Osteonecrosis Induction Using Bisphosphonate and Oxidative Stress Inducer in Mice

Objectives: Aging has been found to be an additional significant risk factor for the development of bisphosphonate-related osteonecrosis (BRONJ). Accumulating evidence suggests that bone aging is associated with oxidative stress (OS), and OS is related to osteonecrosis. To elucidate mechanisms of the onset of BRONJ, we focused on OS and investigated the effects of treatment of pro-oxidant DL-buthionine-(S,R)-sulfoximine (BSO) on healing of the surgically created defects.
Methods: 6-week-old male C57BL/6J mice were randomly divided into four groups; control, treatment with Zoledronic acid (ZOL), treatment with BSO, and combined treatment with ZOL and BSO. The defect of midline palate was created surgically using a root elevator. ZOL (250 μg/kg/day) was injected intraperitoneally from 7 days before the surgical treatment to 4 days after the surgical treatment. BSO (500μg/kg/day) was given at 7 days before the surgical treatment as a single intraperitoneal injection. The maxillae were harvested at 5 days after the surgical treatment. Histological examination, TRAP detection and 8-OHdG staining were performed for these specimens. The imaging of the femurs was performed by micro focus X-ray computed tomography. Our proposed study was approved by the Animal Care and Use Committee of Hyogo College of Medicine (No. 16-078).
Results: The presence of empty osteocyte lacunae in palatal bone was increased by ZOL and BSO treatment. The most number of empty osteocyte lacunae was observed in the ZOL/BSO group. Bone histomorphometric analyses were performed, and BV/TV, Tb.Th and Tb.N were increased by ZOL treatment. The number of TRAP-positive osteoclasts was lower in the ZOL group than in the control group and the BSO group. The number of 8-OHdG-positive osteocytes was higher in the BSO group than in the control group.
Conclusions: OS has been found to be an additional risk factor for the development of BRONJ inducing osteocyte cell death.