[Downloaded free from http://www.jioh.org on Saturday, November 24, 2018, IP: 156.208.79.

12]

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

Demineralization occurs when factors causing demineralization
are not controlled. This process can progress to the point of
degrading the enamel completely, exposing the dentin, and
causing sensitive teeth, and finally, pulp exposure is involved.
Erosion is the chemical destruction of dental tissue without the
presence of microorganisms and is responsible for destroying
tooth enamel.[1] Consumption of acidic soft drinks is a major
cause of erosion,[2,3] and it is necessary to investigate methods
for healing erosive lesions with remineralization compounds.
Fluoride is the most common substance that is used in the
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
dental fluorosis.[6] In addition, the fluoride ion is not able to
remineralize dental demineralization lesions.[7,8] Each unit of
fluoroapatite is composed of calcium and phosphate in addition
to fluoride.[9] Therefore, it is necessary to replace fluoride with
Access this article online
Quick Response Code:
Website:
www.jioh.org
compounds can be used to address dental erosion.
Hydroxyapatite is a major component of biomaterials and
comprises the bulk of the material structure of teeth and
bone.[10] This substance has a high biocompatibility and is
widely used in medical treatment. Nanohydroxyapatite has
unique characteristics such as solubility, higher surface energy,
and good biocompatibility compared to hydroxyapatite.[11] In
addition, nanohydroxyapatite particles have more biological
activity than larger particles.[12] Recent studies have shown
that materials containing nanohydroxyapatite can remineralize
initial caries lesions.[13‑15]
Address for correspondence: Dr. Roza Haghgoo,
Department of Pediatric Dentistry, Dental School, Shahed University, N71,
Italia Street, Vesal Av., Tehran, Iran.
E‑mail: haghgoodent@yahoo.com
This is an open access journal, and articles are distributed under the terms of the Creative
Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to
remix, tweak, and build upon the work non-commercially, as long as appropriate credit
is given and the new creations are licensed under the identical terms.
For reprints contact: reprints@medknow.com

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
[Downloaded free from http://www.jioh.org on Saturday, November 24, 2018, IP: 156.208.79.12]
Yaberi and Haghgoo: Effect of nano‑hydroxyapatite and eggshell on erosive lesions
ES extract contains calcium, fluoride, and strontium and has a
positive effect on metabolism of the bone and teeth.[16]
The aim of this in situ study was to compare the effect of
nanohydroxyapatite and ES extract on microhardness of
healthy third molar teeth after exposure to delestre.
Materials and Methods
Design of this prospective study was parallel and was
conducted with 20 healthy third molar impacted teeth, and this
sample size was selected based on previous studies[3,17] that
were extracted by surgery in the Department of Maxillofacial
Surgery, Faculty of Dentistry, Shahed University.
The study protocol was approved by the Ethics Committee of
Shahed University, Tehran, Iran (Ethics committee reference
number: 41/226702 IRCT2017040733162N2). They were
18–21 years old. Inclusion criteria were healthy participants.
Exclusion criteria were patients did not consume any drug and
were nonsmoker.
The teeth did not have any sign of caries according to the WHO
criteria; no cracks, wear, or hypocalcification.
Procedure
The teeth were kept inside glass containers containing water
at room temperature after cleaning off any blood and saliva.
The water was changed every 2 days to prevent changes in the
surface of the water and to avoid microorganism growth and
water pollution. The surfaces of the teeth were cleaned with
prophylactic pastes containing pumice without fluoride and a
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
carious lesions or any enamel defects and cracks. A tag square
with dimensions of 5 mm × 5 mm was attached to the distal
tooth surface, and all of the remaining surfaces of the tooth
were covered with self‑curing transparent acrylic, and it was
allowed to harden. Samples were kept in cool water to prevent
changes due to heat from the hardening acrylic.
The surfaces of the teeth were polished in water with grit tissue
5000 for measuring microhardness correctly. Then, the surfaces
of the samples were dried, and the initial microhardness was
measured using a Vickers machine (M‑g5037; Shimadzu,
Japan). Consistent with previous studies, 50 g of force was
used for this research.[10]
Based on the studies conducted by the Ministry of Industry and
Behnoosh, lemon delestre was selected as the most popular
delestre among domestic or foreign drinks in the Iranian market.
Teeth were immersed in 40 ml of Behnoosh lemon delestre,
which was placed in the container immediately after opening
the bottle, for a total of 15 min. Then, the microhardness of
the teeth was measured again using the Vickers machine.
In this triple randomized clinical trial (participants, care
providers, and those assessing outcomes), the samples were
randomly (using a coin) sorted into two groups of ten each. For
Journal of International Oral Health  ¦  Volume 10 ¦ Issue 4 ¦ July-August 2018
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. These sections were placed in the vestibule of
the volunteer’s mouth so that it did not interfere with his or
her occlusion. Individuals were not included if they had any
systemic diseases or a history of head and neck radiography.
Persons receiving any type of antibiotics were not included
either.
Each volunteer used this appliance for 10 days.During this
period, the volunteer removed the appliance from her or
his mouth twice a day, and on the one side of the appliance,
containing a section of tooth, applied nano-hydroxyapatite10%
and on the other side, placed a solution containing eggshell
(ES) extract 3% (each package of ES powder contains3 g of dry
matter, which mixes it with 100 ml of distilled water), for 10
min. Then, the volunteer puts the appliance (a Haley appliance
that included a slice of teeth from each group on either side)
back in her or his mouth. After 10 days, the appliances were
collected from the volunteers, and a third measurement of
microhardness of teeth was made by an individual who was
blinded to which tooth received which treatment condition.
The results were analyzed statistically. First, a repeated
measure analysis of variance was performed. In this test, initial
and final microhardness measures were defined as repeated and
each group was defined as a between‑subject factor. Because
the interaction between two factors was significant, the mean
initial and final microhardness values were compared using
paired t‑tests for each group software SPSS Version 16 (IBM
Corp, Armonk, NY).
Results
In this study, the effects of either nanohydroxyapatite or ES
extract on microhardness of the enamel from twenty impacted,
permanent third molars demineralized by exposure to delestre
were investigated. The mean of microhardness of teeth enamel
was calculated before and after the exposure to delestre and
after treatment with nano‑hydroxyapatite or ES.
This study was performed between February and April 2017.
Demographic and clinical characteristics for each group are
shown in Table 1.
The mean ± standard deviations (SDs) of the initial microhardness
for the ten samples in the 1st group (nanohydroxyapatite)
were 548/3 ± 42/27 kgf/mm 2 . The mean + SD was
479/1 ± 46/58 kgf/mm2 after immersion in delestre, which
was 87%/37% of the mean initial microhardness. The mean
of tertiary microhardness of these samples was 496/7 ± 34/93.
A paired t‑test showed that this difference was statistically
significant (P1 = 0.008, P2 = 0.55).
The mean of the initial microhardness for the ten samples in the
2nd Group (ES) was 504/40 ± 35/23 kgf/mm2. The mean ± SD
was 449/80 ± 67/71 after immersion in Behnoosh lemon
delestre, and this mean value was 89%/17% of the mean initial
177
[Downloaded free from http://www.jioh.org on Saturday, November 24, 2018, IP: 156.208.79.12]
Yaberi and Haghgoo: Effect of nano‑hydroxyapatite and eggshell on erosive lesions
Table 1: Demographic and clinical characteristics for
each group
Demographic and Nanohydroxy Egg shell
clinical characteristics apatite
Gender Boy Girl Boy Girl
5 5 5 5
Age 18 19 20 21 18 19 20 21
2 1 4 3 2 1 4
Race Iranian Iranian
Health Statue Healthy Healthy
microhardness. The mean tertiary microhardness of the samples
was 475/20 ± 39/13 kgf/mm2, and a paired t‑test showed
that this difference was statistically significant  (P1 = 0.10,
P2 = 0.86).
Discussion
Teeth are in continuous processes of remineralization and
demineralization in the oral cavity. Normally, these two
processes are in balance. Demineralization becomes dominant
when the oral cavity is exposed to foreign acids, and the speed
of surface damage to the enamel increases progressively. This
imbalance results in erosion. Erosion processes lead to surface
demineralization and then complete destruction of the surface
layer of enamel.[18]
After considering erosion problems due to exposure to acidic
soft drinks, we searched for a substance that could accelerate
remineralization of enamel. In this study for the first time, the
effect of nano‑hydroxyapatite and ES extract on impacted third
molar permanent tooth enamel was investigated in situ after
exposure to an acidic soft drink.
The results of this study showed that enamel microhardness
decreased significantly after immersion in delester and that the
mean microhardness of the teeth demineralized after exposure
to delestre significantly increased after exposing them to either
nanohydroxyapatite or ES extract solution.
This study for the fi rst time compare the effect of ES and
nano-hydroxyapatite on enamel lesions following consumption
of delester and the results of this study showed that these two
materials are able to reconstruct the demineralized enamel, so
this result is one of the strengths of this study. Furthermore,
design of this study was in situ, and the results of such studies
are more documentary.
This study was done in the form of in situ, so one should put
someone else’s tooth in her/his mouth and  finding such people
were very difficult and this was a limitation of the study.
Using the results of this study, it is possible to repair
demineralized lesions resulting from the use of delester.
Hydroxyapatite is an important mineral component of
dental enamel and is bioactive and biocompatible. Many
studies have investigated the use of hydroxyapatite on
178
the dental material, implants, and orthopedic treatment.[19]
Hydroxyapatite at the nanometric level is more similar to
enamel apatite crystals.[13]
Nanohydroxyapatite has hydrophilic properties and a greater
surface area. Therefore, these crystals have moisturizing
properties because of their surface area. When they are used
on dental surfaces, they produce a thin and strong layer on the
3 enamel surface that binds with dental crowns and this thin layer
of crystal is self‑organizing, so this compound can participate
in enamel remineralization.[20]
ES is a rich source of calcium. It contains calcium carbonate,
calcium phosphate, magnesium carbonate, and other elements
such as fluoride and strontium.[21] Hence, according to the
above, the results of this study seem to be reasonable.
The results of Huang’s study are consistent with the results
of the present study, but in Huang’s study, there was another
material included with the nanohydroxyapatite that could
have a synergistic effect along with the nanohydroxyapatite.
Thus, in this study, nanohydroxyapatite was used in pure
form to test if this material by itself can have a positive effect
on the microhardness of the tooth. In the present study, a
10% nanohydroxyapatite solution was used. The results of
Huang’s study, which evaluated different concentrations of
nanohydroxyapatite, showed that 10% and 15% concentrations
had the same effect.[13]
In the present study, 15 min of exposure to delester was chosen
as the duration based on the average daily consumption of
carbonated soft drinks and the 20 s average time of having a
sip of soft drink in the mouth before being cleared by saliva.
In related studies, the application time of the remineralization
materials was different.[13] However, in the present study,
10 min was used because longer application times are
logistically difficult to apply in the clinic.
The results of the present study showed that the microhardness
of tooth enamel, which decreased after exposure to delestre,
also increased after exposure to the ES extract solution. ES
extract contains not only calcium but also other elements such
as fluoride and strontium, and it can have a positive effect on
tooth structure. Therefore, it is logical that the microhardness
of enamel would increase following exposure to ES extract,
considering its composition.[16,22]
The results of the study by Haghgoo et al. showed that there
is no statistically significant difference in the remineralization
of demineralized enamel between 3% and 10% concentrations
of ES extract, so in the present study, a 3% ES extract solution
was used.[21]
The results of the present study are consistent with Haghgoo’s
investigation.[21] However, the present study was done in situ.
This was the first in situ study to compare the effects of
either nano‑hydroxyapatite or ES on the microhardness of
demineralized tooth enamel, so there are no other in situ studies
available for comparison.
Journal of International Oral Health  ¦  Volume 10  ¦  Issue 4  ¦  July-August 2018
[Downloaded free from http://www.jioh.org on Saturday, November 24, 2018, IP: 156.208.79.12]
Yaberi and Haghgoo: Effect of nano‑hydroxyapatite and eggshell on erosive lesions
In this study, we studied effect of nanohydroxyapatite and ES
on the microhardness of demineralized tooth enamel. Future
studies are recommended to do similar assessments using other
remineralizing agents.
Furthermore, it is suggested that, in future studies, the ES is
added to the toothpaste and mouthwash as a remineralizing
agent and its effect on the repair of enamel lesions is surveyed.
Conclusion
The results of the present study showed that microhardness of
the enamel demineralized from delestre exposure significantly
increased after treatment with either 10% nanohydroxyapatite
solution or 3% ES extract solution.
Financial support and sponsorship
This study was supported by the Faculty of Dentistry, Shahed
University, Tehran, Iran.
Conflicts of interest
There are no conflicts of interest.
References
1. Gonçalves GK, Guglielmi Cde A, Corrêa FN, Raggio DP, Corrêa MS.
Erosive potential of different types of grape juices. Braz Oral Res
2012;26:457‑63.
2. Haghgou EH, Haghgoo R, Roholahi MR, Ghorbani Z. Effect of
casein phosphopeptide‑amorphous calcium phosphate and three
calcium phosphate on enamel microhardness. J Contemp Dent Pract
2017;18:583‑6.
3. Haghgou HR, Haghgoo R, Asdollah FM. Comparison of the
microhardness of primary and permanent teeth after immersion in
two types of carbonated beverages. J Int Soc Prev Community Dent
2016;6:344‑8.
4. Swapna G, Sharma S, Soni VP, Tamgadge S. Demineralization adjacent
to orthodontic brackets with fluoride releasing and conventional bonding
agents. Indian J Dent Res 2016;27:426‑32.
5. da Camara DM, Pessan JP, Francati TM, Souza JA, Danelon M,
Delbem AC, et al. Fluoride toothpaste supplemented with sodium
hexametaphosphate reduces enamel demineralization in vitro. Clin Oral
Investig 2016;20:1981‑5.
Journal of International Oral Health  ¦  Volume 10 ¦ Issue 4 ¦ July-August 2018
6. Levy  SM. An update on fluorides and fluorosis. J  Can Dent Assoc
2003;69:286‑91.
7. Selwitz RH, Ismail AI, Pitts NB. Dental caries. Lancet 2007;369:51‑9.
8. Mahantesha T, Dixit UB, Nayakar RP, Ashwin D, Ramagoni NK,
Kamavaram Ellore VP, et al. Prevalence of dental fluorosis and
associated risk factors in Bagalkot district, Karnataka, India. Int J Clin
Pediatr Dent 2016;9:256‑63.
9. Reynolds EC. Calcium phosphate‑based remineralization systems:
Scientific evidence? Aust Dent J 2008;53:268‑73.
10. Haghgoo R, Rezvani MB, Salehi Zeinabadi M. Comparison of
nano‑hydroxyapatite and sodium fluoride mouthrinse for remineralization
11.
of incipient carious lesions. J Dent (Tehran) 2014;11:406‑10.
Suchanek W, Yoshimura M. Processing and properties of
hydroxyapatite‑based biomaterials for use as hard tissue replacement
implants. J Mater Res 1998;13:94‑117.
12. Webster TJ, Ergun C, Doremus RH, Siegel RW, Bizios R. Enhanced
osteoclast‑like cell functions on nanophase ceramics. Biomaterials
2001;22:1327‑33.
13. Huang SB, Gao SS, Yu HY. Effect of nano‑hydroxyapatite concentration
on remineralization of initial enamel lesion in vitro. Biomed Mater
2009;4:034104.
14. de Carvalho FG, Vieira BR, Santos RL, Carlo HL, Lopes PQ,
de Lima BA, et al. In vitro effects of nano‑hydroxyapatite paste on
initial enamel carious lesions. Pediatr Dent 2014;36:85‑9.
15. Huang S, Gao S, Cheng L, Yu H. Remineralization potential of
nano‑hydroxyapatite on initial enamel lesions: An in vitro study. Caries
Res 2011;45:460‑8.
16. Vestergaard P, Jorgensen NR, Schwarz P, Mosekilde L. Effects of
treatment with fluoride on bone mineral density and fracture risk  – A
meta‑analysis. Osteoporos Int 2008;19:257‑68.
17. Haghgoo R, Abbasi F, Rezvani MB. Evaluation of the effect of
nanohydroxyapatite on erosive lesions of the enamel of permanent teeth
following exposure to soft beer in vitro. Sci Res Essays 2011;6:5933‑6.
18. Shenkin JD, Heller KE, Warren JJ, Marshall TA. Soft drink consumption
and caries risk in children and adolescents. Gen Dent 2003;51:30‑6.
19. Haghgoo R, Ahmadvand M, Moshaverinia S. Remineralizing effect
of topical novaMin and nano‑hydroxyapatite on caries‑like lesions in
primary teeth. J Contemp Dent Pract 2016;17:645‑9.
20. Brown CJ, Smith G. The erosive potential of flavoured sparking water
drinks. Int J Pediat Dent 2007;17:86‑91.
21. Haghgoo R, Mehran M, Ahmadvand M, Ahmadvand M.
Remineralization effect of eggshell versus nanohydroxyapatite on caries
lesions in permanent teeth (in vitro). Int J Oral Health 2016;8;435‑9.
22. Meunier PJ, Roux C, Seeman E, Ortolani S, Badurski JE, Spector TD,
et al. The effects of strontium ranelate on the risk of vertebral
fracture in women with postmenopausal osteoporosis. N Engl J Med
2004;350:459‑68.
179