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RESEARCH REPORT |
1 Department of Biomedical Engineering, University Medical Center Groningen, and University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands; and
2 Philips Research, Care & Health Applications, Professor Holstlaan 4, 5656 AA Eindhoven, The Netherlands;
* corresponding author, h.c.van.der.mei{at}med.umcg.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: bacterial adhesion • sonic brushing • rotary brushing • actinomyces • and streptococci
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The formation of dental plaque can be envisaged as a sequence of events that commences with the adsorption of a salivary conditioning film or "pellicle", followed by initial adhesion of early colonizers and late colonizers. Subsequently, co-adhesion occurs, allowing adherent bacteria to start anchoring themselves and finally start to grow and form a biofilm. These events do not occur randomly in space and time, but follow a spatio-temporal sequence, followed by adhesion of more bacteria and growth of the dental plaque (Palmer et al., 2003). We have used a co-adhesion model for initial plaque formation in our studies on mechanical plaque removal and comparison of different toothbrush designs and conditions. In essence, actinomyces were allowed to adhere first to a salivary pellicle in a parallel-plate flow chamber up to a surface density of 1 x 106 cm–2, after which a co-adhering streptococcal strain was allowed to adhere and co-adhere. This yielded a bacterial coverage that consisted of about 30% of singly adhering organisms, while about 40% of the organisms adhered in aggregates comprised of more than 10 bacteria. Note that, for pairs without the ability to co-aggregate, the distribution of single bacteria and aggregates consisting of more than 10 bacteria was 30% and 10%, respectively (Busscher et al., 2003). This model was used to demonstrate the influence of brushing force on the efficacy of sonic brushing, and to prove the concept of non-contact removal of adhering bacteria by sonic brushing (Van der Mei et al., 2004). We were interested in whether the sequence in which the actinomyces and streptococci are applied to the pellicle surface has an impact on removal by various modes of brushing.
Therefore, the aim of this paper was to investigate the non-contact removal by two modes of brushing (sonic and rotary) of co-adhering bacterial pairs from salivary pellicles for two sequences of adhesion, i.e., either the actinomyces first, followed by streptococci or vice versa.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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S. oralis J22 was cultured in Todd-Hewitt Broth (OXOID, Basingstoke, UK) at 37°C in ambient air and A. naeslundii T14V-J1 in Schaedler’s broth supplemented with 0.01 g/L hemin in an anaerobic cabinet (Ruskinn Technology, West Yorkshire, UK) at 37°C. Strains were pre-cultured in an overnight batch culture and inoculated in a second culture which was grown for 16 hrs, harvested by centrifugation for 5 min at 6500 g, and washed twice with adhesion buffer (2 mM potassium phosphate, 50 mM potassium chloride, and 1 mM calcium chloride, pH 6.8). To break bacterial aggregates, we sonicated bacteria intermittently while cooling them in an ice/water bath for 35 sec at 30 W. This procedure was found not to cause cell lysis.
Actinomyces were suspended in adhesion buffer or in adhesion buffer supplemented with 1.5 g/L lyophilized human whole saliva to a concentration of 1 x 108 bacteria per mL. Streptococci were suspended in adhesion buffer supplemented with 1.5 g/L lyophilized human whole saliva to a concentration of 3 x 108 bacteria per mL.
Saliva Collection and Preparation
Human whole saliva from 20 healthy individuals of both genders was collected into ice-cooled beakers after stimulation by the chewing of Parafilm. The saliva was pooled, centrifuged, dialyzed against demineralized water, and lyophilized for storage. For experiments, lyophilized saliva was dissolved at a concentration of 1.5 g/L in adhesion buffer. All participants gave their informed consent to saliva donation, in accordance with the rules set out by the Ethics Committee at the University of Groningen. After centrifugation, saliva contained only very few bacteria, and no growth of these bacteria was noticed during the experiments.
Deposition Protocol and Brushing
Bacterial adhesion was observed on the bottom quartz plate of a parallel plate flow chamber (Busscher and Van der Mei, 2006). The quartz plate was coated with saliva for 16 hrs at room temperature to create a salivary pellicle. Before each experiment, all tubes and the flow chamber were filled with buffer, and 10 min of perfusion with buffer was applied to remove remnants of saliva from the flow chamber.
For co-adhesion experiments, two different sequences were executed: actinomyces first or streptococci first. For actinomyces-first experiments, actinomyces suspended in buffer or saliva were first perfused through the flow chamber until a surface coverage of 1 x 106 bacteria per cm2 was reached, as enumerated by the image analysis system. Thereafter, flow was switched to buffer to remove unattached bacteria from the flow chamber and the tubes for 10 min. Co-adhesion was initiated by switching the flow to the streptococcus in saliva for 2 hrs. For streptococci-first experiments, the same protocol was followed, except that streptococci were allowed to adhere first, until a surface coverage of 1 x 106 per cm2 was achieved, after which actinomyces were passed into the flow chamber for 2 hrs.
After the adhesion phase and before the flow chamber was dismantled for brushing, 10 images were taken from different areas on the pellicle surface, corresponding with the areas to be brushed. The selected areas, after removal of the sample from the flow chamber, were brushed with either a sonic brush (Sonicare® Advance; Philips Oral Healthcare, Snoqualmie, WA, USA) or a rotary brush (Braun Oral-B 3D, Professional Care 7000, D17511, Kronberg, Germany) for 20 sec, with the brush attached to a homemade moving tray, involving 20 strokes back and forth. The distance between the brush, measured from the longest bristle ends to the pellicle surface, was 0, 2, or 4 mm. Surfaces were immersed together with the brush in adhesion buffer, extending 7 mm above the pellicle surface. In addition, experiments were also carried out with a wetted brush and a film-wetted pellicle, i.e., without immersion of the brush. Subsequently, after the brushed pellicle surface was replaced in the parallel-plate flow chamber, the flow chamber was filled again with buffer, and a subsequent series of images of the selected areas was taken.
Throughout the experiments, streptococci were always suspended in saliva. Since actinomyces had the tendency to auto-aggregate in saliva, actinomyces were suspended in buffer in a first series of experiments, but in a second series of experiments, actinomyces were also suspended in saliva to enhance the clinical relevance of the data obtained, while taking for granted the occurrence of aggregates in suspension.
All suspensions were circulated through the system by means of hydrostatic pressure at a wall shear rate of 10 sec–1, which corresponds to physiological conditions of low shear (Dawes et al., 1989) and yields a laminar flow (Reynolds number 0.6). Experiments were carried out at 33°C, a relevant oral surface temperature (Spierings et al., 1984). All data represent averages of triplicate runs with separately cultured bacteria.
Statistics
To evaluate the impact of toothbrush type, toothbrush distance from the surface, bacterial deposition sequence, and the condition "wetted vs. immersed", we have used the independent-sample t test in unified analyses, and accepted p < 0.05 (two-tailed) as statistically significant. We used Student’s t test to compare single conditions.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Quantitative analysis (Fig. 2) indicated that the sequence streptococci followed by actinomyces, both suspended in saliva, yielded most large co-aggregates (p < 0.05).
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), but was significantly (p < 0.05, n = 24) different from non-contact removal. However, both modes of non-contact brushing also yielded removal of co-adhering bacteria. Sonic brushing removed significantly more bacteria under 4 of the 12 non-contact conditions listed, whereas rotary brushing removed significantly more bacteria under one of the conditions. Comparison of sonic and rotary brushing under all conditions, in a unified analysis, showed that the sonic brush removed significantly (p < 0.05, n = 44) more bacteria. Removal under wetted or immersed conditions was not significantly different (p > 0.05, n = 44).
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). In only one experiment involving sonic brushing did the actinomyces-first experiment show significantly more removal than in the streptococci-first experiment. In rotary brushing, bacteria were removed in higher numbers for the ‘actinomyces followed by streptococci’ sequence in 2 of the 4 conditions. Generally, co-aggregation with actinomyces suspended in saliva showed less removal of bacteria compared with co-aggregation with actinomyces suspended in buffer, regardless of sequence or mode of brushing, suggesting that a more strongly adherent initial plaque was formed with actinomyces suspended in saliva than when they were suspended in buffer.
Sonic brushing left very few large co-aggregates adhering on the pellicles after the sequence ‘actinomyces in buffer followed by streptococci in saliva’, while, after the sequence ‘streptococci in saliva followed by actinomyces in buffer’, significantly more large co-aggregates remained on the surface, except for contact brushing (Table 2). Furthermore, it is worth emphasizing that when actinomyces were suspended in saliva, more large aggregates were formed. Rotary brushing generally tended to leave more large aggregates than sonic brushing. In general, in a comparison of the sequence of bacterial adhesion in a unified analysis, streptococci adhering first, followed by actinomyces, left significantly (p < 0.05, n = 12) larger aggregates after brushing.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Bacteria adhering to pellicles in the sequence ‘streptococci in saliva followed by actinomyces in saliva’ appeared more difficult to remove and left more large co-aggregates than when applied in the sequence ‘actinomyces in saliva followed by streptococci in saliva’. It is interesting to speculate on the differences in the mechanisms of bacterial removal exerted by both modes of brushing. Sonic brushing with a rapid side-to-side sweeping motion of the bristles has been described to yield acoustic waves or air displacement, due to bristle action above the wetted pellicle, that may cause fluid pressures in the thin liquid film wetting the pellicles, causing fluid flows toward and over the surface (see video APPENDIX 2, illustrating flowing air bubbles in water generated by sonic brush movement under laser illumination), taking the adhering bacteria through liquid-air interfaces, accompanied by detachment forces several orders of magnitude larger than bacterial adhesion forces (Leenaars and O’Brien, 1989; Gomez-Suarez et al., 2001; Parini et al., 2005). A similar mechanism of alternating fluid pressures toward the surface has been described for immersed conditions, augmented with entrapment of air bubbles from the bristles moving through the liquid-air interface. Although these mechanisms may also be operative during rotary brushing, the rotating brush, with its planar bristle motion, is probably less effective in including air in the fluid flows generated. Moreover, the direction of the fluid flow generated by a sonic brush is toward the surface, while the rotary brush generates a fluid flow direction away from the surface, due to centrifugal action of the brush (APPENDIX 3), causing a fluid flow toward the brush head to replenish the volume of fluid driven away. Clearly, although some efficacy remains for sonic brushing, at a 4-mm distance, both modes of fluid flow occur too far above the surface to have a clear effect on bacterial removal, while at contact, the bristles themselves are accountable for removal. At 2 mm, however, these differences in fluid flow generated appear pivotal to the enhanced efficacy of sonic brushing, likely because the flow toward the surfaces increases the collision probability of air-bubbles included in the flow with adhering bacteria. But when the actinomyces are deposited from saliva, and the salivary layer between the bacteria thickens, which is more like the clinical situation, the fluid flow has problems removing the well-packed bacteria.
A second interesting point is why the sequence of allowing the bacterial pair to co-aggregate on the surface affects removal efficiency (roughly similar for both modes of brushing), and whether this has any clinical implications. Although we have no clear mechanism available to explain these observations, it might be clinically hypothesized that rapid-plaque-forming individuals carry more streptococci than actinomyces, causing their colonization pattern to resemble the ‘streptococci followed by actinomyces sequence’ more closely than the ‘actinomyces followed by streptococci sequence’. Such a hypothesis would be supported by the observation that the morphological features of plaque in slow- and rapid-plaque-forming individuals are different (Zee et al., 1997). Domination of streptococci has also been noticed in flow chambers, regardless of the inoculation order of different bacterial strains (Foster and Kolenbrander, 2004).
Summarizing, both modes of brushing are capable of non-contact removal of adherent co-adhering bacteria, as a model for initial plaque. Clinically, this is important, since many caries-susceptible sites are inaccessible in a contact mode and may thus be better cleaned by sonic or rotary brushing.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received February 15, 2006; Last revision November 27, 2006; Accepted December 19, 2006
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REFERENCES |
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Busscher HJ, Rustema-Abbing M, Bruinsma GM, De Jager M, Gottenbos B, Van der Mei HC (2003). Non-contact removal of coadhering and non-coadhering bacterial pairs from pellicle surfaces by sonic brushing and de novo adhesion. Eur J Oral Sci 111:459–464.[ISI][Medline]
Dawes C, Watanabe S, Biglow-Lecomte P, Dibdin GH (1989). Estimation of the velocity of the salivary film at some different locations in the mouth. J Dent Res 68:1479–1482.
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Gomez-Suarez C, Busscher HJ, Van der Mei HC (2001). Analysis of bacterial detachment from substratum surfaces by the passage of air-liquid interfaces. Appl Environ Microbiol 67:2531–2537.
Leenaars AFM, O’Brien SBG (1989). A new approach to the removal of sub-micron particles from solid (silicon) substrates. In: Particles on surfaces 2: detection, adhesion, and removal. New York: Plenum Press, pp. 361–372.
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Van der Mei HC, Rustema-Abbing M, Bruinsma GM, De Jager M, Gottenbos B, Busscher HJ (2004). Influence of weight on removal of co-adhering bacteria from salivary pellicles by different modes of brushing. Caries Res 38:85–90.[ISI][Medline]
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| Journal of Dental Research ® | Critical Reviews (1990-2004) |
10 on Salivary Pellicle after Non-contact Brushing with a Sonic and a Rotary Brush for a Wetted or Immersed Substratum