Effect of low-intensity ultrasound on the colonization of spermatogonial stem cells

Background: Low-level-intensity ultrasound waves contribute to the proliferation, differentiation, and increasing the number of cells in vitro. However, the interaction of ultrasonic waves involving thermal effects, mechanical vibration, or cavitation as a cell proliferation factor has not been determined. Therefore, the aim of this study was to investigate the effect of cavitation (mechanical index) on spermatogonial stem cell proliferation and colonization by removing heat effects caused by ultrasound radiation. Isolated spermatogonial stem cells from neonatal mice were cultured in a DMEM culture medium with 10% FBS. Spermatogonial stem cells were stimulated by low-level ultrasound for five days and colonization and viability were evaluated on the 7 th day. Results: Regarding the low intensity of ultrasound, the Rayleigh integral model was used for acoustic pressure computation. According to the results of modeling, the intensities of 0.28, 0.45, 0.96, and 1.34 W/cm 2 were selected at 0.5 cm of distance with the mechanical indices of 0.40, 0.51, 0.75, and 0.89. The mechanical indices of 0.40 and 0.89 resulted in 93±4 and 32±4 colonies, respectively. An increase in colony diameter was observed for the mechanical index of 0.40 during all days of the culture. In the culture on the 7 th day, it had the largest average colony diameter of 134.05±1.22 μm in comparison with other groups (p<0.05). Cell viability did not significantly differ across groups (p=0.08). Conclusion: The results suggest that a low-intensity ultrasound of 40 kHz with a 0.40 mechanical index can be effective in increasing the proliferation and colonization of spermatogonial stem cells during culture. In other words, a mechanical index within the threshold of cavitation increases the proliferation and

cavitation typically produces highly localized acoustic streaming which may cause shear stresses on a nearby cell. Inertial cavitation is a transient type of cavitation where the acoustic cavitation oscillates heavily over a single or multiple cycles, eventually leading to a violent collapse/implosion. The collapse is associated with localized high pressures and temperatures and high-velocity liquid jets [15].
Mechanical index (MI) was introduced by Apfel and Holland (1991) as a quantity expressing the biological effects of cavitation [14,16].
There is growing evidence that integrins are promising candidates for sensing extracellular matrix-derived mechanical stimuli and converting them into biochemical signals. Mechanical stimuli can affect both the actin cytoskeleton and cell cycle progression. Direct mechanical stimulation such as the stretching of cells can activate the extracellular-signal-regulated kinase (ERK) pathway in different cell systems, leading to cell proliferation. Acoustic cavitation interaction of low-intensity ultrasound can be regarded as a form of nonphysiological mechanical energy indirectly applying mechanical stress to the cultured cells [17]. Nevertheless, in all studies, specific sonication conditions are determined by considering the availability of the radiation frequency without physical or mechanical reasons or the explanation of the mechanism type [18], and the interaction of low-intensity (lowfrequency) ultrasound has not been studied. In our previous study [19], we calculated the acoustic pressure in the medium by ultrasonic propagation based on a tissue-inducing Rayleigh integral model in which wave equations were linearly calculated in a homogeneous environment. Then, it was used to extract the physical parameters of acoustic pressure and mechanical index. In the present study, the effect of these parameters on SSC viability and colonization was evaluated using a quantitative approach to investigate the effect of the MI of LF and LIUS on spermatogonial stem cell colonization by removing heat effects caused by ultrasound radiation.

Methods
The pressure equation was solved at any point in cylindrical coordinates and extracted minimum acoustic pressure (P m in ). To extract the MI plan, acoustic variables included the frequency of 40 kHz (Ultrasound Laboratory, Medical Physics Department, Tarbiat Modares University, Iran), acoustic intensity of 0 to 1.34 W/cm 2 , and the continuous exposure mode. Modeling was performed in 32°C water (in vitro) with the density of 1000 kg/m 3 , acoustic propagation speed of 1519 m/s, and attenuation coefficient of 0.002 N/MHz [20]. For validation, acoustic pressure was calculated at different distances and compared with the experimental results [19]. Based on the experimental study, the target radius was equal to the hydrophone piston radius (PA124, Precision Acoustics Ltd., Dorchester, UK, 1.3cm sensor radius). The MI according to the intended frequency (f) and the minimum acoustic pressure P m in was defined as: This was applied within the frequency range of 0.5-15 MHz, but Ahmadi [21] showed that it is an appropriate index for frequencies below about 500 kHz.
The present study was conducted under the protocol approved by the Animal Experimental Committee of the Medical Sciences Faculty of Tarbiat Modares University.
Isolation and culture of SSCs from neonatal mice (NMRI, National Medical Research Institute) was performed according to the specified protocol [19]. The animals were euthanized quickly and humanely to avoid any pain by overdose of chemical anesthetics (2-3 times the anesthetic dose) and confirmed by heart tissue harvest [22]. The testicles were extracted for 10 min and collected after washing in Dulbecco's Minimum Essential Medium (DMEM), placed in a new medium. After the removal of the capsule, the testicles were cut into smaller pieces and placed in a culture medium containing 0.5 mg/ml of collagenase IV (Sigma) and incubated at 37° C for 20 min, then centrifuged for 5 min with 1500 rpm. Afterwards, the medium was plaque exchanged with PBS and centrifuged twice for 3 min at 1000 rpm. Next, 0.5 mg/ml of trypsin (Sigma) was added to this solution for 2 min and centrifuged After various stimulation intensities, SSCs were cultured for seven days. The colonies resulting from spermatogonial cells were assayed on the 7 th day with an invert-phase microscope (Zeiss, Germany) equipped with an ocular grid. Cell viability in different groups was compared with Trypan blue. Before measuring the number and diameter of the colonies, the microscope was calibrated.
The data of the viability rate and colonization of stimulated cells are presented as mean ± standard deviation (SD). One-way ANOVA was run to analyze differences between groups at the significance level of 0.05. All the data in this study were analyzed using SPSS statistical software.

Results
Minimum acoustic pressure changes were obtained at different points along the axis in the range of induced cavitation threshold 0.7 (potential hazard) [24], less or more than the threshold at different intensities with a radius of 1.8 cm (Fig. 1).
The axial contours of a 40 kHz ultrasound transducer indicated that the near-field depth is 0.4 cm. As the increasing intensity of ultrasound waves from 0.28 to 1.34 W/cm 2 on the target surface with a radius of 1.8 cm, also showed an increase in acoustic pressure from 54.5 kPa at 1.5 cm to 192 kPa at 0.3 cm from the transducer surface.
The mechanical index changes in the near-field depth showed less volatility. The    Fig. 2a). Figures 2b and 2c respectively illustrate the acoustic pressure changes and the mechanical index changes for the intensity of 0.28 W/cm 2 at the distance of 0.5 cm from the transducer surface along the radial axis of the transducer.
In the center of the transducer, the mechanical index equaled 0.58. It gradually decreases when reaching 0.19 on the sides (Fig. 2a). The color change from blue to red showed an increase in acoustic pressure and mechanical index. In the validation results of a previous study [19], there was a significant correlation between the measured pressure and calculated pressure at 40 kHz (p<0.05).
Thermal control results are given in Fig. 3

Discussion
LIUS is non-physiological mechanical energy which inserts direct mechanical stress into cultured cells and is a type of noninvasive mechanical stimulation. In physiological conditions of 37 °C and using low intensity, non-thermal effects can be dominant, and the main interaction, i.e. acoustic cavitation, is important. According to the studies, stable cavitation without collapse and the ability to apply stress without chemical activities produce beneficial effects in low intensities. The mechanical index parameter is the quantity that will define the phenomenon of acoustic cavitation [14,15,24]. Cavitation threshold for generating the bioeffects of low-frequency ultrasound have been reported, but these vary with the type of cell or tissue being studied. Hill [24] reported that, in 0.25 to 3MHz frequencies, the intensity threshold for inertial cavitation in a liquid suspension of cells was 1 W/cm 2 . At 20 kHz, the threshold to produce observable lesions in human skin was determined to be 2.5 W/cm 2 (MI=1.94) in vitro, which can be considered a hazard limit for contact to human skin [26].
On the other hand, the biological activity of low-frequency ultrasound waves is the result of the stimulation of cells by combining stable cavities and effective acoustic streams in vitro [28]. Ebrahiminia [27]  Furthermore, LIUS is an acoustic pressure wave that can produce localized mechanical stimulation for cells to conduct the activity of cellular membrane tensile receptors, ion channels, and integrin (extracellular messages). There is growing evidence that integrins are promising candidates for sensing extracellular matrixderived mechanical stimuli and converting them into biochemical signals. Thus, there is growing evidence that integrins are promising candidates for sensing extracellular matrix-derived mechanical stimuli and converting them into biochemistry signals [17]. Consequently, low-intensity ultrasound waves can stimulate transmembrane proteins such as integrin to divide and can enter these cells into a mitotic process in order for self-renewal or differentiation pathway. This study is similar to the study by Xu [18] reporting

-Ethics approval and consent to participate
The present study was conducted under the protocol approved by the Animal

Experimental Committee of the Medical Sciences Faculty of Tarbiat Modares
University.

-Consent for publication
Not applicable.

-Availability of data and materials
The analyzed data sets generates during the study are available from the corresponding author on reasonable request.

-Competing interests
The authors declare that they have no competing interests.

-Authors' Contributions
All  The axial contour map of the 40 kHz ultrasonic transducer acoustic pressure (Pa) in the r-z s The enhancement curve of culture medium temperature (mean ± SD) after sonication with di The number of colonies (mean ± SD) from the first 48 hours to seven days in experimental g Figure 6 The diameter of colonies (mean ± SD) from the first 48 hours to the 7th day in experimental

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