Heat shock‐induced necrosis and apoptosis in osteoblasts

Abstract

Damage to bone tissue due to heat shock is one of the main causes of the failure of osseointegration at the bone-implant interface. To investigate the effect of heat shock on regeneration of bone tissue, osteoblasts were exposed to heat shock for 10 minutes at 42, 45, or 48 degrees C or kept at 37 degrees C as a control. After 10 minutes of heat shock, disruption of actin filaments was seen in the cells and the degree of disruption increased with the temperature. The cytoskeleton reassembled after a 12-hour incubation at 37 degrees C in the cells treated at 42 or 45 degrees C, but this reversible recovery did not occur in the cells treated at 48 degrees C. Flow cytometric analysis showed that heat shock at 48 degrees C increased the number of necrotic cells to 15-20% within minutes (p < 0.05 compared with 37 degrees C). Apoptosis, evidenced by annexin V staining, DNA laddering, and caspase 3 activation, started after 6-8 hours of incubation, reached a peak at 12 hours, and gradually declined (p<0.05). Pretreatment with the antioxidant N-acetyl-L-cysteine reduced the necrosis induced at 48 degrees C of heat shock by one-half (p<0.05) but had no significant effect on caspase 3 activation induced by heat shock, suggesting that reactive oxygen species were critical in heat shock-induced necrosis but not in apoptosis. Heat shock at 48 degrees C induced a sustained translocation of p53 into the nucleus and a sustained activation of c-jun N-terminal kinase, whereas that at 42 and 45 degrees C induced only transient p53 translocation and c-jun N-terminal kinase activation. These results suggest that the sustained activation of p53 and c-jun N-terminal kinase pathways may contribute to heat shock-induced apoptosis. On the other hand, heat shock protein 70 increased dramatically in the cells treated at 45 or 48 degrees C, suggesting that the protecting mechanism in the cells was also activated. Such protection was able to prevent apoptosis in cells treated at 45 degrees C but not in those treated at 48 degrees C.