Effect of Audible Sound Waves on Human Umbilical Vein Endothelial Cell Response: Application in Wound and
Fracture Healing
Nafiseh Mirabdolhosseini1, Mehran Kasra1, Elham Rahimtoroghi1
1Amirkabir University of Technology, Tehran, Iran
Disclosures: N. Mirabdolhosseini (None). M. Kasra (None). E. Rahimtoroghi (None)
INTRODUCTION:
Infrasound, sound and ultrasound refer to the three frequency bands in an overall spectrum of acoustic waves. Among these categories, ultrasound has
been studied more than others, and its diagnostic and therapeutic applications have been well identified. Ultrasonic biological effects are used to give
therapeutic benefits in a number of different clinical applications, such as treatment of soft tissue injuries, acceleration of bone repair and cancer treatment
[1]. However, the therapeutic potential of audible sound waves, another category of acoustic waves, still needs to be investigated. Similar to all acoustic
waves, sounds are mechanical waves which require a physical medium to support their propagation, but unlike the ultrasound there is no need for
transmission gel making it easier to use. Also, compared to the ultrasound, sound waves have much lower attenuation; therefore, sound waves can penetrate
deeper causing lower heat in tissue [2]. The objective of this study was to study the effect of audible sound waves at cellular level for angiogenesis
therapeutic purposes. We hypothesized that human umbilical vein endothelial cells, as one of the important cells in tissue repair, will be affected by
mechanical stimulation provided by these waves.
METHODS:
Custom-designed sound generator similar to Sena et al [3] was used to stimulate human umbilical vein endothelial cells (HUVECs). Cell responses
representing angiogenesis and wound healing activities, including viability (MTT assay), migration, and released NO were measured.
MTT assay: The cell viability was examined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The cells were treated
in four groups with three specimens in each group according to the loading stimulation protocol of three groups of 500, 5000, 9000 Hz frequency with
similar intensity of 40.62 mW/cm2 and a control group with no stimulation. Cells were seeded into 35mm dishes, stimulated for 20 minutes, and placed in
the incubator before MTT assay. Finally, the results were read by a plate reader at 490 and 640 nm.
Measurement of nitric oxide (Griess assay): The colorimetric Griess assay has been used to measure the amount of nitrate as representative of released
NO. HUVECs were seeded on 35mm dishes and incubated. All the dishes, except the control dishes, were stimulated for 20 minutes and then were placed in
an incubator for 3 days; all the samples became deproteinized. Finally, the absorption was measured at 540nm using a spectrophotometric plate reader. The
released NO of different groups were compared with that of the control group. The same loading stimulation protocol as the MTT assay was used.
Migration assay (Wound healing assay): The Wound healing assay was used to assess the effect of sound stimulation on cell migration [4]. Cells were
seeded into 35mm dishes and were incubated until a mono cell layer with a density of about 80% was achieved. Then, by using (yellow) pipette tip, straight
scratches simulating a wound were made. At this point, photos from 2 specific position of each dish were taken using CMOS camera and Nikon microscope.
All the dishes except the controls were stimulated for 10 minutes; then they were incubated for 5 hours. Every 90 minutes and 24 hours after making
scratches, the dishes were taken out and the photos from the scratches were taken. The WimScratch and the ImageJ softwares were used for analyzing these
photos as shown in figure 1A. Three different groups were examined, one control group without sound stimulation, and two groups stimulated with 5000Hz
frequency sound waves of 40.62 and 81.24 mW/cm2 intensities. All the data were presented as mean ± standard deviation and analysis of variance
(ANOVA) were used to determine significant differences (p < 0.05).
RESULTS:
The MTT assay results (Fig.2A) showed the 500 Hz frequency, 40.62 mW/cm2 intensity group, having the highest cell viability and proliferation (p <
0.05), compared to the control group and groups stimulated with 5000 and 9000 Hz frequencies. In the study of released NO, as the frequency of sound
waves increased, the amount of released NO also went up; the highest and the least ratio were for 9000 Hz frequency group and the control group
respectively (Fig.2B). Our results indicated that the audible sound waves could affect migration as showed in figure 1B. In this study the 5000 Hz, 81.24
mW/cm2, group had the most significant wound healing rate. In this case, at the first 90 minutes after scratching (time point zero), the control group cells
were entered directly into the linear growth phase and had a high wound healing rate; meanwhile, in the stimulated groups a lag phase occurred until the
cells start migrating; as time passed, cell migration in the stimulated groups accelerated while the migration rate in the control group slowed down.
DISCUSSION:
MTT results of cell viability indicated that the level of stress generated by the sound waves of this study were safe and could produce significantly higher
viability for the 500 Hz frequency group compared to that of the control group (Fig. 2A). Our results of relased NO, as the major regulator of cell migration
and angiogenesis in the endothelial cells, indicated that sound stimulation could have a positive effect on the release of NO molecules. The results of the
wound healing assay also showed a notable influence of the audible sound waves on cell migration (Fig.1B). Other studies using ultrasound systems have
also shown increase in NO levels and up-regulated gene expressions known to induce endothelial cell proliferation and migration[1]; which are crucial for
new blood vessel formation in the healing of fractures. In Conclusion; due to the aforementioned advantages of sound versus ultrasound, if sound could be as
effective as ultrasound or better, for wound and fracture healing purposes, it might be more advantages to use sound frequency instead of ultrasound in
certain applications.
SIGNIFICANCE:
This study introduces the audible sound waves, as an easy and safe treatment tool, having potentials to be used for wound and fracture healing as well as
tissue regeneration purposes.
REFERENCES:
[1] Padilla, F., et al., Ultrasonics, 2014. 54 (5): p. 1125-1145.
[3] Sena, K., et al., Ultrasound in medicine & biology, 2005. 31(5): p.
[2] Laugier, et al. Bone quantitative ultrasound. Vol.576. Dordrecht:
70-703
Springer, 2011.
[4] Rodriguez, et al, Cell Migration: Developmental Methods and
Protocols (2005): 23-29
90 min
40
180 min
20
270 min
0
control
5kHz 40.62 5kHz 81.24
mW/cm2 mW/cm2
Figure 1. (A) Representative pictures of wound healing assay (5KHz, 81.24
mW/cm2 ). (B) Wound healing rate for the control (no stimulation) and the two
sound stimulation groups.
B
150
100
40.62 mW/cm2
Released NO (%)
A
60
Cell Viability (%)
B
Wound Healing (%)
A
*
50
0
500 Hz
5000Hz
9000Hz
control
200
40.62 mW/cm2
150
**
100
50
0
control
500 Hz
5000Hz
9000Hz
Figure 2. (A) Cell viability for control and sound stimulated groups (B) Released NO
for sound stimulated groups and control (*P<0.05, **P<0.01).
ORS 2017 Annual Meeting Poster No.2312