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Ultrasound field characterization and bioeffects in multiwell culture plates.

Patel US, Ghorayeb SR, Yamashita Y, Atanda F, Walmsley AD, Scheven BA - J Ther Ultrasound (2015)

Bottom Line: Calculations were performed using Fourier transform and average intensity plotted against distance from the transducer.The ultrasonic output demonstrated considerable lateral spread of the ultrasound field from the exposed well toward the adjacent culture wells in the multiwell culture plate; this correlated well with the dose-dependent increase in the number of cultured cells where significant biological effects were also seen in adjacent untreated wells.Significant thermal variations were not detected in adjacent untreated wells.

View Article: PubMed Central - PubMed

Affiliation: School of Dentistry, College of Medical and Dental Sciences, University of Birmingham, St Chad's Queensway, Birmingham, B4 6NN UK.

ABSTRACT

Background: Ultrasound with frequencies in the kilohertz range has been demonstrated to promote biological effects and has been suggested as a non-invasive tool for tissue healing and repair. However, many challenges exist to characterize and develop kilohertz ultrasound for therapy. In particular there is a limited evidence-based guidance and standard procedure in the literature concerning the methodology of exposing biological cells to ultrasound in vitro.

Methods: This study characterized a 45-kHz low-frequency ultrasound at three different preset intensity levels (10, 25, and 75 mW/cm(2)) and compared this with the thermal and biological effects seen in a 6-well culture setup using murine odontoblast-like cells (MDPC-23). Ultrasound was produced from a commercially available ultrasound-therapy system, and measurements were recorded using a needle hydrophone in a water tank. The transducer was displaced horizontally and vertically from the hydrophone to plot the lateral spread of ultrasound energy. Calculations were performed using Fourier transform and average intensity plotted against distance from the transducer. During ultrasound treatment, cell cultures were directly exposed to ultrasound by submerging the ultrasound transducer into the culture media. Four groups of cell culture samples were treated with ultrasound. Three with ultrasound at an intensity level of 10, 25, and 75 mW/cm(2), respectively, and the final group underwent a sham treatment with no ultrasound. Cell proliferation and viability were analyzed from each group 8 days after three ultrasound treatments, each separated by 48 h.

Results: The ultrasonic output demonstrated considerable lateral spread of the ultrasound field from the exposed well toward the adjacent culture wells in the multiwell culture plate; this correlated well with the dose-dependent increase in the number of cultured cells where significant biological effects were also seen in adjacent untreated wells. Significant thermal variations were not detected in adjacent untreated wells.

Conclusions: This study highlights the pitfalls of using multiwell plates when investigating the biological effect of kilohertz low-frequency ultrasound on adherent cell cultures.

No MeSH data available.


Effects of 45-kHz ultrasound on MDPC-23 cell proliferation. Cell numbers were determined after 8 days of culture in six-well plates with alternating days of ultrasound treatment. Three groups had ultrasound treatment each with the intensities, 10, 25, and 75 mW/cm2. A sham-treatment control group had no ultrasound applied to the cells. Total viable cell number is shown for each culture well (W0, W1, and W2), and data is expressed as a percentage of the sham-control group (mean ± SD; n = 3). One way ANOVA statistical analysis was carried out, and the statistical significance is indicated (*** p < 0.001; ** p < 0.01; * p < 0.05)
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Fig6: Effects of 45-kHz ultrasound on MDPC-23 cell proliferation. Cell numbers were determined after 8 days of culture in six-well plates with alternating days of ultrasound treatment. Three groups had ultrasound treatment each with the intensities, 10, 25, and 75 mW/cm2. A sham-treatment control group had no ultrasound applied to the cells. Total viable cell number is shown for each culture well (W0, W1, and W2), and data is expressed as a percentage of the sham-control group (mean ± SD; n = 3). One way ANOVA statistical analysis was carried out, and the statistical significance is indicated (*** p < 0.001; ** p < 0.01; * p < 0.05)

Mentions: Application of a 45-kHz ultrasound at the two preset lower intensity levels of 10 and 25 mW/cm2 resulted in cell counts from the directly exposed W0 culture well to be significantly higher than the sham-treated group (p < 0.001 and p < 0.01 respectively) indicating ultrasound-stimulated cell proliferation. The highest preset intensity level, 75 mW/cm2, did not result in a significant difference in cell number (Fig. 6), compared to sham; however, cell viability was reduced to 90 % as shown in Fig. 7. The lower intensity levels of 10 and 25 mW/cm2 reported higher cell viabilities of 98 % and above (Fig. 7). This indicates that higher ultrasound intensities are not as well tolerated by MDPC-23 cells compared to the lower intensities used in this study. This result is statistically significant (p < 0.001).Fig. 6


Ultrasound field characterization and bioeffects in multiwell culture plates.

Patel US, Ghorayeb SR, Yamashita Y, Atanda F, Walmsley AD, Scheven BA - J Ther Ultrasound (2015)

Effects of 45-kHz ultrasound on MDPC-23 cell proliferation. Cell numbers were determined after 8 days of culture in six-well plates with alternating days of ultrasound treatment. Three groups had ultrasound treatment each with the intensities, 10, 25, and 75 mW/cm2. A sham-treatment control group had no ultrasound applied to the cells. Total viable cell number is shown for each culture well (W0, W1, and W2), and data is expressed as a percentage of the sham-control group (mean ± SD; n = 3). One way ANOVA statistical analysis was carried out, and the statistical significance is indicated (*** p < 0.001; ** p < 0.01; * p < 0.05)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4490766&req=5

Fig6: Effects of 45-kHz ultrasound on MDPC-23 cell proliferation. Cell numbers were determined after 8 days of culture in six-well plates with alternating days of ultrasound treatment. Three groups had ultrasound treatment each with the intensities, 10, 25, and 75 mW/cm2. A sham-treatment control group had no ultrasound applied to the cells. Total viable cell number is shown for each culture well (W0, W1, and W2), and data is expressed as a percentage of the sham-control group (mean ± SD; n = 3). One way ANOVA statistical analysis was carried out, and the statistical significance is indicated (*** p < 0.001; ** p < 0.01; * p < 0.05)
Mentions: Application of a 45-kHz ultrasound at the two preset lower intensity levels of 10 and 25 mW/cm2 resulted in cell counts from the directly exposed W0 culture well to be significantly higher than the sham-treated group (p < 0.001 and p < 0.01 respectively) indicating ultrasound-stimulated cell proliferation. The highest preset intensity level, 75 mW/cm2, did not result in a significant difference in cell number (Fig. 6), compared to sham; however, cell viability was reduced to 90 % as shown in Fig. 7. The lower intensity levels of 10 and 25 mW/cm2 reported higher cell viabilities of 98 % and above (Fig. 7). This indicates that higher ultrasound intensities are not as well tolerated by MDPC-23 cells compared to the lower intensities used in this study. This result is statistically significant (p < 0.001).Fig. 6

Bottom Line: Calculations were performed using Fourier transform and average intensity plotted against distance from the transducer.The ultrasonic output demonstrated considerable lateral spread of the ultrasound field from the exposed well toward the adjacent culture wells in the multiwell culture plate; this correlated well with the dose-dependent increase in the number of cultured cells where significant biological effects were also seen in adjacent untreated wells.Significant thermal variations were not detected in adjacent untreated wells.

View Article: PubMed Central - PubMed

Affiliation: School of Dentistry, College of Medical and Dental Sciences, University of Birmingham, St Chad's Queensway, Birmingham, B4 6NN UK.

ABSTRACT

Background: Ultrasound with frequencies in the kilohertz range has been demonstrated to promote biological effects and has been suggested as a non-invasive tool for tissue healing and repair. However, many challenges exist to characterize and develop kilohertz ultrasound for therapy. In particular there is a limited evidence-based guidance and standard procedure in the literature concerning the methodology of exposing biological cells to ultrasound in vitro.

Methods: This study characterized a 45-kHz low-frequency ultrasound at three different preset intensity levels (10, 25, and 75 mW/cm(2)) and compared this with the thermal and biological effects seen in a 6-well culture setup using murine odontoblast-like cells (MDPC-23). Ultrasound was produced from a commercially available ultrasound-therapy system, and measurements were recorded using a needle hydrophone in a water tank. The transducer was displaced horizontally and vertically from the hydrophone to plot the lateral spread of ultrasound energy. Calculations were performed using Fourier transform and average intensity plotted against distance from the transducer. During ultrasound treatment, cell cultures were directly exposed to ultrasound by submerging the ultrasound transducer into the culture media. Four groups of cell culture samples were treated with ultrasound. Three with ultrasound at an intensity level of 10, 25, and 75 mW/cm(2), respectively, and the final group underwent a sham treatment with no ultrasound. Cell proliferation and viability were analyzed from each group 8 days after three ultrasound treatments, each separated by 48 h.

Results: The ultrasonic output demonstrated considerable lateral spread of the ultrasound field from the exposed well toward the adjacent culture wells in the multiwell culture plate; this correlated well with the dose-dependent increase in the number of cultured cells where significant biological effects were also seen in adjacent untreated wells. Significant thermal variations were not detected in adjacent untreated wells.

Conclusions: This study highlights the pitfalls of using multiwell plates when investigating the biological effect of kilohertz low-frequency ultrasound on adherent cell cultures.

No MeSH data available.