Stimulatory Effects of Low-concentration Reactive Oxygen Species on Calcification Ability of Human Dental Pulp Cells

The present study was conducted to investigate the effects of reactive oxygen species (ROS) on the calcification ability of human dental pulp (HDP) cells. HDP cells were treated with 100 μmol/L hydrogen peroxide (H2O2) for 5 or 10 minutes (5-min ROS group and 10-min ROS group) to investigate the mechanism of transmission to cells. Untreated cells were used as controls. Generation of free radicals was quantified by the electron spin resonance spin-trapping method and found to be increased by treatment with ROS. Formation of calcified nodules was also investigated by von Kossa staining and alizarin red S staining. Twenty-eight days after exposure, calcified nodules were present in cell cultures that had been treated with ROS for 5 or 10 minutes. Expression of mRNAs for osteopontin (OPN) and osteocalcin (OCN) was significantly greater in 10-min ROS group 6 and 9 days, respectively, after exposure than in controls. Production of OPN and OCN by 10-min ROS group was also greater 12 and 18 days, respectively, after exposure than in controls. These results suggested that calcification of HDP cells was stimulated by H2O2 and by the ROS it generated.