## 3 Results  
### LIPUS Effect on MIO-M1 Cell Viability  
In vitro experiments were performed to evaluate the acoustic effect on the viability of the cell line MIO-M1. The effect of increasing acoustic intensities and DFs at 1 min treatment length on MIO-M1 viability, 24 h post-treatment, is reported in Figure 2a,b. In the first case (Figure 2a), treatments were performed with the well-on-transducer setup (as detailed in Material and Methods section). We observe that the increment in the acoustic intensity at 10% DF has negligible effects on cell viability. In contrast, at 20% and 50% DF, there are significant effects on the viability of the cells treated at increasing LIPUS intensities. The treatment at 1.6 W/cm\({}^{2}\) and 20% DF produced statistically significant effects on cell viability with only 23.8% of viable cells remaining after stimulation (\({}^{+}\) + \(p\) < 0.01, post-hoc [PERSON]'s test). The increase in the DF to 50% reduces the cell's resistance to increasing acoustic intensities. Substantial cell death is observed at 0.8, 1.2 and 1.6 W/cm\({}^{2}\) at 50% DF, where the percent of cell death was 75.0%, 94.5% y 93.7%, respectively (*** \(p\) < 0.001, post-hoc [PERSON]'s test).  
Figure 2: (**a,b**) Viability of MIO-M1 cells at different acoustic intensities and duty factors (10%, 20% and 50%) for 1 min of treatment when using (**a**) well-on-transducer setup (\({}^{++}\)\(p\) < 0.01, CTL vs. 1.6 W/cm\({}^{2}\), 20% DF; *** \(p\) < 0.001, CTL vs. 0.8–1.6 W/cm\({}^{2}\), 50% DF; post-hoc [PERSON]’s test), and (**b**) well-on-water-surface setup (\({}^{+}\)\(p\) < 0.05, post-hoc [PERSON]’s test; **\(p\) < 0.01, post-hoc [PERSON]’s test). (**c,d**) Viability of MIO-M1 for different treatment durations with 0.4 W/cm\({}^{2}\) at 20% DF evaluated with the water-based setup (**c**) 24 h after treatment, (**d**) 24 h and 48 h post-LIPUS treatment and sub-culturing (\({}^{*}\)\(p\) < 0.05, post-hoc [PERSON]’s test). All experiments were performed in triplicate, except for the viability curves at 10% DF through gel (**a**) and 24 h after LIPUS treatment and cell passaging (**d**), which were performed by duplicate.  
When the setup is changed to a water immersion bath set at 37 \({}^{\circ}\)C (Figure 2b), the acoustic effect on the cell mortality is markedly reduced. Cell death was significant only at 1.6 W/cm\({}^{2}\) at 20% DF (+ \(p\) < 0.05, post-hoc [PERSON]'s test) or 50% DF (**\(p\) < 0.01, post-hoc [PERSON]'s test), with reductions on cell viability to 64.4% and 14.7%, respectively.  
Due to the reduced acoustic effects in the cell viability using a water-based setup, we further evaluate the effect of increasing time exposure by using a fixed acoustic intensity at 0.4 W/cm\({}^{2}\) and 20% DF. Figure 2c illustrates that the MIO-M1 cell viability is not affected with one to ten minutes of ultrasonic treatment under these acoustic conditions. Average water temperature, set at 37 \({}^{\circ}\)C at the beginning of the experiment and measured after 10-min treatments, at three different days, was 36.5 \({}^{\circ}\)C \(\pm\) 0.2 \({}^{\circ}\)C, which should not produce any effect in cell viability  
In order to determine the proliferative capacity of cells stimulated with LIPUS, we exposed MIO-M1 cells to the same acoustic intensity of 0.4 W/cm\({}^{2}\) at 20% DF with also varying treatment lengths. Further, cells were trypsinized, diluted (1:10), and seeded in 24-well plates (Figure 2d). We observe that 24 h after cell exposure and passage, cells seem to maintain their ability to adhere and proliferate as observed in the non-treated condition (CTL). Notwithstanding, cell viability decreases to around 80% at all treatment times 48 h after LIPUS stimulation and passage. This reduction is significant at 3 min and 5 min treatments (* \(p\) < 0.05, post-hoc Dunnett's test), where viability is decreased to 78% and 77%, respectively.