Comparison of Angiogenesis in Bone Defect Healing Process due to the Difference in the Frequency of Low-Intensity Pulsed Ultrasound (LIPUS)

Abstract

Low-intensity pulsed ultrasound (LIPUS) is known to promote bone defect healing and also angiogenesis. It was reported the frequency of the LIPUS was related to directivity and the depth of penetration, but the differences in angiogenesis during bone defected healing remains unknown. The aim of this study is to investigate the effect of low and high frequency LIPUS exposure for the angiogenesis during rat femur bone defect healing process by molecular biological and histomorphological evaluations. Bone defects of 1.6 mm in diameter were created in both femurs of ten-week-old male Long-Evans rats (n=30). Right femur as LIPUS exposure groups were exposed LIPUS (intensity: 30 mW/cm2, burst width: 200 µs, time: 15 min/ day) and divided into a low frequency (1.5 MHz, L15) group and a high frequency (3.0 MHz, L30) group. Left femur as non-exposed LIPUS group were used as control. After 3, 5, and 7 days, femurs were removed and quantitative RT-PCR (qRT-PCR) for vascular endothelial growth factor (VEGF), histomorphological and immunohistochemical evaluations and measurement of new formed capillary vessel ratio in bone defected are were conducted. The results of qRT-PCR were indicated that VEGF expression of L15 at 5 days was significantly higher than that of L30 and control group (p < 0.05). Immune-positive reaction of VEGF was recognized in fibroblasts, endothelial cells, periosteal cells and osteoblast and these expression in LIPUS exposure groups were stronger than control groups. The capillary vessel formation ratio in upper layer of bone defected area in L15 group was significantly increased compared to L30 and control group at 7 days (p < 0.05). In conclusion, 1.5 MHz frequency of LIPUS exposure was more effective to promote VEGF expression and angiogenesis than 3.0 MHz in rat femur bone defected healing.