JIOH on LinkedIn JIOH on Facebook
  • Users Online: 78
  • Home
  • Print this page
  • Email this page
Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contacts Login 

 Table of Contents  
Year : 2018  |  Volume : 10  |  Issue : 4  |  Page : 176-179

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

Department of Pediatric Dentistry, Faculty of Dentistry, Shahed University, Tehran, Iran

Date of Web Publication28-Aug-2018

Correspondence Address:
Dr. Roza Haghgoo
Department of Pediatric Dentistry, Dental School, Shahed University, N71, Italia Street, Vesal Av., Tehran
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jioh.jioh_84_18

Rights and Permissions

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

How to cite this article:
Yaberi M, Haghgoo R. 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. J Int Oral Health 2018;10:176-9

How to cite this URL:
Yaberi M, Haghgoo R. 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. J Int Oral Health [serial online] 2018 [cited 2022 Sep 26];10:176-9. Available from:

  Introduction Top

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 other substances to repair demineralization lesions. Different 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],[14],[15]

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 Top

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.


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 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 Top

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].
Table 1: Demographic and clinical characteristics for each group

Click here to view

The mean ± standard deviations (SDs) of the initial microhardness for the ten samples in the 1st group (nanohydroxyapatite) were 548/3 ± 42/27 kgf/mm2. 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 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 Top

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 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 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.

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 Top

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 Top

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.  Back to cited text no. 1
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.  Back to cited text no. 2
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.  Back to cited text no. 3
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.  Back to cited text no. 4
[PUBMED]  [Full text]  
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.  Back to cited text no. 5
Levy SM. An update on fluorides and fluorosis. J Can Dent Assoc 2003;69:286-91.  Back to cited text no. 6
Selwitz RH, Ismail AI, Pitts NB. Dental caries. Lancet 2007;369:51-9.  Back to cited text no. 7
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.  Back to cited text no. 8
Reynolds EC. Calcium phosphate-based remineralization systems: Scientific evidence? Aust Dent J 2008;53:268-73.  Back to cited text no. 9
Haghgoo R, Rezvani MB, Salehi Zeinabadi M. Comparison of nano-hydroxyapatite and sodium fluoride mouthrinse for remineralization of incipient carious lesions. J Dent (Tehran) 2014;11:406-10.  Back to cited text no. 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.  Back to cited text no. 11
Webster TJ, Ergun C, Doremus RH, Siegel RW, Bizios R. Enhanced osteoclast-like cell functions on nanophase ceramics. Biomaterials 2001;22:1327-33.  Back to cited text no. 12
Huang SB, Gao SS, Yu HY. Effect of nano-hydroxyapatite concentration on remineralization of initial enamel lesion in vitro. Biomed Mater 2009;4:034104.  Back to cited text no. 13
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.  Back to cited text no. 14
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.  Back to cited text no. 15
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.  Back to cited text no. 16
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.  Back to cited text no. 17
Shenkin JD, Heller KE, Warren JJ, Marshall TA. Soft drink consumption and caries risk in children and adolescents. Gen Dent 2003;51:30-6.  Back to cited text no. 18
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.  Back to cited text no. 19
Brown CJ, Smith G. The erosive potential of flavoured sparking water drinks. Int J Pediat Dent 2007;17:86-91.  Back to cited text no. 20
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.  Back to cited text no. 21
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.  Back to cited text no. 22


  [Table 1]

This article has been cited by
1 In Vitro Assessment of Long-Term Fluoride Ion Release from Nanofluorapatite
Katarzyna Herman,Marta Wujczyk,Maciej Dobrzynski,Dorota Diakowska,Katarzyna Wiglusz,Rafal J. Wiglusz
Materials. 2021; 14(13): 3747
[Pubmed] | [DOI]
2 In Vitro Studies concerning Selected Properties of a Composite Material Blended with Nanofluoroapatite Crystals
Marta Zietek, Maciej Dobrzynski, Katarzyna Fita, Dorota Diakowska, Adam Watras, Rafal Jakub Wiglusz
Materials. 2021; 14(23): 7295
[Pubmed] | [DOI]


Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

  In this article
Materials and Me...
Article Tables

 Article Access Statistics
    PDF Downloaded334    
    Comments [Add]    
    Cited by others 2    

Recommend this journal