1. health and fitness
2. disease

Poster Number: 3067
QUANTIFICATION OF PLAQUE REMOVAL
USING A BRUSHING SIMULATOR
J. Latimer1, J. Munday1, S. Forbes1, P. K. Sreenivasan2, and A.J. McBain1
1
Manchester Pharmacy School, University of Manchester, Manchester, England, UK; 2Colgate-Palmolive Technology Center, Piscataway, New Jersey, USA
Abstract
Objective: This study describes the development of methods that incorporate the combined use of plastic
typodonts, simulated plaques and a mechanical brushing simulator to assess the effects of a variety of toothbrush/dentifrice regimens. The development of a custom model with which to cultivate saliva-derived biofilms
on typodont surfaces is also described.
Methods: Plastic typodonts were coated with a layer of colored simulated plaque comprising powder paint and
brushed using a mechanical brushing simulator. Images were captured under controlled light conditions before
and after brushing and color differences were measured using specialist software. This method was used to
compare simulated plaque removal from occlusal surfaces before and after brushing and from interproximal areas
after brushing with water or a 1450 ppm fluoride toothpaste in a silica base (FTP) and a multi-level trim toothbrush.
A custom drip-flow model was developed, using salivary inocula to cultivate oral biofilms on typodont surfaces to
evaluate plaque removal by a new toothbrush in comparison to an old toothbrush with frayed bristles. Biofilms were
stained and brushed with new or worn brushes to simulate 3 months of use to assess differences in biofilm removal.
Results: Image analysis of typodonts brushed under controlled conditions was able to significantly distinguish
between brushed and unbrushed surfaces (p<0.05). Further, this method was able to distinguish between brushing
with water or with FTP on the removal of simulated interproximal plaque, the latter causing significantly greater
reductions (p<0.05). The custom-built drip-flow model was highly effective in cultivating oral biofilms on typodonts.
These model biofilms were effective in demonstrating that new toothbrushes remove significantly more plaque
from surfaces than brushes which had been worn by repeated brushing (p<0.05).
Conclusion: Laboratory-based models using simulated plaque or oral biofilms, in conjunction with a brushing
simulator, were successfully able to quantify a variety of toothbrush/dentifrice regimens.
Introduction
Results
The brushing model was able to detect significant changes in simulated plaque after brushing: We developed a
brushing model in which typodonts were coated with an artificial plaque, brushed in a controlled way on a brushing
simulator and imaged to quantify plaque removal. As a final validation step, we assessed the ability of the model to
clearly distinguish between brushed and unbrushed surfaces. Figure 3 shows that brushed and unbrushed surfaces
were clearly discernible and that image analysis allowed significant distinction of surfaces after brushing.
A fluoride toothpaste (FTP) removed more simulated interproximal plaque than water: Using the brushing
model, we compared the effectiveness of FTP and water to remove simulated plaque from interproximal regions
between the tooth structures of typodonts. FTP removed more plaque than water (93% versus 73%). This difference
was statistically significant (p<0.05)
New brushes remove more plaque than worn brushes. We developed a custom-built drip-flow model for the
cultivation of dental plaque on typodont surfaces. This model was successfully used to cultivate thick biofilms
which were subsequently stained with a disclosing solution. These biofilms were used in the brushing model to
compare the effectiveness of a new toothbrush with an artificially-worn toothbrush. Both toothbrushes removed
statistically significant (p<0.05) levels of plaque from typodont surfaces. However, the new toothbrush removed
statistically significantly (p<0.05) more plaque (93.9%) than the worn toothbrush (71.4%,)
Figure 3. Brushing caused significant changes in simulated plaque. The molars of three plastic typodonts
were coated with simulated plaque and brushed with an unused toothbrush under constant pressure and
brushing pattern. Simulated plaque was then visualised under controlled light conditions. Removal of
simulated plaque is indicated by absence/reduction of green colouration (A). The red channel was isolated,
greyscaled and inverted to differentiate between areas with or without simulated plaque (B). The occlusal
surfaces of each of three molars of each typodont were isolated and light intensity was measured using
ImageJ (B). Significantly less plaque was measured on brushed surfaces (p<0.05)
Unbrushed
Brushed
Unbrushed
Brushed
Oral bacteria continually grow on tooth surfaces, forming complex biofilms termed dental plaque. Plaque bacteria
such as Streptococcus mutans and lactobacilli may overproduce acetic or lactic acids. These acids contribute to the
formation of dental caries, one of the most common diseases globally. If plaque is left uncontrolled, increases in
the relative abundance of gram-negative anaerobes can lead to gingivitis and periodontitis. Regular brushing with
a toothbrush and a dentifrice is a recommended and globally-adopted method of controlling plaque. Inevitably,
brushing techniques differ between individuals in factors including duration, pressure and coverage. These interindividual variations may confound attempts to reproducibly identify and accurately quantify the effects of, for
example, brush type or dentifrice on their ability to remove plaque. We have developed methods by which to
visualise and quantify plaque removal in-vitro, in a reproducible manner, using simulated or real plaque. We
describe how these methods may be used to differentiate the effects of water vs a fluoride toothpaste in a silica
base and worn vs unworn brushes.
Materials and Methods
Development of a reproducible brushing model: Plastic typodont models (Baistra Medical Instruments,
Zhengzhou, China) were evenly coated with simulated plaque. Green marker spray (Occlude, Pascal, Bellevue, WA,
USA) was found during validation studies to be the most suitable substance for use as a simulated plaque (data not
shown). Coated typodonts (up to 8 per experiment) were mounted onto a brushing simulator (model ZM-3.8, SD
Mechatronik, Feldkirchen-Westerham, Germany, Figure 1.). Unused soft, full-head multi-level toothbrushes
(Colgate-Palmolive Company) were mounted on the brushing simulator and used with a pressure of 200 g for 30
sec using a zig-zag motion at 106 strokes per minute. These parameters were found during validation studies to be
the most suitable for consist removal of simulated plaque (data not shown)
Image capture and analysis: Simulated plaque on treated and untreated typodonts was visualised under controlled
light conditions using a digital SLR camera (D3200 with an AF-S DX Micro NIKKOR 40mm f/2.8G lens, Nikon, Tokyo,
Japan). Removal of simulated plaque was indicated by absence or reduction of green colouration. Using specialist
image analysis software (ImageJ, National Institutes of Health, Bethesda, Maryland, USA) each of three top-down
images were greyscaled and mean light signal was calculated for the total occlusal surface or interproximal region
of each of three molars (unless stated otherwise).
Simulated plaque removal: Three molars on each of three typodonts were coated with simulated plaque, as described
above. The occlusal surfaces of the molars were brushed, as described above. Relevant top-down images were
captured and analysed, as above, before and after treatment. Mean levels of simulated plaque remaining on surfaces
with or without brushing were compared. Student’s t-test was applied, reporting significance at p<0.05 (n=9).
Removal of simulated interproximal plaque – water vs dentifrice: Three molars on each of three typodonts were
coated with simulated plaque, as described above. Typodont sections were mounted on the brushing simulator
such that the facial surfaces could be brushed across the teeth, distally to medially. Toothbrush heads were saturated
in distilled water or a 1:3 slurry of a 1450 ppm fluoride toothpaste in a silica base (FTP) before treatment and analysis
as described above. Mean levels of simulated plaque remaining on surfaces treated with water or FTP were compared.
Student’s t-test was applied, reporting significance at p<0.05 (n=6).
Development of a custom-built drip-flow model to cultivate dental plaque on typodont surfaces: Polycarbonate
vacuum-filter units (Sartorius, Goettingen, Germany) were adapted for use as sterile housings in which to contain
typodont sections and a waste vessel (Figure 2.). A medium vessel containing artificial saliva medium (mucin, 2.5
g/liter; bacteriological peptone, 2.0 g/liter; tryptone, 2.0 g/liter; yeast extract, 1.0 g/liter; NaCl, 0.35 g/liter; KCl, 0.2
g/liter; CaCl2, 0.2 g/liter; cysteine hydrochloride, 0.1 g/liter; hemin, 0.001 g/liter; and vitamin K1, 0.0002 g/liter)
was attached via a peristaltic pump running at approximately 4 ml h-1. Medium was delivered drop-wise onto the
typodont for 72 h at 37 oC and surfaces were inoculated daily with the saliva of a healthy adult volunteer (M, 34
years, 2 ml).
Figure 4. A fluoride toothpaste (FTP) removed more simulated interproximal plaque than water. Simulated
plaque was applied evenly to typodont surfaces (n=12), which were then affixed to the brushing machine.
Toothbrushes were saturated with a 1:3 slurry of FTP and used to brush surfaces under constant pressure
and brushing pattern. Distilled water was used a control. Example images are shown. Representative images
(A) and data (B) show removal of simulated plaque from the interproximal regions. FTP removed significantly
more simulated plaque than water (p=0.017).
A
B
Removal of dental plaque – new vs used brush: Dental plaques were cultivated on typodonts using the drip-flow
model described above. Surfaces were immersed in a plaque disclosing solution (Tri Plaque ID gel, GC, Alsip, IL,
USA) diluted 1:2 in distilled water, resulting in plaque stained in pink or purple. Stained typodont sections were
mounted on the brushing simulator such that occlusal surfaces could be brushed, as above. Two types of brush
were mounted on the simulator; new or artificially-worn. All toothbrush heads were saturated in distilled water
before use. Relevant top-down images were captured and analysed, as above, before and after treatment. Mean
levels of plaque remaining on surfaces after brushing with new or worn brushes were compared. Student’s t-test
was applied, reporting significance at p<0.05 (n=2).
Figure 1. A typical experimental set-up. Up to eight typodonts were coated with simulated plaque and
mounted with brushes on the brushing simulator. Typodonts were brushed with a constant brushing pattern
under constant pressure for a specified period of time. Typodonts were then photographed and the images
analysed to quantify removal of simulated plaque.
Figure 5. New brushes remove more plaque than worn brushes. Oral biofilm was cultivated, stained and
brushed with a wetted toothbrush (new or worn) under constant pressure and brushing pattern. Images
show stained plaque before and after brushing. The red channel was isolated, greyscaled and inverted to
differentiate between areas with or without plaque). The occlusal surfaces of two molars were isolated and
light intensity was measured using ImageJ. Data show background-corrected mean signal intensity. Higher
numbers indicate higher levels of plaque. Dark grey bars represent plaque levels before brushing, while light
bars represent plaque levels after brushing. Error bars show standard deviations (n=2). Both brush types
caused significant reductions (* p<0.05). The new brush removed significantly more biofilm than the worn
brush (# p<0.05)
Figure 2. Development of a custom-built drip-flow model for the cultivation of dental plaque on typodont
surfaces. A, medium vessel containing artificial saliva; B, peristaltic pump running at approximately 4 ml h-1;
C, capillary delivering medium, drop-wise, onto a plastic typodont surface (D) in a sterile housing comprising
a growth vessel and a waste vessel (E). The model was run for 72 h at 37oC and surfaces were inoculated daily
with the saliva of a healthy adult volunteer (2 ml). Visible plaque was evident following this growth phase.
Discussion
The current study describes reproducible models with which to visualise and quantify
plaque removal in vitro. These models use a brushing simulator in conjunction with
controlled image analysis to remove simulated plaque or live saliva-derived biofilms from
typodont surfaces. Using these models, we have effectively demonstrated that a standard
fluoride toothpaste is able to remove significantly more simulated plaque than water
alone, and that a new toothbrush with undisturbed brush heads was able to remove
significantly more biofilm than an artificially-worn toothbrush.
IADR/AADR/CADR 2014 Annual Meeting
Boston, Massachusetts March 11-14, 2015
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