Keywords dental plaque - power toothbrush - radiofrequency - toothbrushing - ToothWave - bacterial
viability
Introduction
The oral environment is exposed to permanent colonization by a variety of microorganisms
that are, in normal health circumstances, in balance with the immune system of the
host. Within that environment, the hard dental surfaces are prone to adhesion of a
thin biofilm formed by a highly structured and organized bacterial community.[1 ] When it is not routinely eliminated, this biofilm, which constitutes dental plaque,
can progressively accumulate supra- and subgingivally and become mineralized, forming
calculus.[2 ] These bacterial deposits stimulate a local host response manifested by gingival
inflammation and alter the pH of the saliva and biofilm on tooth surfaces, causing
demineralization of dental tissues.[3 ] Therefore, dental plaque is considered the leading etiological factor of gingivitis
and dental caries.[3 ] Gingivitis is a reversible phenomenon, but if it is left untreated, the inflammation
can propagate to the subjacent periodontium, causing permanent damage to the dental
supporting structures and inducing periodontitis.[4 ] Oral hygiene tools used at home do not currently provide complete elimination of
dental plaque from the oral cavity.[5 ]
[6 ] However, routine and efficient toothbrushing can disrupt the structural integrity
of the biofilm, and restore the normal oral microbial population, thus significantly
reducing the destructive effects of the biofilm, reversing gingival inflammation,
and maintaining good oral health.[5 ]
[7 ]
[8 ] This efficiency is contingent upon the subject's manual dexterity, the type of toothbrush
used, and the brushing technique, time, and frequency.[8 ]
In the market since the early 1960s, powered toothbrushes have been taking up an increasing
share of toothbrush sales, becoming more widely used as an alternative to manual toothbrushes.[9 ] The development of powered toothbrushes aimed to improve many of the parameters
mentioned earlier to achieve a more efficacious brushing routine. Powered toothbrushes
can clean dental surfaces through a direct physical action of the bristles, just like
manual toothbrushes, and through hydrodynamic effects associated with the bristles'
motion and speed superior to manual toothbrushes.[10 ]
[11 ]
[12 ] Thus, the unique characteristics of these powered toothbrushes have established
their efficacy in reducing stains and deposits from dental surfaces.[13 ]
[14 ]
[15 ] Studies have also demonstrated that powered toothbrushes are as safe to use as manual
toothbrushes with no adverse effects on the soft and hard oral tissues when regular
forces are applied during brushing.[16 ]
[17 ]
[18 ]
[19 ]
[20 ] Currently many types of powered toothbrushes are available. They can be categorized
according to the kind of movement of the bristles (vibrational, oscillation-rotational,
or circular); speed of movement (sonic or ultrasonic); type of electric current, direct
or alternating current (electronic or ionic toothbrushes)[9 ]
[21 ]; and whether it is powered by a unique technology (radiofrequency [RF] energy).[14 ]
RF technology has made its way into the medical field through various applications,
one of the latest being its use in the novel powered toothbrush, ToothWave (Home Skinovations,
Israel). This toothbrush uses RF energy, which is a low-power alternating current.
This current oscillates from 3 kHz to 300 GHz and circulates between two electrodes
and over a silicon barrier. Previous studies have shown that this toothbrush using
RF energy significantly reduces extrinsic tooth stains, plaque, calculus, and subsequent
gingivitis.[14 ]
[15 ]
[22 ] It is an effective tool that is easy to incorporate into one's oral hygiene routine
at home while being nonabrasive and well-tolerated.[14 ]
[23 ] As for its mechanism of action, it is hypothesized that during toothbrushing, the
alternating RF current propels the toothpaste's active molecules onto the tooth surface,
compromising the adhesion of bacterial deposits and stains to the enamel surface.
However, no studies have established the mechanism by which RF energy causes a reduction
in dental plaque deposits. Therefore, the aim of this in vitro study was to investigate the effects of a bipolar RF (3 W, 3 MHz) energy applied
through a powered toothbrush on the structural morphology (chain formation) of periodontal
disease-causing dental plaque and the ultrastructure (cell wall and cell components)
of the bacteria components. We hypothesized that the application of 3-MHz bipolar
RF delivered via a powered toothbrush RF electrode after biofilm formation would cause
structural disintegration of dental plaque and disruption of the bacterial cellular
component.
Materials and Methods
Bacterial Strains and Growth Conditions
Three red complex bacteria (Porphyromonas gingivalis ATCC 33277, Treponema denticola ATCC 35404, and Tannerella forsythia ATCC 43037), known to be the major components of dental plaque responsible for causing
periodontal diseases,[3 ] were used in this study. All cultures were grown at 37°C in an anaerobic incubator
containing a gaseous mix of 90% N2 , 5% H2 , and 5% CO2 for 2 to 4 days. P. gingivalis was grown in tryptic soy broth supplemented with hemin (5 μg/mL) and cysteine (0.5 g/L),
T. denticola in modified NOS medium, and T. forsythia in tryptic soy broth supplemented with 0.3% yeast extract, vitamin K (0.4 mg/mL),
and N-acetylmuramic acid (NAM; 10 mg/mL; Sigma Aldrich, St. Louis, Missouri, United
States). After growth, a loop full of individual colonies was transferred from agar
plates to tubes containing oral bacteria growth medium (OBGM) containing brain heart
infusion (12.5 g/L), tryptic soy broth (10 g/L), yeast extract (7.5 g/L), sodium thioglycolate
(0.5 g/L), asparagine (0.25 g/L) D-glucose (2 g/L), ascorbic acid (2 g/L), sodium
pyruvate (1 g/L), sodium bicarbonate (2 g/L), L-cysteine (1 g/L), ammonium sulfate
(2 g/L), thiamine pyrophosphate (6 mg/L), heat-inactivated rabbit serum (5% vol/vol),
hemin (5 mg/L), menadione (1 mg/L), NAM (10 mg/mL), and a volatile fatty acid mix
(0.5% vol/vol). Each tube was incubated at 37°C in an anaerobic chamber (85% N2 , 10% H2 , 5% CO2 ) for 48 hours to attain active exponential growth phase culture to prepare an active
exponential phase multispecies culture.
Experimental Grouping
A total of 360 acrylic disks were fabricated, each measuring ∼6 mm diameter × 0.3 mm
thickness, serving as the substrate on which the biofilm was grown. The disks were
assigned to the following six experimental groups (45 disks/group) consisting of 3
control groups not treated with RF energy (A, B, C) and 3 test groups (D, E, F) treated
with RF energy: (A) bacterial growth medium only, (B) toothpaste slurry (TS) only,
(C) centrifuged toothpaste slurry (CTS) only, (D) RF treatment in the growth medium,
(E) RF and TS treatment, and (F) RF and CTS treatment.
Biofilm Formation in Microbial Plaque Model
The microbial model (MM) is shown in [Fig. 1 ]. The model consists of a compact-sized, custom-made rectangular acrylic culture
bath to accommodate just the toothbrush head and its bristles. A thin circular acrylic
disk served as the substrate on which the dental plaque was grown and placed on the
floor of the well. Attached to the toothbrush head at the two ends of the bristles
are two acrylic stubs positioned to give the bristles 1-mm clearance from the circular
disk on the floor of the well. The compact design ensured that when the toothbrush
was positioned inside the well, the toothpaste was only accommodated within the bristles
and the space between the bristles and the floor of the well, thus ensuring the minimum
amount of toothpaste was used. The liquid volume must be as low as possible because
a high liquid volume will dilute the charged molecules and prevent the formation of
a high concentration of charged molecules on the plaque surface.
Fig. 1 Setup of the microbial model (MM) serving as the culture bath as well as treatment
bath.
Before the start of the study, all components were aseptically set up, following sterilization
in an autoclave for 20 minutes at 121°C (15-lb pressure). The growth medium used in
this system was a 2% sucrose-supplemented OBGM, and the pH was adjusted to 7.2. The
entire assembly was housed inside an anaerobic chamber containing a gaseous mix of
85% nitrogen (N2 ), 10% hydrogen (H2 ), and 5% carbon dioxide (CO2 ) and maintained at a constant physiological temperature of 37°C.
To aid bacterial adhesion for biofilm formation, acquired salivary pellicles were
formed on the enamel disks by a 30-minute immersion in filtered human saliva, with
mild agitation at 70 rpm at 37°C. To initiate bacteria biofilm formation on the acrylic
disks, each culture bath bearing a disk was filled with 20 mL of OBGM inoculated with
the multispecies inoculum of the three red complex bacteria at medium-to-inoculum
ratio of 10:1. Then, each of the culture baths was incubated at 37°C in an anaerobic
chamber (85% N2 , 10% H2 , and 5% CO2 ) for 4 hours (adhesion phase). Following the 4-hour adhesion phase, the OBGM was
replaced with bacteria-free fresh sucrose (2%) supplemented OBGM and incubated anaerobically
(85% N2 , 10% H2 , and 5% CO2 ) for 24 hours at 37°C (biofilm maturation phase). Following this maturation phase
(24-hour biofilm growth), each biofilm-bearing acrylic disk in its respective group
was treated as follows:
Group A: Neither RF device nor toothpaste was used. Biofilm-bearing disks were exposed only
to the growth medium.
Group B: The growth medium was replaced with a slurry of standard fluoride toothpaste (sodium
monofluorophoshate, Colgate-Palmolive, New York, NY, United States) for 4 seconds,
the estimated time for brushing each tooth surface in vivo , and then the slurry was rinsed off with sterile phosphate buffer saline (PBS). The
TS was prepared by mixing one part toothpaste and three parts distilled water using
a laboratory stand-mixer until homogenous.
Group C: A slurry of standard fluoride toothpaste was prepared as described earlier. Then
enough quantity of the slurry was centrifuged for 10 minutes at 10,000 rpm (4,000 g)
at 7°C, and the supernatant fluid (CTS) was used for the treatment of this group.
The enamel disks were covered with CTS for 4 seconds, and then the CTS was rinsed
off with PBS.
Group D: Using a home-use device, Silk'n power toothbrush Model H7001 (ToothWave), each biofilm-bearing
disk was treated with a 3-MHz bipolar RF current for 4 seconds at a 1-mm distance
between the surface of the toothbrush bristles and the biofilm-bearing disk. The toothbrush
was activated on mode 4 (RF only, no vibration). The disks were treated inside the
OBGM with the power toothbrush inserted into the medium.
Group E: The culture bath containing the biofilm-bearing disk was filled with TS, then the
power toothbrush was inserted into the culture bath. The disk was immediately treated
with a 3-MHz bipolar RF current for 4 seconds as described for group D. After treatment,
the TS was rinsed off with PBS.
Group F: The culture bath containing the biofilm-bearing disk was filled with CTS, then the
power toothbrush was inserted into the culture bath. The disk was immediately treated
with a 3-MHz bipolar RF current for 4 seconds as described for group D. After treatment,
the CTS was rinsed off with PBS.
Following each treatment, the OBGM was replaced with a fresh, bacteria-free OBGM.
Over the next 2 days, the OBGM was replaced with an equal volume of fresh bacteria-free
OBGM every 12 hours, and the respective treatment for each group was repeated every
24, 48, and 72 hours of biofilm formation. However, every 24 hours following treatment,
15 disks were aseptically harvested from each group and rinsed thoroughly in sterile
PBS to remove planktonic cells and traces of OBGM, TS, and CTS.
Of the 15 disks from each group, 5 were used for confocal laser scanning microscope
(CLSM) examination to determine the live-to-dead bacteria ratio, 5 for scanning electron
microscope (SEM) examination to determine the effect on dental plaque morphology,
and 5 for transmission electron microscope (TEM) examination to determine the effect
on the bacteria's ultrastructure. These examinations were performed for each time
point (24, 48, 72 hours).
Structural Analysis of the Dental Plaque
Structural Analysis of the Dental Plaque
SEM Examination of the Effect on Dental Plaque Morphology
The biofilm-bearing disks were fixed for 2 hours in 2.5% glutaraldehyde solution at
4°C. After fixation, the disks were rinsed gently in 0.1 M phosphate buffer (pH 7.4;
3 times, 2 minutes each) and then placed in 1% Zetterquist's osmium tetroxide (OsO4 ) for 60 minutes. Afterward, the biofilms were rinsed with 0.1 M phosphate buffer
(1 time, 2 minutes) and then in distilled water (2 times, 2 minutes each) to avoid
contamination by insufficient osmium removal. Subsequently, the samples were dehydrated
in an increasing series of ethanol dilutions (35, 50, 75, 2 × 90, and 2 × 100%) for
30 minutes each time in each solution. Then samples were immersed in hexamethyldisilazane
(HMDS; Polysciences Inc., Warrington, Pennsylvania, United States) for 1.5 hours,
and, finally, HMDS was removed. Biofilm surfaces were airdried in a desiccator for
12 hours. Each sample was coated with gold sputter after critical point drying and
mounted on a glass slide. Finally, the biofilms were examined with an SEM (JCM-5700;
JEOL, Tokyo, Japan) using the secondary electron emission mode with accelerating voltages
of 1, 5, 10, and 20 kV and ×1,500 and ×5,000 magnifications.
TEM Examination of the Effect on Bacteria Ultrastructure
The ultrastructure of the bacteria in the biofilm was examined by TEM analysis. Immediately
upon harvesting and following rinsing with PBS, the biofilm-bearing disks were dropped
into 1.5% paraformaldehyde and 2.5% glutaraldehyde fixative for 2 hours. Disks were
postfixed in 2% osmium tetroxide for 2 hours, dehydrated in ethanol, and embedded
in Araldite M (Merck, Darmstadt, Germany). The routinely performed embedding process
of organic material for TEM examination was designed to transfer dehydrated specimens,
step by step, from a pure propylene oxide solution to the embedding medium of Araldite
M by way of intermediate Araldite–propylene oxide mixtures. In this process, the highly
volatile, lipophilic solvent propylene oxide reduced the viscosity of the Araldite
mixture and ensured a complete wetting and saturation of the structures to be embedded.
The time for hardening lasted 48 hours at 65°C. After embedding, the biofilm on the
disks was decalcified in 4% ethylenediaminetetraacetic acid (EDTA) at pH 7.2 and re-embedded.
Ultrathin sections were cut from all biofilm-bearing disks on an Ultracut E ultramicrotome
(Reichert, Benzheim, Germany) equipped with a Micro Star 55-degree diamond knife (Mikrotechnik,
Benzheim, Germany) specially designed for sectioning materials. Serial ultrathin sections
were mounted on Pioloform-F (Wacker-Chemie, Munich, Germany) coated copper grids,
contrasted with uranyl acetate and lead citrate, and examined in a TEM 201 (Philips,
Eindhoven, The Netherlands) at 80 kV. Representative micrographs were obtained at
a magnification of ×30,000.
CLSM Examination of the Effect on the Viability of the Bacteria in Biofilm
To use the CLSM to assess the plaque bacteria viability on the surface of each disk,
the biofilm was stained with the L7012 Live/Dead BacLight bacterial viability kit
(Molecular Probes, Life Technologies, Carlsbad, California, United States). The biofilm
was stained by incubation in 50 μL of a dye solution for 30 minutes in the dark, at
room temperature. The dye solution was prepared by dissolving 5 μL of SYTO and 5 μL
of propidium iodide (PI) dye in 10 μL of DMSO, and then 980 μL of NaCl buffer (0.89%)
was added to the dye solution to make up the volume of 1 mL. The dye solution was
kept frozen until use. After staining, the excess stain was rinsed with sterile saline
to avoid noise during the acquisition of the image under CLSM. After washing, the
biofilm was immediately examined using an Olympus FLUOVIEW FV 1000 confocal scanning
laser microscope with an FV-10 ASW system (Olympus Corporation, Tokyo, Japan) at excitation
wavelengths of 485 (SYTO-Green Signals) and 543 nm (PI-Red Signals) with a 20X oil
immersion lens. Images were obtained with the software FV10-ASW 4.0 Viewer (Olympus
Corporation) at five random positions of the surfaces within the central periphery
of the disk under the toothbrush, and a stack of maximum slices (1 μm thick each)
was scanned. Finally, the Z Stack (surface topography and three-dimensional architecture)
analysis was performed with FV10-ASW 4.0 Viewer (Olympus Corporation) to evaluate
the number of live and dead bacteria at each location, and the average number for
each disk was calculated.
Statistical Analysis
The following examinations were performed with the data and the images. Using the
SEM micrographs, the structural morphology of the plaque biofilm in all groups was
evaluated and compared among the groups to identify and describe any effects of the
RF treatment on the morphology of the biofilm at each assessment time point (24, 48,
and 72 hours). Also, the effects of RF on biofilm morphology were confirmed by comparing
the morphology of the biofilm in paired RF-treated versus non-RF-treated similar groups,
that is, by comparing A versus D, B versus E, and C versus F at each sampling time
point (24, 48, and 72 hours). Similarly, using the TEM micrographs, the effects of
RF on the ultrastructure of the bacteria (cell wall and its components) were examined
and compared between similar RF-treated and non-RF-treated groups (A vs. D, B vs.
E, and C vs. F) at each sampling time point (24, 48, and 72 hours). Using the results
from the CSLM, the effects of RF on the bacteria viability in each group were determined
by comparing the percentage of viable cells between similar RF-treated and non-RF-treated
groups (A vs. D, B vs. E, and C vs. F) at each sampling time point (24, 48, and 72 hours)
using the two-way analysis of variance (ANOVA) with Bonferroni post-tests.
The primary hypothesis was that the application of 3 -MHz bipolar RF delivered via
a power toothbrush electrode during and after biofilm formation would cause structural
disintegration of dental plaque, disruption of the bacterial cellular component, and
reduction of the number of live bacteria within the plaque. At the same time, the
non-RF-treated control groups would exhibit none of these effects. This was tested
statistically by STATA software version 17.0 (Stata Corp LP, United States) at a significance
level of 5%. All pairwise contrasts (RF-treated vs. non-RF treated and between time
points) were tested with ANOVA, followed by Tukey's honestly significant difference
(HSD) test for multiple hypothesis adjustment.
Results
Confocal Laser Scanning Microscope Analysis
In [Fig. 2 ], significant differences were observed in cell viability between the RF-treated
and non-RF-treated groups. Overall, the biofilm treated with RF (groups D–F) showed
a significant (p < 0.5) reduction in the percentage of viable bacteria cells when compared with the
untreated biofilm (A–C) that underwent a similar protocol. Without the toothpaste,
this effect was not statistically significant at the 24-hour measurement period but
became significant as the thickness of the biofilm increased at 48 and 72 hours. However,
with toothpaste, the effect was significant (p < 0.05) at all time points. At each time point, there was a significant (p < 0.05) reduction in the percentage of viable bacteria cells with CTS than with TS,
with or without RF treatment, except at 48 hours within the non-RF-treated groups
([Fig. 3 ]).
Fig. 2 Combined graph for the three experimental days (24, 48, and 72 hours). Two-way ANOVA
with Bonferroni post-tests was performed using GraphPad prism version 6.04, and all
p values are two-sided and considered significant at p < 0.05. (*p < 0.05, **p < 0.01, ***p < 0.001).
Fig. 3 (a ) Scanning electron microscope images of 24-hour biofilm treated (D–F ) and untreated (A–C ) with radiofrequency (RF) energy and paired groups underwent a similar protocol.
(b ) Scanning electron microscope images of 48-hour biofilm treated (D–F ) and untreated (A–C ) with RF and paired groups underwent a similar protocol. (c ) Scanning electron microscope images of 72-hour biofilm treated (D–F ) and untreated (A–C ) with RF and paired groups underwent a similar protocol.
Scanning Electron Microscope Analysis
The SEM micrographs ([Fig. 3 ]) of the non-RF-treated groups (A–C) showed a well-formed biofilm with intact structural
colonization at each time point (24, 48, and 72 hours). However, in the samples treated
with RF, the biofilm morphology (architecture) was substantially disrupted at each
growth period. The biofilm morphology showed only a little difference among the three
biofilm growth periods (24, 48, and 72 hours) in groups (B–D) treated with either
toothpaste or RF, unlike the groups treated with combined RF and toothpaste (groups
E and F). Basically, the SEM result showed that toothpaste enhanced the effectiveness
of the RF against dental plaque.
Transmission Electron Microscope Analysis
The TEM micrographs ([Fig. 4 ]) showed bacteria in the biofilm grown with neither toothpaste nor RF treatment (group
A) to have a typical shape, intact peritrichous flagella, peptidoglycan layer, and
cytoplasmic membrane. In groups treated with either CTS (C) or RF (D), meager differences
were observed, like lysed cells with broken walls and membranes and huge vacuoles.
On the other hand, the bacteria in the biofilm (group F) treated with combined CTS
and RF demonstrated morphological changes, including compromised cell wall and reduction
and unevenness in electron density in the cytoplasm with the appearance of “ghost
cells.” This again indicates more effectiveness with combined toothpaste and RF treatment.
Fig. 4 (a ) Transmission electron microscope images of bacteria in 24-hour multispecies biofilm
treated (D,F ) and untreated (A,C ) with radiofrequency (RF) energy. (b ) Transmission electron microscope images of bacteria in 48-hour multispecies biofilm
treated (D,F ) and untreated (A,C ) with RF energy. (c ) Transmission electron microscope images of bacteria in 72-hour multispecies biofilm
treated (D,F ) and untreated (A,C ) with RF energy.
Discussions
The efficiency of manual toothbrushing to significantly eliminate plaque biofilm to
maintain good oral health is contingent upon the individuals' manual dexterity, the
type of toothbrush used, and the brushing technique, time, and frequency.[8 ] This has led to varieties of powered toothbrushes becoming more widely used as an
alternative to manual toothbrushes, to provide more efficacious brushing than manual
toothbrushes.[9 ] Among these powered toothbrushes is the ToothWave (Home Skinovations, Israel), which
uniquely uses bipolar RF energy for its effectiveness in reducing exogenous deposits
on tooth surfaces. Previous studies have shown that this RF-utilizing toothbrush significantly
reduces extrinsic tooth stains, plaque, and calculus.[14 ]
[15 ]
[22 ] However, no studies have established the mechanism by which the RF energy causes
the reduction in dental plaque deposits. Thus, the present study investigated the
effects of bipolar RF energy applied through a power toothbrush on the dental plaque's
structural morphology and the bacteria's ultrastructure. In the current study, to
ensure that it is only RF current affecting the changes in the biofilm, both the mechanical
vibration and the oscillating rotatory movements of the RF toothbrush were excluded
by treating each biofilm at a 1-mm distance between the surface of the toothbrush
bristles and the biofilm surface ([Fig. 1 ]). The toothbrush was activated on mode 4 (RF only, no vibration). The study demonstrated,
at every plaque thickness (24, 48, and 72 hours), a substantial disruption of the
plaque architecture (chain formation) in the biofilms treated with RF, while the biofilms
not treated with RF energy showed well-formed biofilm with intact structural morphology
([Fig. 3 ]). This effect was observed to be grossly enhanced when RF treatment was combined
with toothpaste (TS or CTS) treatment but less in the biofilm treated with either
RF or toothpaste only at every plaque thickness ([Fig. 3 ]). This result confirmed our hypothesis that the application of 3-MHz bipolar RF,
delivered via a power toothbrush RF electrode after biofilm formation, would cause
structural disintegration of dental plaque. The RF energy in ToothWave is a low-power
alternating electrical current (AC), which streams back and forth between two electrodes
and over a silicon barrier, providing a localized effect that is limited to the surface
of the substrate on which the plaque is formed. This process is based on the principle
of polarity, which states that every element in nature has a positive or negative
charge.[24 ] The plaque disintegration effect observed in the present study, even at 3-day plaque
thickness (matured plaque), can be attributed to the hypothesized mechanism of action
of the ToothWave toothbrush, which involves a process in which the alternating current
from the RF technology brings charged molecules that originate from the toothpaste
to the dental plaque, to destabilize the electrostatic bonds between the individual
bacterial cells and between the substrate on which the plaque is formed and the plaque
that was attached firmly to the substrate. The high frequency of the AC set by the
RF parameters allows for safely increasing the electrical power as opposed to direct
current, thus achieving significant results.[25 ] Moreover, the RF current tends to flow along the surfaces of electrical conductors,
known as the “skin effect,”[26 ] and thus directs the current toward the plaque surface. The electromechanical silicon
barrier, located between the ToothWave electrodes, additionally contributes to its
increased efficacy. The enhanced efficacy in disintegrating dental plaque observed
with the introduction of toothpaste (TS or CTS) supported our suggested mechanism
of action that the electrically charged toothpaste ingredients take part in the process
that occurs in the biofilm. Toothpaste is a water-based complex mixture of abrasives
and surfactants, humectants, binders, and other active ingredients. As such, it contains
charged molecular compounds that act as electrolytes in the medium once the RF is
activated, carry the charges within the plaque, and achieve the desired effect. The
fact that the effect of the RF energy was observed at varying plaque thicknesses,
even at 3-day plaque thickness, indicated that the effect of the RF energy was not
limited to the surface of the plaque but penetrated deep into the entire thickness
of a matured plaque.
The observed action of RF energy on the plaque in the present study is strongly believed
to be responsible for the report of a previous comparative 6-week study that examined
the effect of an RF-utilizing toothbrush on calculus, plaque, and gingival inflammation
and reported a statistically more significant reduction in plaque and gingivitis in
the test group that used RF toothbrush compared with the control group that used sonic
vibrating toothbrush.[15 ] It is believed that the process described earlier by which RF energy disintegrates
plaque architecture would apply to any exogenous materials (e.g., stains) that are
firmly attached to a substrate layer, such as tooth surface, when an RF-utilizing
toothbrush is used on such surface. For this reason, the result of the present study
can be presumed to be responsible for the observation in another previous study that
investigated the effect of this RF-utilizing toothbrush on stain deposits on the tooth
surface and reported a more significant reduction in extrinsic dental stains with
RF-utilizing toothbrush compared with the control sonic vibratory toothbrush.[14 ] These previous studies and the present study further support the beneficial effects
of this unique technological feature, which utilizes RF energy that streams on the
tooth surface during brushing.
We further visualized the established and treated biofilms with TEM to examine the
ultrastructure of individual bacteria. TEM is a standard histology technique for viewing
the ultrastructure of bacteria cells. It enables the cellular structures that allow
the cell to function properly within its specific environment to be examined at an
ultrastructural level.[27 ] TEM analysis of the dental plaque showed the bacteria in the biofilm grown with
neither RF treatment nor toothpaste treatment to have all the typical structures and
characteristics of live bacteria. At the same time, the bacteria components of the
biofilm treated with either CTS or RF alone displayed all characteristics of lysed
cells, such as broken walls and membranes and huge vacuoles ([Fig. 4 ]). Again, this effect was more pronounced with combined CTS and RF in which the bacterial
cells demonstrated more morphological changes, including compromised cell wall and
reduction and unevenness in electron density in the cytoplasm with the appearance
of “ghost cells” ([Fig. 4 ]). This demonstrated that the cells' inner structure seemed strikingly affected by
RF treatment. This observation can be attributed to the long-established effect of
RF energy. RF is a type of dielectric heating that has the potential for uniform and
rapid heating of samples through ionic conduction.[28 ] In the presence of an alternating electric field induced by RF, the displacement
of ions with opposite charges leads to an increase in the kinetic energy of the molecules
and, thus, to the rise in the temperature of the samples, in this case, the plaque
biofilm. The DNA of microorganisms absorbs the heat generated inside the biofilm because
of RF radiation, which subsequently changes their physical structure and reduces function.[29 ] For this reason, the RF has the capacity to lyse the bacterial cell wall and degenerate
the cellular structures, with the charged molecules from the toothpaste being contributing
factors. Furthermore, RF energy can also generate active molecules like singlet oxygen
that have strong oxidative properties for action on stains and the destruction of
cells.[30 ] This bactericidal action of the RF current, which was evident with the three plaque
thicknesses and more significant in the presence of toothpaste, was confirmed by the
result of the cell viability analysis with CLSM that showed a significant reduction
in the percentage of viable bacteria cells in the biofilm treated with RF when compared
with the biofilm not treated with RF energy. This result also accepted our hypothesis
that the application of 3-MHz bipolar RF delivered via a power toothbrush RF electrode
after biofilm formation would disrupt the bacterial cellular component. The antimicrobial
action of the RF indicates that the RF energy acts on multiple targets, bacterial
chain formation, and bacterial components. This must undoubtedly have contributed
to the reduction in plaque observed with the RF treatments.
Although the present study used the periodontal disease-causing three red complex
bacteria (P. gingivalis , T. denticola , and T. forsythia ) to develop a multispecies dental plaque, it is strongly believed that the ability
of the RF energy to kill the bacteria and disintegrate its structural morphology is
broad spectrum and not species specific. The result of the present study indicates
that the approach of using an RF-utilizing power toothbrush has the potential to contribute
toward a better oral hygiene procedure and could be a useful device to eradicate biofilm
formation within the oral cavity and, as such, control the two major oral diseases,
dental caries and periodontal diseases. Furthermore, considering that calculus is
formed by calcification of undisturbed dental plaque by calcium in the saliva, undoubtedly,
the destruction of bacteria and their associated biofilm will reduce the rate of calculus
formation on tooth surfaces in individuals using the RF-utilizing toothbrush for their
routine oral hygiene.
The present study demonstrated that the application of 3-MHz bipolar RF delivered
via a power toothbrush RF electrode can disrupt dental plaque morphology and kill
its component bacteria cells. The study further demonstrated that the effect of RF
energy on plaque was enhanced by the combined application of RF and toothpaste applied
as either TS or supernatant of CTS. Furthermore, the effect of RF energy in the present
study can be presumed to be the mechanism by which the RF-utilizing toothbrush caused
more reductions in plaque and gingivitis as well as extrinsic dental stains in groups
that used RF toothbrushes compared with the control group in previously reported studies.
One of the limitations of this study was not comparing the RF-utilizing toothbrush
with a non-RF toothbrush. However, we considered the experimental groups that were
subjected through the same protocol but not treated with RF energy as adequate control
groups for the study objectives. Other limitations were not including a simple thermal
camera to show whether or not heating takes place and lack of exhaustive control of
confounding factors, such as having more groups with (1) RF electrodes without bristles;
(2) nonworking toothbrush without RF; (3) only a vibrating toothbrush, with no RF;
and (4) RF generated by a different means. We are presently investigating these factors
in our current ongoing study.
