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.

Effect of Low-intensity Pulsed Ultrasound on Healing of Bone Defects in Rat Tibia as Measured by Reconstructed Three-dimensional Analysis of Micro CT Images

“…Sasaki et al (15) reported accelerated capillary vessel formation in LIPUS-stimulated bone. Based on our results, we hypothesize that LIPUS induces angiogenesis that affects bone metabolism in defect areas of the tibia.…”

A Comparison of 1‐ and 3.2‐MHz Low‐Intensity Pulsed Ultrasound on Osteogenesis on Porous Titanium Alloy Scaffolds: An In Vitro and In Vivo Study

“…LIPUS is a type of mechanical energy that is transmitted through and into biologic tissues as an acoustic pressure wave at frequencies above the limit of human fracture healing. LIPUS is used widely in medicine as a therapeutic, operative, and diagnostic tool and promotes healing of fractures and bone defects . In this study, the effects of different frequencies of LIPUS for bone formation on scaffolds were investigated by molecular biologic and histomorphologic evaluation.…”

“…Takebe et al reported increased new bone growth in the low‐frequency (1‐MHz) LIPUS group compared with the control group and high‐frequency (3‐MHz) LIPUS group in rabbit sinus augmentation models. Sasaki et al concluded that 1.5 MHz of LIPUS exposure more effectively promoted vascular endothelial growth factor expression and angiogenesis compared with 3 MHz for rat femur bone defect healing. On the other hand, Monden et al reported no significant differences in the gene expression of bone differentiation markers and the volume of newly formed bone in the rat femur bone defective area between the 1.5‐ MHz and 3‐ MHz groups.…”

Low‐intensity pulsed ultrasound stimulation for mandibular condyle osteoarthritis lesions in rats

“…LIPUS exposure is known to accelerate not only osteoblastic cell maturation and bone fracture healing but also angiogenesis (Chen et al., ). Angiogenesis plays a role in early bone repair and ossification by creating new blood vessels, which bring oxygen, inflammatory cells and bone precursor cells to the injury site (El‐Bialy et al., ; Hankenson, Dishowitz, Gray, & Schenker, ; Sasaki, Monden, Yoshinari, & Yajima, ). We suggest that the most effective time point for LIPUS application corresponds to the angiogenesis stage, and a constant but non‐significant increase was shown from 18 to 20 weeks (Figure ad,bd and cd).…”