UNIVERSIDADE ESTADUAL PAULISTA
“JÚLIO DE MESQUITA FILHO”
Instituto de Ciência e Tecnologia
Campus de São José dos Campos
ORIGINAL ARTICLE DOI: https://doi.org/10.4322/bds.2024.e4015
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Braz Dent Sci 2024 Jan/Mar;27 (1): e4015
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Can Scanning Electron Microscopy be used to quantitatively
measure dentine roughness?
A microscopia eletrônica de varredura pode ser usada para medir quantitativamente a rugosidade da dentina?
Shivaughn Maria Marchan1 , Marsha IVEY2 , Amrita RAJHBEHARRYSINGH1 , Kelee BASCOMBE3
1 - The University of the West Indies, School of Dentistry, Unit of Restorative Dentistry, Mount Hope Medical Complex, Champs Fleurs,
Trinidad and Tobago
2 - The University of the West Indies, Public Health and Primary Care, Unit of Paraclinical Sciences, Mount Hope Medical Complex,
Champs Fleurs, Trinidad and Tobago
3 - The University of the West Indies, School of Dentistry, Unit of Child Dental Health, Mount Hope Medical Complex, Champs Fleurs,
Trinidad and Tobago
How to cite: Marchan SM, Ivey M, Rajhbeharrysingh A, Bascombe K. Can Scanning Electron Microscopy be used to quantitatively
measure dentine roughness. Braz Dent Sci. 2024;27(1):e4015. https://doi.org/10.4322/bds.2024.e4015
ABSTRACT
Objective: The aim of this study was to determine if SEM used with imaging software could be used to
quantitatively determine the extent of dentine roughness due to tooth-brushing. Material and methods: Flat,
polished dentine surfaces were subjected to 25,000 cycles of simulated tooth-brushing at 2 strokes per second
with a load of 200 g. At the end of the brushing cycle, dentine surfaces were rst assessed using prolometry
and then subjected to SEM analysis. Ra (average roughness) readings were obtained for prolometric
assessment. Using imaging software, the horizontal distance between adjacent characteristic grooves noted on
micrographs was measured, ensuring that the middle of each crest of the associated groove was used as the
reference point, the Crest-to-Crest distance (C-C distance). These two parameters were examined statistically
for correlation. Results: When Spearman Rank tests were utilized the correlation between average Ra and
crest to crest measurements was 0.709 (p <0.01). The Bland Altman plot, however, showed poor agreement
between the two test parameters. Conclusion: Further work is needed to validate the use of this methodology
in the quantitative assessment of tooth surface loss due to abrasion.
KEYWORDS
Abrasion; Bland-Altman; Dentine roughness; Prolometry; Scanning electron microscopy.
RESUMO
Objetivo: O objetivo deste estudo foi determinar se o MEV usado com software de imagem poderia ser
usado para determinar quantitativamente a extensão da rugosidade da dentina devido à escovação dentária.
Material e Métodos: Superfícies dentinárias planas e polidas foram submetidas a 25.000 ciclos de escovação
dentária simulada a 2 movimentos por segundo com uma carga de 200g. No nal do ciclo de escovação, as
superfícies dentinárias foram primeiro avaliadas por perlometria e depois submetidas à análise MEV. Leituras
de Ra (rugosidade média) foram obtidas para avaliação perlométrica. Utilizando um software de imagem, foi
medida a distância horizontal entre sulcos característicos adjacentes observados nas micrograas, garantindo
que o meio de cada crista do sulco associado fosse usado como ponto de referência, a distância crista a crista
(distância CC). Esses dois parâmetros foram examinados estatisticamente para correlação. Resultados: Quando
foram utilizados os testes de Spearman Rank, a correlação entre a média de Ra e as medidas de crista a crista foi
de 0,709 (p<0,01). O gráco de Bland Altman, no entanto, mostrou fraca concordância entre os dois parâmetros
de teste. Conclusão: Mais trabalhos são necessários para validar o uso desta metodologia na avaliação quantitativa
da perda da superfície dentária devido à abrasão.
PALAVRAS-CHAVE
Abrasão dentária; Escovação dentária; Dentina; Propriedades de superfície; Microscopia eletrônica de varredura.
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Braz Dent Sci 2024 Jan/Mar;27 (1): e4015
Marchan SM et al.
Can Scanning Electron Micr oscopy be used to quantitatively measure dentine roughness?
Marchan SM et al. Can Scanning Electron Microscopy be used to quantitatively
measure dentine roughness?
Can Scanning Electron Microscopy be used to quantitatively
measure dentine roughness?
INTRODUCTION
Methodologies that assess tooth brushing
abrasion are well described in the literature.
Such in vitro tests of dental hard tissue abrasion
are based on an assessment of surface roughness
and the differences between surface roughness
measurements before and after an abrasive
challenge [1]. Using profilometric methods,
differences in roughness between worn and
unworn surfaces can be calculated to understand
the amount of tooth structure lost [1]. In vitro
methods of determining tooth surface loss
(abrasion and/or erosion) have included
measurements of changes in either linear or
volumetric surface roughness [2,3]. Increases
in surface roughness indicate increasing tooth
surface loss [4,5].
While contact profilometry can be used
to assess tooth wear due to abrasion, the
use of contact profilometry to assess tooth
wear due to a combination of both abrasion
and erosion is limited, since contact with an
acid-softened enamel or dentine surface may
produce erroneous roughness measurements [6].
With mixed abrasive/erosive lesions, surface
roughness should be assessed using non-contact
profilometry in assessments of roughness to
mitigate misleading results on already softened
enamel or dentine surfaces caused by acidic
challenges [6]. However, the use of contact
prolometry to assess the effects of tooth-brushing
abrasion is still relevant due to the nature of bulk
tissue removal caused by prolonged interaction
and motion of toothbrush bristles with tooth
structure, mediated by toothpaste [4].
Visual analysis of micrographs obtained from
scanning electron microscopy (SEM) has also
been used to assess erosive, abrasive, or mixed
challenges on tooth structure [7]. The use of SEM
has been mainly qualitative with descriptions of
the alterations of either dentin or enamel [7,8].
A review of the literature reveals few descriptions
of quantitative methods assessing dentin abrasion
using micrographs obtained from SEM.
As part of a larger study, the authors
demonstrated a distinct and characteristic
pattern of grooves on dentin surfaces due to
simulated toothbrushing with a soft-bristled
toothbrush and toothpastes with known
abrasives. Such characteristic grooves: with
distinct and identiable peaks and depressions,
were previously described, but not visualized, in a
prolometric study of dentine abrasion [9]. Initial
comparisons of micrographs revealed variations
in created groove patterns in terms of relative
width when brushed with different toothpastes.
This study aims to validate a methodology for
quantifying roughness changes in dentin as a
result of long-term tooth-brushing abrasion
using micrographs obtained from SEM. The null
hypothesis stated there would be no signicant
linear relationship different from zero between
average roughness Ra and C-C measurements
when used to measure the surface roughness.
MATERIALS AND METHODS
An exemption to ethical approval was given by
the university’s ethics committee prior to the start
of the larger study (CREC-SA.0181/02/2020).
The power analysis (G Power, Universitat Kiel,
Germany) was performed as part of a larger
study, on toothbrushing abrasion with various
types of toothpastes, that calculated the amount
of samples in each experimental group at 6 given
an effect size of 0.25, and a power of 0.8.
The occlusal thirds of human molars were
removed using a diamond water-cooled saw
to reveal dentine with a periphery of enamel.
Exposed dentin was polished using decreasing
grits of silicon carbide paper, 600, 800, 1000,
1200 grit, and culminating in 1500 grit. Baseline
average Ra measurements were taken using a
prolometric technique using Mitutoyo Surftest
401 surface roughness analyzer (Mitutoyo America
Corporation, Aurora, IL, USA) with a cutoff of
0.25 mm, a transverse length of 1.6 mm, a sample
length of 1.25 mm, and a vertical bandwidth of
50µm. For each evaluated surface, 3 random
readings were taken to give a mean Ra for each
specimen. Only samples with mean roughness
≤ 0.4µm were included. Samples were either
polished until the required value for inclusion
was obtained or the specimen was replaced
by another altogether. The specimens were
allocated to one of 5 groups; one negative control
(distilled water) and four experimental groups
of charcoal containing toothpastes with known
abrasive particles (Table I). Auto-polymerizing
polymethylmethacrylate (Dentsply, NC, USA)
was mixed according to the manufacturer’s
instructions and poured into PVC cylinders
(20mmX12mm). The prepared teeth were
subsequently mounted into the auto-polymerizing
resin and placed in a simulated tooth-brushing
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Marchan SM et al.
Can Scanning Electron Micr oscopy be used to quantitatively measure dentine roughness?
Marchan SM et al. Can Scanning Electron Microscopy be used to quantitatively
measure dentine roughness?
machine (Toothbrushing Simulator MEV 4X-3D,
Odeme Dental Research, Brazil) and subjected to
brushing for 25,000 cycles using a soft, circular
bristled toothbrush (Colgate Extra-Clean Full
Head, Colgate Palmolive Company, NY, USA)
at 2 strokes per second with a load of 200 g.
All specimens in each of the ve groups were
brushed for 25,000 cycles. Mounted specimens
were brushed perpendicular to the direction
in which baseline Ra readings were taken.
The entire surface of each specimen was exposed
to toothbrush bristles and toothpaste slurry
(1:1 ratio with distilled water by volume) at all
times during simulated brushing. Specimens were
thoroughly washed with distilled water, lightly
blotted and Ra readings were immediately taken
using the same prolometric standards used for
baseline measurements.
All the specimens were vacuum desiccated
(Ted Pella Inc, Redding, CA, USA) for 30 minutes,
mounted on aluminum stubs (Pelco, Redding,
CA, USA), and sputter coated (Denton Vacuum
LLC, Moorestown, NJ, USA) with a homogenous
coating of gold. Micrographs were obtained using
a scanning electron microscope (Philips SEM 515,
Eindhoven, Netherlands) operating at 30kV and
at an operating distance of 12mm. Micrographs
were taken at various magnications. Micrographs
with at least 3 of these distinctive grooves were
chosen for analysis. Imaging software (Image J,
National Institutes of Health, Stapleton, NY) was
used to attain linear horizontal measurements
from the middle of each crest to the middle of
the adjacent crest, the Crest-to-Crest distance
(C-C distance). Prior to quantifying linear
measurements, the scale bar for each micrograph
was used to calibrate the ImageJ software as it
related to linear measurement in micrometers
of that micrograph. Only one C-C measurement
was taken for each distinctive groove. Three
measurements were randomly taken of each
selected micrograph for each specimen.
Using SPSS (Statistical Package for Social
Sciences) Version 24 (IBM, Chicago) the means
and standard deviations of C-C (Crest to Crest)
distance and Ra (Average Roughness) were
calculated for each tested toothpaste. Scatter plots
followed by tests of correlation (Spearman-Rank)
were used to look for an association between the
two quantitative measurements. A Bland-Altman
plot was used to look for agreement between
mean Ra values and mean C-C distance.
RESULTS
Mean Ra and C-C distance together with
their standard deviations can be seen in Table II.
Micrographs of the dentine surfaces brushed
with various charcoal containing toothpastes are
shown in Figures 1-5. The characteristic grooves
Table I - Materials used in the study
Group Brushing Agent Ingredients Manufacturer
1 Distilled Water
2 Active Wow
Purified Water, Xylitol, Diatomite Diatomaceous Earth,
Baking Soda, Activated Charcoal, Tea Tree Oil, Organic
Coconut Oil, Xantham Gum, Citric acid, Natural Flavors
Active Wow
Toronto, Ontario, Canada
3 Crest 3D White with Charcoal
Sodium Fluoride 0.243%, Water, Sorbitol, Hydrated
Silica, Disodium Pyrophosphate, Sodium Lauryl
Sulphate, Flavor, Cellulose Gum, Sodium Hydroxide,
Sodium Saccharin, Carbomer, Charcoal Powder,
Polysorbate 80, Mica, Titanium Dioxide
Procter & Gamble, Cincinnati
OH, USA
4 Curaprox
Water, Sorbitol, Hydrated Silica, Glycerin, Charcoal
Powder, Aroma, Decyl Glucoside, Cocamidopropyl
Betaine, Sodium Monofluorophosphate, Tocopherol,
Xanthan Gum, Maltodextrin, Mica, Hydroxyapatite
(NANO), Potassium Acesulfame, Titanium Dioxide,
Microcrystalline Cellulose, Sodium Chloride, Potassium
Chloride, Citrus Limon, Peel Oil, Sodium Hydrochloride,
Zea Mays, Starch, Amyloglucosidase, Glucose
Curaden USA Inc
Mesa, Arizona, USA
5 Colgate Essentials with Charcoal
Sodium Monofluorophosphate 0.76%, Water, Hydrated
Silica, Sorbitol, Calcium Pyrophosphate, Glycerin,
PEG-12, Pentasodium Triphosphate, Tetrapotassium
Pyrophosphate, Flavor, Sodium Lauryl Sulphate, Cellulose
Gum, Sodium Saccharin, Charcoal Powder, Xanthan Gum,
Cocamidopropyl Betanine, Blue 1, Red 40, Yellow 5
Colgate-Palmolive Company,
New York, NY, USA
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Can Scanning Electron Micr oscopy be used to quantitatively measure dentine roughness?
Marchan SM et al. Can Scanning Electron Microscopy be used to quantitatively
measure dentine roughness?
were notably absent from the control dentine
samples brushed with distal water only, as shown
in Figure 1. Dentine samples brushed with the
various toothpaste brands look machined in
appearance as shown in Figures 2 to 5.
Figure 6 shows the scatter plot of the
average Ra versus C-C distance. From the graph,
it can be noted that there is a low positive
correlation between the two measurements.
Data was normally distributed for Ra, however,
C-C distances were not normally distributed,
therefore a Spearman-Rank test was used.
However, when Spearman Rank tests were
performed, the correlation between average Ra
and C –C measurements was 0.709 at p <0.01.
Figure 7 shows the Bland-Altman plot for average
Ra versus C-C Measurements. Even though
correlation values were above 0.5, the results
of this plot revealed that there was not good
agreement between the two measured tests in
assessing tooth brushing abrasion.
DISCUSSION
Prolometric methods are very popular in the
assessment of tooth surface loss due to abrasion.
Prolometric measurements also can examine
the complexity of tooth brushing abrasion where
the interplay of factors such as behavioral,
mechanical, chemical, and biological aspects are
important in individual toothbrushing [10].
Linear profilometry was the methodology
employed in the larger study that examined the
abrasive effect of charcoal containing toothpastes on
exposed dentine at varying time points i.e. brushing
cycles. The use of SEM in qualifying the appearance
of abraded dentine happened at the terminal point
of experimentation since surfaces had to be sputter
coated. For this reason, SEM images were obtained
of abraded dentine at the end of a cumulative
abrasive challenge of 25,000 cycles.
In linear prolometric analysis, the vertical
displacement of the contact stylus across the
abraded or roughened surface provides various
roughness parameters with average Ra being
the mean of such deflections over the profile
length [11]. Prolometry measures the crest to
valleys of grooves that are formed because of the
abrasive action of toothbrushing with toothpaste.
In attempting to use SEM to quantify the abrasive
change a horizontal crest to crest measurement
was utilized. The authors compared vertical
Table II - Mean (S.D.) of Dentine Roughness and Crest to Crest Distance
Toothpaste
Brand
Mean (S.D.) Ra
Value
Mean (S.D) Crest
to Crest Distance
Distilled (Control) 0.31 (0.05) 37.81 (6.04)
Active Wow 3.22 (0.83) 92.36 (4.01)
Crest 3D White
with Charcoal 4.03 (1.66) 147.85 (5.10)
Curaprox 4.86 (1.21) 276.57 (13.02)
Colgate Essentials
with Charcoal 4.13 (0.63) 173.69 (18.11)
Figure 1 - SEM of dentine brushed with distilled water (control) X125.
Figure 2 - SEM of dentine brushed with Active Wow Charcoal
Toothpaste X 75.
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Can Scanning Electron Micr oscopy be used to quantitatively measure dentine roughness?
Marchan SM et al. Can Scanning Electron Microscopy be used to quantitatively
measure dentine roughness?
dentine loss with horizontal dentine loss within
the characteristic groves that were formed by
simulated toothbrushing.
While a comparison of the methods showed
a positive correlation with a coefcient ratio for
the Spearman-Rank statistical tests above 0.5,
correlation values approaching 1 do not necessarily
imply good agreement between the two methods
for measuring the effect of toothbrushing on
exposed dentine. In statistical methods, agreement
measures the level of concordance between two
or more sets of measurements [12]. Agreement
is often used to demonstrate if newer methods or
techniques to assess a measurement parameter
can be substituted for known or proven methods.
In this instance, the authors wanted to understand
the use of quantifying crest-to-crest distance may
be an appropriate method for assessing dentine
roughness caused by toothbrushing.
A Bland and Altman plot is a meaningful way
to assess bias between mean differences between
the two methods and to estimate an agreement
interval within which 95% of the differences of the
second method compared to the rst one fall [13].
A Bland-Altman plot is attained by plotting the
difference between the methods versus the mean
of method A and method B (Figure 7). Bland
and Altman concluded that a high correlation
between any two methods designed to measure
Figure 3 - SEM of dentine brushed with Crest 3D white with
Charcoal X137.
Figure 4 - SEM of dentine brushed with Curaprox X156.
Figure 5 - SEM of dentine brushed with Colgate Essentials with
Charcoal X 150.
Figure 6 - Scatter plot of Ra versus Crest to Crest distance.
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Can Scanning Electron Micr oscopy be used to quantitatively measure dentine roughness?
Marchan SM et al. Can Scanning Electron Microscopy be used to quantitatively
measure dentine roughness?
the same property could be attributed to the effect
of widespread sampling [14].
In this study, a typical Bland Altman plot
showing agreement was not achieved despite
good correlation and only three readings taken
for each of the C-C readings. What is not yet clear
is if there would be both good correlation and
agreement between the two methods at earlier
assessment periods less than 25000 cycles of
brushing or if additional C-C readings across each
crest would produce agreement.
Further work would see an expansion of
the methodology in an attempt to validate the
use of SEM to quantitatively measure dentine
roughness due to tooth-brushing abrasion. This
would involve a large sample being brushed with
a single toothpaste and removing a xed number
of samples at each time point to be subjected to
both profilometric and SEM analysis until the
endpoint of the experiment. In this way, data could
be obtained at each time-point for comparison
of the methods. This can yield useful results on
whether these characteristic grooves are produced
at earlier time points and whether they can be
easily measured and positively correlated with Ra
compared with the grooves produced with longer
brushing cycles as seen in this work.
This proposed method holds promise for the
assessment of dental tissue where the combined
effect of erosion and abrasion challenges are being
evaluated where contact prolometry can introduce
error into the methodology because of softened
dental tissue caused by the erosive challenge [15].
Future work will also involve the use of this method
in quantifying changes in dentine caused by
combined erosive and abrasive challenges.
A cost analysis on the use of SEM compared
to prolometry in quantifying roughness caused
by abrasion alone or combined abrasive/
erosive challenges should be considered before
widespread use and acceptance of this method
for the in vitro studies of tooth wear. However,
those institutions that already have a scanning
electron microscope may find this method a
useful methodological adjunct in assessing tooth
wear.
CONCLUSIONS
Within the limitations of this study the
following conclusions may be drawn:
(1) The combination of tooth brushing
with toothpastes produced defects with
characteristic grooves on exposed dentine,
which were visualized using SEM;
(2) Specimens of dentine brushed with water
alone did not show these characteristic
grooves;
(3) The null hypothesis was rejected since a
positive correlation was noted between the
measured C-C distances and Ra;
(4) Poor agreement, between average
roughness (Ra) and C-C distance
underscores the need for further validation
of the study’s ndings.
Acknowledgements
The authors would like to acknowledge the
efforts of Mr. David Hinds, the developmental
engineer at the Department of Physics, The
University of the West Indies for his invaluable
assistance in obtaining the micrographs for
this study.
Author’s Contributions
SMM was involved in project conceptualisation
and development of the methodology. KB and
AR were involved in data curation under the
supervision of SMM. MI completed the formal
analysis. SMM produced the original draft of the
manuscript. All authors reviewed and edited the
nal draft of the manuscript.
Conict of Interest
The authors declare that they have no
conicts of interest with respect to this research.
Figure 7 - Bland Altman Plot of Ra versus Crest to Crest distance.
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Braz Dent Sci 2024 Jan/Mar;27 (1): e4015
Marchan SM et al.
Can Scanning Electron Micr oscopy be used to quantitatively measure dentine roughness?
Marchan SM et al. Can Scanning Electron Microscopy be used to quantitatively
measure dentine roughness?
Can Scanning Electron Microscopy be used to quantitatively
measure dentine roughness?
The authors did not use AI or AI-assisted
writing technologies in the writing process for
this manuscript.
Funding
This research was made possible with a
grant from the Campus Research and Publication
Fund of the University of the West Indies, St.
Augustine (CRP3Nov18.8). The funders had no
role in the planning or execution of the study nor
reviewed any versions of the manuscript prior to
submission for publication.
Regulatory Statement
This study was conducted in accordance
with all the provisions of the local human
subjects oversight committee guidelines and
policies of The University of the West Indies, St.
Augustine. The approval code for this study is
CREC-SA.0181/02/2020.
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Shivaughn Maria Marchan
(Corresponding address)
The University of the West Indies, School of Dentistry, Unit of Restorative Dentistry,
Mount Hope Medical Complex, Champs Fleurs, Trinidad and Tobago
Email: Shivaughn.marchan@sta.uwi.edu
Date submitted: 2023 Aug 26
Accept submission: 2023 Dec 02
