Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 9  |  Issue : 1  |  Page : 28-33

Comparative evaluation of Nano-hydroxyapatite and casein Phosphopeptide-amorphous calcium phosphate on the remineralization potential of early enamel lesions: An in vitro study


1 Paedodontics & Preventive Dentistry, Manipal College of Dental Sciences, Manipal University, Mangalore, Karnataka, India
2 Public Health Dentistry, Manipal College of Dental Sciences, Manipal University, Mangalore, Karnataka, India

Date of Web Publication14-Jun-2017

Correspondence Address:
Arathi Rao
Paedodontics & Preventive Dentistry, Manipal College of Dental Sciences, Manipal University, Light House Hill Road, Mangalore - 575 001, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0975-8844.207941

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  Abstract 


Background: Benefits of remineralizing agents in a wide variety of formulations have been proved beneficial in caries management. Casein phosphopeptide-amorphous calcium phosphate (CPP–ACP) nanocomplex has been recommended and used as remineralizing agent. Nano-hydroxyapatite (n-HAp) is one of the most biocompatible and bioactive material having wide range of application in dentistry, but does it excel better compared to CPP-ACP. Aims: To evaluate and compare the remineralizing efficiency of the paste containing hydroxyapatite and casein phosphopeptide-amorphous calcium phosphate. Settings and Design: The study was an in vitro single blinded study with lottery method of randomization approved by the Institutional Ethics Committee. Materials and methods: 30 non carious premolar teeth. The teeth were demineralized and divided into 2 groups and subjected to remineralization. The samples were analysed for surface hardness and mineral content. Statistical Analysis: Student t’ test and repeated measures of ANOVA was applied. Results: Average hardness in Nano-hydroxyapatite group increased to 340 ± 31.70 SD and 426 ± 50.62 SD for 15 and 30 days respectively and that of (CPP–ACP), 355.83 ± 38.55 SD and 372.67 ± 53.63 SD. The change in the hardness values was not statistically significant with P value of 0.39 (P > 0.05). Calcium and Phosphorous levels increased in both the groups but was not significant. Conclusion: Both the agents used are effective in causing remineralization of enamel. Nano-hydroxyapatite is more effective as compared to Casein phosphopeptide-amorphous calcium phosphate, in increasing the Calcium and Phosphorus content of enamel, and this effect is more evident over a longer treatment period. Key Message: Remineralizing agents are a boon for caries management. With the advent of many formulations it is difficult to clinically select the agent. This study compares the remineralizing potential of Casein phosphopeptide-amorphous calcium phosphate nanocomplex and Nano-hydroxyapatite and found that both are effective as remineralizing agents.

Keywords: CPP–ACP, nano-hydroxyapatite, remineralization


How to cite this article:
Sharma A, Rao A, Shenoy R, Suprabha BS. Comparative evaluation of Nano-hydroxyapatite and casein Phosphopeptide-amorphous calcium phosphate on the remineralization potential of early enamel lesions: An in vitro study. J Orofac Sci 2017;9:28-33

How to cite this URL:
Sharma A, Rao A, Shenoy R, Suprabha BS. Comparative evaluation of Nano-hydroxyapatite and casein Phosphopeptide-amorphous calcium phosphate on the remineralization potential of early enamel lesions: An in vitro study. J Orofac Sci [serial online] 2017 [cited 2021 Jun 16];9:28-33. Available from: https://www.jofs.in/text.asp?2017/9/1/28/207941




  Introduction Top


Dental caries is a highly prevalent dental disease and is a major public health problem.[1] Up to recent times, a surgical approach was being followed for the management of carious lesion. With clear understanding regarding the demineralization–remineralization theory of caries initiation, emphasis in present-day dentistry is on remineralization of carious lesion. The benefits of remineralizing agents in a wide variety of formulations have been proved.[2],[3]

Casein phosphopeptide–amorphous calcium phosphate (CPP–ACP) nanocomplex is found to remineralize subsurface lesions in the human enamel.[4] Nano-hydroxyapatite (n-HAp) is one of the most biocompatible and bioactive materials having a wide range of application in the field of dentistry. Its application in the remineralization of subsurface lesions is recently being explored.[5]

The purpose of this study was to evaluate and compare the remineralizing efficiency of the pastes containing HAp and CPP–ACP. The null hypothesis was that there was no difference between the two materials.


  Materials and Methods Top


The study was an in vitro, single-blinded study with lottery method of randomization and was approved by the Institutional Ethics Committee. (Letter ref MCODS/198/2011 dated 17th October 2011 issued by Institutional Ethics Committee, Manipal College of Dental Sciences, Mangalore).

Sample size and selection

The power of the study was kept at 0.90 and confidence level at 95%. Estimated sample size was 30 in each experimental group [Table 1] (Stata 10 was used to calculate the sample size) (Stata Data Analysis and Statistical Software, StataCorp LLC, Texas).
Table 1: Sample distribution

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Sample collection

A sample of 30, non-carious, premolar teeth was collected for this study on the basis of the criteria below.

Inclusion criteria

Healthy premolars extracted for orthodontic purposes were included for the study.

Exclusion criteria

  1. Carious teeth
  2. Fractured teeth
  3. Defective teeth
  4. Restored teeth


The contaminants and surface debris were removed by manually cleaning the tooth and rinsing with hydrogen peroxide, after which the tooth was stored in saline at room temperature. The tooth was stored in saline till the study began.

Sample preparation

The crown portion of the tooth was subjected to prophylaxis with a polishing paste of medium grit (Propol, DPI India) using a rubber polishing cup and then washed with distilled water. The tooth were cut in longitudinal sections into two equal halves with a diamond disc using micromotor handpiece to obtain a sample of 60 sectioned teeth (n = 60). After the application of pastes according to the protocol, one half of the samples were used for quantitative and qualitative analysis using scanning electron microscope with energy dispersive X-ray (SEM–EDAX) analysis. The other half was embedded in a diestone block measuring 4 cm × 3 cm. This half was used to evaluate the surface hardness.

Demineralization of the samples

After pre-experiment evaluation was done in Group 1 (n = 6) for mineral content and microhardness using SEM–EDAX and Vickers microhardness tester, respectively, the remaining samples (n = 54) were subjected to demineralization using demineralizing solution, which consisted of CaCl2 (2.2 mM), NaH2PO4 (2.2 mM), lactic acid (0.05 M), fluoride (0.2 ppm); the demineralizing solution was adjusted with 50% NaOH to a pH of 4.5.[6] The specimens were allowed to stay in the solution for 48 h for adequate demineralization.

Experimental steps

Samples belonging to Group 1 (n = 6) and Group 2 (n = 6) were analyzed for surface hardness and mineral content before and after demineralization, respectively, to obtain the baseline data.

All the remaining samples (n = 48) were brushed daily at 24 h interval for 2 min with the respective pastes using a manual toothbrush with mild pressure so as to just stabilize the bristles on the surface of the teeth.

Samples belonging to Group 3 (n = 24) were brushed with HAp paste (ReminPro, VOCO) for 2 min using 1 cm of the paste.

Samples belonging to Group 4 (n = 24) were brushed with CPP–ACP paste (GC Tooth Mousse, RECALDENT) for 2 min using 1 cm of the paste.

Each time after brushing, the samples were cleaned with distilled water and immersed in Fusayama Meyer’s artificial saliva, which was composed of NaCl (0.400 g/l), KCl (0.400 g/l), CaCl2 · H2O (0.906 g/l), NaH2PO4 · 2H2O (0.690 g/l), Na2S · 9H2O (0.005 g/l) and urea (1 g/l) with a pH of 7.1. The samples were left immersed in the artificial saliva for 24 h at room temperature, and the solution was replenished every 24 h. The samples were tested for surface hardness and mineral content after 15 and 30 days.

Hardness testing

Vickers hardness testing (microhardness) was done using Matsuzawa Digital Micro Hardness Tester MMT-X7A (Matsuzawa Co. Ltd., Japan). The software used for measurement was Clemex software designed by Matsuzava. The samples were embedded on diestone block and placed on a stage. The samples were positioned with the assistance of a digital optical microscope (magnification of 400×) so that the indent fell on the enamel portion of the section. A load of 200 gf for 15 s was applied, the rhomboid indent was measured for length and depth digitally and the hardness value was calculated.

Quantitative and qualitative analysis by SEM–EDAX

The samples were ensured to be moisture-free, and care was taken to avoid any direct contact with air or moisture. These samples were placed on a metal mounting block and then kept inside the gold sputter coater (Polaron SC7640, Quorum Technologies Ltd., U.K.). These samples were then analyzed using a scanning electron microscope (Carl Zeiss EVO 40, Germany) for surface morphology and a energy dispersive X-ray analyser (Bruker XFlash Detector 3010, U.S.A.) for quantification of calcium and phosphorous content from the amount of calcium ions and phosphate ions present. The images were obtained with a magnification of 5000× and 20 kV voltage for comparison. The enamel thickness was also measured simultaneously by drawing a tangent from the dentinoenamel junction to the surface enamel, and the measurements were given in micron units.

Statistical analysis

Descriptive statistics were calculated. Student’s paired t-test and repeated measures of analysis of variance (ANOVA) were applied to compare between the two groups. Statistical analysis was performed using the Statistical Package for the Social Sciences version 16 software (SPSS Inc., Chicago, IL, USA).


  Results Top


A total number of 30 teeth were taken for the study, which were sectioned into two equal halves giving a sample size of 60 sections. Six sections were evaluated for hardness, surface characteristics and mineral content to obtain baseline data. The remaining 54 samples were subjected to demineralization for 48 h, and six samples were evaluated to obtain data post demineralization. Finally, the remaining 48 samples were divided into two equal groups of 24 each, and the pastes were applied daily for 1 month. The samples were evaluated for hardness, surface characteristics and mineral content at 15 and 30 days interval, and comparison between the two groups was done.

[Table 2] describes the descriptive statistics for GC Tooth Mousse and ReminPro for Vickers hardness number (VHN), as well as calcium and phosphorus levels at 15 and 30 days interval. [Table 3] gives the comparison between groups for VHN, as well as calcium and phosphorus levels at 15 and 30 days interval.
Table 2: Descriptive statistics for GC Tooth Mousse and ReminPro for Vickers hardness number, as well as calcium and phosphorus levels at 15 and 30 days interval

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Table 3: Comparison between groups for Vickers hardness number, as well as calcium and phosphorus levels at 15 and 30 days interval

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Estimation of hardness of the enamel

Vickers hardness of the enamel was tested before the beginning of the study. The hardness value at baseline, that is before demineralization, was found to be 329.60 ± 42.18 standard deviation (SD). The hardness value fell considerably following demineralization (203.03 ± 43.46 SD).

Mean hardness values after remineralization

VHN in Group 3 (ReminPro) after 15 days of remineralization was 340 ± 31.70 SD, and after 30 days, it was 426 ± 50.62 SD. In Group 4 (CPP–ACP), VHN after 15 days of remineralization was 355.83 ± 38.55 SD, and after 30 days of remineralization, it was 372.67 ± 53.63 SD.

Comparison of hardness values between the two groups

The two groups were compared for the change in hardness values by applying a statistical analysis of repeated measures of ANOVA, and the following observations were recorded for intergroup comparison. The change in the hardness values was not statistically significant with P value of 0.39 (P > 0.05) for the time factor (before demineralization, after demineralization and 15 and 30 days after remineralization) taken as the dependent variable.

When Student’s t-test for pairing was applied to find the hardness value relation in between the groups, it was noted that the difference in hardness values was not significant between the two groups after 15 days (P = 0.46) and also after 30 days (P = 0.11).

Mean calcium value at baseline

The amount of calcium present in the tooth samples was assessed by energy dispersive X-ray analysis, and the values were presented in weight percentage (wt%). The mean baseline content of calcium in the normal enamel was 34.84 ± 4.21 SD, and after demineralization there was a decrease in the content, making it to 29.86 ± 1.56 SD.

Mean calcium value after remineralization

In Group 3, there was a negligible increase in the content of calcium after 15 days (29.87 ± 2.53 SD), and then a significant increase was observed after 30 days of remineralization (37.93 ± 2.11 SD).

In Group 4 also, there was a negligible increase in the content of calcium after 15 days (30.94 ± 1.23 SD), and then a slight increase was observed after 30 days of remineralization (32.76 ± 3.26 SD).

Mean phosphorous value at baseline

The amount of phosphorus present in the tooth samples was assessed by energy dispersive X-ray analysis, and the values were presented in wt%. The mean baseline content of phosphorous in the normal enamel was found to be 14.77 ± 0.55 SD, and after demineralization, there was only a slight decrease in the content making it to 14.73 ± 0.58 SD.

Mean phosphorous value after remineralization

In Group 3, there was an increase in the content of phosphorus after 15 days (15.53 ± 0.57 SD), and a further increase was observed after 30 days of remineralization (15.73 ± 1.04 SD).

In Group 4 also, there was an increase in the content of phosphorus after 15 days (15.27 ± 0.57 SD), and then a decrease was observed after 30 days of remineralization (13.21 ± 1.74 SD).

Analysis of the enamel surface

SEM analysis of a sound enamel revealed a smooth and intact surface [[Figure 1]a]. Following demineralization, the surface appeared rough and irregular [[Figure 1]b]. It was observed that following remineralization procedure in both the groups, the surface characters improved and resembled the sound enamel [Figure 2] and [Figure 3]. This resemblance was more closer to a sound enamel in tooth treated with ReminPro.
Figure 1: (a) SEM image of normal enamel before demineralization showing smooth and intact enamel surface. (b) SEM image after demineralization showing increased roughening and surface irregularities on the enamel surface

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Figure 2: (a) SEM image showing effect of CPP–ACP after 15 days revealing smoothening of the enamel surface. (b) SEM image showing effect of ReminPro after 15 days revealing smoothening of the enamel surface

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Figure 3: (a) SEM image showing effect of CPP–ACP after 30 days revealing deposits on the enamel surface. (b) SEM image showing effect of ReminPro after 30 days revealing deposition and smooth enamel surface

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  Discussion Top


The enamel is the hardest and most mineralized tissue of the human body. It is structured to resist mechanical injuries, abrasion and chemical attack.[7] However, it is constantly subjected to the processes of remineralization and demineralization within the oral cavity. If the demineralization phase continues for a long period of time, it results in excessive loss of minerals, which leads to loss of the enamel structure and cavitation − the typical characteristics of dental caries.[8] In the modern era of minimal intervention dentistry, a variety of agents have been developed to promote remineralization of the demineralized enamel. This study evaluated the remineralization potential of two commercially available pastes, containing CPP–ACP and HAp, on the demineralized enamel.

Clinical evidence indicates that the CPP–ACP complex reduces the formation of caries through remineralization.[9] CPP–ACP nanocomplexes have been shown to be readily soluble in saliva, creating a diffusion gradient that allows them to localize in supragingival plaque. CPP–ACP in plaque releases ions into the plaque fluid and then diffuses into the enamel.[10] Therefore, it could be inferred that the use of CPP–ACP might enhance remineralization of the early enamel lesions. However, there are newer materials that are being developed to serve a similar purpose for remineralizing initial lesion.

Carbonate HAp nanocrystals, having size, morphology, chemical composition and crystallinity comparable to that of enamel, are said to remineralize enamel.[8] HAp-containing toothpaste might have a beneficial effect on the remineralization of incipient dental caries. It is possible for nano-sized HAp particles to fill the fine pores of the demineralized enamel surfaces.[11] Toothpastes containing n-HAp have been shown to remineralize early enamel lesions.[12]

Vickers hardness testing was used, as it provides a relatively simple, non-destructive and rapid method for comparative studies of materials and their properties. In this study, the average VHN value of normal enamel was found to be 329.60 ± 42.18 SD, which was in agreement with earlier published data.[13],[14] In Group 3, the VHN value increased to 340 ± 31.70 SD after 15 days of remineralization and further to 426 ± 50.62 SD after 30 days. In Group 4, VHN after 15 days of remineralization was 355.83 ± 38.55 SD, and after 30 days, it was 372.67 ± 53.63 SD. On comparison, however, it was found that the results for microhardness were not statistically significant at different time intervals within the same group (P = 0.39), and also between the two groups after 15 days (P = 0.46) and after 30 days (P = 0.11).

Lata et al.[6] compared remineralization potential of fluoride and ACP–CPP on enamel lesions. The results indicated that the increase in the VHN value after treatment with CPP–ACP was not significant. They concluded that CPP–ACP cream was effective, but to a lesser extent than fluoride in remineralizing early enamel caries at surface level.

Although statistically not significant, in this study, ReminPro yielded better results with regard to microhardness than CPP–ACP cream. The difference between the results of the two materials was substantially more after 30 days, indicating that ReminPro had better long-term effect as compared to GC Tooth Mousse.

SEM analysis allowed the investigation of the morphology of both demineralized enamel and the features observed after remineralization procedures induced by in vitro application of biomimetic carbonated HAp nanocrystals.[7] Remineralization could be estimated in terms of calcium and phosphorous content of enamel also. EDAX results indicated that there was a decrease in the wt% of both calcium and phosphorus following the demineralization of samples. Furthermore, after the treatment for 15 days, there was no significant increase of the two elements. However, after 30 days of treatment, ReminPro yielded results that were significantly better than GC Tooth Mousse. The results of this study indicated that ReminPro was significantly more effective in increasing the mineral content (calcium and phosphorus) as compared to GC Tooth Mousse over longer treatment duration. This difference in the results can be attributed to the fact that ReminPro acts differently than GC Tooth Mousse. ReminPro consists of additional components of fluoride and xylitol, which may aid in increasing the remineralization potential of the paste.


  Conclusion Top


CPP–ACP is known to be a source of calcium and phosphate close to the sites of possible demineralization, and this is likely to inhibit demineralization, enhance remineralization or possibly both. A number of studies have demonstrated CPP–ACP to have anticariogenic activity in laboratory, animal and human in situ experiments. HAp is a material with high tissue biocompatibility with structure similar to enamel.

Within the limitations of our in vitro study, we conclude the following:
  1. Both the agents used, that is ReminPro and GC Tooth Mousse, were effective in causing remineralization of enamel.
  2. ReminPro was more effective as compared to GC Tooth Mousse in increasing the calcium and phosphorus content of enamel, and this effect was more evident over a longer treatment period.
  3. Hardness of the tooth structure improved following application of ReminPro than GC Tooth Mousse.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Pradeep K, Rao PK. Remineralizing agents in the non-invasive treatment of early carious lesions. Int J Dent Case Rep 2011;1:73-84.  Back to cited text no. 1
    
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Zero DT. Dentifrices, mouthwashes, and remineralization/caries arrestment strategies. BMC Oral Health 2006;6(Suppl 1):S9.  Back to cited text no. 2
    
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Walsh LJ. Contemporary technologies for remineralization therapies: A review. Int Dent SA 2009;11:6-16.  Back to cited text no. 3
    
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Kumar VL, Itthagarun A, King NM. The effect of casein phosphopeptide-amorphous calcium phosphate on remineralization of artificial caries-like lesions: An in vitro study. Aust Dent J 2008;53:34-40.  Back to cited text no. 4
    
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Hannig M, Hannig C. Nanomaterials in preventive dentistry. Nat Nanotechnol 2010;5:565-9.  Back to cited text no. 5
    
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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. 6
[PUBMED]  [Full text]  
7.
Roveri N, Battistella E, Foltran I, Foresti E, Iafisco M, Lelli M et al. Synthetic biomimetic carbonate-hydroxyapatite nanocrystals for enamel remineralization. Adv Mater Res 2008;47:821-4.  Back to cited text no. 7
    
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Rao A, Malhotra N. The role of remineralizing agents in dentistry: A review. Compend Contin Educ Dent 2011;32:26-33.  Back to cited text no. 8
    
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Gupta R, Prakash V. CPP-ACP complex as a new adjunctive agent for remineralisation: A review. Oral Health Prev Dent 2011;9:151-65.  Back to cited text no. 9
    
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Cochrane NJ, Reynolds EC. Calcium phosphopeptides − Mechanisms of action and evidence for clinical efficacy. Adv Dent Res 2012;24:41-7.  Back to cited text no. 10
    
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Rimondini L, Palazzo B, Iafisco M, Canegallo L, Demarosi F, Merlo M et al. The remineralizing effect of carbonate-hydroxyapatite nanocrystals on dentine. Mater Sci Forum 2007;539-43: 602-5.  Back to cited text no. 11
    
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Tschoppe P, Zandim DL, Martus P, Kielbassa AM. Enamel and dentine remineralization by nano-hydroxyapatite toothpastes. J Dent 2011;39:430-7.  Back to cited text no. 12
    
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Chuenarrom C, Benjakul P, Daosodsai P. Effect of indentation load and time on Knoop and Vickers microhardness tests for enamel and dentin. Mater Res 2009;12:473-6.  Back to cited text no. 13
    
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Ryge G, Foley DE, Faorhurst CW. Micro-indentation hardness. J Dent Res 1961;40:1116-26.  Back to cited text no. 14
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]


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