The timing of glucocorticoid-induced osteocytic cell necrosis under hypoxia

Background: Osteocytic cell necrosis is induced when dexamethasone (Dex) administration and hypoxia are superimposed. We investigated the time needed for osteocytic cell necrosis to develop in cultured osteocytic cells. Materials and Methods: Cultured murine MLO-Y4 osteocytic cells were used. After the addition of 1 mu M Dex they were cultured in 1% O-2 (hypoxia) in an incubator for 6 h (6 h group), 9 h (9 h group), 12 h (12 h group), or 24 h (24 h group) respectively, and then cultured under normoxia (20% O-2) until the respective total culture time reached a total of 24 h. Viability assays were then performed using an Apoptotic/Necrotic Cells Detection Kit according to the manufacturer's instructions, and the percentages of apoptotic and necrotic cells relative to the total cell number were determined. As a Control group cultured osteocytic cells to which Dex was added and cultured for 24 h under normoxia were used. Results: The Control group, 6 h group, 9 h group, and 12 h group showed only slight increases in apoptosis, with no significant differences found between any of them. In contrast, in the 24 h group apoptosis was significantly increased, while necrosis, which did not show any increase in any of the groups up to the 12 h one, was significantly increased in the 24 h group. Discussion: In the presence of Dex and hypoxia the development of osteocytic cell necrosis appears to require a period of at least 12-24 h. This suggests that the development of osteocytic cell necrosis might be interrupted if hypoxia was suppressed or blocked within the critical time limit of 12 h.