Low-dose X-irradiation Promotes Callus Mineralization By Stimulation Of Angiogenesis Through VEGF Up-regulation

Objectives: Low-dose X-irradiation (LDI) can promote callus mineralization, but the mechanism remains unclear. This study is aimed to investigate the molecular mechanisms of LDI induced callus mineralization by stimulation of angiogenesis through VEGF up-regulation.Methods: One hundred and seventy six male Sprague–Dawley rats were subjected to standard closed fracture on right femur. Eighty eight of which were irradiated by X-ray irradiation of 1Gy right after fracture models establishment. The remaining rats without irradiation were regarded as control group. The serum and callus samples were obtained at weeks 1, 2, 3 and 4. blood vessel casting with a radiopaque silicone rubber compound containing lead chromate and MicroCT scanning were performed on randomly selected rats(n=6). 3-D vascular images were reconstructed and then calculated for vessel volume, average diameter and vessel volume fraction. The serum vascular endothelial growth factor (VEGF) was measured by ELISA kits. Reverse transcription-polymerase chain reaction (RT-PCR) and western-blot were employed to quantify the expression patterns of VEGF. The remaining callus was assessed by using immunohistochemisty for observation of the expression of VEGF, VEGF receptor 2 (VEGFR-2/flk-1), Platelet endothelial cell adhesion molecule-1 (CD31/PECAM-1) and CD34.Results: At 1 weeks post fracture, MicroCT images visibly showed reduced early neovascularization in LDI group,whereas at 2 weeks, the quantitative analysis revealed increased vessel volume and volume fraction in LDI group (p<0.05). In both groups, the serum VEGF continuously increased post fracture and peaked at 3 weeks,then decreased at 4 weeks. But at 2 and 3 weeks, the serum VEGF in the LDI group were higher than those of control groups (p<0.05). Elevated mRNA and protein expression of VEGF were seen at all time points, with peak present at 2 and 3 weeks respectively in the LDI group compared with that of control group (p<0.05). Immunohistochemistry of callus sections revealed stronger expression of VEGF, VEGFR2, CD31 and CD34 in the LDI group at 2 weeks (p<0.05).Conclusion: The results indicate that LDI promotes callus mineralization by stimulation of angiogenesis through VEGF up-regulation. Moreover, weeks 2 and 3 after model establishment may be the key angiogenic time points in the development of rat fracture callus.