Journal of Dentistry 56 (2017) 105111

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Journal of Dentistry
journal homepage: www.intl.elsevierhealth.com/journals/jden

Micro-CT analysis of naturally arrested brown spot enamel lesions

Mahdi Shahmoradia , Michael V. Swaina,b,*
a
Biomaterials and Bioengineering, Faculty of Dentistry, The University of Sydney, Australia
b
Faculty of Dentistry, Health Sciences Centre, Kuwait University, Kuwait

A R T I C L E I N F O A B S T R A C T

Article history: Objectives: The aim of this study was to characterize the mineral density parameters through natural
Received 25 July 2016 enamel brown spot lesions (BSLs) and to visualize and map their mineral distribution pattern in
Received in revised form 9 November 2016 comparison to enamel whitespot lesions (WSLs).
Accepted 19 November 2016
Methods: Study specimens included seventeen proximal WSLs (ICDAS 1, 2), seventeen proximal BSLs and
seventeen sound proximal specimens (ICDAS 0) collected from The Oral Surgery Department at Sydney
Keywords: Dental Hospital, Sydney, Australia. Imaging was undertaken using a high resolution, desktop micro-
Enamel caries
computed tomography system. A calibration equation was used to transform the grey level values of the
Remineralization
Micro-computed tomography
images into true mineral density values. The qualitative analysis and the quantication of the lesion
Caries arrest parameters including the mineral density and the thickness of the enamel lesion surface layer were
performed using mineral density proles plotted in FIJI and the visualized mineral maps in MATLAB
respectively.
Results: The maps of brownspot lesions revealed irregular demineralization patterns with faint
boundaries and outlines. The regular triangular shape of proximal lesions was recognizable only in some
parts of individual BSLs or was completely unrecognizable within the entire lesion. Scattered free-form
areas of high density enamel were observed within or close to the surface of BSLs. A layer of high density
enamel with a mineral density close to that of sound enamel was observed in all of the BSLs. The mean
mineral density of the body of BSLs, including the scattered areas of high mineral density, was
signicantly higher than the corresponding values in white-spot lesions. The mean thickness of the
surface layer in BSLs (79  15 mm) was also signicantly higher than white-spot lesions (51 11 mm)
(p < 0.05).
Conclusion: This study demonstrated that the mineralization parameters such as density and the
thickness of the surface layer as well as distribution patterns through natural enamel brown spot lesions
(BSLs) are different from enamel white-spot lesions (WSLs). The higher mineral density of the body of the
lesion and the increased thickness of the surface layer in brown spot enamel lesions may suggest possible
subsurface remineralization in the majority of naturally arrested BSLs.
2016 Elsevier Ltd. All rights reserved.

1. Introduction
Enamel caries is the result of the disruption of the microbial
homeostasis of dental plaque and the consequent breakdown of
the mineral equilibrium between enamel and the biolm uid.
Enamel lesions have been classied using a range of criteria based
on their activity, staging and location [1]. Staging criteria such as
International Caries Detection and Assessment System (ICDAS) [2]
* Corresponding author at: Biomaterials and Bioengineering, Faculty of Dentistry,
The University of Sydney, Australia Address: Westmead Center for Oral health, Rm.
2043, Level 2 Westmead Hospital, Westmead, NSW, 2145, Australia.
E-mail address: michael.swain@sydney.edu.au (M.V. Swain).
not only are utilized for the classication of caries, but also provide
the basis for the choice of an adequate therapy. Among various
activity criteria such as color, luster, hardness, roughness and
overlying microbiota [3,4], the color of the lesion represents a
simple and clinically perceived index for the classication of non-
cavitated enamel caries. Accordingly, dull and chalky white enamel
lesions are considered as active demineralization areas [5],
whereas brown-spot lesions are assumed to reect naturally
arrested or hardened enamel caries [68].
Brown-spot lesions (BSLs) are localized brown to black
discoloration of enamel usually located gingival to the contact
area of proximal tooth surfaces. These lesions are commonly
formed at a previously stagnant and plaque retentive region which
has turned into a highly cleansable free-smooth surface such as in

http://dx.doi.org/10.1016/j.jdent.2016.11.007
0300-5712/ 2016 Elsevier Ltd. All rights reserved.
106 M. Shahmoradi, M.V. Swain / Journal of Dentistry 56 (2017) 105111
the proximal surfaces adjacent to extraction sites which have not
been restored for a long time [9]. Due to the absence of the adjacent
tooth in these cases, clinicians can perform direct visual and tactile
examination of proximal BSLs and therefore directly recognize the
possible arrest of the carious process and the hardening of the
demineralized enamel in these lesions.
Some literature has mentioned the faint radiographic evidence
of remineralization in brown-spot lesions and the subsequent
challenges for the radiographic diagnosis of these lesions [10].
Several studies have also shown the increased mineralization and
resistance of the external surface of BSLs [1113]. Despite these
observations, the internal structure and mineral distribution
patterns of brown-spot lesions have not been investigated
extensively or with high resolution methods.
In fact there is a knowledge gap regarding the mineral structure
of the hardened surface layer and the supposed remineralized
subsurface area in these lesions. Concerning the current interest in
the remineralization [14] and non-invasive management of dental
caries [15], understanding the mineral content and microstructural
characteristics of naturally arrested enamel lesions can provide
valuable insights about caries arrest and remineralization pro-
cesses in vivo. Therefore, the aim of this study is to characterize the
mineral density parameters through natural enamel BSLs and to
visualize and map their mineral distribution pattern in comparison
to enamel white-spot lesions (WSLs).
2. Materials and methods
2.1. Study specimens
Extracted molar and premolar teeth were collected from The
Oral Surgery Department at Sydney Dental Hospital, The Universi-
ty of Sydney (Ethics approval protocol No X12-0065 & HREC/12/
RPAH/106). The majority of the teeth with white spot enamel
lesions were extracted for orthodontic purposes from young
individuals aged 1418. Teeth with brown spot enamel lesions
were extracted for periodontal or periapical disease from mature
aged patients. Following extraction, teeth were partially sterilized
in MILTON anti-bacterial solution (0.95% (w/w) sodium hypochlo-
rite, Milton Australia PTY LTD.) for 15 min and then brushed clean
under running water to eliminate any plaque or removable stain.
After decontamination, the samples were stored in Hanks Balanced
Salt Solution (HBSS) at 4  C prior to use. Thymol granules (Sigma
Aldrich, Australia) were added to the solution for disinfection and
prevention of fungal growth.
Two calibrated clinicians visually assessed the specimens and
seventeen proximal WSLs (ICDAS 1, 2) [16], seventeen proximal
BSLs and seventeen sound proximal areas (ICDAS 0) were chosen as
study and control groups. The inclusion criteria for brown spot
enamel lesions, included being non-cavitated with localized dark
Fig. 1. Digital photographs of a non-cavitated BSL showing the intrinsic dark brown
discoloration of enamel. (For interpretation of the references to colour in this gure
legend, the reader is referred to the web version of this article.)
brown discoloration which is intrinsic to enamel and is not an
external surface stain (Fig. 1).
Three hydroxyapatite discs with known low (1.240 g/cm3),
medium (1.406 g/cm3) and high (1.666 g/cm3) mineral densities
were used for grey level calibration and determination of the
mineral density values. Hydroxyapatite discs were located and
xed on top of each specimen before imaging. Details for the
fabrication process and the materials used for constructing the
phantoms are described elsewhere [17].
2.2. Parameters for x-ray micro-tomography scanning and
tomographic reconstruction
Imaging of the teeth and phantoms was performed using a high
resolution fourth generation micro-computed tomography system
(Skyscan 1172, Skyscan, N.V, Aartsellar, Belgium) with an
accelerating source voltage of 100 keV, a source current of
100 mA and an exposure time of 885 ms. Before the scan, teeth
were stored in Hanks Balanced Salt Solution (HBSS) and during
scan they were stabilized in a plastic tube using polystyrene foam,
preventing dehydration of the specimens. Low energy x-rays were
eliminated using an inbuilt lter equal to 1.0 mm thickness of
aluminum and 0.05 mm of copper to restrict spectral bandwidth of
the polychromatic radiation. The equivalent monochromatic
energy spectrum of ltered x-ray had an effective mean energy
of 60 keV. The long axes of the teeth were parallel with the center
of rotation of the mounting device. During the scanning process,
the samples were rotated over 360 at angular increments of 0.14
generating 2570 two-dimensional shadow projections with an
image matrix of 2000 pixels  1048 pixels. These images were
saved as 16 bit Tagged Image File Format (TIFF) and consequently
exported to a 3-D cone beam reconstruction program (NRecon
software, version 1.4.4; SkyScan) for the reconstruction of the 3-D
object. The tomographic reconstruction produced a dataset of slice
views in 16 bit TIFF format, which were perpendicular to the
specimen rotation axis and had a voxel size resolution of 8.82 mm.
The reconstructed volumes of the whole image stacks were
vertically re-sliced in Fiji (W.S. Rasband, U. S. National Institutes of
Health, Bethesda, Md, USA) to produce vertical tomographic
images.
2.3. De-noising and mineral mapping
The produced tomographic images were consequently
imported into MATLAB (MatLab R2012b 8.0.0.783, Mathworks,
Natick, MA, USA) for the de-noising process and for increasing the
signal to noise ratio of micro-CT images. De-noising was performed
using a method based on total variation regularization [18,19], with
the following parameters:
m (regularization parameter) = 0/04
rr (initial penalty parameter) = 3 and ro = 40
Following de-noising, lesions were color-coded for improved
visualization and mapping of mineral density using colormape-
ditor command in MATLAB by choosing Jet color map with xed
RGB (Red, Green, Blue) index values for all of the colorized images.
The color codes were based on the grey level values and
corresponding mineral densities of each specimen, producing
calibrated mineral maps.
The calibration of the mineral density was implemented by
measuring and averaging the grey level values of 10 points on
selected images of each hydroxyapatite phantom, followed by
 M. Shahmoradi, M.V. Swain / Journal of Dentistry 56 (2017) 105111 107

plotting the obtained grey level values against the mineral density
value of that phantom. Based on the plotted values, the calibration
equation was calculated and used to transform the grey level
values of the images into true mineral density values.
2.4. Quantitative analysis
For each specimen, a total of 6 slices corresponding to the
beginning, middle and the end of the lesion were selected from the
image stack. For calculating the mineral density of each region of
interest (ROI) including the surface layer and the body of the lesion,
the values of all pixels in the specic region were measured and
averaged. In the sound specimens, the ROIs were selected at the
outer layer as well as the inner layer of enamel.
The thickness of the surface layer of the carious lesions was
measured using line scans of the mineral content prole across the
surface layer of the lesions (Fig. 2B and D). The beginning of the
surface layer was dened on the external enamel surface and the
end of the surface layer was dened as the position where the inner
slope of the mineral curve of the surface layer undergoes a radical
change. This denition is in accordance with the method of
Groeneveld and Arends [20].
The qualitative analysis and the quantication of the lesion
parameters including the mineral density and the thickness of
the surface layer, were performed using visualized mineral
maps in MATLAB and mineral density proles plotted in Fiji
respectively.
2.5. Statistical analysis
The value of mineral density and the thickness of the surface
layer for each sample were calculated by averaging the measured
values in several slices from various representative locations of the
lesion. The measurements were checked for normal distribution.
Statistical analysis was performed using statistical software
GraphPad Prism (Graphpad Software.San Diego, CA) to test for
differences between the means. The results of mineral density
quantication in different groups (WSL, BSL and sound enamel)
were analyzed by One-way analysis of variance (ANOVA). Multiple
comparisons between groups were performed by posthoc Tukey
test. Comparison of the thickness of the surface layer between
white and brown-spot lesions was performed using unpaired
Students t-test. P-values less than 0.05 were considered to be
statistically signicant.
3. Results
3.1. Mineral mapping and qualitative characterization
The mineral maps of proximal WSLs, in most of the specimens,
revealed the characteristic triangular pattern of enamel lesions
with the tip towards the dentin enamel junction (DEJ) and the wide
base at the external surface of the tooth (Fig. 2E). In these lesions, a
high density surface layer with fairly uniform shape on the external
surface of the lesion was observed as reported by previous
researchers [21,22]. Most of the studied WSLs had a dened,
regular outline and were commonly limited to the outer two-thirds
of the enamel (Fig. 2E).
In BSLs, the mineral maps demonstrated various degrees of
enamel demineralization in the region of interest, corresponding
to the physical location of the lesions. Most of these lesions had
irregular demineralization patterns with faint boundaries and
outlines (Fig. 3AD). While the lesion boundary in WSLs usually
had a steep demineralization gradient, the boundary of BSLs had a
more gradual density gradient (Fig. 3).
The demineralization pattern of BSLs varied among specimens
and it was not uniform within different parts of the same lesion.
The regular triangular shape of proximal lesions was observed only
in one of the BSLs through the entire volume, whereas it was
recognizable only in some parts of individual BSLs or was

Fig. 2. (A) Original micro-CT image (B) de-noised image and (C) colorized image of a tooth with brown spot lesion. (D) Gray level prole of the de-noised image of a tooth with
brown-spot lesion, corresponding to the broken (yellow) line plotted in the micro-CT image. Note the presence of a thick and well-mineralized surface layer with near sound
enamel density on top of the brown-spot lesion. (E) Colorized micro-CT image of a proximal white-spot lesion, displaying the characteristic triangular pattern of enamel
lesions with the tip towards the dentin-enamel-junction and the wide base at the external surface of the tooth. The enamel lesion follows the direction of the enamel prisms.
(For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)
108 M. Shahmoradi, M.V. Swain / Journal of Dentistry 56 (2017) 105111

Fig. 3. De-noised micro-CT images (AD) and Mineral maps (EH) of brown-spot lesions showing the extension of the lesion to DEJ, the considerable demineralization of
underlying dentin and the undened and irregular shape of the enamel lesion. Note the presence of scattered free-form high density areas within and close to the surface of
the lesions. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

completely unrecognizable within the whole lesion. Scattered
free-form areas of high density enamel were observed within or
close to the surface of BSLs (Figs. 3 D, and 4 A). Although the outline
and boundary of some BSLs were faint and not as well-dened as
WSLs, a distinguishable demineralization pattern including a
diverging pattern was identied in parts or in the entire structure
of BSLs (Fig. 4).
A layer of high density enamel with a mineral density close to
that of sound enamel was observed in all of the BSLs. This well-
mineralized layer generally extended into the body of the lesion
and in some cases was not recognizable as a separate entity from
other mineralized areas of the lesion body (Fig. 3A and B).
A consistent feature, observed in sixteen of the seventeen teeth
with BSLs, was the involvement of the entire thickness of enamel
and the extension of the lesion into the DEJ area. In most of these
cases (N = 14) there were considerable amounts of mineral loss
(demineralization scar), up to 80% of the mineral density of the
dentin beneath the enamel lesions (Figs. 3 and 4).
The sagittal tomographic images and the three-dimensional
volumetric reconstruction of BSLs also displayed the different and
irregular mineral patterns as well as the signicant mineralization
in subsurface areas of these lesions (Fig. 5A and B).
3.2. Quantitative characterization
The mineral density of the surface layer of the lesion was in the
range of 2.122.79 g/cm3 in BSLs and was from 2.01 to 2.6 g/cm3 in
WSLs. The mineral density of the lesion body was in the range of
 M. Shahmoradi, M.V. Swain / Journal of Dentistry 56 (2017) 105111 109

Fig. 4. De-noised micro-CT images of brown-spot lesions showing different pattern of demineralization including a diverging pattern. (B,C). Note the mineralization of the
body of the lesion and the presence of scattered free-form high density areas within and close to the surface of the lesions.

the scattered areas of high mineral density, was signicantly

higher than the corresponding values in WSLs (p < 0.05). Although
the mean mineral density of the surface layer of BSLs was higher
than WSLs, no signicant difference was found between the two
groups (Table 1).
The thickness of the surface layer exhibited considerable
variation among white-spot and brown-spot lesions. It ranged
from 0 to 90 mm in WSLs and 20 to 250 mm in BSLs (Figs. 2 and 3).
The mean thickness of the surface layer in BSLs (79  15 mm),
averaged from three line scans from the surface, was signicantly
higher than in WSLs (51 11 mm) (P-value < 0.05).

4. Discussion

As a reliable high resolution approach, micro-CT has been

employed for the non-destructive study of mineral distribution of
dental tissues [21,23,24], however it had not been previously
utilized for studying arrested brown-spot enamel lesions in a
sizable sample. In the current study, using high resolution micro-
CT images and calibrated mineral maps, new insight was gained
regarding the shape, extent and mineral content of BSLs as well as
the indications for considerable remineralization in subsurface
areas of naturally arrested enamel lesions.
Fig. 5. (A) The 3-dimensional reconstruction and (B) the sagittal tomographic
The initial macroscopic manifestations of enamel lesions on
image of a brown-spot lesion displaying different and irregular mineral pattern as smooth surfaces are rough surfaced, milky-white opacities called
well as the considerable mineralization in subsurface areas of these lesions. (For white-spot lesion. In the later stages, the whole or parts of these
interpretation of the references to colour in this gure legend, the reader is referred active lesions may remain active and progress, turn inactive
to the web version of this article.)
without considerable remineralization, arrest and partially remi-
neralize or may totally disappear [25,26]. The disappearance of
Table 1 smooth surface white spot enamel lesions after several years has
Mean  SD value of mineral density (g/cm3) of carious brown and white proximal been attributed to remineralization, surface abrasion or a
lesions. combination of these two processes [6,27,28].
White-spot lesion BSLs have been presumed clinically as arrested and partially
Surface layer of the lesion Body of the lesion remineralized lesions [6,7], although there have been some
Mineral density 2.31  0.13 g/cm3 1.88  0.15 g/cm3* controversies among researchers about the color and the
remineralization of these lesions. Regarding the color of BSLs,
Brown-spot lesion
Surface layer of the lesion Body of the lesion some researchers have considered it as incidental [29] or have
Mineral density 2.46  0.14 g/cm3 2.18  0.12 g/cm3* ascribed the browning to the absorption of organic materials on
*
the surface of the lesion [30]. On the other hand, some literature
P < 0.05.
has attributed the color of BSLs to the chronic absorption and
entrapment of organic debris or metallic molecules during
1.412.67 g/cm3 in the enamel area of the BSLs and was from 1.52 numerous episodes of demineralization and remineralization
to 2.41 g/cm3 in WSLs. [10]. In the current study, lesions with brown and dark
The mean mineral density of the surface layer and body zones of discoloration were found to have higher mineral density and a
brown-spot and white-spot enamel lesions are presented in different demineralization pattern supposedly due to remineral-
Table 1. The mean mineral density of the body of BSLs, including ization activity and not as incidental discoloration.
110 M. Shahmoradi, M.V. Swain / Journal of Dentistry 56 (2017) 105111
Regarding mineralization of BSLs, it has been suggested that the
clinical observations of smoother and harder surface of arrested
lesions is the result of abrasion or polishing of the dull, partly
dissolved surface of the active lesion causing exposure of more
tightly packed crystals [26,31]. An AFM study also found that the
surface layer of BSLs were less rough compared to WSLs [12].
However, a detailed ultrastructural TEM study of enamel lesions
has shown the surface layer of WSLs to have poorly packed and
distributed needle shaped crystals, while in the surface layer of
some BSLs the crystals have more uniformity and a concentrated
distribution. Precipitated clusters of new small crystals and
amorphous materials occluding the demineralization voids and
inter-prismatic spaces, as well as enlargement of existing crystals
occluding the spaces at prism boundaries, were also more common
in brown-spot lesions [11]. In the current study, considerable
surface attrition was observed in several BSLs (Figs. 3 C and D and 4
A) which conrm the previous observations in regressed enamel
lesions [6,27,28,32]. However, the mineral density of the surface
layer was not signicantly higher in BSLs compared with WSLs
despite having greater thickness.
BSLs have demonstrated higher demineralization resistance
compared to neighboring unaffected enamel [10,13] and WSLs [12].
This higher resistance has been attributed to the presence of
organic materials on the tooth surface [30] or to the incorporation
of uoride in the remineralized crystals [10], with uoride
amounts directly related to the degree of discoloration [33].
Several mechanisms have been suggested to explain the role of
organic materials in the lesion (especially proteins) in protecting
the enamel against demineralization. These demineralization
protection mechanisms include the alteration of the attachment
and colonization of cariogenic microorganisms [34], the formation
of an acid resistant ion permeable selective membrane on the
enamel surface [35,36], the mechanical blocking of dissolution
sites on enamel [37] and the restriction of the entry of ions into
enamel lesion [38]. Buffering the hydroxyapatite against acid
attack by regulating the electrostatic properties of the enamel
surface, is also a suggested mechanism for the role of enamel
matrix proteins [39]. Although other studies, however, have
implied that organic materials such as albumin can interfere with
the remineralization of the hydroxyapatite crystals [40,41], the role
of organic materials in demineralization inhibition seems more
dominant.
Considering the increased resistance of BSLs against deminer-
alization, it is intuitive to assume a shift in the equilibrium of the
mineralization reactions towards remineralization and an increase
over time, in the mineral content of the lesion. Currently there is
not sufcient evidence showing substantial remineralization of the
body of the lesion in natural enamel caries occurs. In fact, there are
signicant uncertainties about the possibility of remineralization
of subsurface areas of advanced enamel lesions due to structural
and chemical challenges in the remineralization process [26,42].
However, based upon observations made from micro-CT mineral
maps of BSLs (Figs. 24), various specic factors indicate
remineralization of subsurface areas in naturally arrested enamel
lesions:
Firstly, in most of the studied BSLs, the depth and extent of the
lesion was relatively large, involving the entire thickness of the
enamel and causing the demineralization of dentin. In addition, the
location of the lesion body which was observed to be in the inner
half or inner third of the enamel in BSLs, indicates the formation of
a deep lesion in these cases, expected to cause considerable
amount of demineralization in enamel. However comparison of
the mineral density of the body of BSLs with those of WSLs
indicates that, despite the greater extent and deeper progression,
BSLs present higher mineral density in different parts compared to
less advanced WSLs.
Secondly, the presence of a thicker, well mineralized surface
layer in BSLs compared with WSLs, implies signicant reminer-
alization in the surface areas of BSLs. The thickness of the surface
layer of BSLs was considerably higher than previously reported
mean values of 45  29 mm [43] and the range of 3560 mm [21]
and 1050 mm [22] for the thickness of the surface layer in WSLs.
Thirdly, the shape of BSLs, which showed faint boundaries along
with high density areas within the lesion, and dissimilar patterns
compared to WSLs, suggests possible considerable remineraliza-
tion in the surface and subsurface areas of BSLs. The triangular
pattern of WSLs which usually follows the direction of the enamel
prisms, has been suggested to represent multiple individual
lesions each at a different stage of progression [44]. We assume the
unique shape of BSLs observed in some specimens is the result of
different levels of remineralization in the surface, middle and deep
areas of these lesions.
The combined ndings from the above mentioned quantitative
characterization and mineral mapping of brown and white spot
enamel lesions, which also corroborate the clinical observations of
re-hardening and arrest of BSLs, suggest possible subsurface
remineralization in the majority of naturally arrested BSLs and
highlight the importance of BSLs as natural models of caries
remineralization. Further studies, however are required to reveal
the underlying mechanisms, favorable conditions and likely
obstacles of a natural remineralization process which may provide
clues for the development of biomimetic remineralization
methods and non-invasive techniques [27] for the management
of dental caries in future.
From a clinical perspective, in view of the signicant level of
mineralization in BSLs, it seems rational to recommend that in a
BSL without any clinical signs such as surface cavitation or plaque
retention and symptoms suggestive of pulpal involvement such as
thermal sensitivity, the lesion should remain non-restored unless
esthetically essential. Moreover, non-symptomatic arrested lesions
may be treated with non-invasive approaches such as caries
inltration with unlled resin [27] to ensure the sealing of possible
enamel scars and hampering potential demineralization in future.
5. Conclusion
This study demonstrated that the mineralization parameters
such as density and the thickness of the surface layer as well as
distribution patterns through natural enamel brown spot lesions
(BSLs) are different from enamel white-spot lesions (WSLs). The
higher mineral density of the body of the lesion and the increased
thickness of the surface layer in brown spot enamel lesions may
suggest possible subsurface remineralization in the majority of
naturally arrested BSLs.
Acknowledgements
The authors would like to thank the Australian Dental Research
Foundation for ADRF grants 84-2012 and 71-2013, Colgate
Australia, Mr. Mojtaba Lashgari, Dr. Matthew Foley, Ms. Gina
Browne and the Australian Center for Microscopy and Microanal-
ysis (ACMM).
The authors declare no potential conicts of interest with
respect to the authorship and/or publication of this article.
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