LM8 mouse osteosarcoma and MC3T3-E1 mouse osteoblastic cells were obtained from the Japanese Collection of Research Bioresources Cell Bank (Ibaraki, Japan) and were maintained in Eagle's minimal essential medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) containing 10% heat-inactivated fetal calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a humidified incubator containing 5% CO₂.

The Sonic Accelerated Fracture Healing System ultrasound apparatus (Teijin Ltd., Osaka, Japan) was used with 1.5 MHz frequency pulses, with a pulse width of 200 µs, repeated at 1 kHz, at a spatial average and temporal average intensity of 30 mW/cm, in all sonication experiments. The LIPUS transducer was placed horizontally on each plate of cells for different times (1, 12, 18 or 24 h). In control experiments, the cells were treated in the same manner without LIPUS exposure.

LM8 cells were seeded into 96-well black/clear plates (Falcon; Corning Incorporated, Corning, NY, USA) at a density of 1×10⁴ cells/well. MC3T3-E1 cells were seeded into black/clear 96-well plates at a density of 7.5×10³ cells/well. Following incubation overnight at 37°C, the cells were treated with or without LIPUS for 1, 12, 18 or 24 h. The WST-8 reagent (Cell Counting Kit-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) The WST-8 reagent (Cell Counting Kit; Dojindo Lab, Tokyo, Japan) was added to each well, and the cells were cultured for 2 h according to the manufacturer's protocol. Absorbance in conditioned medium (Eagle's minimal essential medium: Sigma-Aldrich; Merck KGaA) was monitored at 490 nm using a microplate reader (Molecular Devices LLC, Sunnyvale, CA, USA). IC₅₀ values were calculated using the Softmax Pro software 6 (Molecular Devices LLC).

LM8 cells were seeded into 96-well black/clear plates at a density of 4×10⁴ cells/well. MC3T3-E1 cells were seeded into black/clear 96-well plates at a density of 2×10⁴ cells/well. Following incubation overnight at 37°C, the cells were treated with or without LIPUS for 48 h. The mitochondrial membrane potential of the cells were measured using a membrane potential cytotoxicity kit (Mito-ID; Enzo Life Sciences, Inc., Farmingdale, NY, USA) and fluorescence microscopy (IX73; Olympus Corporation, Tokyo, Japan).

LM8 cells (1×10⁶ cells/well) were cultured in a 35-mm dish (Lumox dish 35; Sarstedt K.K., Tokyo, Japan) and stimulated with LIPUS at 37°C for 48 h. The LM8 cells were then treated with trypsin-EDTA, washed with PBS and resuspended in binding buffer (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan) according to the manufacturer's protocol. Cell suspension (85 µl) was then incubated with 10 µl Annexin V/fluorescein isothiocyanate and 5 µl protein iodide. After a 15-min incubation at room temperature in the dark, 400 µl binding buffer was added. The cells were analyzed by flow cytometry (FACSCanto II; BD Biosciences, San Jose, CA, USA) and the results were analyzed using software (FlowJo version 10.2; Tomy Digital Biology, Tokyo, Japan).

An Apoptosis In Situ Detection kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was used to measure apoptosis using the TUNEL staining method, according to the manufacturer's protocol, following treatment of LM8 (1×10⁶) cells with LIPUS at 37°C for 48 h. TUNEL-positive and TUNEL-negative cells were counted in four random high-power fields (magnification, ×400) of each section. The rate of apoptosis was calculated using the following equation: TUNEL-positive cell number/total cell number ×100 (%).

LM8 cells were seeded into a 35-mm dish (Lumox dish 35) at a density of 1×10⁶ cells. Following incubation overnight at 37°C, the cells were exposed to LIPUS for 24 or 48 h. The cells were then treated with trypsin-EDTA, rinsed with cold PBS and solubilized in cell lysate buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) containing a complete inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) and 1 mM PMSF (phenylmethyl sulfonyl fluoride; Sigma-Aldrich; Merck KGaA) buffer. Lysates were subsequently rocked gently at 4°C for 30 min. Following centrifugation at 14,000 × g for 5 min at 4°C, the supernatants were transferred to test tubes. Sample protein levels were quantified using Bradford method (Protein Assay; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and then diluted to a concentration of 1.0 mg/ml and used with the PathScan Stress and Apoptosis Signaling Antibody Array kit (Cell Signaling Technology, Inc.) according to the manufacturer's protocol. The detected dots were visualized using the supplied LumiGLO reagent and enumerated with the ImageQuant LAS-4000 instrument (GE Healthcare, Chicago, IL, USA). The relative dot densities were determined with ImageJ version 1.48 software (National Institutes of Health, Bethesda, MD, USA), normalized to the relative density of α-tubulin.

The significance of differences between groups was evaluated by a paired t-test. Data are presented as the mean ± standard deviation of 6–10 replications performed. In all analyses, P<0.05 was considered to represent a statistically significant difference. All analyses were performed using the Statview statistical software package (version 5.0; Abacus Concepts, Berkley, CA, USA).

Treatment with LIPUS for 18 or 24 h significantly inhibited the growth of LM8 cells, compared with no treatment (18 h, P=0.0133; 24 h, P=0.0022). There was no significant difference in cell growth when treated for 1 or 12 h compared with no treatment (1 h, P=0.3762; 12 h, P=0.1858; Fig. 1). Treatment with LIPUS for 1 or 12 h significantly inhibited the growth of MC3T3-E1 cells, compared with no treatment (1 h, P=0.0048; 12 h, P<0.0001). There was no significant difference in cell growth when treated for 18 or 24 h compared with no treatment (18 h, P=0.6574; 24 h, P=0.3606; Fig. 2).

LM8 cells treated with LIPUS for 48 h had a significantly lower mitochondrial membrane potential compared with cells without treatment (P=0.0019), but there were no significant differences in mitochondrial membrane potential between MC3T3-E1 cells with or without LIPUS treatment (P=0.2437; Fig. 3).

Flow cytometry analysis revealed that the LM8 cells treated with LIPUS had significantly higher numbers of apoptotic and necrotic cells compared with cells without treatment (apoptotic cells, P<0.0001; necrotic cells, P=0.0091; Figs. 4 and 5). TUNEL staining analysis indicated that the LM8 cells treated with LIPUS had significantly higher numbers of apoptotic cells compared with cells without LIPUS treatment (P<0.0001; Figs. 6 and 7).

As determined with a stress and apoptosis signaling array (Fig. 8), LIPUS treatment of LM8 cells for 24 h significantly enhanced the levels of phosphorylated (p)-mitogen-activated protein kinase 3/1 (ERK1/2), p-Akt and NF-κB inhibitor α (IκBα; p-ERK1/2 P<0.0001; p-Akt; P<0.0001; IκBα: P<0.0001) and reduced the levels of p-mitogen-activated protein kinase 7 (TAK1) and p-checkpoint kinase 1 (Chk1; TAK1, P<0.0001; Chk1, P=0.0006). However, there were no effects on the levels of p-Bcl-2-associated agonist of cell death (Bad) (P=0.6926; Fig. 9). LIPUS treatment of the LM8 cells for 48 h significantly increased p-Akt and IκBα levels (Akt, P<0.0001; IκBα, P=0.0001) and reduced p-TAK1 and p-Chk1 levels (p-TAK1, P<0.0001; p-Chk1, P=0.0008). However, there were no effects on the levels of p-ERK1/2 or p-Bad (p-ERK1/2, P=0.2437; Bad, P=0.9837; Fig. 10).