Microstructure and Mineral Composition of Dental

Enamel of Permanent and Deciduous Teeth

MARIA ANGE

LICA HUEB DE MENEZES OLIVEIRA,

1
CAROLINA PAES TORRES,
2
JACIARA MIRANDA GOMES-SILVA,
2
MICHELLE ALEXANDRA CHINELATTI,
3
FERNANDO CARLOS HUEB DE MENEZES,
4
REGINA GUENKA PALMA-DIBB,
5
AND MARIA CRISTINA BORSATTO
6
*
1
Department of Pediatric Dentistry, Dental School of Uberaba, University of Uberaba, Uberaba, Minas Gerais, Brazil
2
Department of Pediatric Clinics, Preventive and Community Dentistry, Dental School of Ribeira o Preto,
University of Sa o Paulo, Ribeira o Preto, Sa o Paulo, Brazil
3
Department of Restorative Dentistry, Dental School of Ribeira o Preto, University of Sa o Paulo, Ribeira o Preto, Sa o Paulo, Brazil
4
Department of Restorative Dentistry, Dental School of Uberaba, Uberaba, Minas Gerais, Brazil
5
Department of Restorative Dentistry, Dental School of Ribeira o Preto, University of Sa o Paulo, Ribeira o Preto, Sa o Paulo, Brazil
6
Department of Pediatric Clinics, Preventive and Community Dentistry, Dental School of Ribeira o Preto,
University of Sa o Paulo, Ribeira o Preto, Sa o Paulo, Brazil
KEY WORDS dental enamel; scanning electron microscopy; X-ray diffraction; energy
dispersive X-ray spectrometer
ABSTRACT Purpose: This study evaluated and compared in vitro the microstructure and min-
eral composition of permanent and deciduous teeths dental enamel. Methods: Sound third molars
(n 5 12) and second primary molars (n 5 12) were selected and randomly assigned to the following
groups, according to the analysis method performed (n 5 4): Scanning electron microscopy (SEM),
X-Ray diffraction (XRD) and Energy dispersive X-ray spectrometer (EDS). Qualitative and quanti-
tative comparisons of the dental enamel were done. The microscopic ndings were analyzed statis-
tically by a nonparametric test (Kruskal-Wallis). The measurements of the prisms number and
thickness were done in SEM photomicrographs. The relative amounts of calcium (Ca) and phospho-
rus (P) were determined by EDS investigation. Chemical phases present in both types of teeth
were observed by the XRD analysis. Results: The mean thickness measurements observed in the
deciduous teeth enamel was 1.14 mm and in the permanent teeth enamel was 2.58 mm. The mean
rod head diameter in deciduous teeth was statistically similar to that of permanent teeth enamel,
and a slightly decrease from the outer enamel surface to the region next to the enamel-dentine
junction was assessed. The numerical density of enamel rods was higher in the deciduous teeth,
mainly near EDJ, that showed statistically signicant difference. The percentage of Ca and P was
higher in the permanent teeth enamel. Conclusions: The primary enamel structure showed a lower
level of Ca and P, thinner thickness and higher numerical density of rods. Microsc. Res. Tech.
73:572577, 2010. V VC 2009 Wiley-Liss, Inc.
INTRODUCTION
The crowns of teeth are covered by dental enamel,
which is the hardest tissue in the human body, and is
composed of 9296% inorganic matter or mineral
phase, and 4% of organic material and plasma by
weight. The mineral phase consists primarily of cal-
cium phosphate in the form of hydroxyapatite crystals,
which are carbonated or uoridated (Gwinnett, 1992;
Ten Cate, 1989). The primary crown average growth is
from 6 to 14 months, whereas the permanent teeth
average growth is from 3 to 4 years. So, primary teeth
present lesser thickness of enamel than its successor
teeth (Arau jo et al., 1995; Mortimer, 1970).
The enamel microstructure consists of crystals
arranged in prisms or rods, which run approximately
perpendicular from the dentine-enamel junction
towards the tooth surface (Fava et al., 1999; Ten Cate,
1989). The interfacial area between prisms is protein-
rich, and termed interprismatic enamel (Fejerskov
et al., 1984). In the prismless enamel layer, the
hydroxyapatite crystals are disposed parallel to each
other and perpendicular to the enamel surface (Fava
et al., 1997).
Studies have described the presence of a prismless
enamel layer that is thicker and uniform in deciduous
teeth compared to permanent teeth (Fava et al., 1997).
According to Mortimer (1970), the thinness of the
enamel in deciduous teeth and its lower level of miner-
alization (80.6% in primary teeth enamel and 89.7% in
permanent teeth enamel) are the main differences
between the primary teeth enamel when compared to
the permanent teeth.
As the chemical, morphological, and physiological
aspects vary between permanent teeth and deciduous
*Correspondence to: Profa Dra. Dr. Maria Cristina Borsatto, Faculdade de
Odontologia de Ribeirao Preto/USP, Departamento de Cl nica Infantil, Odontolo-
gia Preventiva e Social, Avenida do Cafe, s/n, Monte Alegre. CEP: 14040-904
Ribeira o Preto, SP, Brasil. E-mail: borsatto@forp.usp.br
Received 13 August 2009; accepted in revised form 4 October 2009
DOI 10.1002/jemt.20796
Published online 23 November 2009 in Wiley InterScience (www.interscience.
wiley.com).
V VC
2009 WILEY-LISS, INC.
MICROSCOPY RESEARCH AND TECHNIQUE 73:572577 (2010)
teeth (Mortimer, 1970; Sonju-Clasen and Ruyter,
1997), the behavior of primary teeth seems to be differ-
ent under conditions such as caries, erosion process,
and bond strength (Hunter et al., 2000; Marquezan
et al., 2008; Wang et al., 2006).
The majority of studies are performed solely in per-
manent or in deciduous teeth, and frequently the
results obtained with permanent teeth are extrapo-
lated to the primary teeth. Presently, the literature is
still scarce in data comparing the microstructure and
composition between the permanent and deciduous
teeth and the available studies shows contradictory
results.
The analysis of the primary and permanent teeth
enamel in the same research is important in order to
provide the methodology standardization and, there-
fore, a reliable comparison between these substrates.
New and improved research techniques have enable
investigators to improve understanding of the dental
structures.
In this way, it is of a great scientic relevance the
accomplishment of a comparison between these sub-
strates in order to establish specic preventive and
restorative protocols. Therefore, the purpose of this
study was to evaluate and compare in vitro the micro-
structure and the mineral composition of permanent
and deciduous teeths dental enamel.
MATERIALS AND METHODS
Sample Selection
This study was approved by the Ethics in Research
Committee of the School of Dentistry of Ribeira o Preto,
University of Sa o Paulo.
Freshly extracted sound third molars (n 5 12) with
complete root formation and second primary molars
(n 5 12) with at least 2/3 of the dental root length
(from the Human Tooth Bank of the School of Dentistry
of Ribeira o Preto, University of Sa o Paulo, Brazil),
were hand scaled and cleaned with water/pumice
slurry in rotating bristle brushes to remove calculus
and surface-adhered debris, and were examined under
a 320 magnier to discard those with structural
defects. The selected teeth were randomly assigned to
the following groups, according to the analysis method
performed to analyze the substrates (n 5 4): Scanning
Electron Microscopy (SEM), X-Ray diffraction (XRD)
and Energy dispersive X-ray spectrometer (EDS).
SEMAnalysis
The roots were sectioned 3 mm below the cement-
enamel junction. Crowns were xed with wax in plex-
glass
1
plates and bisected longitudinally in a mesiodis-
tal direction using a double-faced diamond disk (KG
Sorensen, 7015, Barueri-SP, Brasil) mounted at a low-
speed handpiece. The cut surfaces were then attened
and polished with #600 and #1200-grit silicon carbide
paper (Norton/Saint-Gobain Abrasivos, Guarulhos, SP,
Brazil) and 0.5-lm alumina paste (Struers A/S, Copen-
hagen, Denmark). Next, the enamel thickness was
measured in a surface-junction direction using a digital
electronic paquimeter (Digimatic Caliper/code number
071-467B, Mitutoyo, Suzano, Brazil), with 0.01 mm
resolution.
Then, the specimens were immersed in 3% glutaral-
dehyde (Sigma-Aldrich, St. Louis, MO) in 0.05 M
sodium cacodylate buffer at pH 7.4 (Merck KGaA,
Darmstadt, Germany) for 24 h at 48C. After xation,
the samples were rinsed with distilled water several
times for 1 h.
The enamel surfaces were etched with a 35% phos-
phoric acid gel (Scotchbond etchant, 3M/ESPE) for 15 s
and rinsed thoroughly for 15 s. Then, the specimens
were immersed for 10 min in a ultrasonic cleaner (T-
1449-D, Odontobra s Ind. e Com, Ribeira o Preto, SP,
Brazil) containing distilled water and sequentially
dehydrated in an ascending ethanol (Labsynth Produ-
tos para Laboratorio, Diadema-SP, Brasil) series
(25% for 20 min; 50% for 20 min; 75% for 20 min; 90%
for 30 min; 100% for 60 min). Next, the specimens were
immersed in hexamethyldisizilane (HMDS; Merck
KGaA, Darmstadt, Germany), for 10 min, placed on
absorbing paper inside glass plates and left drying in
an exhaust system.
Specimens were mounted on stubs, sputter-coated
with gold and examined with a scanning electron
microscope (Philips-FEG SEM, Philips Electron Optics,
Netherlands) belonging to the Laboratory of Structural
CharacterizationDepartment of Materials Engineer-
ingFederal University of Sa o Carlos (UFSCar, Sa o
Carlos, SP, Brazil) operating at 20.0 kV. The following
enamel regions were analyzed (the mean rod head
diameter and the numerical density of enamel rods): a
region near the outer surface and a region near the
inner surface, next to the dentinoenamel junction
(EDJ). The most representative areas were recorded
surfaces at magnications of 5003, 1,5003, and
5,0003and a single previously calibrated examiner an-
alyzed the morphological ndings, blinded to the
groups to which the specimens belonged, by performing
a visual and qualitative comparison of the dental
enamel. In a previous data analysis since a non-normal
distribution was detected, the microscopic ndings
were analyzed statistically by a nonparametric test
(Kruskal-Wallis).
The measurements of the prisms number and thick-
ness were done in SEM photomicrographs by mean of
the computer software AxioVision (Zeiss).
EDS Analysis
The same sample that had been used in the SEM
investigation was used for the EDS analysis and in the
same regions: near the outer surface and near the
inner surface, next to the dentinoenamel junction. For
the analysis of the percentage component composition
of the enamel, the X-ray detector system attached to a
scanning electron microscope (Philips-FEG SEM,
Philips Electron Optics, Netherlands) belonging to the
Laboratory of Structural CharacterizationDepart-
ment of Materials EngineeringFederal University of
Sa o Carlos (UFSCar, Sa o Carlos, SP, Brazil) operating
at 20.0 kV, spot size of 5 nm, was used. This method
allowed the relative amounts of calcium (Ca) and phos-
phorus (P) by volume percent to be determined.
XRDAnalysis
X-Rays are useful in identifying crystal structures
since X-ray radiation represents the spectrum of
radiation around 0.1 nm, which is an acceptable
approximation of the size of atoms and ions. For the
X-rays analysis the roots were sectioned 3 mm below
Microscopy Research and Technique
573 PERMANENT AND PRIMARY ENAMEL MICROSTRUCTURE
the cemento-enamel junction. Crowns were xed with
wax in plexglass
1
plates and bisected longitudinally in
a mesio-distal direction using a double-faced diamond
disk (KG Sorensen, 7015, Barueri -SP, Brasil) mounted
at a low-speed handpiece. In the cut fragments, the
dentin substrate was removed using high-speed dia-
mond dental burs with water-cooling, resulting in slabs
measuring at least 1-mm thick.
To determine the crystal structure of enamel, XRD
was carried out using a diffractometer powder system
(Rigaku Geigerex, Woodland, TX) with Ni-ltered
CuKa radiation and a source operating at 40 kVand 25
mA, belonging to the Laboratory of Structural Charac-
terization, Department of Materials Engineering, Fed-
eral University of Sa o Carlos (UFSCar, Sa o Carlos, SP,
Brazil).
RESULTS
SEMAnalysis
The mean thickness measurements observed in the
deciduous teeth enamel was 1.14 mm and in the per-
manent teeth enamel was 2.58 mm.
The morphological analysis showed that the mean
rod head diameter in deciduous teeth was statistically
similar to that of permanent teeth enamel. It was
observed a slightly decrease in the rod head diameter
from the outer enamel surface to the region next to the
dentinoenamel junction in both types of teeth, but
no statistically signicant difference was assessed (P >
0.05; Table 1).
The numerical density of enamel rods was higher in
the deciduous teeth than that recorded in the perma-
nent teeth, mainly near EDJ, that showed statistically
signicant difference (P < 0.05). Additionally, It was
observed a slightly increase in the number of rods from
the outer enamel surface to the region next to the den-
tinoenamel junction in both types of teeth, but no stat-
istically signicant difference was assessed (P > 0.05;
Table 2).
SEM micrographs representative of the two regions
analyzed are illustrated in Figure 1.
EDS Analysis
The results of EDS analysis of the two regions in pri-
mary and permanent enamel are shown in Table 3.
The percentage of calcium and phosphorus was higher
in the two regions analyzed of the permanent teeth
enamel when compared to the deciduous teeth.
XRDAnalysis
The XRD patterns obtained from primary and per-
manent enamel are shown in Figure 2. Phase analysis
of the XRD patterns indicated that the phases present
in deciduous teeth enamel, evidenced by the intensity
of peaks, were hydroxyapatite, calcium silicate,
hydrated calcium phosphate, calcium phosphate sili-
cate, and potassium calcium phosphate. Phase analysis
of the XRD patterns indicated the phases present in
permanent teeth enamel, evidenced by the intensity of
peaks, were hydroxyapatite, calcium silicate, hydrated
calcium phosphate, and hydrated calcium silicate.
DISCUSSION
Concerning the morphological aspect of the dental
enamel, it was observed that the mean rod head diame-
ter in deciduous teeth was statistically similar to that
of permanent teeth enamel, with a slightly decrease
from the outer enamel surface to the region next to the
dentinoenamel junction. The mean rod head diameter
varies from 3.22 lm (60.45) to 3.47 lm (60.48) for the
primary teeth and from 3.84 lm (60.73) to 4.34 lm
(60.95) lm for the permanent teeth. Different results
were reported by Fosse (1968) and Mortimer (1970)
that noted higher enamel prism diameter values in
canines permanent teeth (6 lm) and deciduous teeth
(47 lm).
In the current investigation, the mean numerical
density of rods in the permanent teeth enamel was
13.582/mm
2
(6600) in the outer region and 14.010/
mm
2
(6391) in the region nearest the dentinoenamel
junction. In contrast, Fosse (1964) calculated a mean
number of 21,904 prisms per mm
2
on the outer surface
and a mean number of 47,089 prisms per mm
2
on the
inner enamel surface.
Moreover, the numerical density of enamel rods was
higher in the deciduous teeth than that recorded in the
permanent teeth and increased from the supercial
surface to the deep layer nearest the junction. Consist-
ent with the present study, Fosse (1964) found that the
total number of prism on the inner surface of the
enamel exceeded the number on the outer surface. This
characteristic may cause more interprismatic region in
the permanent teeth enamel, which forms diffusion
pathways. The diffusion rate of substances, ions, and
molecules through the pores in the enamel plays a cru-
cial role in the dynamics of the caries process (Linden
et al., 1986).
As regards the amount of calcium and phosphorus,
their percentage was higher in the permanent teeth
enamel when compared to the deciduous teeth. In this
context, Derise et al. (1974) reported that the mean
concentration of Ca and P were 37.1 and 18.1% in the
permanent teeth enamel, while Lakoma and Rytomaa
(1977) did not observed differences in the amount of
theses ions between primary and permanent teeth
enamel. The different values of Ca and P reported
in the current study may be due to factors such as
the region where the teeth were collected, variations
between individual teeth, the type, age and ethnicity
and even the methodology performed at the
researches.
As the mineralization level is related to the crystals
density (Gwinnett, 1992), it might be speculated that
TABLE 1. Mean head diameter of the enamel rods (lm) to permanent
and deciduous teeth in the different regions
Outer surface
Means (SD)
Near EDJ
Means (SD)
Deciduous teeth 3.47 (60.48) 3.22 (60.45)
Permanent teeth 4.34 (60.95) 3.84 (60.73)
TABLE 2. Mean number of rods (per square mm) to permanent and
deciduous teeth in the different regions
Outer surface
Means (SD)
Near EDJ
Means (SD)
Deciduous teeth 14.149 (61.009) 15.244 (6648)
Permanent teeth 13.582 (6600) 14.010 (6391)
Microscopy Research and Technique
574 M.A.H. DE MENEZES OLIVEIRA ET AL.
the overall crystals density should be lower in primary
enamel when compared to the permanent teeth. It has
been disclosed that overall mineral density was lower
in the outermost layers, but showed no signicant dif-
ferences closer the dentinoenamel junction (Wilson and
Beynon, 1989).
The thinness of the enamel in deciduous teeth and
its lower level of mineralization could be responsible to
its whiter appearance compared to their permanent
counterparts. Additionally, it could play some roles to
the faster and higher rate of dental caries and erosion
progression in primary teeth enamel reported in previ-
ous investigations (Amaechi et al., 1999; Featherstone
and Mellberg, 1981; Johansson et al., 2001; Wang
et al., 2006).
It has been suggested that the mechanical properties
of a calcied tissue are generally linked to its mineral
content (Kodaka et al., 1992).Therefore, the mineral
content and the crystals arrangement differences
between primary and permanent enamel might
account to the lower level of mechanical properties
observed in the deciduous teeth (Low et al., 2007). In
this context, Kerebel et al. (1979) described that the
enamel crystallites thickness are 26.3 nm in perma-
nent teeth and 16.0 nm in primary teeth, while Low
et al. (2006, 2007) observed larger and coarser crystals
in primary teeth (185 nm) when compared to the per-
manent teeth (94 nm).
In this study, the deciduous teeth showed a thinner
enamel layer when compared to the permanent one.
Accordingly, Mortimer (1970) observed that the thick-
ness of deciduous enamel, in general, was about half of
that found on permanent teeth. Furthermore, Low
Fig. 1. Qualitative analysis of the enamel regions by scanning electron microscopy. A: Deciduous teeth
enamel near the outer surface. B: Permanent teeth enamel near the outer surface. C: Deciduous teeth
enamel near the enamel-dentine junction. D: Permanent teeth enamel near the enamel-dentine junction.
TABLE 3. Results of EDS analysis
Permanent teeth Deciduous teeth
Outer
surface (%)
Near
EDJ (%)
Outer
surface (%)
Near
EDJ (%)
P 21.19 21.11 17.23 17.36
Ca 52.50 56.62 35.11 35.80
Microscopy Research and Technique
575 PERMANENT AND PRIMARY ENAMEL MICROSTRUCTURE
et al. (2007) reported that the deciduous teeth enamel
was thinner, softer and more prone to fracture when
compared to the permanent teeth.
Several studies (Fava et al., 1999, 1997) have docu-
mented the prismless enamel layer, in which the hy-
droxyapatite crystals are disposed parallel to each
other and perpendicular to the enamel surface on
teeth. They also demonstrated that this layer shows a
greater and uniform thickness in deciduous teeth.
The presence of aprismatic enamel may interfere
with the dental enamel acidic demineralization process
(Kuhar et al., 1997). Therefore, some authors have sug-
gested the prolonging of the primary enamel phos-
phoric acid-etching time in restorative procedures in
order to obtain the dissolution of the prismless layer
(Bozalis et al., 1979; Hosoya, 1991). Others demon-
strated that the acid etching during 15 s seems suf-
cient to cross the prismless layer, resulting in adequate
Fig. 2. XRD patterns of primary and permanent enamel.
Microscopy Research and Technique
576 M.A.H. DE MENEZES OLIVEIRA ET AL.
retention of the resin materials to the enamel surface
(Garcia-Godoy and Gwinnett, 1991; Gwinnett et al.,
1992). In the present research, the prismless layer
could not be observed since the teeth selected probably
did not present prismless layer due to physiological
dental wear as described by Fava et al. (1999).
The conicting data reported in studies may be due
to factors such as the variations between individual
teeth, the type, age, and ethnicity, the region where the
teeth were collected, the number of teeth analyzed and
even the methodology performed at these researches.
Moreover, the literature is scarce in data comparing
the deciduous to the permanent teeth enamel. There-
fore, the lack of studies testing the same methodology
tested in this study was a hindrance to stating a reli-
able comparison with outcomes of previous investiga-
tions.
Although the ndings obtained from permanent
teeth have been assumed to apply to primary teeth, the
existence of remarkable differences between primary
and permanent enamel substrates must be taken into
account, reinforcing the need of specic and biological-
based preventive and restorative protocols to be deter-
mined in order to provide effective treatments to these
different tissues.
CONCLUSIONS
Within the limitations of this study, the following
conclusions can be drawn:
The numerical density of enamel rods was higher in
deciduous teeth when compared to permanent teeth,
mainly near the amelo-dentinal junction;
the percentage of calcium and phosphorus was
higher in permanent teeth enamel when compared
to deciduous teeth enamel;
deciduous teeth showed a thinner enamel layer
when compared to the permanent one.
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