Low-intensity pulsed ultrasound induces osteogenic differentiation of human periodontal ligament cells through activation of bone morphogenetic protein-smad signaling.
OBJECTIVE
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/cm(2) 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/cm(2) 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.
Affiliated Hospital of Stomatology, Chongqing Medical University, 426 Songshi North St, Yubei District, 401147 Chongqing, China. soongjl@163.com.
Zun
Z
Yang
Leixi
L
Ren
Feng
F
Deng
Zhibiao
Z
Wang
Jinlin
J
Song
D000293:Adolescent; D019485:Bone Morphogenetic Proteins / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology / Q000528:radiation effects; D002478:Cells, Cultured; D005260:Female; D019220:High-Energy Shock Waves; D006801:Humans; D008297:Male; D010006:Osteoblasts / Q000166:cytology / Q000502:physiology / Q000528:radiation effects; D010012:Osteogenesis / Q000502:physiology* / Q000528:radiation effects; D010513:Periodontal Ligament / Q000166:cytology* / Q000502:physiology* / Q000528:radiation effects; D011829:Radiation Dosage; D015398:Signal Transduction / Q000502:physiology / Q000528:radiation effects; D051785:Smad Proteins / Q000378:metabolism*; D013010:Sonication / Q000379:methods*
D019485:Bone Morphogenetic Proteins; D051785:Smad Proteins
Smad; basic science; bone morphogenetic protein; human periodontal ligament cells; low-intensity pulsed ultrasound; osteogenic differentiation; signal transduction