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 Table of Contents  
Year : 2022  |  Volume : 14  |  Issue : 4  |  Page : 377-381

Efficacy of domestically manufactured remineralizing agent on artificially created surface lesions: A comparative ex-vivo study

1 Department of Conservative Dentistry and Endodontics, Adhiparasakthi Dental College and Hospital, Melmaruvathur, Tamil Nadu, India
2 Department of Conservative Dentistry and Endodontics, Sri Ramakrishna Dental College, Coimbatore, Tamil Nadu, India
3 Consultant Endodontist, Iyyapanthangal, Chennai, Tamil Nadu, India
4 Department of Conservative Dentistry and Endodontics, Sri Venkateshwaraa Dental College, Ariyur, Puducherry, India

Date of Submission12-Jan-2022
Date of Decision25-Jul-2022
Date of Acceptance25-Jul-2022
Date of Web Publication29-Aug-2022

Correspondence Address:
Dr. Chakravarthy S Vineetha
Department of Conservative Dentistry and Endodontics, Sri Ramakrishna Dental College, Coimbatore 641006, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/JIOH.JIOH_14_22

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Aim: To compare and evaluate the efficacy of domestically manufactured remineralizing agents on artificially created carious lesions. Materials and Methods: Freshly extracted 60 human maxillary first premolars were used for our in-vitro comparative study. The samples were embedded in acrylic resin such that the surface of enamel is visible for study. These samples were stored in deionized water for 1 month. The surfaces were demineralized to create a subsurface lesion. Samples were then divided using random sampling method into four groups. Each group had a sample size of 15, according to the remineralizing agent used. Group 1—tri-calcium phosphate (β-TCP), Group 2—domestically manufactured nano hydroxyapatite (NHA), Group 3—bioactive glass (BAG), and Group 4—artificial saliva (control). These remineralizing agents were applied on the samples of respective groups for 4 min, at every 24th hour for a duration of 7 days. After which, incubation of the samples was carried out by placing them in artificial saliva. This was done at a temperature of 37°C between every cycle. After completion of seven cycles, Vickers microhardness test was used to measure the surface microhardness. Data were collected and HV values among the groups were compared using one-way analysis of variance. Multiple pairwise comparisons were analyzed using Tukey’s honestly significant difference post hoc tests. Results: Improved surface remineralization was observed in all the three groups. Nano-hydroxyapatite showed highest remineralization. TCP showed better remineralization compared to BAG. Conclusion: Remineralizing efficacy of NHA toothpaste was higher when compared with BAG and β-TCP.

Keywords: Bioactive Glass, Nano Hydroxyapatite, Remineralization, Tricalcium Phosphate

How to cite this article:
Tamil S, Vineetha CS, Prasad R, Jayakumar S, John BM, Varghese R. Efficacy of domestically manufactured remineralizing agent on artificially created surface lesions: A comparative ex-vivo study. J Int Oral Health 2022;14:377-81

How to cite this URL:
Tamil S, Vineetha CS, Prasad R, Jayakumar S, John BM, Varghese R. Efficacy of domestically manufactured remineralizing agent on artificially created surface lesions: A comparative ex-vivo study. J Int Oral Health [serial online] 2022 [cited 2023 Mar 24];14:377-81. Available from:

  Introduction Top

Alternating episodes of demineralization and remineralization caused dental caries.[1] When the oral pH decreases below the critical pH of 5.5, it leads to demineralization.[2] This is prevented when there is a favorable environment for remineralization.[3] Early enamel lesions can be remineralized by further increasing the mineral content of the tooth, thereby increasing resistance to the acids, using remineralizing treatments.[4]

Fluoride is considered to be one of the highly effective remineralizing agents in the prevention of caries. Several alternatives to fluorides have been proposed in recent years because of their high anticariogenic characteristics.[5],[6]

N-Hap has smaller particle size that is similar to hydroxyapatite structure present in the enamel. It is a bioactive material and has a high affinity to the enamel surface.[7],[8] Since it has a unique potential for remineralization, it is widely used as an effective anti-cariogenic agent.[9],[10]

Bioactive glass (BAG) (Novamin) is another novel remineralizing agent in which the calcium sodium phosphosilicate disintegrates and leads to the release of minerals like calcium and phosphate which increases the mineralized content of the tooth structure.[11],[12],[13]

In recent days, biomimetic material has gained attention as it prevents the need for invasive treatment. Several studies have used various remineralizing agents and methods for early tooth structure loss,[14] but not many studies have compared the efficacy of these agents on artificially created surface carious lesion.

Hydroxyapatite is the major hard tissue component of teeth and bones. Nanosized hydroxyapatite has various medical and dental applications. The commercially available remineralizing agents are expensive and hence in this study, we have compared the domestically manufactured nanohydroxyapatite with commercially available tricalcium phosphate (TCP) and BAG.

The null hypothesis for the current study is that there is no difference in remineralizing efficacy of the three remineralizing agents. The outcome of this study will aid in selecting the appropriate remineralizing agent for the treatment of early enamel caries.

  Materials and Methods Top

Ethical clearance was obtained (VDCW/IEC/2016) from the Institution’s Ethics Board for an ex-vivo study with a study duration of 6 months. Caries-free teeth, extracted for orthodontic or periodontal reasons, were set as the inclusion criteria and teeth with evidence of caries, hypoplasia, or fracture were not included in the study. The samples were grouped based on random sampling method. Double blinding method was followed to avoid bias. Sample size was calculated using Cochran’s formula. Sixty freshly extracted maxillary first premolars were selected following inclusion and exclusion criteria. The samples were cleaned from external debris and mounted in self-cure acrylic such that the buccal aspect of the enamel was visible outside. The samples were immersed in 50 mL of demineralizing solution for 48 h and incubated at 37°C to create a subsurface lesion.[15] Then, the demineralized samples were rinsed with deionized water, dried, and were then segregated as n = 15 based on the remineralizing agent used:

  • Group 1—β-tri-calcium phosphate (β-TCP)

  • Group 2—domestically manufactured nanohydroxyapatite

  • Group 3—BAG

  • Group 4—control (only artificial saliva)

Preparation of nanohydroxyapatite

Hydroxyapatite nano powders (20–60 nm) were synthesized in the Chemistry Department Laboratory, Vivekanandha Dental College for Women, with a sol–gel route using calcium nitrate and phosphoric acid. Double distilled water was used as a diluting medium for HA sol preparation and ammonia was used to adjust the pH.[16]

Sample preparation

After treating the samples with the specific remineralizing agent for 4 min using polishing cup, they were stored in artificial saliva. This process was carried out at every 24th hour for 7 days. The samples in the control group were immersed only in artificial saliva with no other remineralizing agent.

A universal incubator was used to incubate the samples at 37°C between each remineralizing cycle. Vickers microhardness test was carried out to assess the surface micro hardness after seven remineralization cycles [Figure 1].
Figure 1: Vickers microhardness test on the mounted sample

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The values were tabulated and analyzed using SPSS [IBM Version 23.0, Armonk]. Level of significance was set as α = 0.05. Shapiro–Wilks tests and normality tests showed normal distribution of the variable.

  Results Top

Based on the mean values, the control group showed 54.02 ± 2.582 MPa, which was the lowest. In the experimental group, the highest mean of 181.05 ± 2.963 MPa was noted in the nano hydroxyapatite (NHA) group, TCP had a mean value of 143.05 ± 3.295 MPa, and the lowest mean value was seen in the BAG group which was 92.23 ± 2.067 MPa [Table 1].
Table 1: One-way ANOVA to compare mean HV values between groups

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F-statistic of 6123.315 was arrived using one-way analysis of variance (ANOVA), which showed that the mean microhardness among all the groups was significantly different. After this, pairwise comparison was performed [Table 2]. One-way ANOVA and Tukey’s honestly significant difference (HSD) post hoc tests were carried out for multiple pairwise comparisons [Table 3].
Table 2: ANOVA test

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Table 3: Tukey HSD post hoc tests for multiple pairwise comparisons

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The mean value of NHA showed that it had the highest microhardness than other two groups. There was a statistically significant difference from each other in the mean values of all the three groups.

  Discussion Top

Salivary calcium and phosphate ions play a major role in compensating demineralization.[17] The current study focusses on analyzing the efficacy of BAG, β-TCP, and domestically manufactured-NHA, which would aid in selecting an effective remineralizing agent for clinical use. As BAG and TCP were extensively used in several studies and not many evidence is available on nanohydroxyapatite as remineralizing agent, the efficacy of nanohydroxyapatite in comparison with the other two agents was evaluated in this study.

BAG[18] has a remarkable capacity in the formation of hydroxycarbonate apatite.[19],[20] Blending of β-TCP, sodium laurylsulfate, and fumaric acid increases the fluoride remineralization.[21],[22] NHA has gained attention in the field of medicine and dentistry.[23] The use of hydroxyapatite has also been mentioned as biomimetic[24] advancement in rebuilding the lost enamel structure in a recent published study by Acharya et al.[25]

A recent study by Juntavee et al.[26] showed a potential lesion depth reduction and forming a new enamel layer using nanohydroxyapatite compared with TCP, which was similar to the results of the current study.

After remineralization, there was a gradual increase in microhardness values in enamel to 251.08 VHN at the 28th day which was similar to the control values (268.38 VHN). This was also similar to the results of a study by Rana et al.,[27] which showed the penetration depth and improved surface microhardness with the use of Novamin on enamel demineralization.

From the results of the present study, it can be seen that the microhardness of tooth was significantly increased in all the three experimented remineralizing agents. A study by Amaechi et al.[28] showed a similar remineralizing efficacy for fluoridated toothpaste and nanohydroxyapatite, but in our study, NHA toothpaste had higher remineralizing ability than that of fluoride toothpaste. This may be due to the use of artificial saliva in our study which better simulates the oral environment.

Currently, the cost of all the above-mentioned remineralizing agents in the form of toothpastes would take a toll on patients from the economically weaker sector. Thus, evaluation of the efficacy of a domestically made toothpaste containing NHA is more affordable than similar foreign products. Thus, it would help patients opt for an indigenous tooth paste. Hence, in our study, we manufactured nanohydroxyapatite which costed nearly one-third of the expense of a commercially available remineralizing agent.

Since this is an in vitro study, it cannot completely simulate oral environment. Thus, in-vivo studies using variable concentrations and duration need to be carried out to further enhance the conclusion drawn from the present study. The scope of the current study can also be expanded by comparing various other remineralizing agents and using different modes of application.

Manufacture of other remineralizing agents domestically could also play a major role in decreasing the cost factor. Comparison of commercial and domestically manufactured remineralizing agents has a wider scope for further research.

  Conclusion Top

Within the limitations of this study, the null hypothesis was rejected. Remineralization was evident in all the three experimental groups. However, there was no complete remineralization within a period of 7 days. This study showed that domestically manufactured NHA paste had better ability to remineralize the surface than the other two experimental groups. Thus, traditional invasive dental treatment can be avoided with early diagnosis and the use of newer and indigenous remineralizing toothpaste.


Not applicable.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

Authors’ contributions

Not applicable.

Ethical policy and Institutional Review Board statement

Ethical clearance was obtained (VDCW/IEC/2016) fromthe Institution’s Ethics Board for an ex-vivo study

Patient declaration of consent

Not applicable.

Data availability statement

Data is available on reasonable request to corresponding author mail.

  References Top

Mehta AB, Kumari V, Jose R, Izadikhah V Remineralization potential of bioactive glass and casein phosphopeptide-amorphous calcium phosphate on initial carious lesion: An in-vitro pH-cycling study. J Conserv Dent 2014;17:3-7.  Back to cited text no. 1
Palaniswamy UK, Prashar N, Kaushik M, Lakkam SR, Arya S, Pebbeti S A comparative evaluation of remineralizing ability of bioactive glass and amorphous calcium phosphate casein phosphopeptide on early enamel lesion. Dent Res J (Isfahan) 2016;13:297-302.  Back to cited text no. 2
Vinod D, Gopalakrishnan A, Subramani SM, Balachandran M, Manoharan V, Joy A A comparative evaluation of remineralizing potential of three commercially available remineralizing agents: An in vitro study. Int J Clin Pediatr Dent 2020;13:61-5.  Back to cited text no. 3
Hicks J, Garcia-Godoy F, Flaitz C Biological factors in dental caries: Role of saliva and dental plaque in the dynamic process of demineralization and remineralization (part 1). J Clin Pediatr Dent 2003;28:47-52.  Back to cited text no. 4
Kalra DD, Kalra RD, Kini PV, Prabhu CA Nonfluoride remineralization: An evidence-based review of contemporary technologies. J Dent Allied Sci 2014;3:24.  Back to cited text no. 5
Abbas HM, Hamza HMA Minimal intervention approaches in remineralizing early carious lesions. J Am Sci 2012;8:709-17.  Back to cited text no. 6
Li L, Pan H, Tao J, Xu X, Mao C, Gu X, et al. Repair of enamel by using hydroxyapatite nanoparticles as the building blocks. J Mater Chem 2008;18:4079-84.  Back to cited text no. 7
Talaat DA, Abdelrahman AA, Abdelaziz RH, Nagy D Effect of two remineralizing agents on initial caries-like lesions in young permanent teeth: An in vitro study. J Contemp Dent Pract 2018;19:1181-8.  Back to cited text no. 8
Balasundaram G, Sato M, Webster TJ Using hydroxyapatite nanoparticles and decreased crystallinity to promote osteoblast adhesion similar to functionalizing with RGD. Biomaterials 2006;27:2798-805.  Back to cited text no. 9
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. 10
Khamverdi Z, Kordestani M, Panahandeh N, Naderi F, Kasraei S Influence of CO2 laser irradiation and CPPACP paste application on demineralized enamel microhardness. J Lasers Med Sci 2018;9:144-8.  Back to cited text no. 11
Lata S, Varghese NO, Varughese JM Remineralization potential of fluoride and amorphous calcium phosphate-casein phospho peptide on enamel lesions: An in vitro comparative evaluation. J Conserv Dent 2010;13:42-6.  Back to cited text no. 12
Sathe N, Chakradhar Raju RV, Chandrasekhar V Effect of three different remineralizing agents on enamel caries formation—An in vitro study. Kathmandu Univ Med J (KUMJ) 2014;12:16-20.  Back to cited text no. 13
Arifa MK, Ephraim R, Rajamani T Recent advances in dental hard tissue remineralization: A review of literature. Int J Clin Pediatr Dent 2019;12:139-44.  Back to cited text no. 14
Gangrade A, Gade V, Patil S, Gade J, Chandhok D, Thakur D In vitro evaluation of remineralization efficacy of different calcium- and fluoride-based delivery systems on artificially demineralized enamel surface. J Conserv Dent 2016;19:328-31.  Back to cited text no. 15
Sanosh KP, Chu MC, Balakrishnan A, Kim TN, Cho SJ Preparation and characterization of nano-hydroxyapatite powder using sol-gel technique. Bull Mater Sci 2009;32:465-70.  Back to cited text no. 16
Narayana SS, Deepa VK, Ahamed S, Sathish ES, Meyappan R, Satheesh Kumar KS Remineralization efficiency of bioactive glass on artificially induced carious lesion: An in-vitro study. J Indian Soc Pedod Prev Dent 2014;32:19-25.  Back to cited text no. 17
Jagga U, Paul U, Padmanabhan V, Kashyap A, Guram G, Keswani K Comparative evaluation of remineralizing effect of Novamin and tricalcium phosphate on artificial caries: An in vitro study. J Contemp Dent Pract 2018;19:109-12.  Back to cited text no. 18
Suryani H, Gehlot PM, Manjunath MK Evaluation of the remineralisation potential of bioactive glass, nanohydroxyapatite and casein phosphopeptide-amorphous calcium phosphate fluoride-based toothpastes on enamel erosion lesion—An ex vivo study. Indian J Dent Res 2020;31:670-7.  Back to cited text no. 19
Wu Q, Mei ML, Wu X, Shi S, Xu Y, Chu CH, et al. Remineralising effect of 45S5 bioactive glass on artificial caries in dentine. BMC Oral Health 2020;20:49.  Back to cited text no. 20
Bhadoria N, Gunwal MK, Kukreja R, Maran S, Devendrappa SN, Singla S An in vitro evaluation of remineralization potential of functionalized tricalcium phosphate paste and CPP-ACPF on artificial white spot lesion in primary and permanent enamel. Int J Clin Pediatr Dent 2020;13:579-84.  Back to cited text no. 21
Hamba H, Nakamura K, Nikaido T, Tagami J, Muramatsu T Remineralization of enamel subsurface lesions using toothpaste containing tricalcium phosphate and fluoride: An in vitro µCT analysis. BMC Oral Health 2020;20:292.  Back to cited text no. 22
Wierichs RJ, Wolf TG, Campus G, Carvalho TS Efficacy of nano-hydroxyapatite on caries prevention—A systematic review and meta-analysis. Clin Oral Investig 2022;26: 3373-81.  Back to cited text no. 23
Wakwak M Biomimetic remineralization with nano-hydroxyapatite treatment of enamel erosion (an in vitro study). Al-Azhar J Dent Sci 2021;24:125-31.  Back to cited text no. 24
Acharya G, Agrawal P, Patri G Recent biomimetic advances in rebuilding lost enamel structure. J Int Oral Heal 2022;8:527.  Back to cited text no. 25
Juntavee A, Juntavee N, Sinagpulo AN Nano-hydroxyapatite gel and its effects on remineralization of artificial carious lesions. Int J Dent 2021;2021:7256056.  Back to cited text no. 26
Rana N, Singh N, Shaila , Thomas AM, Jairath R A comparative evaluation of penetration depth and surface microhardness of resin infiltrant, CPP-ACPF and Novamin on enamel demineralization after banding: An in vitro study. Biomater Investig Dent 2021;8:64-71.  Back to cited text no. 27
Amaechi BT, AbdulAzees PA, Alshareif DO, Shehata MA, Lima PPCS, Abdollahi A, et al. Comparative efficacy of a hydroxyapatite and a fluoride toothpaste for prevention and remineralization of dental caries in children. BDJ Open 2019;5:18.  Back to cited text no. 28


  [Figure 1]

  [Table 1], [Table 2], [Table 3]


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