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To study the potential effects of low-intensity pulsed ultrasound (LIPUS) on cell response in vitro, the ability to alter LIPUS parameters is required. However, commercial LIPUS systems have very little control over parameter selection. In this study, a custom LIPUS system was designed and validated by exploring the effects of using different pulse repetition frequency (PRF) parameters on human adipose derived adult stem cells (hASCs) and bone marrow derived mesenchymal stem cells (hMSCs), two common stem cell sources for creating bone constructs in vitro. Changing the PRF was found to affect cellular response to LIPUS stimulation for both cell types. Proliferation of LIPUS-stimulated cells was found to decrease for hASCs by d 7 for all three groups compared with unstimulated control cells (P = 0.008, 0.011, 0.014 for 1 Hz, 100 Hz and 1 kHz PRF, respectively) and for hMSCs by d 14 (donor 1: P = 0.0005, 0.0002, 0.0003; donor 2: P = 0.0003, 0.0002, 0.0001; for PRFs of 1 Hz, 100 Hz, and 1 kHz, respectively). Additionally, LIPUS was shown to strongly accelerate osteogenic differentiation of hASCs based on amount of calcium accretion normalized by total DNA (P = 0.003, 0.001, 0.003, and 0.032 between control/100 Hz, control/1 kHz, 1 Hz/1 kHz, and 100 Hz/1 kHz pulse repetition frequencies, respectively). These findings promote the study of using LIPUS to induce osteogenic differentiation and further encourage the exploration of LIPUS parameter optimization. The custom LIPUS system was successfully designed to allow extreme parameter variation, specifically PRF, and encourages further studies. [ABSTRACT FROM PUBLISHER]