The effect of glycosaminoglycan loss on chondrocyte viability: a study on porcine cartilage explants.
OBJECTIVE
Loss of glycosaminoglycan (GAG) is an early event in osteoarthritis. Recent findings showed increased cell death in arthritic cartilage and linkage with extracellular matrix degradation. The aim of this study was to analyze the direct effect of GAG loss on chondrocyte survival and cell death following mechanical injury.


METHODS
In full-thickness cartilage explants from porcine knee joints, GAG was depleted by digestion with chondroitinase ABC. Explants were subjected to single-impact mechanical injury. Cell viability and the types of cell death were analyzed by Live/Dead cell assay, staining for active caspase 3, and sensitivity to caspase inhibitor.


RESULTS
GAG depletion did not directly lead to increased cell death. In chondroitinase ABC-treated explants, but not in control explants, mechanical injury caused an immediate reduction in cell viability (from 84.6% to 71.0%); the reduction was prominent in the superficial zone. This immediate cell death was not inhibited by the pancaspase inhibitor Z-VAD-FMK, suggesting cell necrosis. During subsequent culture, viability in these explants decreased further, to 50.5% on day 3. The second wave of cell death was reduced by the addition of Z-VAD-FMK in chondroitinase ABC-treated explants and was also associated with activation of caspase 3, suggesting apoptotic mechanisms of cell death.


CONCLUSIONS
These results indicate that GAG loss alone does not directly lead to chondrocyte death. In response to mechanical injury, there is an immediate induction of necrotic cell death that is seen only in GAG-depleted explants and primarily in the superficial zone. During subsequent culture, cell death spreads via apoptotic mechanisms.
Otsuki
Shuhei
S
Division of Arthritis Research, The Scripps Research Institute, La Jolla, California 92037, USA.
Brinson
Diana C
DC
Creighton
Lilo
L
Kinoshita
Mitsuo
M
Sah
Robert L
RL
D'Lima
Darryl
D
Lotz
Martin
M
D000818:Animals; D002358:Cartilage, Articular / Q000503:physiopathology*; D016923:Cell Death / Q000502:physiology*; D002470:Cell Survival; D002478:Cells, Cultured; D019902:Chondrocytes / Q000502:physiology*; D019765:Chondroitin ABC Lyase; D004195:Disease Models, Animal; D006025:Glycosaminoglycans / Q000502:physiology*; D066298:In Vitro Techniques; D009336:Necrosis; D020370:Osteoarthritis, Knee / Q000503:physiopathology*; D034421:Sus scrofa
D006025:Glycosaminoglycans; D019765:Chondroitin ABC Lyase