor-mediated effects of 5-7 x 10(5)MW hyaluronan (HA, Hyalgan) on cell viability in normal and damaged human chondrocyte primary cultures isolated from articular cartilage.


METHODS
Primary cultures of human chondrocytes were established from normal articular biopsies and expanded to the second culture passage. The dose-response effects of HA on the viability of normal cultures were identified. Chondrocytes were then treated with either hypoxanthine (2 mM) and xanthine oxidase (20-60 mU), or with activated polymorphonuclear leukocytes (PMNs) to induce injury. Damaged and control cells were then treated with 5-7 x 10(5)HA in the previously identified optimal dose of 0.05 mg/ml. Viability was assessed at specific time periods for the chemically and PMN-damaged cells. To identify if HA effects were mediated by the CD44 receptor, chondrocytes were incubated with anti-CD44 antibody at saturating concentrations (5 microg/ml for 100,000 cells) to produce a maximum inhibition of HA binding. Cells were evaluated using the MTT viability assay, histology, electron microscopy and immunohistochemistry.


RESULTS
Direct addition of HA (optimal dose, 0.5 mg/ml) significantly increased cell survival in normal chondrocyte primary cultures (P<0.05). Similarly, addition of this same dose of HA to cultures of free radical-damaged chondrocytes, restored the viability to baseline conditions. Cell viability rates dropped significantly (P<0.05) when CD44 receptor binding was inhibited, indicating that cell growth was mediated by the CD44 receptor.


CONCLUSIONS
HA (0.5 mg/ml of 5-7 x 10(5)) significantly increased the viability of normal human chondrocytes in primary culture and restored cell viability to near normal levels after oxidative cell injury.
Brun
P
P
Department of Histology, Microbiology and Medical Biotechnology, Faculty of Medicine, University of Padova, Italy.
Panfilo
S
S
Daga Gordini
D
D
Cortivo
R
R
Abatangelo
G
G
D002358:Cartilage, Articular / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000494:pharmacology*; D019271:Hypoxanthine / Q000494:pharmacology; D007150:Immunohistochemistry / Q000379:methods; D008854:Microscopy, Electron; D009504:Neutrophils / Q000502:physiology; D014969:Xanthine Oxidase / Q000494:pharmacology
D018960:Hyaluronan Receptors; D019271:Hypoxanthine; D006820:Hyaluronic Acid; D014969:Xanthine Oxidase