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RESEARCH ARTICLE
Enamel demineralization model in primary teeth: Micro-CT and
SEM assessments of artificial incipient lesion
Arlete González-Sotelo
1
| Rosalía Contreras-Bulnes
1
| Laura E. Rodríguez-Vilchis
1
|
Maria de los Angeles Moyaho-Bernal
2
| Efraín Rubio-Rosas
3
| Jorge R. Cerna-Cortez
4
1
Universidad Autónoma del Estado de México,
Facultad de Odontología, Centro de
Investigación y Estudios Avanzados en
Odontología (CIEAO), Jesús Carranza esq.
Paseo Tollocan, Col. Universidad, Toluca,
Estado de México, C.P. 50130, México
2
Benemérita Universidad Autónoma de
Puebla, Facultad de Estomatología, Av. Manuel
Espinosa Yglesias 31 Pte. 1304, Col. Los
Volcanes, Puebla, Puebla, C.P. 72570, México
3
Benemérita Universidad Autónoma de
Puebla, Dirección de Innovación y
Transferencia de Conocimiento, Prolongación
de la 24 Sur y Av. San Claudio, Ciudad
Universitaria, Col. San Manuel, Puebla, Puebla,
C.P. 72570, México
4
Benemérita Universidad Autónoma de
Puebla, Facultad de Ciencias Químicas, Centro
Avanzado de Pruebas Analíticas No
Destructivas, Blvd Valsequillo y esquina Blvd
Municipio libre S/N, Cd Universitaria, Col. San
Manuel, Puebla, C.P. 72570, Mexico
Correspondence
Rosalía Contreras-Bulnes, Universidad
Autónoma del Estado de México, Facultad de
Odontología, Centro de Investigación y
Estudios Avanzados en Odontología, Paseo
Tollocan Esq. Jesús Carranza. Toluca, Estado
de México, C.P. 50130, Mexico.
Email: rcontrerasb@uaemex.mx; rcb0209@
yahoo.com.mx
Review Editor: Paul Verkade
Abstract
Many studies have analyzed different tooth demineralization models, which generate
artificial incipient lesions; however, most of them are complex, slow, not clear and
results could not be employed in both primary and permanent teeth because of
chemical content differences among them. This study evaluates a demineralization
model on primary enamel, under three incubation periods; quantifying artificial incipi-
ent lesions formation, and depth by micro-CT, complementing with SEM for morpho-
logical characterization. Sixteen healthy human anterior primary teeth extracted for
prolonged retention and orthopedic/orthodontic reasons were included in this study,
previous informed consent. The sample was randomly assigned to four groups n=4:
G_Control, G_2D, G_4D, and G_7D. Micro-CT and SEM were performed during two
stages: before demineralization (BD) and after demineralization (AD). A t-student test
was carried out to determine differences among groups (p≤.05). No incipient lesions
were observed in control group. Artificial lesion depth was similar among experimen-
tal groups; values were from 38.16 ± 05.40 μm to 42.61 ± 04.75 μm. An amount of
14 to 17 artificial incipient lesions were formed per group, the extension and distribu-
tion were different for each incubation period. Five erosive lesions were produced in
G_7D. All experimental groups were able to form incipient artificial lesions in primary
enamel. SEM characterization revealed more pronounced changes on the enamel sur-
face, as the days of immersion in the demineralization solution increased. The 4-day
incubation period is the most recommended for the demineralization model, due to
the formation of incipient lesions only and its extension, which facilitates their
assessment.
KEYWORDS
artificial incipient lesion, demineralization model, micro-CT, primary enamel, SEM
1|INTRODUCTION
Dental caries is a progressive and multifactorial disease deter-
mined by a continual imbalance between protective factors (saliva
flow, proteins, calcium, phosphate) and pathological factors
(acidogenic bacteria, carbohydrates, reduced salivary function)
alternating periods of demineralization and remineralization.
(Cochrane, Cai, Huq, Burrow, & Reynolds, 2010; Featherstone,
2004; Selwitz, Ismail, & Pitts, 2007). Demineralization produces
the dissolution of calcium, phosphate, carbonate, and other ions
from the tooth (Featherstone, 2004; Selwitz et al., 2007). The ini-
tial clinical caries is a visible white-spot lesion that can be reversed
if the carious lesion is non-cavitated (Featherstone, 2008; Selwitz
et al., 2007).
Received: 13 September 2020 Revised: 24 December 2020 Accepted: 16 January 2021
DOI: 10.1002/jemt.23718
Microsc Res Tech. 2021;1–9. wileyonlinelibrary.com/journal/jemt © 2021 Wiley Periodicals LLC 1
The use of laboratory models has been introduced to obtain infor-
mation on the mechanisms responsible for this phenomenon of inter-
est (Moreno & Zahradnik, 1974; Wang, Tang, Bonstein, Bush, &
Nancollas, 2006). Many studies have analyzed different tooth demin-
eralization models, which generate artificial incipient lesions; however,
most of them are complex, slow, not clear and use different deminer-
alization solutions. Moreover, provided that the enamel of primary
teeth is more susceptible to caries development than permanent teeth
due to the lower mineral content and higher organic content, (Bajaj
et al., 2016; Itthagarun, King, & Rana, 2007) the behavior of primary
teeth seems to be different under conditions like caries, erosion pro-
cess and bond strength (Hunter, West, Hughes, Newcombe, &
Addy, 2000; Marquezan, da Silveira, Burnett Jr., Rodrigues, &
Kramer, 2008). Consequently, models could not be employed in both
primary and permanent teeth because of chemical content differences
among them (Marquezan et al., 2008; Moreno & Zahradnik, 1974;
Wang et al., 2006).
Additionally, conventional techniques have been used to evaluate
the characteristics of artificial caries lesions on the enamel surface, as
well as preventive protocols. These procedures include scanning elec-
tron microscopy (SEM) (Elkassas & Arafa, 2014; Whittaker, 1982),
microradiography, (Itthagarun et al., 2007; ten Cate &
Duijsters, 1982), microhardness (Mohd Said, Ekambaram, &
Yiu, 2017), polarized light microscopy (PLM) (Itthagarun et al., 2007;
Itthagarun, Wei, & Wefel, 2000; Salman, ElTekeya, Bakry, Omar, & El
Tantawi, 2019; Tuloglu, Bayrak, Tunc, & Ozer, 2016) and Energy dis-
persive X-ray spectrometer (EDS), (De Menezes Oliveira et al., 2010);
however, some of these methods require destructive sample
preparation.
Micro- computer tomography (micro-CT) was developed in the
early 1970's and has progressed over time (Swain & Xue, 2009). The
imaging process is a nondestructive, innovative, and noninvasive
approach for the experiments that explore the structure mineral tis-
sues maintaining the enamel integrity (Ozgul, Orhan, & Oz, 2015;
Swain & Xue, 2009; Zan et al., 2018). Furthermore, micro-CT creates
a three- dimensional (3D) image of the tooth and permits the qualita-
tive and quantitative analysis of internal tooth structure (Chałas
et al., 2017). However, this technique has its limitations. For this rea-
son, SEM (2D) and micro-CT (3D) have been used together in order to
take advantage of their complementary assessment (Furat
et al., 2018).
This method has not been utilized to evaluate dental enamel
demineralizing models. In addition, considering the current need for
developing a specific model of demineralization in primary teeth to
use in future research protocols, the aim of this study was to evaluate
a demineralization model on primary enamel, under three incubation
periods; to quantify artificial incipient lesions formation, and depth by
micro-CT, and to complement the study with SEM for morphological
characterization.
The working hypothesis was that the artificial lesion depth would
increase with a longer incubation period; additionally, more evident
morphological changes on enamel surface would appear after the
demineralization model.
2|MATERIALS AND METHODS
The research protocol was reviewed and approved by the Research Ethics
Committee of Dental Research and Advances Studies Center, School of
Dentistry, at the Autonomous University of the State of Mexico (UAEM).
2.1 |Tooth selection and sample preparation
Human anterior primary teeth from the mandibular and maxillary
arches were obtained after getting a written informed consent from
the patients and their parents.
Healthy teeth extracted for prolonged retention, and orthopedic/
orthodontic reasons were included in this study. Teeth with obvious
decay, evidence of fluorosis, fracture, and any sign of restoration were
excluded from the sample.
After extraction, the specimens were stored in a 0.2% thymol
solution at 4C, within 3 months from the time of extraction, until the
experiment began.
Selected teeth were cleaned with deionized water, traces of soft
tissue were removed with a scalpel, and remnants of the root were
separated with a diamond disc (BesQual, New York, NY) mounted on
a low-speed motor (Brasseler, Savannah, GA).
The specimen chamber space was filled with composite resin
(3 M ESPE, Filtek™Z350XT), previously conditioned with a self-
etching agent (Single Bond Universal 3 M ESPE, St. Paul, MN). Then,
teeth were washed in an ultrasonic bath for 10 min in separated con-
tainers with deionized water (Quantrex Q140 L&R Ultrasonics, NJ) to
remove any residues from the surfaces.
2.2 |Study design
Sixteen teeth were randomly assigned to four groups (n= 4 per
group): G_Control (untreated), G_2D, G_4D, and G_7D (2, 4, and
7 days under demineralization incubation period, respectively).
Micro-CT and SEM analyzes were performed during two stages:
before the demineralization (BD) and after the demineralization (AD). All
procedures were performed in isolated experimental units (Figure 1).
2.3 |Demineralization model for artificial lesion
formation
The demineralizing solution employed was composed of 2.2 mM cal-
cium chloride, 2.2 mM potassium dihydrogen phosphate, 0.050 M
acetic acid, and 1 M potassium hydroxide pH 4.4 (Kumar, Itthagarun, &
King, 2008). Teeth were incubated in the demineralizing solution for
2 days, 4 days (Kumar et al., 2008), and 7 days (ten Cate, Buijs, &
Damen, 1995) at 37C to create artificial enamel lesions.
After the incubation period, each specimen was washed with
deionized water from the demineralizing solution and cleaned in an
ultrasonic bath. Finally, they were dried at room temperature.
2GONZ
ALEZ-SOTELO ET AL.
2.4 |Micro-CT
2.4.1 |Scanning procedure
A high-resolution desktop micro-CT system (Nikon Metrology NV),
based on combination microscopy and tomographic data reconstruc-
tion was used. The scanning conditions were focal spot size 5.8 μm,
2000×2000-pixel size and rotation in 0.3steps. The scans were per-
formed at 100 kV, 100 μA, voxel size of 5.8 x 5.8 x 5.8 μm. Thus,
1,200 projection views were employed for each tooth.
Reconstruction was performed with the software interface CT
Pro 3D which had been provided by the manufacturer of the scanner.
To minimize ring artifacts, the detector was air-calibrated before each
scan. Each sample was rotated 360within an integration time
of 5 min.
In order to evaluate dental images, the teeth were scanned with
micro-CT system and the segmented regions were saved in DICOM
format.
Subsequently, segmented regions were extracted and converted
into digital images, immediately. For the covering procedures, RadiAnt
DICOM Viewer (https://www.radiantviewer.com) was utilized to cre-
ate a 3D volume model and multiplanar reconstructions.
2.4.2 |Images analysis
An incipient enamel lesion (artificial or not) was defined as the transi-
tion between the light grey values of the surrounding healthy enamel
tissues and the darker grey levels due to demineralization (Ogawa,
Yamashita, Ichijo, & Fusayama, 1983; Schulte, Wittchen, Stachniss,
Jacquet, & Bottenberg, 2008). For this task, a total of 4,800 images
per group were evaluated to identify these incipient lesions.
BD, the enamel of each sample was verified as healthy (without
loss of continuity and the presence of incipient lesions).
AD, lesion formation by group was identified, as well as the num-
ber of artificial lesions for each incubation period.
The depth of 10 lesions per group was estimated, the analysis
was carried out in the segmented image data at a specific slice, the
bottom of the lesion was delimited with a continuous black line and
the deepest lesion area was selected and registered in μm, as shown
in Figure 2.
2.5 |SEM
Two specimens from each group were randomly selected to obtain
SEM images before and after the demineralization model. The mor-
phological changes were observed by a scanning electron microscope
(JEOL, JSM-6610 LV, Japan), under the following conditions: low vac-
uum mode, 10 Pa of chamber pressure, an electron acceleration volt-
age of 20 kV, detecting back- scattered electrons at 200×, 500×,
1,000×, and 1,500×magnifications.
2.6 |Statistical analysis
All data were analyzed using SPSS software (SPSS IBM, New York,
NY) version 25. A Kolmogorov–Smirnov test was applied to assess
the data distribution and t-student test was carried out to determine
differences among groups (p≤.05).
3|RESULTS
3.1 |Micro-CT
As expected, no incipient lesions were observed in control group, as
well as in baseline micro-CT images of all groups (BD). After the
FIGURE 1 Study design
GONZ
ALEZ-SOTELO ET AL.3
experiments (AD), micro-CT analysis revealed an amount of,
16, 14 and 17 enamel artificial lesions in G_2D, G_4D and G_7D,
respectively. All artificial lesions observed in G_2D and G_4D were
incipient, while G_7D showed 12 incipient lesions and 5 erosive ones.
Regarding the artificial incipient lesion depth, it was similar among
experimental groups, values ranged between 38.16 ± 05.40 μmto
42.61 ± 04.75 μm, no statistically significant differences were found
(p> .05). See Table 1.
The representative micro-CT images of the specimens showed in
Figure 3, revealed that extension and distribution of artificial lesions
were specific by group. In G_2D,no loss of enamel continuity was
observed, only grayish areas along its thickness, corresponding to
incipient lesions marked with white triangles, which were observed as
small and localized, with similar dimensions at the bottom and surface
(Figure 3b).
As can be seen in Figure 3c, G_4D did not show loss of enamel
continuity either, very extensive incipient artificial lesions were
formed in a large area of the enamel surface, as shown by the grayish
zone indicated by the white triangles, in the enamel thickness.
Finally, as indicated by the black triangles in Figure 3d, G_7D was
the only group where artificial enamel erosive lesions occurred, char-
acterized by the loss of continuity of this surface, a very irregular
lesion surface is observed.
3.2 |SEM
Morphological characterization of enamel surface by study group is
observed in representative micrographs (Figure 4). BD, a smooth surface
appearance, isolated enamel microporosities, wear grooves and homo-
geneous scratches formation are evident in all groups (a, d, g, and j).
AD, no changes are seen in control group (b and c), while variations
in enamel surface morphology were revealed from mild to more pro-
nounced according to each incubation period. After 2 days, enamel sur-
face micrographs showed new pits and cracks (e and f), after 4 days, a
fish-scale pattern was observed (h and i). After 7 days, an irregular sur-
face was produced, characterized by abundant microporosities (k and l).
Figure 5 exhibited evident erosive lesions that occurred on pri-
mary enamel surface, after the 7 days' incubation period. BD, the
enamel surface showed open prisms and wear grooves, characteristic
of a temporary enamel in an exfoliated tooth (a). After the experiment,
G_7D micrograph (b) revealed well-defined eroded areas, also irregu-
larities were observed on the enamel surface.
4|DISCUSSION
The present study used micro-CT and SEM to evaluate a deminerali-
zation model on primary enamel, under three incubation periods (2, 4,
and 7 days).
FIGURE 2 Micro-CT image of
primary enamel surface, including
measurements of demineralization area
TABLE 1 Mean ± SD value of lesion depth (μm) by group
Group Lesion depth Statistical analysis
Control 00.00 ± 00.00 μmA
2D 42.61 ± 04.75 μmB
4D 39.52 ± 06.93 μmB
7D 38.16 ± 05.40 μmB
Note: Capital letters in a row are for the comparison of lesion depth in
different groups after demineralization model. Same capital letters follow
means that do not differ statistically.
*t-student test, p> .05.
4GONZ
ALEZ-SOTELO ET AL.
Although several methods have been used to demineralize teeth,
most of them have experimented with demineralization permanent
enamel and bovine enamel (Kumar et al., 2008; ten Cate et al., 1995;
Whittaker, 1982), and there are scarce reports related to deciduous
enamel. A specific demineralization model for primary enamel is nec-
essary since differences between permanent and deciduous teeth
have been demonstrated, among them, that the latter has aprismatic
zones in their surface (Ripa, 1966; Whittaker, 1982). Even more, an
important relationship with caries susceptibility has been attributed to
them. Another difference is the lower inorganic content of deciduous
enamel (Bajaj et al., 2016). For these reasons, based on the literature
review and the results of a pilot study, a 4.4 pH demineralization
model under three incubation periods to produce artificial enamel
lesions was evaluated, unlike those proposed for permanent teeth
(Elkassas & Arafa, 2014; Kumar et al., 2008; ten Cate et al., 1995; ten
Cate & Duijsters, 1982). Due to the previously mentioned differences,
as well as a thinner layer of enamel in the deciduous dentition
(De Menezes Oliveira et al., 2010), it was decided to evaluate the
effects of incubation time on the formation and depth of the lesions,
as well as whether these would occur from a period as short as 2 days
(proposed incubation time), achieve a depth that involves only the
enamel at 4 days (Kumar et al., 2008) or even produce deeper lesions
after 7 days (ten Cate et al., 1995).
Lesion formation was visually examined (Oliveira et al., 2014) at
the end of each incubation period; subsequently, all the assessment
techniques proposed for this study were carried out.
This study is the first to use micro-CT to assess the formation of
artificial incipient lesions by a demineralization model on primary
enamel, under three incubation periods. Furthermore, it is the first to
strength the evaluation with additional assessments such as SEM,
contrary to the use of bovine incisors evaluated by microradiographs
previously reported (Itthagarun et al., 2007; ten Cate et al., 1995; ten
Cate & Duijsters, 1982).
Studies using a comparison of micro-CT and histology to evaluate
caries detection have shown that the results correlate well with 2D
histological sections and micro-CT evaluation (Boca et al., 2017),
hence, is an alternative to evaluate artificial lesions especially in pri-
mary teeth, which have a small surface area for analysis, and also, this
surface is more fragile than that of permanent teeth
(Whittaker, 1982). Although, as mentioned, one advantage is that
micro-CT is a non-destructive technique (Ozgul et al., 2015), subse-
quent analysis with other non-destructive vacuum techniques (SEM)
caused fractures in the samples, which was the reason why indepen-
dent samples were used in this study.
There are various characterization methods for enamel surface
evaluation, most of them have been applied on human permanent
teeth, bovine enamel surface, and scarcely on human primary teeth.
Some of them include: microhardness (Mohd Said et al., 2017; Tuloglu
et al., 2016), polarized light (Bajaj et al., 2016; Kumar et al., 2008; Sal-
man et al., 2019), transversal microradiography and microradiography
(Kumar et al., 2008; ten Cate et al., 1995; ten Cate & Duijsters, 1982).
However, the analysis of the samples using these methods requires
cutting them into thin sections, before evaluating the same surface,
before and after certain procedures (Mellberg, Castrovince, &
Rotsides, 1986). Even further, some methods are destructive tech-
niques, resulting in even more fragile (Ozgul et al., 2015) and non-
reusable samples during follow-up evaluations of proposed protocols.
On the other hand, in the last 5 years, micro-CT assessment has been
used in some studies regarding dental caries and treatments, including
silver diamine fluoride therapy (Li et al., 2019), dentifrices
FIGURE 3 Representative images of artificial enamel lesions by study groups. White triangles indicate artificial lesions; GC showed no
lesions, small lesions were produced at 2D incubation period. Large lesion areas were observed in group 4D. Enamel artificial lesions, as well as
loss of enamel structure (black triangles), are seen in 7D incubation period [Color figure can be viewed at wileyonlinelibrary.com]
GONZ
ALEZ-SOTELO ET AL.5
remineralization potential (Bijle et al., 2019), dental varnish efficacy
(Sleibi, Tappuni, Davis, Anderson, & Baysan, 2018) and caries detec-
tion methods (Ribeiro et al., 2015). This method has shown advan-
tages in comparison with the previously mentioned characterization
techniques.
In this study, representation of the demineralized area revealed
by the micro-CT image of a specimen, was comparable to that
described by Ozgul et al., (2015). In this way, micro-CT analysis rev-
ealed a similarity in the depth and number of artificial lesions formed
by group. However, they were shallower than artificial lesions in pri-
mary teeth reported in negative control groups evaluated by polarized
light or micro-CT. Conversely, a lower lesion depth was reported in
preventive protocols groups (Ozgul et al., 2015; Tuloglu et al., 2016).
Furthermore, depth lesion per se does not provide a complete
analysis of the effects produced by different incubation periods, since
each one presented a specific morphological characterization of the
enamel surface, as revealed by additional observations in micro-CT
and SEM images. With micro-CT, 3D morphology and location of arti-
ficial lesions was detected. Even though the whole enamel surface
was immersed in a demineralizing solution, artificial lesions were
formed at determined sites, and also limited to the buccal or palatal
surfaces of the anterior teeth, probably because the enamel is neither
structurally nor chemically homogeneous (Robinson et al., 2000).
Furthermore, a previous study found variations not only between
different teeth but among different sites on the same tooth
(Whittaker, 1982).
FIGURE 4 Representative scanning electron microscopic (SEM) micrographs. Before demineralization model, enamel surface showed smooth
appearance, microporosities, wear grooves and homogenous scratches (a, d, g, and j). No morphological changes were observed in control group
(b and c). After 2 days demineralization, enamel surface has new pits and cracks (e and f), 4 days demineralization showed fish-scale pattern
(h and i); after 7 days demineralization, an irregular surface was produced, with abundant microporosities (k and l). Original magnification: 500×;
scale bar = 50 μm (a, b, d, e, g, h, j, and k) and 1,500×scale bar = 10 μm (c, f, i, and l)
6GONZ
ALEZ-SOTELO ET AL.
Variations according to the incubation period in the demi-
neralizing solution were evident in relation to artificial lesions exten-
sion and types. Artificial lesions occurred in demineralization model as
short as 2 days incubation, as reported by Wang et al. (2006) who
concluded that deciduous enamel dissolved considerably faster than
permanent enamel, and could be more pronounced if a longer incuba-
tion period is set as revealed by the micro-CT analysis. Each period
presented specific characteristics: small incipient lesions after 2 days
incubation, larger ones associated to 4 days and 2 types of artificial
lesions (incipient and erosive) were formed after 7 days, resulting in a
new mixed lesion formation model, a not expected important finding.
In order to nurture the micro-CT findings, complementary SEM
micrographs were made on enamel surface, and provided valuable
additional information on the morphological characterization. It is
important to notice that there are no previous reports that comple-
ment micro-CT studies with SEM characterization of enamel artificial
lesion models in primary teeth. A study by Yu et al. (2018) evaluated
the remineralizing effect of silver diamine fluoride and sodium fluoride
application on permanent enamel caries lesions and reported that
enamel surfaces treated with fluorides remained relatively dense and
intact compared with other group micrographies. Furthermore, lesion
depths obtained from micro-CT images ranged from 129 to 181 μm
(higher than our results), without significant differences in the lesion
depths among experimental groups, as we reported. It should be
emphasized that although they used micro-CT and SEM to character-
ize permanent teeth, they did not relate both techniques in their
results. In comparison, our findings revealed a relation between the
micro-CT continuous enamel surface and the SEM smooth appear-
ance of the tissue, comprised of microporosities and homogenous
scratches; both morphologies described are characteristics of func-
tional healthy enamel, previously seen before the demineralization
model for artificial lesion formation. Even though experimental groups
showed multiples incipient lesions along the enamel surface in micro-
CT analysis, the surface appearance varied among groups in the SEM
characterization: G2_D showed small lesions as enamel rough appear-
ance, while more extensive incipient lesions were observed in G_4D
as a regular fish scale pattern on enamel surface. The loss of enamel
surface continuity observed by micro-CT in G_7D was characterized
as an eroded enamel surface as revealed by SEM analysis.
The fish scale pattern observed in G_4D was similar to results
reported by Elkassas and Arafa (2014), where lesion formation was
produced by 5 days' incubation period under a different demineraliza-
tion solution.
The findings of our study suggest that the use of 4-day incubation
period for artificial incipient lesions formation model, under the specific
conditions employed, is the best one, since it produces a greater number
of homogeneous lesions, which could increase the quality of evaluation
of future protocols for the prevention or treatment of incipient lesions.
The 7-day incubation period could be used in simultaneously evaluating
protocols for the prevention or treatment of not only incipient lesions
but also enamel erosion, in addition to providing the basis for the devel-
opment of new erosion models in primary teeth.
Although the use of the 2-day incubation period is not rec-
ommended due to the formation of small artificial lesions that are
more complex to locate and assess, it highlights the susceptibility of
deciduous enamel to the development of incipient caries lesions, as
previously mentioned (Bajaj et al., 2016; Itthagarun et al., 2007) even
in short periods of demineralization, emphasizing the need for preven-
tion and treatment of these lesions from very early stages in children.
The main limitations in this study were difficulties in collecting
the samples (healthy primary teeth extracted for therapeutic reasons),
reduced access to micro-CT equipment (highly demanded because of
the wide range of materials analysis it can perform, since it is able to
modulate the amount of X-rays), time spent for scanning, reconstruc-
tion and analysis of the images, among others; in addition the sample
analysis during two stages (before and after the demineralization
model), doubled the number of images required. Although the number
of samples per group was reduced, the micro-CT scanning per sample
FIGURE 5 SEM micrographs from enamel surface. BD, enamel surface showed open prisms and wear grooves (a). After 7 days of
demineralization, well-defined eroded areas and irregularities were observed on the enamel surface (b). Original magnification: 200×; scale
bar = 100 μm
GONZ
ALEZ-SOTELO ET AL.7
and study stage was comprehensive (1,200 per sample in each stage),
resulting in the assessment of 9,600 images. However, additional
studies are recommended in relation to mineral density and porosity
of enamel artificial lesions.
Even though this in vitro model included exfoliated teeth (repli-
cating a constant demineralization to produce an artificial incipient
enamel lesion) it does not reflect the exact conditions in the oral
cavity. Therefore, this variable should be considered in other in vitro
studies when evaluating incubation periods of a demineralization
model related to materials and methods for dental caries prevention.
5|CONCLUSIONS
Within the limitations of this study, the results support that:
Although all in vitro evaluated model under three incubation periods
forms incipient artificial lesions similar in depth, more pronounced mor-
phological changes were observed as the period increased; therefore,
the 4-day incubation is the most recommended period due to the forma-
tion of incipient lesions only in primary enamel and its extension.
ACKNOWLEDGMENTS
The authors acknowledge the Centro Universitario de Vinculación
(CUV), Benemérita Universidad Autónoma de Puebla (BUAP) and Cen-
tro Avanzado de Pruebas Analíticas No Destructivas-BUAP. Special
thanks for the assistance of Gabriela Esquina-Arenas, BS Chem., in
the micro-CT scanning.
CONFLICT OF INTEREST
The authors declare no potential conflict of interest.
AUTHOR CONTRIBUTIONS
The responsibility of acquisition, analysis, interpretation of data,
drafting the work and final approval of the version to be published
was to Arlete González-Sotelo.
The responsibility of design, acquisition, analysis, interpretation of
data, drafting the work and final approval of the version to be publi-
shed was to Rosalía Contreras-Bulnes.
The responsibility of design, analysis, interpretation of data, drafting
the work and final approval of the version to be published was to
Laura Emma Rodríguez Vilchis.
The responsibility of analysis, interpretation of data, revising it criti-
cally and final approval of the version to be published was to Maria de
los
Angeles Moyaho-Bernal.
The responsibility of acquisition, analysis of data and final approval of
the version to be published was to Efrain Rubio-Rosas.
The responsibility of analysis, interpretation of data, revising it criti-
cally and final approval of the version to be published was to Jorge
Raúl Cerna-Cortez.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
ORCID
Rosalía Contreras-Bulnes https://orcid.org/0000-0003-1760-2000
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How to cite this article: González-Sotelo A, Contreras-
Bulnes R, Rodríguez-Vilchis LE, Moyaho-Bernal MdlA, Rubio-
Rosas E, Cerna-Cortez JR. Enamel demineralization model in
primary teeth: Micro-CT and SEM assessments of artificial
incipient lesion. Microsc Res Tech. 2021;1–9. https://doi.org/
10.1002/jemt.23718
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ALEZ-SOTELO ET AL.9
