JADA Continuing Education
Removal of oral biofilms by bubbles
The effect of bubble impingement angle and sonic waves
MICHAEL R. PARINI, M.S. and
WILLIAM G. PITT, Ph.D.
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ABSTRACT
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Background. Previous research showed that the collision of bubbles with biofilm could remove biofilm from a surface. However, the effectiveness of biofilm removal by bubbles and sonic waves had not been determined.
Methods. The authors mounted Streptococcus mutans biofilms in a chamber containing artificial saliva and exposed them to bubbles and sonic waves. They generated sonic waves via an oscillator at the frequencies and acoustic intensities of sonic toothbrushes. They also mounted biofilms at different angles to measure the effect of a bubble’s impingement angle.
Results. The presence of sound had no significant effect on the amount of biofilm removed (F = 0.51). There was no statistically significant difference in the amount of biofilm removed with respect to the angle at which bubbles impinged on the biofilm (F = 0.65). The authors performed analysis of variance tests to determine whether the difference in the amount of biofilm removal was significant (F < 0.05) in the presence or absence of sound and at the different angles tested.
Conclusions. The collision of a bubble at any angle between 5 and 45 degrees was equally effective in removing biofilm. The addition of sound to a bubble stream at the tested intensities had a negligible effect.
Clinical Implications. Bubbles produced by dental instruments are effective in removing biofilm from the surface of the teeth, regardless of the angle at which the bubble impinges on the biofilm.
Key Words: Streptococcus mutans; biofilm; bubbles; sonic vibration; toothbrush
Does the addition of sound or ultrasound to a toothbrush enhance its performance? This question has generated a decade of research and, yet, remains largely unanswered.1–5 An ultrasonic toothbrush uses a transmitter that emits low-amplitude pressure waves above the frequency of human hearing, while a sonic toothbrush generates audible sound as its head and bristles vibrate. Most sonic toothbrushes vibrate at frequencies near 260 hertz.1 Conventional electric toothbrushes operate at lower frequencies than those of sonic toothbrushes—between 25 and 62 Hz.6
The collision of a bubble at any angle between 5 and 45 degrees was equally effective in removing biofilm.
Published data have shown that bacteria can be damaged by the application of acoustic phenomena, but data on the removal of an oral biofilm—such as plaque—by exposure to sound waves have not been reported.7
In a previous study, we found that a stream of bubbles striking an oral biofilm efficiently removes bacteria from the surface.8 We conducted these experiments, however, in the absence of sound waves, and such acoustic pressure waves cause bubbles to oscillate in size.9 The question remains as to whether an oscillating bubble such as that found in the oral cavity during sonic toothbrushing is more efficient in removing oral bacteria than is a nonvibrating bubble. The experiments we describe in this article attempt to answer this question.
Another characteristic of sonic toothbrushes is that they usually generate high fluid velocities at the bristle tip owing to the rapid movement of the brush head.6 This motion propels bubbles at the surfaces of the teeth, but the bubbles strike the surface at different angles. It is plausible that a bubble striking the biofilm on the tooth at a glancing angle is less efficient at removing bacteria than is a bubble striking the tooth more directly. We conducted this study to test the hypothesis that a more direct angle of impingement removes more biofilm.
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MATERIALS AND METHODS
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We have reported previously on the growth of the oral biofilm, construction of the experimental apparatus and measurement of biofilm removal.8 In this previous study, we grew biofilms of Streptococcus mutans (ATCC 700610) on glass cover-slips in a drip flow reactor. We clipped these coverslips to a stage that could be rotated to any desired angle. We placed the stage in a plexiglass chamber that was filled with an artificial saliva solution so that the biofilm was completely submersed. The artificial saliva consisted of water and the polysaccharide scleroglucan (Clearogel CS 11D, MMP, South Plainfield, N.J.). In the bottom of the chamber directly beneath the stage was a septum through which we passed a flat-tipped needle. Gas and artificial saliva were mixed in a chamber below the needle, and the mixture exited in a stream of gas bubbles and saliva. We controlled the stream’s velocity, gas fraction and bubble diameter as described previously.8
We exposed the biofilm on the stage to the artificial saliva and bubble stream for five seconds, after which we removed the coverslip from the chamber and placed it in a polystyrene Petri dish, biofilm side down. We scanned the biofilms through the Petri dish on a flatbed scanner and analyzed the images to determine the area and the amount of biofilm removed using image analysis software.
Experiments to determine the effect of the impingement angle on biofilm removal.
To determine the effect of the fluid impingement angle, we exposed biofilms to one of two bubble stream conditions at one of three angles (Table 1
). The conditions of one of the bubble streams were a velocity of 1.5 meters per second, a gas fraction of 0.04 and a median bubble diameter of 205 micrometers (experiments A–C). The other bubble stream consisted of bubbles that were the same size as those of the other bubble stream but that had a velocity of 4.5 meters per second and a gas fraction of 0.41 (experiments D–F). The impingement angles we used in this study were 45-, 30-and 5-degree angles. We replicated all combinations of bubble streams and angles four times.
Acoustic wave production.
We mounted a Ling oscillator (V203, Ling Dynamic Systems, Royston, Herts, England) into a plexiglass stand that was clamped on top of the plexiglass chamber (Figure 1). The oscillator was powered by a 25-watt integrated stereo amplifier, which received a signal from a waveform generator. A complete description of the Ling oscillator has been published.10
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Figure 1. Ling oscillator on chamber. 1. Ling oscillator (V203, Ling Dynamic Systems, Royston, Herts, England). 2. Hydrophone. 3. Septum. 4. Needle. 5. Biofilm on glass coverslip. 6. Plexiglass chamber.
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In our study, we reproduced the acoustic energy to be received by a biofilm 1 millimeter from the tip of the bristles of a standard sonic toothbrush. We placed the tip of a hydrophone (8103, Brüel & Kjær, Nærum, Denmark) in the plexiglass chamber in which the biofilm was mounted during experiments. We positioned a fully charged Sonicare Elite 7800 toothbrush (Philips Oral Healthcare, Snoqualmie, Wash.) 1 mm from the tip of the hydrophone. We measured and recorded the acoustic intensity and frequency of toothbrush, and then we removed the toothbrush from the tank. We then attached the Ling oscillator to the tank and adjusted the input voltage going to the Ling oscillator until the acoustic signal received by the hydrophone from the oscillator was the same as the signal from the toothbrush. We defined this acoustic intensity value as 1 toothbrush equivalent (TBE).
Experiments to determine the effect of sound on biofilm removal.
To maximize the consistency of the bubble streams during the experiments, we started the bubble stream before we inserted the biofilm and protective shield in the plexiglass chamber. Once the bubble stream had reached a steady state at the desired parameters, we opened the shield for five seconds to expose the biofilm. We removed the biofilm from the chamber while the bubble stream continued to flow; next, we activated the acoustic oscillator. We then placed a second biofilm and shield in the chamber and exposed them to the bubble stream and sound for five seconds. We then analyzed the second biofilm.
The frequencies we used in this experiment were based on those emitted by common sonic toothbrushes: the fundamental frequency was 260 Hz, the second harmonic frequency was 520 Hz, and the primary subharmonic frequency was 150 Hz. At each frequency, we used various intensities to determine whether the frequency or the intensity of the sound was more dominant in removing biofilm. The acoustic intensities used with each of these frequencies were 0.2, 1 and 2 TBE, respectively; owing to the mechanical limitations of the Ling oscillator, the maximum acoustic intensity achievable at a frequency of 520 Hz was 0.2 TBE.
We used two different bubble streams in these experiments. The parameters of one stream were a velocity of 10.5 meters per second, a gas fraction of 0.27 and a median bubble diameter of 205 µm (experiments M–V) (Table 2). The parameters of the other stream were a velocity of 12 meters per second, a gas fraction of 0.65 and a median bubble diameter of 300 µm. We set the impingement angle to 45 degrees for all acoustic experiments. We also performed experiments without a bubble stream to measure the amount of biofilm removal caused by sound alone (experiments G–L).
Statistical analysis.
We used analysis of variance (ANOVA) to determine whether the removal of biofilm under different conditions of impingement angle and acoustic frequencies and intensities was significant.
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RESULTS
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Effect of angle.
The data showed that the angle at which the bubbles impinged on the biofilm had no significant effect on the amount of biofilm removed (Table 1
, experiments A–F). As shown in Figure 2, however, the shape of the biofilm removed differed as the angle changed; the removal area was narrow and elongated when bubbles struck the biofilm at a low-angle impingement and circular when bubbles struck the bioflm at a high-angle impingement. ANOVA showed that the amount of biofilm removal was not different at varying impingement angles (F = 0.65).
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Figure 2. Removal of biofilm by bubbles at different angles of impingement. All bubble streams had a velocity of 1.5 meters per second, a gas fraction of 0.04 and a median bubble diameter of 205 micrometers. The angles of impingement were 5 degrees (left), 30 degrees (center) and 45 degrees (right). These are selected photographs from experiments A, B and C, respectively. The scale bar is 2.54 centimeters.
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Effect of sound.
Figure 3 shows a paired comparison of S. mutans biofilm removal by bubble stream with and without the addition of 1 TBE of acoustic pressure. Figure 4 shows a similar comparison but with a bubble stream with a larger gas fraction and velocity. As these figures and the data summarized in Table 2 indicate, the presence of sound had no significant effect on the amount of biofilm removed (F = 0.51).
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Figure 3. Two Streptococcus mutans biofilms after exposure to a bubble stream at a 45-degree angle. The border of disturbed biofilm evident on the top and the sides was caused by the apparatus to which the coverslip was mounted. All bubble streams had a velocity of 10.5 meters per second, gas fraction of 0.27 and bubble diameter of 205 micrometers. The biofilm in the left-hand image (from experiment O) also was exposed to 260 hertz of sound at twice the output of a Sonicare Elite 7800 toothbrush (Philips Oral Health Care, Snoqualmie, Wash.) on "high." The biofilm in the right-hand image (from experiment R) was not exposed to sound. The scale bar is 2.54 centimeters.
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Figure 4. Two Streptococcus mutans biofilms after a five-second exposure to a bubble stream at an impingement angle of 45 degrees. All bubble streams had a velocity of approximately 10.5 meters per second, gas fraction of approximately 0.65, bubble diameter of approximately 250 micrometers. The biofilm in the left-hand image (from experiment M) was exposed to sound at the same frequency and intensity as that produced by the Sonicare Elite 7800 toothbrush (Philips Oral Health Care, Snoqualmie, Wash.), while the biofilm in the right-hand image (from experiment N) was not exposed to sound. The scale bar is 2.54 centimeters.
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We compared biofilms exposed to sonic waves at a frequency of 260 Hz and 1 TBE in the absence of any fluid or bubble flow with biofilms in sham experiments. In these sham experiments, we placed the biofilm in the holder in the chamber for only five seconds in the absence of fluid flow or exposure to sound. The handling process in our study removed some biofilm from the edges of the coverslip, but not any measurable amount from the center test area; neither did exposure to sound alone remove any measurable amount of biofilm from the test area.
In experiments using a sound frequency of 260 Hz and 2 TBE, no additional biofilm removal could be attributed to the higher intensity of the sound. Likewise, in experiments using a sound frequency of 520 Hz and 0.2 TBE or a frequency of 150 Hz and 2 TBE, no additional biofilm removal could be attributed to these variations in frequency and intensity.
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DISCUSSION
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We hypothesized that bubbles that impinged on the biofilms at more direct angles (45 degrees) would remove more biofilm than those that impinged on the biofilm at more glancing angles (5 degrees). These experiments, however, proved that the angle at which the bubbles impinge on the biofilm does not affect the amount removed; a bubble stream does not have to be perpendicular to the surface of the biofilm to effectively remove it. For example, if the labial, buccal and lingual surfaces of the teeth are exposed to a stream of bubbles, the bubbles that pass through the interproximal spaces of the teeth will remove biofilm on the surfaces with which they come into contact.
More importantly, in the design of a powered toothbrush, other parameters such as velocity, gas fraction and mean bubble diameter are more significant than the angle of collision, as long as the fluid flow is directed somewhere toward the teeth and gingivae.8
Sound has been shown to reduce the adhesion of planktonic bacteria to surfaces and to cause damage to the fimbria on exposed cells.7,11 However, we found that exposing the biofilm to audible sound at the same intensity as that of a sonic toothbrush did not remove biofilm or enhance the removal of biofilm in the presence of a bubble stream. Although it is possible for sonic pressure waves to remove bacteria, if biofilm removal occurred in our study, it was such a small amount that the biofilm removal caused by sound was negligible compared with the biofilm removal caused by the action of bubbles striking the surface of a biofilm. Likewise, Adams and colleagues12 and Heersink and colleagues13 have shown that biofilm removal caused by bubble and fluid action is less than that caused by direct contact with the bristles of the toothbrush.
Although sound is not an effective means of biofilm removal, it often is a byproduct of instruments vibrating at frequencies and intensities sufficient to displace the surrounding fluid and generate shear forces that remove biofilm. For example, the head of a sonic toothbrush vibrates quickly and creates a region of turbulent fluid flow near the tips of the bristles. Air trapped in the fluid creates bubbles that are propelled away from the tips of the bristles.13 These bubbles can remove biofilm from the surfaces of the teeth, including the proximal surfaces.
Although in our study sound alone did not remove measurable amounts of biofilm, another study showed that high-intensity sound without bubbles can remove a simulated oral biofilm.10 However, that study used a sound intensity that was much greater than any sound produced by any sonic toothbrushes available.
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CONCLUSIONS
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The results of our study show that a stream of artificial saliva and bubbles are effective in removing S. mutans biofilm from a surface. In addition, the bubble stream’s efficiency at removing biofilm is not dependent on the angle with which it strikes a biofilm, at least not between the angles of 5 and 45 degrees. Thus, a bubble that impinges at a more glancing angle is not necessarily less efficient than a bubble that impinges at a more direct angle.
Sonic waves at frequencies and of intensities similar to those generated by commercially available sonic toothbrushes do not appear to produce a measurable amount of S. mutans biofilm removal from a surface in the absence of a bubble stream. Furthermore, when such sound is employed in combination with a bubble stream, the sound does not measurably increase the removal of biofilm any more than the amount normally removed by the bubbles alone. Further studies will need to be conducted to determine whether sonic waves of higher intensities are able to remove biofilm from a surface.
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FOOTNOTES
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DISCLOSURE
Philips Oral Health Care (Snoqualmie, Wash.) and the National Institutes of Health (Bethesda, Md.) grant R01 CA98138 provided funding for this study.
Mr. Parini was a graduate student in chemical engineering, Brigham Young University, Provo, Utah, when this article was written. He now lives in Fairfield, Calif.
Dr. Pitt is a professor, Department of Chemical Engineering, 350 Clyde Building, Brigham Young University, Provo, Utah 84602, e-mail "pitt{at}byu.edu". Address reprint requests to Dr. Pitt.
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REFERENCES
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ABSTRACT
MATERIALS AND METHODS
