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Original Research

A Comparative Study of the Effect of Nanohydroxyapatite and


Eggshell on Erosive Lesions of the Enamel of Permanent Teeth
Following Soft Drink Exposure: A Randomized Clinical Trial
Maede Yaberi1, Roza Haghgoo1
1
Department of Pediatric Dentistry, Faculty of Dentistry, Shahed University, Tehran, Iran

Abstract
Aims: The purpose of this in situ study was to compare the effects of nanohydroxyapatite or eggshell (ES) extract on the microhardness of
healthy third molar tooth enamel following soft drink exposure. Materials and Methods: This study was performed as a randomized clinical
trial. This experimental study was conducted with ten participants and involved twenty extracted, healthy, permanent third molar teeth. The
initial microhardness values of the teeth were measured. The teeth were then exposed to delestre for 10 min, and the microhardness was
measured again. Samples were divided into two groups of 10. For each appliance, two teeth were placed, one from each group and one on
either side of the appliance. For the in situ study, sections of two teeth, one from each group, were embedded in an appliance and placed in
the mouth of the volunteer. Each volunteer used this appliance for 10 days. After 10 days, the microhardness of the teeth was measured again.
Finally, data were analyzed using analysis of variance and paired t‑tests. Results: The mean tooth enamel microhardness was significantly
decreased following exposure to delestre and the mean microhardness of demineralized tooth enamel significantly increased after exposure to
both nanohydroxyapatite and eggshell extract. Conclusion: Nanohydroxyapatite and ES have the potential to remineralization erosive lesions.

Keywords: Demineralization, dental enamel, eggshell, erosion, microhardness, nano‑hydroxyapatite, third molar

Introduction other substances to repair demineralization lesions. Different


compounds can be used to address dental erosion.
Demineralization occurs when factors causing demineralization
are not controlled. This process can progress to the point of Hydroxyapatite is a major component of biomaterials and
degrading the enamel completely, exposing the dentin, and comprises the bulk of the material structure of teeth and
causing sensitive teeth, and finally, pulp exposure is involved. bone.[10] This substance has a high biocompatibility and is
Erosion is the chemical destruction of dental tissue without the widely used in medical treatment. Nanohydroxyapatite has
presence of microorganisms and is responsible for destroying unique characteristics such as solubility, higher surface energy,
tooth enamel.[1] Consumption of acidic soft drinks is a major and good biocompatibility compared to hydroxyapatite.[11] In
cause of erosion,[2,3] and it is necessary to investigate methods addition, nanohydroxyapatite particles have more biological
for healing erosive lesions with remineralization compounds. activity than larger particles.[12] Recent studies have shown
that materials containing nanohydroxyapatite can remineralize
Fluoride is the most common substance that is used in the
initial caries lesions.[13‑15]
remineralization of demineralization lesions.[4,5] However,
there are some concerns about respiratory and digestive
problems from chronic exposure to low doses as well as Address for correspondence: Dr. Roza Haghgoo,
dental fluorosis.[6] In addition, the fluoride ion is not able to Department of Pediatric Dentistry, Dental School, Shahed University, N71,
remineralize dental demineralization lesions.[7,8] Each unit of Italia Street, Vesal Av., Tehran, Iran.
E‑mail: haghgoodent@yahoo.com
fluoroapatite is composed of calcium and phosphate in addition
to fluoride.[9] Therefore, it is necessary to replace fluoride with
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How to cite this article: Yaberi M, Haghgoo R. A comparative study of the


DOI: effect of nanohydroxyapatite and eggshell on erosive lesions of the enamel
10.4103/jioh.jioh_84_18 of permanent teeth following soft drink exposure: A randomized clinical
trial. J Int Oral Health 2018;10:176-9.

176 © 2018 Journal of International Oral Health | Published by Wolters Kluwer - Medknow
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Yaberi and Haghgoo: Effect of nano‑hydroxyapatite and eggshell on erosive lesions

ES extract contains calcium, fluoride, and strontium and has a the in situ study, sections of two teeth, one from each group,
positive effect on metabolism of the bone and teeth.[16] were embedded in an appliance and placed in the mouth of
the volunteer. These sections were placed in the vestibule of
The aim of this in situ study was to compare the effect of
the volunteer’s mouth so that it did not interfere with his or
nanohydroxyapatite and ES extract on microhardness of
her occlusion. Individuals were not included if they had any
healthy third molar teeth after exposure to delestre.
systemic diseases or a history of head and neck radiography.
Persons receiving any type of antibiotics were not included
Materials and Methods either.
Design of this prospective study was parallel and was
Each volunteer used this appliance for 10 days.During this
conducted with 20 healthy third molar impacted teeth, and this
period, the volunteer removed the appliance from her or
sample size was selected based on previous studies[3,17] that
his mouth twice a day, and on the one side of the appliance,
were extracted by surgery in the Department of Maxillofacial
containing a section of tooth, applied nano-hydroxyapatite10%
Surgery, Faculty of Dentistry, Shahed University.
and on the other side, placed a solution containing eggshell
The study protocol was approved by the Ethics Committee of (ES) extract 3% (each package of ES powder contains3 g of dry
Shahed University, Tehran, Iran (Ethics committee reference matter, which mixes it with 100 ml of distilled water), for 10
number: 41/226702 IRCT2017040733162N2). They were min. Then, the volunteer puts the appliance (a Haley appliance
18–21 years old. Inclusion criteria were healthy participants. that included a slice of teeth from each group on either side)
Exclusion criteria were patients did not consume any drug and back in her or his mouth. After 10 days, the appliances were
were nonsmoker. collected from the volunteers, and a third measurement of
microhardness of teeth was made by an individual who was
The teeth did not have any sign of caries according to the WHO
blinded to which tooth received which treatment condition.
criteria; no cracks, wear, or hypocalcification.
The results were analyzed statistically. First, a repeated
Procedure
measure analysis of variance was performed. In this test, initial
The teeth were kept inside glass containers containing water
and final microhardness measures were defined as repeated and
at room temperature after cleaning off any blood and saliva.
each group was defined as a between‑subject factor. Because
The water was changed every 2 days to prevent changes in the
the interaction between two factors was significant, the mean
surface of the water and to avoid microorganism growth and
initial and final microhardness values were compared using
water pollution. The surfaces of the teeth were cleaned with
paired t‑tests for each group software SPSS Version 16 (IBM
prophylactic pastes containing pumice without fluoride and a
Corp, Armonk, NY).
low‑speed handpiece with a rotation range of 500–1500 rpm
followed by brushing. Then, the teeth were examined with a
stereomicroscope using × 40 magnification for the presence of Results
carious lesions or any enamel defects and cracks. A tag square In this study, the effects of either nanohydroxyapatite or ES
with dimensions of 5 mm × 5 mm was attached to the distal extract on microhardness of the enamel from twenty impacted,
tooth surface, and all of the remaining surfaces of the tooth permanent third molars demineralized by exposure to delestre
were covered with self‑curing transparent acrylic, and it was were investigated. The mean of microhardness of teeth enamel
allowed to harden. Samples were kept in cool water to prevent was calculated before and after the exposure to delestre and
changes due to heat from the hardening acrylic. after treatment with nano‑hydroxyapatite or ES.
The surfaces of the teeth were polished in water with grit tissue This study was performed between February and April 2017.
5000 for measuring microhardness correctly. Then, the surfaces
Demographic and clinical characteristics for each group are
of the samples were dried, and the initial microhardness was
shown in Table 1.
measured using a Vickers machine (M‑g5037; Shimadzu,
Japan). Consistent with previous studies, 50 g of force was The mean ± standard deviations (SDs) of the initial microhardness
used for this research.[10] for the ten samples in the 1st group (nanohydroxyapatite)
were 548/3 ± 42/27 kgf/mm 2 . The mean + SD was
Based on the studies conducted by the Ministry of Industry and
479/1 ± 46/58 kgf/mm2 after immersion in delestre, which
Behnoosh, lemon delestre was selected as the most popular
was 87%/37% of the mean initial microhardness. The mean
delestre among domestic or foreign drinks in the Iranian market.
of tertiary microhardness of these samples was 496/7 ± 34/93.
Teeth were immersed in 40 ml of Behnoosh lemon delestre,
A paired t‑test showed that this difference was statistically
which was placed in the container immediately after opening
significant (P1 = 0.008, P2 = 0.55).
the bottle, for a total of 15 min. Then, the microhardness of
the teeth was measured again using the Vickers machine. The mean of the initial microhardness for the ten samples in the
In this triple randomized clinical trial (participants, care 2nd Group (ES) was 504/40 ± 35/23 kgf/mm2. The mean ± SD
providers, and those assessing outcomes), the samples were was 449/80 ± 67/71 after immersion in Behnoosh lemon
randomly (using a coin) sorted into two groups of ten each. For delestre, and this mean value was 89%/17% of the mean initial

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Yaberi and Haghgoo: Effect of nano‑hydroxyapatite and eggshell on erosive lesions

the dental material, implants, and orthopedic treatment.[19]


Table 1: Demographic and clinical characteristics for
Hydroxyapatite at the nanometric level is more similar to
each group
enamel apatite crystals.[13]
Demographic and Nanohydroxy Egg shell
clinical characteristics apatite Nanohydroxyapatite has hydrophilic properties and a greater
Gender Boy Girl Boy Girl surface area. Therefore, these crystals have moisturizing
5 5 5 5 properties because of their surface area. When they are used
Age 18 19 20 21 18 19 20 21 on dental surfaces, they produce a thin and strong layer on the
2 1 4 3 2 1 4 3 enamel surface that binds with dental crowns and this thin layer
Race Iranian Iranian of crystal is self‑organizing, so this compound can participate
Health Statue Healthy Healthy in enamel remineralization.[20]
ES is a rich source of calcium. It contains calcium carbonate,
microhardness. The mean tertiary microhardness of the samples calcium phosphate, magnesium carbonate, and other elements
was 475/20 ± 39/13 kgf/mm2, and a paired t‑test showed such as fluoride and strontium.[21] Hence, according to the
that this difference was statistically significant  (P1 = 0.10, above, the results of this study seem to be reasonable.
P2 = 0.86).
The results of Huang’s study are consistent with the results
of the present study, but in Huang’s study, there was another
Discussion material included with the nanohydroxyapatite that could
Teeth are in continuous processes of remineralization and have a synergistic effect along with the nanohydroxyapatite.
demineralization in the oral cavity. Normally, these two Thus, in this study, nanohydroxyapatite was used in pure
processes are in balance. Demineralization becomes dominant form to test if this material by itself can have a positive effect
when the oral cavity is exposed to foreign acids, and the speed on the microhardness of the tooth. In the present study, a
of surface damage to the enamel increases progressively. This 10% nanohydroxyapatite solution was used. The results of
imbalance results in erosion. Erosion processes lead to surface Huang’s study, which evaluated different concentrations of
demineralization and then complete destruction of the surface nanohydroxyapatite, showed that 10% and 15% concentrations
layer of enamel.[18] had the same effect.[13]
After considering erosion problems due to exposure to acidic In the present study, 15 min of exposure to delester was chosen
soft drinks, we searched for a substance that could accelerate as the duration based on the average daily consumption of
remineralization of enamel. In this study for the first time, the carbonated soft drinks and the 20 s average time of having a
effect of nano‑hydroxyapatite and ES extract on impacted third sip of soft drink in the mouth before being cleared by saliva.
molar permanent tooth enamel was investigated in situ after In related studies, the application time of the remineralization
exposure to an acidic soft drink. materials was different.[13] However, in the present study,
10 min was used because longer application times are
The results of this study showed that enamel microhardness
logistically difficult to apply in the clinic.
decreased significantly after immersion in delester and that the
mean microhardness of the teeth demineralized after exposure The results of the present study showed that the microhardness
to delestre significantly increased after exposing them to either of tooth enamel, which decreased after exposure to delestre,
nanohydroxyapatite or ES extract solution. also increased after exposure to the ES extract solution. ES
extract contains not only calcium but also other elements such
This study for the fi rst time compare the effect of ES and
as fluoride and strontium, and it can have a positive effect on
nano-hydroxyapatite on enamel lesions following consumption
tooth structure. Therefore, it is logical that the microhardness
of delester and the results of this study showed that these two
of enamel would increase following exposure to ES extract,
materials are able to reconstruct the demineralized enamel, so
considering its composition.[16,22]
this result is one of the strengths of this study. Furthermore,
design of this study was in situ, and the results of such studies The results of the study by Haghgoo et al. showed that there
are more documentary. is no statistically significant difference in the remineralization
of demineralized enamel between 3% and 10% concentrations
This study was done in the form of in situ, so one should put
of ES extract, so in the present study, a 3% ES extract solution
someone else’s tooth in her/his mouth and  finding such people
was used.[21]
were very difficult and this was a limitation of the study.
The results of the present study are consistent with Haghgoo’s
Using the results of this study, it is possible to repair
investigation.[21] However, the present study was done in situ.
demineralized lesions resulting from the use of delester.
This was the first in situ study to compare the effects of
Hydroxyapatite is an important mineral component of either nano‑hydroxyapatite or ES on the microhardness of
dental enamel and is bioactive and biocompatible. Many demineralized tooth enamel, so there are no other in situ studies
studies have investigated the use of hydroxyapatite on available for comparison.

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