American Journal of Dentistry, February, 2006
EUGENIO BRAMBILLA, MARIA GRAZIA CAGETTI, GIUSEPPE BELLUOMO, LUIGI FADINI, FRANKLIN GARCÍA-GODOY
Dental plaque is a microbial community growing on the tooth surface as a biofilm. An ecological imbalance of this ecosystem leads to the predominance of cariogenic species and an increased caries development risk.1-4 Caries prevention and treatment are based on the elimination of risk factors for the disease. Since dental plaque is the most important risk factor for caries, plaque control, using both chemical and mechanical methods, represents a cornerstone of caries prevention.2,5-8
From the mechanical methods point of view, a large num- ber of manual and powered toothbrushes are available on the market for this purpose. Particularly, powered toothbrushes seem to have better efficacy in removing plaque than manual ones, improving, by regular use, the oral hygiene level.9-13
Powered toothbrushes use vibrating, rotating or oscillating brush heads that achieve plaque removal primarily through direct physical contact between bristles and tooth surface. One generation of powered toothbrushes, also called sonic tooth- brushes, was designed to combine bristle contact with the pro- duction of localized hydrodynamic shear forces that dra- matically improve the efficacy of plaque removal.14-18 When the bristle tips are placed in an air/fluid environment, the frequency of brush head oscillation (around 260 Hz), produces fluid activity and cavitation. The effect of this action near the tooth surface generates shear forces that disgregate and disperse the biofilm adherent to tooth surface.19-21
Experimental studies showed that sonic energy generated by sonic toothbrushes can damage surface cell structures, interfering with microbial adhesion in vitro.22-24 Few data about the effect of the exposure to low frequency acoustic energy on the viability of microbial biofilms are available.20-22,25,26 Fur- thermore there is no evidence of a selective or differential effect of this energy form on the different oral microorganism species. The microbial structures of the different species of oral microorganisms (for example Gram-positive and Gram-nega- tive) may have different reactions to acoustic energy exposure. Differences in the in vivo activity of a sonic toothbrush (Sonicarea), against three different groups of cariogenic bacteria was demonstrated.27,28 Sonicare showed the higher activity in the reduction of mutans streptococci concentration in interdental plaque compared with activity against Lactobacillus species and Candida albicans.27,28
The present study compared the in vitro effect of sonic energy on adhesion and on the first stages of colonization of two bacterial groups: cariogenic (Streptococcus mutans, Lactobacillus acidophilus) and non-cariogenic (Streptococcus salivarius, Veillonella alcalescens) microorganisms.
MATERIALS AND METHODS
Microorganisms – Four oral microorganisms were selected for the study. Two were cariogenic bacteria (Streptococcus mu- tans, Lactobacillus acidophilus) and two were non-cariogenic microorganisms (Streptococcus salivarius, Veillonella alcales- cens). Streptococcus mutans (OSP 236) and Streptococcus coccus salivarius (OSP 248) were cultured in brain-heart infusion broth (BHIb), supplemented with 2% sucrose for 48 hours at 37°C in a 5% supplemented CO2 environment. Lactobacillus acidophilus (OSP 211) was cultured in Rogosa SL brothb for 48 hours at 37°C. Veillonella alcalescens (OSP 403) was cultured in Bacto Veillonella brothb for 48 hours at 37°C.
Colonization model – Bovine enamel disks (diameter 5 mm and 1 mm thick), coated with artificial saliva formed the sub- stratum for microbial biofilm development. Ten volunteers were asked to collect whole paraffin-stimulated saliva in a sterile container which was treated as follows: saliva was pooled and clarified by centrifugation (14,000 x g for 30 minutes), then heated to 60°C for 30 minutes and centrifuged again (14,000 x g for 30 minutes), just before the addition of 0.04% wt/vol. of sodium azide to prevent microbial growth. The enamel disks were immersed in KCl buffer for 1 hour prior to a 12-hour incubation with artificial saliva at room temperature. At the end of the incubation, each disk was placed in a single well of a 24-well polystyrene plate (Multi- dish 24-well Nunclonc) and washed three times with sterile PBS.d 2.5 ml of TSB (Trypticase soy brothb) and 100 µl of microbial suspension of the previously described micro- organisms were added to each well and a 36-hour incubation was performed to allow the development of a multilayer biofilm. At the end of the incubation each disk was rinsed carefully with sterile PBS to remove the non adherent cells and transferred in a well of a new 24-well plate containing 1 ml of sterile PBS. 120 disks were used for the study: 90 disks were used for the experimental and 30 for the control group.
Acoustic exposure – The exposure of the biofilms to acoustic energy was performed using a Sonicare Advance 4700 tooth- brush,a and two toothbrush heads with only two tufts. A small stainless steel wire fitted tightly against the well wall was used to lock the disk against the well bottom during Sonicare action. The wires were sterilized by autoclaving. The tooth- brush handle was manually placed in the right position to align the tufts with the center of the disk. The distance between the end of the bristles and the disk surface was 7 mm. This ensured that the biofilm removal was simply due to fluid forces and not bristle contact during the brushing cycle.
The disks were divided into four groups of 30 disks each. The first (Group A) was exposed to Sonicare action for 5 seconds, the second (Group B) for 15 seconds, the third (Group C) for 30 seconds. The control group disks were left untreated.
Table. ANOVA performed to assess the significance of the differences among the microbial species considered.
Figure. Effect of time exposure to acoustic energy of the monospecific biofilms.
Viable biomass assay (MTT-assay) – After the acoustic exposure, both experimental (Groups A, B, C) and control disks were washed twice with 1 ml of sterile PBS, then 1 ml of sterile PBS was added to each well.
The MTT solution was prepared by dissolving 5 mg of (3- [4,5]dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromided)/ ml of sterile PBS.
A lysing solution (LS) was prepared by dissolving 10% V/V of sodium dodecyl sulfated (SDS) and 50% V/V of dimethylformamided (DMF) in distilled water.
MTT solution (100 µl) was placed in each well and the plates were incubated for 3 hours at room temperature in the dark (during the incubation, microbial redox systems convert the yellow salt to an intracellular insoluble purple formazan).
Formazan crystals were dissolved by addition of 0.5 ml of lysing solution and, after a second 3-hour incubation at room temperature, 100 µl of the suspension of each well were placed in the wells of a 96-well plate (96-well Microtiter platec). Absorbance (OD) was measured by a dual wavelength spectrophotometer at 620-550 nm (LP200e). The mean blank OD for the same 96 wells-plate was subtracted from each test OD value.
Statistical analysis – All statistical analyses were performed using statistical software (JMP4f). The comparison among the biomass adherent to the disk surfaces at the four exposure times evaluated was performed for each bacterial species with ANOVA. Differences between means were considered significant at P< 0.05.
After the 36-hour growth period, all the tested bacterial strains formed an extensive biofilm on enamel disks.
The Table shows the results of ANOVA performed to assess the significance of the differences among the microbial species considered. The data showed that Sonicare exposure significantly reduced the biomass adherent to the disks of S. mutans and S. salivarius, while L. acidophilus and V. alcalescens seemed to remain basically unaffected.
The effects of time exposure to acoustic energy of the monospecific biofilms for 5, 15 and 30 seconds are shown in the Figure. In the two streptococcal groups the increase of the exposure time led to different reduction trends, while S. salivarius exhibited a progressive decrease over time, S. mutans shows a rapid reduction of the adherent biomass with a 15-second exposure to Sonicare.
Dental caries is an infectious disease caused by an eco- logical imbalance in the dental plaque ecosystem.29-31 When cariogenic species become predominant on the bacterial pop- ulation of plaque, environmental conditions of tooth surface become favorable to the demineralization processes.32,33
Every preventive or therapeutic strategy, aimed at the reduction of caries risk, has to consider, as its primary target, the re-equilibrium of the plaque ecosystem, preferably by means of a selective suppression of the cariogenic part of the flora. The final aim should be the correction of the imbalance and the restoration of physiological status of homeo- stasis.29,31,33
Research9,11,34-39 demonstrated that powered toothbrushes were more efficient than manual toothbrushes in plaque removal. Both manual and powered toothbrush action is based on direct contact between the tips of the bristles and the tooth surface. For this reason plaque removal is limited to the areas only reached by the bristle tips. Furthermore, the toothbrush action on dental plaque is non-selective, affecting both cario- genic and saprophyte microorganisms.27
As observed in the two previous clinical studies, a selective effect on some cariogenic microorganisms was demon- strated.27,28 The results indicate that the hydrodynamic fluid forces generated by Sonicare are able to cause the in vitro disruption of Streptococcus mutans and Streptococcus Sali- varius biofilms. These microorganisms respectively showed a reduction of 40.9% and 29.8% after a 30-second exposure to Sonicare action. On the other hand, the data showed little influence of the same treatment on Lactobacillus acidophilus and Veillonella alcalescens biofilms.
The exposure time had a significant influence on biofilm removal. When the exposure time was set to 5 seconds, some effect, mainly on Streptococcus salivarius, was seen; 15 seconds appeared to be the best exposure time to highlight the inter-specific differences. A 30-second exposure seemed to achieve the best removal of the adherent microorganisms from enamel surfaces.
As the distance between the bristle ends and the enamel surface was set at 7 mm, it must be pointed out that, in the clinical situation, this parameter could be significantly lower and the effect of the sonic energy on the cariogenic microorganisms could be much more intense than in the experimental conditions.
Although a new generation of Sonicare, the Elite, is currently on the market, the sonic nature of its action is similar to the Advance and therefore, the results should be similar. This needs further evaluation.
Our results demonstrated that cariogenic microorganisms showed different sensitivity to acoustic energy exposure. Specific studies about the intensity and the frequency of the acoustic vibration of the sonic toothbrushes should be planned to maximize the effects on cariogenic bacteria and to develop sonic toothbrushes specifically targeted to selectively reduce cariogenic bacteria concentration in dental plaque.
- Philips Oral Healthcare, Snoqualmie, WA,
- Difco-BD, Sparks, MD,
- Nunc, Kamstrup,
- Sigma-Aldrich s.r.l. Italia, Milan,
- Diagnostic Pasteur, Milan,
- SAS Institute Inc., Cary, NC,
Dr. Brambilla is Professor, Dr. Cagetti, Dr. Belluomo and Dr. Fadini are Researchers, Department of Restorative Dentistry, University of Milan, Department of Medicine, Surgery and Dentistry, Milan, Italy. Dr. García- Godoy is Professor, Associate Dean for Research, and Director, Bioscience Research Center, College of Dental Medicine, Nova Southeastern University, Fort Lauderdale, FL, USA.
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