journal of dentistry 35 (2007) 343349

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Caries susceptibility of human fluorosed enamel and dentine

P.G.K. Waidyasekera a,*, T. Nikaido a, D.D.S. Weerasinghe a, K.A. Wettasinghe b,
J. Tagami a,c
a

Cariology and Operative Dentistry, Department of Restorative Sciences, Graduate School, Tokyo Medical and Dental University,
1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan
b
Department of Restorative Dentistry, Faculty of Dental Sciences, University of Peradeniya, Peradeniya, Sri Lanka
c
Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone,
Tokyo Medical and Dental University, Tokyo 113-8549, Japan

article info

abstract

Article history:

Objective: Objective of our laboratory study was to determine the impact of dental fluorosis

Received 31 July 2006

severity on the formation of caries in the human enamel and dentine.

Received in revised form

Materials and methods: Thirty-three human molars were grouped according to modified

23 October 2006

ThylstrupFejerskov index (TFI) into normal (N, TFI 0), mild fluorosis (ML, TFI 13) and

Accepted 27 October 2006

moderate fluorosis (MD, TFI 46). Three mesio-distal sections were made in corono-apical
axis of the tooth, giving enamel and dentine samples. They were embedded in an epoxy
resin, and polished. Half of the polished surface was covered with an acid resistant varnish

Keywords:

and immersed in standard acidified buffer solution (pH 4.5) for 48 h to create artificial caries

Dental fluorosis

lesions. They were treated with 5% NaOCl for 45 min and sectioned longitudinally along the

Enamel caries

center into two halves. Cut surfaces were polished and observed under a confocal laser

Dentinal caries

scanning microscope for depth of demineralization. Morphology of the demineralized zones

was observed under a field emmision scanning electron microscope (FE-SEM). Data were
analyzed using one-way ANOVA and Sheffe test ( p = 0.05).
Results: Statistically significant difference in depth of demineralization was found between
N and MD groups ( p = 0.046) in the enamel, and between N and ML ( p = 0.002), N and MD
( p < 0.001), ML and MD ( p = 0.029) in dentine. FE-SEM observation of the normal enamel
showed direct dissolution with large fissures. Spongy appearance of intertubular dentine
gradually disappeared from N to MD.
Conclusions: Moderately fluorosed enamel showed a significant caries resistance. In contrast, mild and moderately fluorosed dentine was significantly caries susceptible in vitro.
# 2006 Elsevier Ltd. All rights reserved.

1.

Introduction

Fluoride has been used through out the world to prevent

dental caries despite its link with dental fluorosis.1 A tooth
malformation is believed to be caused by chronic ingestion of
fluoride during tooth development,1,2 which is the only known
side effect of systemic fluoride use in caries prevention.3

Recent literatures have revealed increase of prevalence of

dental fluorosis (DF) ranging between 7.7 and 80.9% in the
areas with fluoridated water and between 2.9 and 42% in areas
without water fluoridation.46
Fluorosed enamel is characterized by outer hyper mineralization and subsurface hypomineralization. The pores in the
subsurface enamel are occupied by water as well as enamel

* Corresponding author. Tel.: +81 3 5803 5483; fax: +81 3 5803 0195.
E-mail address: kanchanapgkw@yahoo.com (P.G.K. Waidyasekera).
0300-5712/$ see front matter # 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jdent.2006.10.008

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journal of dentistry 35 (2007) 343349

secretary proteins which are retained due to the effect of the

excessive fluoride level on ameloblasts.7 Distinct changes in
mineralization pattern are not confirmed in the fluorotic
enamel, while the dentine laid down exhibits accentuation of
the incremental pattern. After cessation of enamel environment secretion, a substantial variation of mineral content can
be observed in dentine with occasional bands of interglobular
dentine.8,9
Several epidemiological studies have been carried out to
assess the relationship between dental fluorosis and caries
experience.10,11 However, the relationship varies because of
the additional social factors such as malnutrition, poor tooth
cleaning and unfavorable dietary habits. In vitro studies have
not been carried out to evaluate caries susceptibility of the
fluorosed enamel and dentine in comparison with the normal
teeth.
Therefore, the purpose of the present study was to evaluate
the caries susceptibility of the enamel and dentine with
different levels of fluorosis against artificial caries formation
in vitro.
The nulhypothesis was that severity of fluorosis did not
affect on the caries susceptibility of human enamel and
dentine.

2.

Materials and methods

2.1.

Sample preparation

Thirty-three extracted human molars from subjects between

the age of 40 and 60 living in endemic areas for fluorosis in Sri
Lanka were used for this study. The teeth were caries free and
had been extracted due to periodontal reasons. Informed
consent was obtained from the subjects before the extraction
to use the extracted teeth for the research. They were cleaned,
stored in distilled water in a refrigerator at 4 8C separately with
the details of the date of extraction, age and gender of the
patient. The teeth were used less than 4 months after extraction.
Before being prepared, a total of 86 teeth were washed
under running water, quickly dried and analyzed for dental
fluorosis severity according to the modified Thylstrup
Fejerskov index (TFI).12 This was done by two investigators
independently. Intra-examiner reproducibility gave a Cohens
k statistic of 0.95.13 TFI is the only index that attempts to
correlate the clinical appearance of dental fluorosis with the
pathological changes in the tissue2 and is normally the index
of choice for the evaluation of dental fluorosis severity.14
Teeth used in the present study were categorized according to
the TFI as demonstrated in Table 1. Teeth of TFI 7, 8 and 9 were
not available in this study. Classified teeth were grouped into

Table 1 Number of teeth examined according to their

TFI classification
Classification
TFI
Number of specimens

Normal Mild fluorosis

0
11

1
4
11

2
3

3
4

Moderate
fluorosis
4
3
11

5
3

TFI 0-normal, TFI 13-mild fluorosis and TFI 46-moderately

fluorosis.15
After the dental fluorosis severity assessment, teeth were
sectioned by means of a slow rotating diamond bur (Isomet,
Buehler, Lake Bluff, IL, USA) under water lavage in a mesiodistal direction to remove the outer most occlusial part of the
enamel (Fig. 1). This was done to remove the mechanically
damaged, abraded outer porous fluorotic enamel.16 Two
sections were made in the mesio-distal direction following
the coronal-apical axis. The first in a plane at 2 mm distance
from the buccal surface and the second in a plane at 2 mm
distance from the lingual surface (enamel samples). Another
section was made along the same axis dividing the remaining
central part of the tooth into two halves (dentine samples).
Roots were cut and separated at the cemento-enamel
junction. Previous studies have shown that there is no
significant difference in the fluoride concentration between
the buccal and lingual aspects of the same tooth.17 Therefore,
one enamel and one dentine sample were selected without
any visible damage out of the four prepared samples from
each tooth. The remaining samples were used to observe the
polished surfaces of the enamel and dentine without an acid
challenge, under FE-SEM.
Samples were embedded in an epoxy resin (Epoxycure resin,
Buehler, USA), having the buccal or lingual suface enamel of the
enamel samples and the occlusal surface of circumpulpal
dentine of the dentine samples facing the surface that is to be
polished later. Resin was allowed to polymerize at room
temperature over night. After curing of the epoxy resin, the
samples were ground with a series of water proof abrasive SiC
papers (280, 800, 1000, 1200 and 1500-grit) under water coolant.
This was proceeded by polishing with diamond pastes (Struers,
Japan) successively from 6 mm down to 0.25 mm grit size to
produce a high gloss surface. Grinding and polishing of all these
samples was done by the same investigator to ensure that all
were subjected to the same procedure. After each step of
polishing, ultrasonically cleaning in distilled water was carried
out to remove polishing paste debris.
After a quick air dry, an acid resistant nail varnish was
applied covering half of each specimen which is to be
demineralized, to provide a reference surface for measurement of the depth of demineralization. Enamel and dentine
samples of each group was separately immersed in 100 ml of
standard acidified buffer solution for 48 h at 37 8C and stirred
to obtain caries like lesions. The solution contained 2.2 mmol/
L CaCl2, 2.2 mmol/L NaH2PO4 and 50 mmol/L acetic acid
adjusted to pH 4.5 with NaOH.18,19 The solution was changed
after 24 h to ensure that the under saturated conditions
remained constant.20 After removing the specimens from the
demineralizing solution, they were thoroughly rinsed in
distilled water and then both enamel and dentine samples
were immersed in an ultrasonic bath of 5% NaClO for 45 min in
an attempt to remove the denatured surface collagen fibrils of
the dentine.

2.2.
Confocal laser scanning microscopic (CLSM)
observation
6
5

The samples were rinsed in distilled water for 60 s. A cut was

made through the center of each specimen parallel to the long

journal of dentistry 35 (2007) 343349

345

Fig. 1 Schematic diagram of the specimen preparation.

axis by means of a slow rotating diamond bur (Isomet) under

water lavage, resulting in two halves. One of the half sections
from each specimen was polished to high gloss with abrasive
discs and diamond pastes successively from 6 mm down to
0.25 mm grit size. From the remaining halves randomly
selected, three enamel and dentine samples from each group
were prepared for field emission scanning electron microscopic (FE-SEM) examination.
Polished surfaces were ultrasonically cleaned in distilled
water and observed using a CLSM (1LM15W, Nikon, Japan) at
500 magnifications (20 objective lens). The CLSM used in this
study was a video rate instrument, achieving a frame time of
33 ms by the use of an accousto-optic deflector for high speed
scanning in one axis. This allows rapid three-dimensional
assessments of samples without damage from prolong
drying. The utility of this technique for imaging the depth
and the shape of lesions is well demonstrated elsewhere.19
We evaluated the depth of the formed artificial carious lesion
from the surface of the undemineralized acid resistant
varnish covered tooth part to the deepest demineralized
front (Fig. 2).
The depths of the lesions were statistically analyzed with
one-way ANOVA and Scheffe post hoc comparison test using
SPSS for windows Version 11 ( p = 0.05).

2.3.

observed under a FE-SEM (S4500, Hitachi, Tokyo, Japan) in an

accelerating voltage of 3 kV under a magnification of 3000.

3.

Results

3.1.

Depth of demineralization

The mean values and standard deviations of the depth of

demineralization in micrometers (mm) are shown in Table 2.
One-way ANOVA indicated a statistically significant difference
between the different sample groups and the mean depths of
demineralization in enamel (F = 3.773, p = 0.038) and in dentine
(F = 21.396, p < 0.001). Furthermore, Scheffe post hoc comparison showed no significant difference in depth of demineralization between normal and mild fluorosed enamel. However, a

FE-SEM observation

In order to observe the morphology of the demineralized

surface zone of the caries lesion and the polished undemineralized surface, the samples were dried, and sputter-coated
with platinum and palladium for 90 s (thickness of 4 nm) and

Fig. 2 Schematic diagram of measuring the depth of

demineralization.

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journal of dentistry 35 (2007) 343349

Table 2 Depth of demineralization in enamel and dentine (mm)

Classification
Enamel
Dentine

Normal

Mild fluorosis

15.7  3.9 a
19.7  2.1

12.0  2.2 a,b

27.2  6.0

Moderate fluosis
11.4  2.5 b
32.5  4.9

Statistical comparison is done with in each substrate. n = 11, mean  S.D. Same letters in each row are statistically similar ( p < 0.05).

statistically significant difference in the depth of demineralization was found between normal and moderately fluorosed
enamel groups ( p = 0.046). Demineralization of the normal
enamel was deeper than the moderate fluorosed group. A
significant difference in the depth of demineralization was
found in dentine between normal and mild fluorosed ( p = 0.002),
normal and moderately fluorosed ( p < 0.001), mild and moderately fluorosed ( p = 0.029) groups, with a gradual increase in the
depth from normal to moderately fluorosed group.

3.2.
Field emission scanning electron microscopic (FE-SEM)
observation
Non demineralized surfaces of the normal, mild fluorosed
and moderately fluorosed enamel appeared to be flat and
smooth (Fig. 3AC). Whereas the normal demineralized
enamel showed a pronounced direct dissolution forming
large fissure-like spaces and steps corresponding to the
location of the cross-striations of the enamel in addition to
the enlarged interprismatic regions (Fig. 3A1). For mild
fluorosed enamel, a distinct dissolution of the outer surface

was clearly seen with many surface irregularities in terms of

shallow fissures and other defects (Fig. 3B1). For moderately
fluorosed enamel, loss of the outer microsurface was visible
with a relatively defects free, undisturbed, smooth surface
(Fig. 3C1). For normal dentine, the demineralized dentine
exhibited a porous appearance with clearly evident dentinal
tubular branch orifices. The remaining intertubular dentine
structure gave the appearance of a spongy network (Fig. 4A1).
The intertubular dentine of the undemineralized surface was
homogenous (Fig. 4A). For mild fluorosed demineralized
dentine, the porous appearance was visible. The demineralized intertubular dentine appeared as an eroded rough
surface (Fig. 4B1). Whereas the intertubular dentine of the
undemineralized surface appeared even (Fig. 4B). The
undemineralized moderately fluorosed dentine showed
areas of interglobular dentine in the region of the intertubular
dentine (Fig. 4C). For moderately fluorosed demineralized
dentine, the peritubular dentine was dissolved giving an
irregular appearance to the dentinal tubular orifices. Intertubular area was rough with chipping off of the surface in
some areas (Fig. 4C1).

Fig. 3 SEM images of the enamel surfaces. (A) Non demineralized polished surface of the normal enamel. (B) Non
demineralized polished surface of the mild fluorosed enamel. (C) Non demineralised polished surface of the moderately
fluorosed enamel. (A1) Normal demineralized enamel showing the large fissure like spaces and distinct steps. (pointer). (B1)
Mild fluorosed demineralized enamel showing shallow fissures on a relatively smooth surface (pointer). (C1) Artificial
caries lesion of the moderately fluorosed enamel.

journal of dentistry 35 (2007) 343349

347

Fig. 4 SEM images of the dentine surfaces. (A) Non demineralized polished surface of the normal dentine. (B) Non
demineralized polished surface of the mild florosed dentine. (C) Non demineralized polished surface of the moderately
fluorosed dentine showing an area of interglobular dentine (pointer). (A1) Normal demineralized dentine showing the
spongy appearance of the intertubular dentine (pointer). (B1) Mild fluorosed demineralized dentine. (C1) Moderately
fluorosed demineralized dentine showing irregular shaped tubular orifices (pointer).

4.

Discussion

The classification of the fluorosed teeth was done according

to the modified Thylstrup and Fejerskov index, which is based
on the clinical changes in fluorosed teeth.21 This classification
has a reported reproducibility.12,15 Although the results of
this study were based on a relatively limited number of teeth,
the samples covered a range of dental fluorosis severity from
TFI 0 to TFI 6. Human molars were used to avoid the effect of
the variation of fluoride concentration in different tooth
types.15
In the present study, the caries susceptibility of the teeth in
the scale of TFI 06 was evaluated by measuring the depth of
demineralized enamel, dentine and FE-SEM observation for the
surface morphology before and after challenge of the standard
acidified buffer solution (pH 4.5). The solution is reported to be a
successful tool in the artificial caries production in dentine in
vitro.19 A different pH value was reported to be successful in
enamel caries formation,18 and different time periods were
been used in the previous studies for the acid challenge
depending on the purpose of the study and the type of the tooth
tissue exposed to the caries attack.19,22,23 The use of a single
artificial caries technique and an acid challenge period of 48 h
were useful for this study since both enamel and dentine
specimens of the fluorosed and normal teeth could be subjected
to an identical acid attack. Although the 5% NaOCl treatment for
45 min was necessary only for the dentine samples, enamel
samples were also treated in the same manner to create same
laboratory conditions.

In the fluorosed teeth, the highly mineralized enamel

surface layer is composed of a mixture of many large and
extremely small crystals, and the hypomineralised subsurface
area is composed of fairly sparsely arranged large crystals with
a few small crystals. Some of the crystals in the subsurface
hypomineralised layer exhibit defects such as perforations.24
The extent and the severity of the subsurface hypomineralization are in keeping with the TFI scores assigned to the
teeth. Thus in teeth with the highest TFI scores the width of
the hypomineralized lesions extends almost to the enamel
dentine junction. And in these teeth the subsurface pore
volume exceeds 25%.25 Whereas the well mineralized surface
zone is reported to have a varying thickness.24 In our study the
samples were ground polished and ultrasonically cleaned
prior to the acid challenge. In this way, flat surfaces without
surface contaminations were obtained. Although this procedure may have disturbed or removed the hypermineralized
surface layer of the fluorosed enamel, lesions produced in this
type of subsurface enamel are reported to be much more
reproducible than the lesions created in enamel at the
anatomical surface.18
The epidemiological studies revealed controversial results
on relation of the dental caries and dental fluorosis. Mainly a
positive relationship is documented between dental fluorosis
and dental caries.11,26,27 A study by McInnes et al. indicated a
negative relationship between dental fluorosis and dental
caries.10 This controversy may be possibly due to the social
and environmental factors which are said to be having a great
impact on DMFT of a population. We carried out our in vitro

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journal of dentistry 35 (2007) 343349

study to eliminate these parameters on the process of caries

formation.
TFI index which we used in this study correlates the clinical
appearance of dental fluorosis with the pathological changes
in the tissue.2 Therefore Fluorosis tends to be consistent with
in a particular tooth. And we used each tooth specimen as the
unit of comparison According to the findings of the present
study, the mean depth of the demineralized enamel decreased
with the increasing level of the severity of fluorosis, with
although a small value but a significant difference between
normal and moderate fluorosed groups. In the FE-SEM
photomicrographs of the demineralised surface zone, deep
fissures were observed in the normal enamel. A similar
observation with distinct steps corresponding to the location
of the striae of retzius was observed by Holman et al. (1985)
after 5 days exposure of the human enamel to lactate buffer of
pH 5.23 However, the interval between two fissures was in
accordance with the interval between cross striations of the
enamel in the present study. These may have resulted by a
shift in the crystal orientation23 or by enlargement of laminar
pores which were reported to be more important diffusion
pathways than interprismatic enamel during the acid attack.28
In addition, shallow fissures with other irregularities were
observed in mild fluorosed enamel. The surface tends to get
smooth with the severity of fluorosis. These results confirm a
gradual worsening of surface etching relative to the reduction
of the TFI value or the lower demineralization potential of the
fluorosed enamel in the artificial caries attack. Previous
studies revealed that the histological features of the lesions
produced with an artificial caries attack in fluorosed and
normal enamel were similar in water under the polarized
light.22 Despite the initial porosity of fluorosed enamel, it was
found to be remarkably resistant against in vitro lesion
formation.22 In fluorosed specimens, even the removal of
the superficial intact surface layer had not accelerated the
demineralization.22
The fluoride content of the enamel is increased with
increasing severity of fluorosis.29 This may exert an effect
either by reducing the solubility rate of the tissue30 or by
encouraging the remineralization at the point of dissolution.31
It is also possible to assume that the high protein content of
the abnormal organic enamel matrix of the fluorosed enamel
may exert a protective effect.32 Such protein may reduce
access of ions to the crystallite surface33 or prevent the spread
of small ions responsible for dissolving minerals.34 These
increased levels of fluoride and enamel proteins in the
fluorosed enamel may play a major role in this protection
against acid challenge observed in the fluorosed enamel.
On the other hands, our study showed an increase of the
depth of demineralized dentine with the increasing severity of
dental fluorosis. The FE-SEM observations indicated that the
most vigorous artificial caries attack was observed in the
moderate fluorosed dentine in three groups. The intertubular
area of the normal dentine appeared to be spongy in nature.
Loss of this intertubular spongy appearance could be seen in
the mild fluorosed specimens. This can be explained by the
simultaneous penetration of the artificial caries solution
through dentinal tubules and intertubular dentine in mild
fluorosed dentine, while this process may have been mainly
confined to the dentinal tubules with a weak acid penetration

through the intertubular dentine in the normal dentine.

Therefore, in normal dentine, the intertubular collagen network may have not been distorted. This prediction is
supported by the findings of Kierdorf et al.9 and Fejerskov
et al.,8 where areas of interglobular dentine was observed in
the dentine following high fluoride administration. Interglobular dentine (Fig. 4C) is a hypocalcified area where the
globular laid out minerals in the dentine; calcopherites do not
completely fuse.35 Although interglobular dentine is a structure which may persist in the normal mature dentine, in
fluorosed dentine extensive areas of hypomineralized, angular patches of interglobular dentine was reported to be visible
with an overall inhomogenous mineral distribution.8 This may
be an easy pathway for transportation of the artificial
demineralizing solution, resulting in the dissolution of the
intertubular dentine. Intertubular dentine is demineralized
more slowly than peritubular dentine in a demineralization
attack.36 In compared with the enamel, a significantly higher
fluoride concentration and a positive correlation between
fluoride concentration and dental fluorosis severity are found
in the dentine.12 Although fluoride is said to be exerting an
inhibitory effect for demineralization, the vulnerability of the
dentine for the acid attack showed to be increasing with the
increased severity of fluorosis. In moderately fluorosed
dentine, the peritubular dentine appeared to be dissolved
forming irregular dentinal tubular orifices. The intertubular
dentine had chipped off regions forming some depressed
areas. This may be a result of change in the acid penetration
routes due to changed morphology of the moderately
fluorosed dentine in a much vigorous manner than the mild
fluorosed dentine.
In conclusion with the evidence of our in vitro study, human
moderately fluorosed enamel showed a significant caries
resistance, in contrast to the mild and moderately fluorosed
human dentine which was significantly caries susceptible.
However, a much more detailed micro morphological analysis
is necessary for the further understanding of the behavior of
human fluorosed dental tissue against a caries attack.

Acknowledgment
This work was granted by the center of excellence program
(Tokyo Medical and Dental University) for Frontier research on
molecular destruction and reconstruction of tooth and bone.

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