journal of dentistry 38 (2010) 868–874
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Impact of two toothpastes on repairing enamel erosion
produced by a soft drink: An AFM in vitro study
Claudio Poggio a,*, Marco Lombardini a, Marco Colombo b, Stefano Bianchi b
a
b
Department of Operative Dentistry, University of Pavia, Policlinico ‘‘San Matteo’’, Piazzale Golgi 3, 27100 Pavia, Italy
Department of Endodontics, University of Pavia, Italy
article info
abstract
Article history:
Objectives: The aim of the present in vitro study was the evaluation of two toothpastes
Received 2 May 2010
(Sensodyne Pronamel and Biorepair Plus on repairing enamel erosion produced by a soft
Received in revised form
drink (Coca Cola), using Atomic Force Microscopy (AFM).
13 July 2010
Methods: Fifty extracted human central incisors free of caries were selected and divided in a
Accepted 20 July 2010
treatment and a control half; they were kept in artificial saliva during whole experimentation. The treatment halves were divided into five groups; group 1: demineralization with soft
drink; group 2: demineralization with soft drink + Pronamel; group 3: demineralization with
Keywords:
soft drink + Biorepair Plus; group 4: intact enamel + Pronamel; group 5: intact enamel + -
Enamel
Biorepair Plus. Specimen demineralization was carried on in 4 intervals of 2 min. In groups 2,
Surface properties
3, 4, and 5 the toothpastes were applied for 3 min at 0, 8, 24 and 36 h. The surface of each
Toothpastes
specimen was imaged by AFM and Rrms, root-mean-square roughness, and Maximum Depth
Atomic Force Microscopy (AFM)
of the cavities were registered.
Results: Amongst treatment specimens of groups 1, 2, and 3 a statistically significant
difference (P < 0.01) in Rrms and Maximum Depth values was registered: the toothpastes
reduced enamel demineralization. No statistical differences in Rrms values were registered
between the two toothpastes.
Conclusions: The toothpastes tested (Pronamel and BioRepair Plus) offer a degree of protection from erosive drinks.
# 2010 Elsevier Ltd. All rights reserved.
1.
Introduction
Surface demineralization and remineralization processes
have an important role for mineralized tissues, such as bones
and teeth. Dental enamel is the hardest substance of the body
because of its high mineral content. The ultrastructure of
enamel has been widely investigated, showing that the
inorganic components are distributed in the form of rods or
prisms,1 which are composed of hexagonal hydroxyapatite
crystals. Both aprismatic and prismatic crystals can be found
at enamel surface; aprismatic enamel is generally more
mineralized and more resistant to acid challenge.2
In adults, enamel does not contain any cells and is therefore
not capable of self-regeneration. Any damage is irreversible, as
there is no biological process capable of repairing damaged
enamel. For this reason, any reparative action must be provided
by materials or substances that are extraneous to dental tissue
metabolism. These substances are either synthetic or are
precipitated from saliva.3 Tooth structure undergoes continuous demineralization and remineralization: if this balance is
interrupted, demineralization will lead to a progressive deterioration of tooth.4,5 Erosive tooth wear in the oral environment
has gained more importance from the dental practice since the
decline in dental caries in many industrialized countries.6
* Corresponding author. Tel.: +39 0382 516257/39 3398124925; fax: +39 0382 516224.
E-mail address: [email protected] (C. Poggio).
0300-5712/$ – see front matter # 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jdent.2010.07.010
journal of dentistry 38 (2010) 868–874
Changes in dietary habits have led to excessive consumption of
acidic food and beverages, which are the most widespread
extrinsic factors that cause dental erosion.7 Typical acid sources
come from the diet, medications, occupational exposure and
lifestyle activities.8,9
The characteristics of erosion may be due to several factors,
including the chemical properties of the erosive medium and
the frequency and method of contact between acid and tooth.
Biological and chemical factors in the oral environment
influence the progress of dental erosion. Saliva provides
protective effects by neutralizing and clearing the acids; it is
also a source of inorganic ions necessary for the remineralization process10; this is the reason why patients with diminished
salivary flow are more exposed to dental erosion and decay.11
Many studies have demonstrated that the early stages of
erosion result in the softening of the enamel surface up to a
depth of less than 1 mm.12–14 Surface softened enamel is not
capable of complete rehardening by exposure to saliva although
rehardening of surface softened enamel by the exposure to
calcifying solutions in vitro and in vivo has been reported.
Fluoride dentifrice had a protective effect on eroded
enamel subjected to brushing abrasion.15 Currently, conventional fluoride-containing toothpastes do not appear to be able
to protect efficiently against erosion.16
Recently, two toothpastes have been introduced: Pronamel
and BioRepair Plus. Their main characteristic is the ability to
contrast enamel erosion.
Sensodyne Pronamel (GlaxoSmithKline, Brentford, Middlesex, UK) is a derivative of Sensodyne toothpaste; it is made
of bioavailable sodium fluoride (1450 ppm fluoride) and also
contains potassium nitrate to help prevention against enamel
erosion and mitigate the effects of dentinal hypersensitivity.17
Its biocompatibility has been enhanced by removing sodium
lauryl sulphate from the composition.
BioRepair Plus (Coswell S.p.A., Bologna, Italy) is a fluoride
free toothpaste made of hydroxyapatite nanocrystals, which
have been introduced because of their excellent biological
properties, lack of toxicity and inflammatory and immunological responses. The hydroxyapatite microparticles are
completely identical to the mineral that forms dentine and
enamel. In the case of the enamel, the microparticles action
takes place via their ability to bond to natural tissues, thus
filling microgaps in the enamel.18
Atomic Force Microscopy gives images with atomic
resolution with minimal sample preparation. This technique
has been widely used to characterize the erosion of enamel
and dentin.19–21 In a previous study AFM have been used to
investigate the protective effect of a mouthrinse on enamel
softened by a demineralizing beverage.22
In the present in vitro study Atomic Force Microscopy was
used to investigate the surface roughening during dental
erosion caused by a soft drink. Our chief aim was to study the
role played by two toothpastes (Pronamel and BioRepair Plus)
on repairing enamel erosion.
cleansed of soft tissue debris and inspected for cracks,
hypoplasia and white spot lesions; they were disinfected in
5.25% sodium hypochlorite solution for 1 h1 and stored in
artificial saliva (pH 7.0, 14.4 mM NaCl; 16.1 mM KCl; 0.3 mM
Cl26H20; 2.9 mM K2HPO4; 1.0 mM CaCl22H2O; 0.10 g/100 ml
sodium carboxymethylcellulose) during the whole experimentation.5 The specimens were cut longitudinally, with a
high-speed diamond rotary bur with a water-air spray; one
half served as a control, and the other half as treatment. The
labial surfaces were ground using silicon carbide papers
(grades 600–1200) under water irrigation to remove 50–100 mm
and produce flat surfaces.7 Samples were placed into Teflon
moulds measuring 10 mm 8 mm 2 mm, embedded in
flowable composite resin and polymerized. A soft drink (Coca
Cola, Italy) was chosen for the demineralization process. The
pH at 20 8C, buffering capacity, concentration of calcium and
phosphate of the beverage were measured.22 Measurements
were performed in triplicate and average values calculated.
Two toothpastes were used: Pronamel and BioRepair Plus.
The samples were randomly assigned to 5 groups, each
made of 10 teeth (Fig. 1):
group
group
group
group
group
1:
2:
3:
4:
5:
soft drink,
soft drink + Pronamel,
soft drink + BioRepair Plus,
intact enamel + Pronamel,
intact enamel + BioRepair Plus.
The treatment specimens of groups 1, 2, and 3 were
immersed in 6 ml of the soft drink for 2 min at room
temperature before rinsing with deionized water. Four consecutive intervals of the immersion procedure were carried out.6
The toothpastes were applied neat onto the surface of the
specimens to cover the enamel surface without brushing in
the treatment groups 2, 3, 4, and 5 and wiped off with distilled
water washing; the matching control specimens received no
treatment. The toothpaste was applied to the enamel surfaces
for 3 min at 0, 8, 24 and 36 h; during these intervals the
specimens were kept in artificial saliva.
2.1.
Atomic Force Microscopy (AFM) observations
AFM images were collected with an Atomic Force Microscopy
AutoProbe CP 100 (Themormicroscopes, Veeco), equipped with
a piezoelectric scanner, which can cover an area of 100 mm 100 mm with a range of 7 mm in the z-direction. The root-meansquare roughness, Rrms, was obtained from the AFM investigations by testing, for each sample, at least 10 different film areas
of 30 mm 30 mm with a resolution of 256 256 pixels. From the
analyses of the AFM height profiles, it was also possible to
estimate the erosion cavities depth of the enamel surface. The
data were obtained by averaging on at least 20 selected lines of
the image.23 Measurements were performed on the treatment
specimens and on the matching controls.
2.2.
2.
869
Statistical analysis
Materials and methods
Specimens were prepared from 50 extracted human incisors
free of caries and defects. After the extraction, the teeth were
Differences in the averaged values amongst the groups were
analyzed by ANOVA test. Statistical difference was set at
P < 0.01.
870
journal of dentistry 38 (2010) 868–874
[(Fig._1)TD$IG]
Fig. 1 – Flow chart.
Table 1 – Mean roughness values (Rrms) and Maximum Depth of the cavities obtained in the five groups on the exposed
and the not-exposed specimens.
Groups
Treatment
Rrms
Group 1: soft drink
Exposed
Not-exposed
Exposed
Not-exposed
Exposed
Not-exposed
Exposed
Not-exposed
Exposed
Not-exposed
0.32 0.01
0.05 0.01
0.15 0.02
0.06 0.02
0.17 0.02
0.07 0.03
0.07 0.04
0.07 0.02
0.06 0.05
0.07 0.02
Group 2: soft drink + Pronamel
Group 3: soft drink + BioRepair Plus
Group 4: intact enamel + Pronamel
Group 5: intact enamel + BioRepair Plus
3.
Results
The pH of the soft drink at 20 8C was 2.44; the buffering
capacity was 0.0056.
Concentration of calcium was 20.83 mg/ml, concentration
of phosphate 175.7 mg/ml.
Table 1 reports mean Rrms values and Maximum Depth (MD)
of the cavities obtained through the demineralization process.
In groups 1–5 the not-exposed halves of the specimens
provided similar Rrms values (no statistical difference),
suggesting that the groups may be comparable. Comparing
exposed specimens of groups 1 (softened enamel) with group 2
(softened enamel + Pronamel) and 3 (softened enamel + BioRepair Plus), a not statistically significant difference (P > 0.01) in
Rrms values was registered.
Comparing groups 2 and 3, Pronamel and BioRepair Plus
gave no statistically significant Rrms values, thus demonstrating a comparable remineralizing ability.
In groups 4 and 5, no statistically significant difference was
registered between exposed and not-exposed halves of the
specimens and between the two groups.
Maximum Depth (mm)
0.50 0.15
0.17 0.07
0.20 0.09
0
0
As for the Maximum Depth of the cavities, there was a
statistical significant difference between group 1 (softened
enamel) and groups 2, 3 (softened enamel repaired by
toothpastes).
Fig. 2(a) shows an untreated specimen surface. Fig. 2(b)
shows the demineralized surface of a specimen (group 1). Fig. 3
displays a specimen surface demineralized and then exposed
to the protective effect of Pronamel (a) and BioRepair Plus (b)
toothpastes (groups 2, 3). Fig. 4 shows an intact enamel surface
exposed to the protective effect of Pronamel (a) and BioRepair
Plus (b) toothpastes (groups 4, 5).
The surfaces of the untreated teeth appear quite smooth
(Fig. 1). A remarkable increase of enamel surface alterations
was observed after the exposure to the acid drink, which
causes a loss of material from the surface (Fig. 2). The final
enamel morphology recalls a typical prismatic structure, with
prism size ranging between 3 and 5 mm: the etching process
involves mainly the inner area of the prism creating a
honeycomb-like structure.22–24
There is not a statistically significant difference in Rrms
between the exposed surfaces of groups 2 and 3 and between
the not-exposed halves of groups 2 and 3. Regarding the tooth
journal of dentistry 38 (2010) 868–874
[(Fig._2)TD$IG]
871
[(Fig._3)TD$IG]Fig. 2 – Intact enamel, untreated specimen surface (a); demineralized surface of a specimen (b).
Fig. 3 – Specimen surface demineralized and exposed to Pronamel (a) and BioRepair Plus (b) toothpastes (groups 2, 3).
[(Fig._4)TD$IG]
Fig. 4 – Intact specimen surface exposed to the protective effect of Pronamel (a) and BioRepair Plus (b) toothpastes (groups 4, 5).
surfaces treated with the toothpastes after exposure to the
soft drink (groups 2, 3), the interprismatic cavities appear
largely filled in, with a consequent decrease of their depth.
Zones of intact enamel within the test area were interspersed
with superficial adherent irregularities, appearing as granular
or globular deposits. Moreover, the observation and the
comparison of the exposed halves of groups 2 and 3 show
that the treatment with the two toothpastes causes a different
remineralizing process. After treatment with Pronamel, bulk
material loss was observed: both interprismatic and prismatic
enamel structures still appear evident. BioRepair Plus, through
its hydroxyapatite nanocrystals, showed a progressive microparticles deposition: the interprismatic and prismatic enamel
structures appear to be completely hidden by a thick
homogeneous apatitic layer.
Concerning the Maximum Depth of the cavities, the
treatment with the two toothpastes tested resulted in a
statistically significant depth reduction.
4.
Discussion
This study compared the effect of two toothpastes on
human enamel demineralized by a soft drink. Atomic Force
Microscope has been used to image and comparatively
analyze enamel surface. AFM provides high-resolution
872
journal of dentistry 38 (2010) 868–874
images and is an important tool as a source of new structural
information.1
In a previous study, enamel surfaces treated with demineralizing solutions were imaged by tapping mode AFM,
showing a net boundary between exposed and unexposed
enamel areas. AFM was used to study the effects of bleaching
agents on enamel, as it is a suitable microscopic method to
analyze biological objects under natural conditions.25,26 The
authors concluded that AFM imaged non-dehydrated enamel
surfaces.
AFM nanoindentation was used to investigate the demineralization and remineralization of surface softened enamel,27,28 which provided a very sensitive measurement. An AFM
nanoindentation study has shown that the early stage of
enamel dissolution in situ results in the softening of the
enamel surface up to a depth of less than 1 mm.29 Other studies
showed that the amount of mineral loss increased in the
presence of a demineralizing solution with increasing exposure time.
Enamel surface is often aprismatic and more highly
mineralized than enamel subsurface; however, enamel
surface is completely removed by the polishing process and
the resulting flat surface is not present in the oral cavity. This
prismless enamel arises at the end of amelogenesis. Although
this layer of enamel is more frequent on the surface of
deciduous teeth, it can also be found on the surface of
permanent teeth.30 It is known that the prism-free enamel is
gradually worn off during mastication but it is retained in
protected areas. Flat and polished specimens were used in the
present study in an attempt to standardize specimens: nontreated halves gave very similar Rrms results, allowing us to
compare the groups to each other. However, it should be noted
that natural tooth surfaces erode more slowly than polished
surfaces.31 Specimens were cleaned with 5.25% NaOCl for 1 h,
which could not alter enamel surface.1
It was demonstrated that erosion is correlated to pH;
moreover, there was a negative correlation between calcium
concentration and erosion, but no clear relationship between
phosphate concentration and erosion.10 In a previous study,
Coca Cola was the beverage with the lowest pH and highest
concentration of calcium, so it was chosen for its highest
erosion potential. There is a clear correlation between erosion
and temperature of the beverages.5 In the present study, the
beverages were kept at a constant temperature of 20 8C, in
order to stress their demineralizing potential. Although
erosion proceeds more slowly in vivo than in vitro owing to
the protective effect of saliva and acquired pellicle, the effect
of temperature can be expected to be significant.32
Acquired pellicle is a protective layer that covers enamel
surface and consists mainly of salivary proteins: it has the
ability to slow down the overall remineralization process. We
did not intend to simulate the acquired pellicle as this barrier
for acid diffusion could masque the potential of a toothpaste to
protect from erosion. In an in vivo model, Hara et al.33
concluded that the acquired pellicle reduced dental erosion,
but that this effect was limited to the less severe erosive
challenge on enamel surfaces.
Previous research suggests that human saliva has the
ability to reharden the enamel surface in the oral environment, moreover enamel rehardening has often been reported
in vitro studies by various calcifying solutions.34,35 These
calcifying solutions often possess a higher remineralization
potential than human saliva, as their calcium and phosphate
concentrations are higher. Surface precipitation of calcium
phosphates is believed to occur in the form of an amorphous
precursor phase, which undergoes a rapid transformation to
crystalline apatite.36
Devlin et al.37 demonstrated that Coca Cola reduced the
mean indentation hardness of enamel in the teeth, but the
hardness was partially restored with artificial saliva. Lippert
et al.27 stated that enamel rehardening by saliva was not
observed. The authors added that a complete recovery of the
initial surface hardness was not observed so far.
In the present study, artificial saliva was not used during
the erosion phase; its use would buffer the acidity of the cola
drink and limit the demineralization process.6 For the
remineralization phase natural saliva and acquired pellicle
might influence the results provided. Soft drink tested showed
high erosive potential on human enamel. In the oral
environment, many factors can enhance or prevent demineralization such as mineral concentration, pellicle and plaque
formation, salivary factors (flow rate and buffering capacity).
Another important factor that must be taken into consideration is toothbrushing. Hemingway et al.10 demonstrated by
optical profilometry that enamel softened by erosion is readily
susceptible to abrasion trough toothbrushing. Lippert et al.27
investigated the toothbrush abrasion of surface softened
enamel using AFM. They concluded that the amount of
enamel lost due to toothbrushing was independent of the
demineralization time and was lower compared to the mineral
loss caused by demineralization treatments. We decided to
avoid toothbrushing in order to stress the demineralizing
potential of the soft drink chosen.
In the present study, a sodium fluoride/potassium nitrate
toothpaste (Pronamel) and a hydroxyapatite nanocrystals
toothpaste (Biorepair Plus) were compared by AFM.
After exposure to a demineralizing soft drink, the initial
Rrms values were not regained after exposure to the two
toothpastes. Though they provided similar Rrms values before
and after treatment, the images obtained by AFM showed a
different remineralizing pattern for the two toothpastes.
Lussi et al.16 compared 5 fluoride-containing toothpastes
on the prevention of erosion by a microhardness test. They
underlined that the range of free-fluoride availability was
quite similar in the 5 groups, which provided similar values of
microhardness.
Using an in situ erosion remineralization model and a
microhardness test, Zero et al.38 concluded that fluoride
dentifrice containing potassium nitrate significantly enhanced the remineralization of enamel previously subjected
to an erosion challenge.
After treatment with the demineralizing solution followed by
Pronamel, both interprismatic and prismatic enamel structures
still appear evident. Pronamel favors mineral deposition, thus
providing a demineralization reduction, but in case of further
acid challenges, no protective action will be obtained.
On the contrary, BioRepair Plus showed a progressive
microparticles deposition: the interprismatic and prismatic
enamel structures appear to be completely hidden by a thick
homogeneous apatitic layer. According to Roveri et al.18 this
journal of dentistry 38 (2010) 868–874
coating formation corresponds to an apatite deposition inside
the demineralized areas of enamel surface.
The treatment of the teeth surface with the CPP-ACP paste
causes the formation of a layer that fills the interprismatic
cavities, and partially cover the prisms faces. It is well known
that the localization of ACP at the tooth surface buffers the free
calcium and phosphate ion activities, thus helping to maintain
a state of super saturation which depresses demineralization
and enhances remineralization of the enamel, thus providing
a protective action against further acid attack.
5.
12.
13.
14.
15.
Conclusions
16.
This in vitro study demonstrated that both toothpastes tested
(Pronamel and BioRepair Plus) offer a degree of protection
from erosive drinks, without statistical difference. The
morphological interpretation of AFM images suggested that
this protective effect is considered more evident for Biorepair
Plus.
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