Low‐Intensity Pulsed Ultrasound Induces Osteogenic Differentiation of Human Periodontal Ligament Cells Through Activation of Bone Morphogenetic Protein–Smad Signaling

Objectives Low‐intensity pulsed ultrasound (US) can accelerate fracture healing and osteogenic differentiation. The aim of this study was to investigate the osteogenic effect of low‐intensity pulsed US on human periodontal ligament cells and to determine whether bone morphogenetic protein (BMP)‐Smad signaling was involved. Methods Human periodontal ligament cells were exposed to low‐intensity pulsed US at a frequency of 1.5 MHz and intensity of 90 mW/cm2 for 20 min/d. Osteogenic differentiation was determined by assaying alkaline phosphatase (ALP) and calcium deposition. Expression of BMP‐2, BMP‐6, and BMP‐9 was detected by real‐time polymerase chain reaction analysis. Phosphorylated Smad was detected by western blotting; Smad in the cells was labeled by an immunofluorescent antibody and observed by laser‐scanning confocal microscopy. Results The optical density of ALP stimulated by US at 1.5 MHz and 90 mW/cm2 for 20 min/d was significantly higher than in other groups (P < .01); therefore, this dosage was considered optimal for promoting osteogenic differentiation. After 13 days of US exposure, ALP increased gradually after 5 days, peaked at 11 days, and decreased at 13 days, with a significant difference compared with the control group (P < .05). Osteocalcin production increased from 9 to 13 days and peaked at 15 days, with a significant difference compared with the control group (P < .05). BMP‐2 and BMP‐6 increased dynamically after exposure for 13 days. BMP‐2 increased 6.07‐fold at 3 days, 6.39‐fold at 11 days, and 5.97‐fold at 13 days. BMP‐6 expression increased 6.82‐fold at 1 day and 51.5‐fold at 3 days and decreased thereafter. BMP‐9 was not expressed. Phospho‐Smad1/5/8 expression was significantly increased after exposure (P< .05) and transferred from the cytoplasm into the nuclei. Conclusions Low‐intensity pulsed US effectively induced osteogenic differentiation of human periodontal ligament cells, and the BMP‐Smad signaling pathway was involved in the mechanism.