Objectives: It is well known that bone formation steadily declines with age, resulting in a significant loss of bone mass. Cellular senescence-elevated oxidative stress has been shown to be related to the regulation of cellular functions in physiological and pathological conditions, and it has been clearly demonstrated that oxidative stress inhibits osteoblast differentiation and leads to a reduction in bone formation. However, the precise mechanism of the effect of reactive oxygen species on the function of osteoblastic cells is not clear. In this study, we examined the effect of oxidative stress in bone nodule formation and confirmed the mRNA expression of osteogenic markers, Runx2, Osx and Msx2.
Methods: The mouse osteoblast-like cell line, MC3T3-E1, were cultured in α-MEM containing 10% FBS and antibiotics. The cells were treated with α-MEM containing 400 μM H
2O
2 for 3 hrs at confluent. After treatment with H
2O
2, the cells were cultured with α-MEM supplemented with 50 μg/ml ascorbic acid and 10 mM Na β-glycerol-phosphate containing 10 % FBS and antibiotics. Mineral deposition was detected by von Kossa staining on day 1, 7, 14, 21, 30, 40 and 50. Bone nodule formation was evaluated by NIH image software. The mRNA expressions of osteogenic markers at 3hrs after treatment with H
2O
2 were evaluated by real time-PCR.
Results: The number of bone nodules per well, total area of bone nodule formation, and single bone nodule size were significantly decreased by treatment with H
2O
2, as compared with the control (
p <0.05). The mRNA expressions of Runx2 and Osx were significant lower than those in control. In contrast, mRNA expression of Msx2 was significant higher than that in control.
Conclusion: We demonstrated that the oxidative stress suppresses the osteoblastic differentiation to mature osteoblast of MC3T3-E1 cells via Msx2, and suggested that the oxidative stress negatively regulates the bone formation.