International Dental & Medical Journal of Advanced Research (2016), 2, 1–5
ORIGINAL ARTICLE
Remineralization of bleached enamel with novel
nutraceutical agents: An in vitro study
Girija S. Sajjan, Revathi Indukuri, Tanikonda Rambabu, Kanumuri Madhu Varma, Rajulapati Kalyan Satish
Department of Conservative Dentistry and Endodontics, Vishnu Dental College, Bhimavaram, West Godavari District, Andhra Pradesh, India
Keywords
Energy dispersive X-ray spectroscopy,
nutraceutical, remineralization,
vital bleaching
Correspondence
Dr. Girija S. Sajjan, Vishnu Dental College,
Vishnupur, Bhimavaram, West Godavari
District - 534 202, Andhra Pradesh, India.
Phone: 91-8179846088.
Email: [email protected]
Received 09 March 2016;
Accepted 11 April 2016
doi: 10.15713/ins.idmjar.41
Abstract
Background/Purpose: The effect of bleaching agents on dental hard tissues was
uncertain. Moreover, reverting the side effects caused by bleaching using nutraceuticals
as remineralizing agents remained uninvestigated. Hence, the aim of this study was to
evaluate the effect of 22% carbamide peroxide (CP) on the mineral content of enamel
and to evaluate the remineralization of bleached enamel after treatment with three
different remineralizing agents.
Materials and Methods: A total of 30 extracted human maxillary incisors were selected,
and their initial mineral content was analyzed using scanning electron microscopy with
energy dispersive X-ray spectroscopy (EDX). All the specimens were bleached with
22% CP and their mineral content was reanalyzed. They were then randomly divided
into three groups. Group A: Remineralization with casein phosphopeptide-amorphous
calcium phosphate (CPP-ACP), Group B: Remineralization with a 6.5% grape seed
extract (GSE) solution prepared in phosphate buffer, and Group C: Remineralization
with 6.5% wheat grass solution in phosphate buffer. Remineralization was carried out for
10 min, and EDX analysis was carried out again. Statistical analysis was done using oneway ANOVA and Student’s t-test.
Results: Bleaching with 22% CP significantly decreased the mineral content of the
enamel. All the three remineralizing agents significantly increased the mineral content.
Group A (CPP-ACP) showed the highest remineralization than Group B and C but was
not statistically significant.
Conclusion: Within the limitations of the present study, it can be concluded that
bleaching with CP decreases the mineral content of enamel and agents such as CPPACP, GSE, and wheat grass help in remineralizing the bleached surface.
Vital bleaching is considered as an easy, effective, and a
conservative way to lighten the discolored teeth. The etiology
of tooth discoloration is complicated and is broadly classified as
intrinsic, extrinsic, or internalized in nature.[1]
Bleaching agents such as hydrogen peroxide or carbamide
peroxide (CP), undergo ionic dissociation when applied to the
tooth structure and give rise to the formation of free radicals such
as nascent oxygen, hydroxyl radical, per hydroxyl, and superoxide
anions, which are unstable free radicals. These highly reactive
free radicals reach-out for electron rich regions of pigment inside
the dental structure and cause breakdown of the large pigmented
molecules into smaller, less pigmented ones.[2]
However, the side effects of bleaching on enamel such as
decrease in mineral content and hardness, increased roughness
and morphological alterations remain quite a debatable topic in
the literature due to conflicting results from previous studies.[3]
One of the most common clinical complaints associated
with bleaching is the sensitivity of teeth. The incidence of
hypersensitivity was reported to be 10-90% with the inoffice bleaching procedure. Although it could be mostly
mild or moderate, it is at times so severe that the treatment
is discontinued.[4] Enamel is generally considered to be
impermeable. Nevertheless, the oxidative free radicals generated
from hydrogen peroxide have low molecular weight and are able
to denaturize proteins. These free radicals create porosities on
the enamel structure which may thus permit diffusion through
International Dental & Medical Journal of Advanced Research ● Vol. 2 ● 2016
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Introduction
Remineralization of bleached enamel with novel nutraceutical agents
the organic matrix of dentin. From dentine hydrogen peroxide
reaches the pulp, where it directly activates A - δ nerves which
thereby results in pain.[5] Thus, the surface morphological changes
may contribute to the sensitivity associated with bleaching.
Along with surface remineralization, the surface requires to
be free of oxygen free radicals as the residual free radicals can
cause pulpal inflammation,[6] compromised bond strength for
immediate composite restoration[3] and altered optical properties
of bleached enamel. So, it would be beneficiary if remineralizing
agent also has antioxidant property. In this view, grape seed
extract (GSE) and wheat grass, which are known antioxidants,
were tried as surface remineralizing and desensitizing agents.
GSE, a nutraceutical, is a rich source of proanthocyanidin
(PA), mainly composed of monomeric catechin and epicatechin,
gallic acid, and polymeric and oligomeric procyanidins.[7]
GSE as a remineralizing agent was evaluated by confocal laser
scanning microscopy. It was concluded in this morphological
analysis that GSE has a positive effect on remineralization
process of the artificial carious lesion.[8] However, chemical
analysis of the bleached surface, regarding changes in the
chemical composition of the surface has not been studied in any
of the previous studies.
Wheatgrass is another nutraceutical with many health
benefits. It is a good source of Vitamin A, Vitamin C, Vitamin E,
Vitamin K, thiamin, riboflavin, niacin, Vitamin B6, pantothenic
acid, calcium, phosphorus, potassium, iron, zinc, copper,
manganese, and selenium.[9] Wheat grass has anti-inflammatory,
anticancer, antiaging, and antioxidant property.[10]
However, no studies regarding the remineralization potential
of these novel nutraceutical agents on bleached enamel can be
found in the literature. Hence, the aims of the study were:
1. To evaluate the effect of 22% CP on the mineral content of
enamel
2. To evaluate the remineralizing potential of casein
phosphopeptide-amorphous calcium phosphate (CPPACP), GSE, and wheat grass on the bleached enamel.
The mineral content was assessed using energy dispersive
X-ray spectroscopy (EDX) (ZEISS, EVO18, Special Edition,
Department of Physics, Osmania University, Hyderabad.)
Sajjan, et al.
Preparation of 6.5% w/v of wheat grass solution in
phosphate buffer (pH 7.4)
About 6.5 g of wheat grass powder was dissolved in 100 ml of
phosphate buffer solution.
The samples were randomly divided into three groups
(n = 10) based on the treatments as follows:
Group A: Remineralization with CPP-ACP.
Group B: Remineralization with 6.5% (w/v) GSE solution.
Group C: Remineralization with 6.5% (w/v) wheat grass
solution.
Remineralizing agents were gently agitated on the bleached
enamel with an applicator tip for 10-min and cleaned thoroughly
using deionized water. The mineral content of bleached enamel
after the remineralization regimen was assessed using EDX.
Statistical analysis was done using one-way ANOVA and
Student’s t-test.
Results
EDX was used to determine the calcium and phosphorus content
in % weight of sound, bleached and remineralized enamel in
each group. The calcium (Ca) and phosphorus (P) content was
converted into Ca/P ratios. The EDX graphs of the highest Ca/P
ratio attained among sound enamel samples, bleached samples,
and Group A, B, and C samples are shown in Graphs 1-5,
respectively. Table 1 shows a comparison of the mean Ca/P
ratios of sound and bleached enamel using t-test. There was a
statistically significant decrease in the mineral content after
bleaching (P = 0.021). Table 2 represents the mean Ca/P ratio
of bleached and remineralized samples analyzed by t-test. There
was a significant increase in the mineral content after surface
treatment with remineralizing agents in all the groups (P = 0.00).
One-way ANOVA was applied to compare the mean Ca/P ratios
of the study groups after remineralization shown in Table 3
which showed no statistically significant difference between the
three study groups (P = 0.633). Graph 6 represents relationship
between the remineralization potential of study groups.
Discussion
Materials and Methods
About 30 extracted human maxillary incisors were cleaned using
ultrasonic scaler and analyzed using EDX (ZEISS, EVO18,
Special edition. Department of Physics, Osmania University,
Hyderabad) to determine the initial mineral content. 22% CP
(Philips Zoom, M & M Dental Associates, Mumbai, India) was
applied to the labial surfaces 8 h/day for 2-week. Then, samples
were washed and stored in artificial saliva. Post-bleaching EDX
analysis was done.
Bleaching agents produce free radicals which cause
fragmentation of pigmented molecules into smaller species and
alter the light absorption and thereby reduce or eliminate the
stain.[10] It was stated in a review that H2O2 induces alteration
of the chemical composition of enamel by reducing the Ca/P
Table 1: Comparison of mean Ca/P ratios of sound and bleached
enamel using t-test
Group
Number
Mean
Preparation of 6.5 % w/v of GSE solution in phosphate
buffer (pH 7.4)
Sound enamel
30
1.8973
Standard
deviation
0.17924
t value
P value
5.203
0.021*
After bleaching
30
1.7284
0.14705
About 6.5 g of GSE powder was dissolved in 100 ml of phosphate
buffer solution.
*The mean difference is significant at the 0.05 level. Ca/P: Calcium/
phosphorus
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International Dental & Medical Journal of Advanced Research ● Vol. 2 ● 2016
Sajjan, et al.
Remineralization of bleached enamel with novel nutraceutical agents
Table 2: t-test values to analyze the mean Ca/P ratio of bleached and remineralized samples in study groups
Group
A (CPP-ACP)
B (GSE)
C (wheat grass powder)
Procedure
Bleached enamel
Mean
1.6999
Standard deviation
0.07893
Remineralized enamel
1.8550
0.12169
Bleached enamel
1.7286
0.20867
Remineralized enamel
1.8474
0.30024
Bleached enamel
1.7567
0.13456
Remineralized enamel
1.7767
0.12429
t value
68.105
P value
0.00*
0.00*
26.195
0.00*
41.287
*The mean difference is significant at the 0.05 level. CPP-ACP: Casein phosphopeptide-amorphous calcium phosphate, Ca/P: Calcium/phosphorus.
GSE: Grape seed extract
Table 3: Comparison of mean Ca/P ratios of study groups using one-way ANOVA
Group
A (CPP-ACP)
Procedure
Remineralized enamel
Number of mean samples
10
Mean
1.8550
Standard deviation
0.12169
B (GSE)
10
1.8474
0.12429
C (wheat grass powder)
10
1.7767
0.30024
F value
0.464
P value
0.633
CPP-ACP: Casein phosphopeptide-amorphous calcium phosphate, GSE: Grape seed extract, Ca/P: Calcium/phosphorus
Graph 1: Elemental analysis of sound enamel by energy dispersive
X-ray spectroscopy. Calcium/phosphorus ratio: 2.2185
Graph 3: Elemental analysis of enamel sample by energy
dispersive X-ray spectroscopy after remineralization with casein
phosphopeptide amorphous calcium phosphate. Calcium/
phosphorus ratio: 2.7313
Graph 2: Elemental analysis of bleached enamel by energy
dispersive X-ray spectroscopy.. Calcium/phosphorus ratio: 1.3185
ratio.[11] Raman spectroscopic study showed that exposure to
10% CP for more than 7 days resulted in a significant decrease
in phosphate concentration in the bleached enamel.[5] On the
contrary, a study did not report any mechanical, morphologic, or
chemical changes following vital bleaching with CP.[12] Hence,
the effect of bleaching on dental enamel is not yet clear and is still
a controversial subject.
International Dental & Medical Journal of Advanced Research ● Vol. 2 ● 2016
Graph 4: Elemental analysis of enamel sample by energy dispersive
X-ray spectroscopy after remineralization with grape seed extract.
Calcium/phosphorus ratio: 1.9859
EDX is used for elemental characterization. The ratio of Ca
and P is also indicative of chemical changes since change can
suggest that the mineral phase was altered or that a significant
substitution of ions may have occurred.[12]
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Remineralization of bleached enamel with novel nutraceutical agents
Sajjan, et al.
In the current study, the elemental analysis revealed a
statistically significant decrease in the mean Ca/P ratio from
1.8973 to 1.7284 after bleaching.
CP breaks down to produce water, oxygen, carbon dioxide,
and ammonia, which would result in a slight lowering of pH of
the bleaching agent. This reduction in pH might have an effect
on the dissolution of the mineral content of enamel.[13]
Bleaching procedure may have potential side effects such as
sensitivity which could be due to surface enamel loss and decrease
in the mineral content. There is decrease in the hardness of teeth
and increase in roughness. It was also reported that the residual
peroxide on the surface interferes with resin attachment, inhibits
resin polymerization, and the bond strength of composite
resin to the tooth is compromised if bonding is performed
immediately.[3] This could also be because of decreased mineral
content of the surface.
So, an attempt to remineralize the enamel surface may be
useful in the prevention of sensitivity to the patient and also
increase the success of adhesive restorations if required to be
done immediately after the bleaching.
A newer concept for remineralization is the use of milk and
milk products, which appear to have a protective effect against
the mineral loss.[14] Results of the current study showed that
there was a significant increase in the mean Ca/P ratio from
1.6999 to 1.8550 after remineralization with CPP-ACP.
CPP binds to pellicle and plaque at the tooth surface and
stabilizes high concentrations of calcium, phosphate, and
fluoride ions. This nano complex attaches to dental plaque and
tooth surfaces and acts as a calcium and phosphate reservoir.
The attached CPP-ACP maintains a supersaturated mineral
environment by releasing calcium and phosphate ions, and
thereby helps in enhancing remineralization of enamel.[15] The
results obtained also correlated with a morphological study in
which a combination of CPP-ACP and bleaching agent prevented
negative changes of hardness, roughness, and morphology.
In the case of immediate bonded restorations, the oxygen free
radicals need to be taken care of as they can cause compromised
bond strength. Thus, antioxidants may have a beneficiary effect.
The present study tested the benefits of GSE and wheat grass
for their remineralizing potential, both of which are basically
very good antioxidants.[3,16] A very few studies were reported
on the effects of natural products or nutraceuticals on the
remineralization processes of dental hard tissues.
GSE has been recently advocated for its beneficial
antioxidant, antibacterial, and free radical scavenging properties.
The biologically active constituents of GSE are polyphenols,
mainly PAs.
The results of the current study show a significant increase
in the mean Ca/P ratio from 1.7286 after bleaching to 1.8474
after remineralization with GSE. The results of the present
study are in accordance with a scanning electron microscopic
study that showed scaffolding deposits on the enamel surface
with cluster-like structures resembling remineralization process
initiation.[7] The remineralization potential of GSE on artificial
root caries was also evaluated using a polarized light microscope
and confocal laser scanning microscope.[8]
Gallic acid, one of the major constituents of GSE and Galla
Chinensis, were assumed to facilitate mineral deposition,
predominately on the surface layer.[7]
Remineralization with wheat grass solution showed a
statistically significant increase in the mean Ca/P ratio from
1.7567 to 1.7767. The oxygen radical absorption capacity of
wheatgrass was found to be very satisfactory in phytotherapy
research studies.[16]
The major clinical utility of wheat grass in diseased conditions
might be due to the presence of biologically active compounds
and minerals in it and due to its antioxidant potential. The
antioxidant potential of wheat grass could be by virtue of the
high content of bioflavonoids in it such as apigenin, quercetin,
and luteolin. Furthermore, its therapeutic potential may also be
related to the presence of indole compounds namely choline and
laetrile.[9] However, the exact mechanism by which wheat grass
solution increased the mineral content of enamel is not well
understood.
According to the results obtained in the present study, all the
three remineralizing agents significantly increased the mineral
content of the bleached surface, with no statistically significant
difference between the groups.
To our best knowledge, this is the first attempt to evaluate
the remineralizing capacity of GSE and wheat grass on bleached
enamel. These two novel nutraceutical agents were able to
significantly contribute to the remineralization process.
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International Dental & Medical Journal of Advanced Research ● Vol. 2 ● 2016
Graph 5: Elemental analysis of enamel sample by energy dispersive
X-ray spectroscopy after remineralization with wheatgrass.
Calcium/phosphorus ratio: 1.8653
Graph 6: Remineralization between the remineralization potential
of study groups
Sajjan, et al.
Although CPP-ACP will help in remineralization, it does
not have the antioxidant property. The presence and diffusion
of oxygen free radicals might lead to pulpal inflammation and
decreased resin bond strength. Nutraceutical agents tested in
the present study also possess antioxidants which might help in
neutralizing the oxygen free radicals. Hence, these products can
be considered as surface remineralizing agents post-bleaching.
Although bleaching is an effective procedure, it has its own
limitations like alteration in the mineral contents. Clinicians
should be aware of the role of remineralizing to overcome the
side effects of bleaching.
Further, there is a need for long-term studies as well as ex vivo
and in vivo studies in future to evaluate the efficiency of these
remineralizing agents in a clinical scenario. Future studies are
required to elaborate the mechanism of action of these newer
agents and the clinical protocol of their application. Spectroscopic
analysis of the color change followed by application these novel
remineralizing agents to be done.
Conclusion
Within the limitations of the present study, the following
conclusions were drawn:
1. There was a significant decrease in the mineral content after
bleaching with 22% CP
2. CPP-ACP, GSE, and wheat grass significantly increased the
remineralization of bleached enamel with no statistically
significant difference between the three groups
3. The added advantage of the anti-oxidant property of GSE and
wheat grass powder can be utilized along with remineralizing
potential.
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How to cite this article:Sajjan GS, Indukuri R, Rambabu T,
Varma KM, Satish RK. Remineralization of bleached enamel
with novel nutraceutical agents: An in vitro study. Int Dent Med
J Adv Res 2016;2:1-5.
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