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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.


Experimental setup for ultrasound-field characterization. Annotated diagram describing the setup of equipment for measuring the ultrasound field generated from the DuoSon ultrasound machine
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Fig1: Experimental setup for ultrasound-field characterization. Annotated diagram describing the setup of equipment for measuring the ultrasound field generated from the DuoSon ultrasound machine

Mentions: A vacuum degassing chamber was constructed from plastic (Applied Vacuum Engineering, Bristol, UK) with a curved internal surface to reduce ultrasonic reflections. An acoustically absorbing base was constructed of a combination of rubber and Apltile SF5048 (Precision Acoustics, Dorchester, UK). A 1.0-mm needle hydrophone probe (Model 1452; Precision Acoustics, Dorchester, UK) connected to a HP Series Submersible Preamplifier (PA09022, Precision Acoustics, Dorchester, UK) was held in place vertically by the Apltile SF5048 material. The chamber was filled with 12 L of double distilled deionized water and air evacuated to achieve a vacuum. The water was degassed for 12 h with a vacuum of 0.95 bar. The DuoSon transducer was positioned vertically in line over the hydrophone, with their central axes aligned, and its movement was controlled by an XYZ manual travel translation stage (Thorlabs Inc., Newton, NJ, USA) as shown in Fig. 1. Both the transducer and needle hydrophone probe were submerged for 4 h. This mimicked the conditions present when the hydrophone was calibrated. Voltage measurements were recorded using a PC oscilloscope (PicoScope 5203; Pico Technology, St Neots, UK). The hydrophone and preamplifier were connected to a DC Coupler (DCPS038; Precision Acoustics, Dorchester, UK), and the signal was passed through a 50-Ω Terminator (TA051 Feed-Through Terminator; Pico Technology, St Neots, UK) prior to connecting to the PC oscilloscope (Fig. 1). The transducer face was positioned 50 mm below water level, and maximum voltage measurements and frequency were recorded at ten vertical points from the transducer at 1-mm intervals from the transducer face. The transducer was displaced horizontally and ten vertical measurements were taken at a further five positions from the transducer face at 5-mm intervals.Fig. 1


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)

Experimental setup for ultrasound-field characterization. Annotated diagram describing the setup of equipment for measuring the ultrasound field generated from the DuoSon ultrasound machine
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Experimental setup for ultrasound-field characterization. Annotated diagram describing the setup of equipment for measuring the ultrasound field generated from the DuoSon ultrasound machine
Mentions: A vacuum degassing chamber was constructed from plastic (Applied Vacuum Engineering, Bristol, UK) with a curved internal surface to reduce ultrasonic reflections. An acoustically absorbing base was constructed of a combination of rubber and Apltile SF5048 (Precision Acoustics, Dorchester, UK). A 1.0-mm needle hydrophone probe (Model 1452; Precision Acoustics, Dorchester, UK) connected to a HP Series Submersible Preamplifier (PA09022, Precision Acoustics, Dorchester, UK) was held in place vertically by the Apltile SF5048 material. The chamber was filled with 12 L of double distilled deionized water and air evacuated to achieve a vacuum. The water was degassed for 12 h with a vacuum of 0.95 bar. The DuoSon transducer was positioned vertically in line over the hydrophone, with their central axes aligned, and its movement was controlled by an XYZ manual travel translation stage (Thorlabs Inc., Newton, NJ, USA) as shown in Fig. 1. Both the transducer and needle hydrophone probe were submerged for 4 h. This mimicked the conditions present when the hydrophone was calibrated. Voltage measurements were recorded using a PC oscilloscope (PicoScope 5203; Pico Technology, St Neots, UK). The hydrophone and preamplifier were connected to a DC Coupler (DCPS038; Precision Acoustics, Dorchester, UK), and the signal was passed through a 50-Ω Terminator (TA051 Feed-Through Terminator; Pico Technology, St Neots, UK) prior to connecting to the PC oscilloscope (Fig. 1). The transducer face was positioned 50 mm below water level, and maximum voltage measurements and frequency were recorded at ten vertical points from the transducer at 1-mm intervals from the transducer face. The transducer was displaced horizontally and ten vertical measurements were taken at a further five positions from the transducer face at 5-mm intervals.Fig. 1

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.