Effect of Low-intensity Pulsed Ultrasound (LIPUS) with Different Frequency on Bone Defect Healing

Abstract

Low-intensity pulsed ultrasound (LIPUS) is known to promote bone defect healing. The frequency of the LIPUS is known to influence directivity and depth of penetration, but the differences on bone healing remains unknown. This study was to investigate the effect of LIPUS with different frequencies on bone defect healing. Bone defects of 1.6 mm in diameter were created in femurs of ten-week-old male Long-Evans rats (n=36). Experimetal groups were exposed LIPUS (intensity: 30 mW/cm2, burst width: 200 μs, time: 15 min/day) and divided into a low frequency (LF, 1.5 MHz) group and a high frequency (HF, 3.0 MHz) group. Non-exposed LIPUS group were used as control. After 3, 5, 7, and 10 days, femurs were removed and radiological, histomorphological and molecular biological evaluations were conducted. Micro-CT images showed that the depression in cortical bone was reduced in LF and HF groups. 3D bone morphological analysis at 10 days revealed that LIPUS increased cortical bone volume / tissue volume (BV/TV) and decreased BV/TV in the lower layer of cancellous bone (P<0.05). Histomorphologically, clot retraction was seen in the both LIPUS groups but not in the control group at 3 days. These effects were observed at a deeper layer in the LF group than in the HF group. No significant difference in osteopontin (OPN) gene expression was observed. However, osteocalcin (OCN) gene expression was significantly elevated in the HF group relative to the control group at 10 days (P<0.05). Immunohistochemical staining revealed that newly formed bone exhibited a positive reaction to OPN and OCN in both LIPUS groups. Healing of the bone defect area was noted in both LIPUS groups, but there was no clear difference in histomorphology between the LF and HF groups. LIPUS frequencies of 1.5 MHz and 3.0 MHz promote increased cortical bone mass and remodeling of cancellous bone in rat femurs with bone defects.