Osteogenic Differentiation of Human Adipose-Derived Stem Cells Can Be Accelerated by Controlling the Frequency of Continuous Ultrasound

Objectives-The purpose of this study was to demonstrate that the effects of continuous ultrasound on the osteogenic differentiation of human adipose-derived stem cells (hASCs) are dependent on the frequency in vitro. Methods-Before stimulation, we characterized the hASCs using cluster of differentiation marker profiles and tridifferentiation. Then we selected effective frequencies in the range of 0.5 to 1.5 MHz (with a peak negative pressure of 52 kPa), which upregulated runt-related transcription factor 2 messenger RNA expression. Next, the effects of ultrasound at the selected frequencies on the osteogenic differentiation were evaluated at the protein level. Alkaline phosphatase activity and the formation of mineralized nodules were measured. We additionally identified the cellular mechanisms underlying the effects of ultrasound stimulation using Western blotting. Results-The hASCs showed general cluster of differentiation marker profiles of stem cells and confirmed their potentials to yield adipogenic, chondrogenic, and osteogenic differentiation. Frequencies of 0.5, 1.0, and 1.5 MHz were selected for higher runt-related transcription factor 2 expression in the range of 0.5 to 1.5 MHz. Among the 3 groups, alkaline phosphatase activity and the formation of mineralized nodules were increased in cells exposed to 1.5-MHz ultrasound compared with cells exposed to 0.5-or 1.0-MHz ultrasound and nontreated control cells. We additionally confirmed that this acceleration of osteogenic differentiation was related to p38 and protein kinase B signaling pathways. Conclusions-In this study, we found that, in the selected range, 1.5 MHz was the most effective frequency for inducing the osteogenic differentiation of hASCs.