Consecutive Low-Intensity Pulsed Ultrasound Mediated Osteogenic Differentiation in Rat Osteblasts

Objectives: There have been no studies investigating the effects of the mechanical stimulation provided by consecutive low-intensity pulsed ultrasound (LIPUS) treatment for bone augmentation on non-sufficient bone volume performing dental implants. LIPUS is known to accelerate mineralization and bone regeneration, but the precise cellular mechanism is unclear. Our purpose was to determine how consecutive LIPUS affects cell proliferation, alkaline phosphatase (ALPase) activity, osteogenesis-related gene and protein expressions, and mineralized nodule formation in a rat osteosarcoma cell line.

Methods: Cells of ROS 17/2.8, a typical osteoblastic cell line, were cultured with or without the addition of consecutive LIPUS stimulation. The ultrasound signal consisted of 1.5 MHz at an intensity of 30 mW/cm² for 20 minutes daily for 14 days for all cultures. The expression of mRNA encoding the osteogenesis-related transcription factors were measured using real-time PCR. The protein expression of these transcription factors was also determined by Western blotting.

Results: Consecutive LIPUS stimulation affects the rate of cell proliferation. ALPase activity was increased at day 10 of culture by consecutive LIPUS stimulation. Real-time PCR analysis indicated that consecutive LIPUS significantly increased the expression of mRNA for the transcription factors Runx2, Msx2, Dlx5, Osterix and bone sialoprotein (BSP), whereas the mRNA expression of AJ18 was significantly reduced. Runx2 and BSP protein expressions were increased substantially at 14 day after consecutive LIPUS stimulation. The mineralized nodule formation was markedly increased on day 14 of culture by consecutive LIPUS stimulation.

Conclusion: Our study demonstrates that consecutive LIPUS stimulation directly affects osteogenic cells, leading to mineralized nodule formation. It is likely that consecutive LIPUS has an important influence on key functional activities of osteoblasts in alveolar bone.