Journal of Conservative Dentistry
Home About us Editorial Board Instructions Submission Subscribe Advertise Contact e-Alerts Login 
Users Online: 633
Print this page  Email this page Bookmark this page Small font sizeDefault font sizeIncrease font size
 


 
ARTICLE Table of Contents   
Year : 2007  |  Volume : 10  |  Issue : 1  |  Page : 19-25
Remineralization of enamel sub-surface lesion using casein phosphopeptide amorphous calcium phosphate (CPP-ACP) - a quantitative energy dispersive X-ray analysis (EDAX)


Department of Conservative Dentistry & Endodontics, A.B Shetty Memorial Institute of Dental Sciences, Deralakatte, Mangalore- 574160, India

Click here for correspondence address and email
 

   Abstract 

This study aimed at quantitatively evaluating the remineralization potential of casein phosphopeptide -amorphous calcium phosphate (CPP-ACP) paste on artificial enamel sub-surface lesion using Energy Dispersive X-rayAnalysis (EDAX).
60 enamel specimens were prepared from extracted human molars. All specimens were evaluated for mineral (calcium and phosphorous) content (wt %) using EDAX. The specimens were placed in demineralizing solution for 48 hrs to produce artificial carious lesions. Mineral content was again measured using EDAX. The specimens were then randomly assigned to 3 study and 1 control group (incubated in artificial saliva after demineralization), in which each group except the control was treated with remineralizing paste (10 % CPP-ACP paste) for 1. 5 and 10 days twice daily for 3 minutes, followed by incubation in artificial saliva at 370°C The control group received no treatment with remineralizing paste. After the remineralization treatment mineral content (wt %) of samples was measured using EDAX.
The study groups showed an increase in the mineral content as compared to demineralized samples. No change was seen in the control group. 10 % CPP­ACP paste could significantly remineralize the artificial enamel sub-surface lesion in vitro and the remineralization potential was dose dependent with.

How to cite this article:
Hegde MN, Shetty S, Pardal D. Remineralization of enamel sub-surface lesion using casein phosphopeptide amorphous calcium phosphate (CPP-ACP) - a quantitative energy dispersive X-ray analysis (EDAX). J Conserv Dent 2007;10:19-25

How to cite this URL:
Hegde MN, Shetty S, Pardal D. Remineralization of enamel sub-surface lesion using casein phosphopeptide amorphous calcium phosphate (CPP-ACP) - a quantitative energy dispersive X-ray analysis (EDAX). J Conserv Dent [serial online] 2007 [cited 2019 May 19];10:19-25. Available from: http://www.jcd.org.in/text.asp?2007/10/1/19/42276

   Introduction Top


Dental caries is the localized destruction of tooth tissue by specific dental plaque bacteria that ferment dietary sugar to organic acids. A substantial volume of literature now exists demonstrating an anticariogenic effect of dairy products [milk, milk concentrates, powders and chesses] [3],[5],[7] . This anticariogenic effect has been attributed to the multiphosphoseryl-containing sequences of casein. These sequences can be released as casein phosphopeptides (CPP) from an enzymatic digest of casein. The CPP have a remarkable ability to stabilize calcium phosphate in solution as amorphous calcium phosphate (ACP) nanocomplexes. Through their multiple phosphoseryl residues, the CPP bind to ACP in metastable solution preventing their growth to the critical size required for nucleation and phase transformation to an insoluble crystalline calcium phosphate [9],[10] .

The casein phosphopeptide -amorphous calcium phosphate nanocomplexes complexes (CPP-ACP) have been shown to localize at the tooth surface and prevent enamel demineralization in laboratory, animal and human in situ trials [8],[11] . The CPP-ACP has also been shown to remineralize enamel subsurface lesions in situ when delivered in oral care products. The proposed anticariogenic mechanism for CPP­-ACP is the localization of amorphous calcium phosphate (ACP) at the tooth surface which buffers the free calcium and phosphate ion activities, thereby helping to maintain a state of supersaturation with respect to tooth enamel, preventing demineralization and enhancing remineralization. The casein phosphopeptide- amorphous calcium phosphate interacts with fluoride ions to produce an amorphous calcium fluoride phosphate stabilized by the CPP at the tooth surface. This provides soluble calcium, fluoride and phosphate ions to promote remineralization with fluorapatite that is more resistant to future acid challenge.

Modern prospective caries studies require the measurement of small changes in tooth mineral content. Quantitative measurements of changes in mineral content in a single caries lesion are desirable [1],[2] . One of the most recent techniques is Scanning Electron Microscope with Energy Dispersive X-ray analysis (EDAX) attachment. It is a microanalytical technique that is employed to estimate quantitatively the amounts of mineral in a given tooth sample [1],[4] .

This study aimed at quantitatively evaluating the remineralization potential of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) paste on artificial enamel sub-surface lesion using energy dispersive X-ray analysis (EDAX).


   Materials and Methods Top


60 enamel specimens were prepared from 30 freshly extracted human molars by sectioning from buccal and lingual surfaces using low speed diamond discs. Each enamel slab was carefully shaped into a dimension of 5mm x 5mm x 1mm.

All the specimens were evaluated for mineral content (calcium and phosphorus content in wt %) using Scanning Electron Microscopy with Energy Dispersive X-ray Analysis (SEM-EDAX) attachment. The specimens were placed in the demineralizing solution containing 20 ml of acid buffer containing 2 mmol/1 Ca, 2 mmol/l PO 4 ,and 0.075 mol/l acetate at pH 4.3 (White and Featherstone 1982) [17] for 48 hrs to produce artificial carious lesions. Mineral content was again measured using EDAX.

The specimens were then randomly assigned to 3 study and 1 control group. Each study group was treated with remineralizing paste [10 %-CPP-ACP paste (GC-Tooth Mousse)] for 1(group 1), 5(group 11) and I0(group 111) days twice daily for 3 minutes, followed by incubation in artificial saliva at 37°C .The control group (group IV) received no treatment with remineralizing paste and was incubated in artificial saliva at 37°C for a period of 10 days. After remineralization mineral content (wt %) of samples was measured using EDAX.


   Results Top


Energy dispersive X-ray analysis was used to determine Calcium and Phosphorus content in weight % of sound, demineralized and remineralized enamel in each group [Figure 1],[Figure 2],[Figure 3],[Figure 4]. The calcium and phosphorus content was then converted into Ca/P ratios in each group [Table 1],[Table 2],[Table 3],[Table 4]and a range of Ca/P ratio was calculated from the obtained data [Table 5]. Ca/P ratio of sound enamel was 1.77+0.06. In the demineralized enamel the Ca/P ratio fell to 1.49±0.04. The ratio kept on increasing to reach a value of 1.71+0.03 at the 10th day.

Statistical analysis was done using one way ANOVA test to compare the Ca/P ratios of demineralized and remineralized specimens in each group [Table 6]. The value of significance was set at p<0.05. It was seen that for all the study groups the comparison revealed very highly significant results with the greatest difference observed in group III. Group IV on the other hand did not show any difference between the Ca/P ratio of demineralized and remineralized samples.


   Discussion Top


The results of this study showed that 10% CPP-ACP paste remineralized subsurface lesions in human enamel in vitro. The remineralization was maximum in the samples kept for 10 days. Thus it could be said that the remineralization was dose dependent. Although the remineralization was greatest at the 10"' day, the Ca/P ratios did not reach the values of sound enamel. This could be because of the short duration (10 days) of remineralization treatment.

The CPP have a substantial ability to stabilize calcium phosphate in solution. The peptide was found to bind 21 calcium and 14 phosphorous ions per molecule. In neutral and alkaline supersaturated calcium phosphate solutions ACP nuclei spontaneously form. It is proposed that the peptide binds to the forming ACP nano-clusters producing a metastable solution preventing ACP growth to the critical size required for nucleation and phase transformation. A 1.0% w/v CPP solution can stabilize 60 mM CaCI, and 36 mM sodium phosphate at pH 7.0 to form colloidal amorphous calcium phosphate-CPP nanocomplexes [11] . Remineralization process involves diffusion of calcium and phosphate ions through the protein/H 2 0-filled pores of carious surface enamel into the body of the enamel lesion. Once in the body of the enamel lesion, these calcium and phosphate species, increase the activities of Ca 2+ and PO4 3- , thereby increasing the degree of saturation with respect to hydroxyapatite [11],[12] . The formation of hydroxyapatite in the lesion would lead to the generation of acid and phosphate, which would diffuse out of the lesion down a concentration gradient. The CPP, by stabilizing calcium phosphate in a metastable solution, facilitate high concentrations of calcium and phosphate ions, which can diffuse into the enamel subsurface lesion. The CPP will also maintain the high activities of the free calcium and phosphate ions during remineralization through the reservoir of bound ACP. The bound ACP, by being in dynamic equilibrium with free calcium and phosphate ions, will maintain the concentrations of the species involved in diffusion into the lesion. Furthermore, dissociation of the CPP-bound ACP will be facilitated by the acid generated during enamel remineralization. This would explain why the CPP ­supported metastable calcium phosphate solutions are such efficient remineralizing solutions, since they would consume the acid generated during enamel lesion remineralization by generating more calcium and phosphate ions, thus maintaining their high concentration gradients into the lesion [11],[12],[13],[16]

The remineralization treatment regimen of 3 minutes twice daily application was employed as per the manufacturer's recommendations to make it clinically relevant. The specimens were incubated in artificial saliva at 37°C during hiatus period to stimulate the oral conditions.

Energy dispersive X-rays analysis (EDAX) has been used for elemental analysis at the ultrastructural level. It is one of the latest microanalytical technique that is used in conjunction with scanning electron microscope (SEM) wherein SEM does the structural analysis and elemental analysis is done by EDAX [1] . The principle is based on the energy emitted in the form of x-ray photons when the electrons from external sources hit the atoms in a material, thus generating characteristic x-rays of that element [6],[15] ,. When the sample is bombarded by the electron beam of the SEM, electrons are ejected from the atoms on the specimen's surface (secondary electrons). A resulting electron vacancy is filled by an electron from a higher shell, and an x-ray is emitted (characteristic x-rays) to balance the energy difference between the two electrons. The EDAX x­ray detector measures the number of emitted x-rays versus their energy. The energy of the x-ray is characteristic of the element from which the x-ray was emitted. A spectrum of the energy versus relative counts of the detected x-rays is obtained [Figure 2]&[Figure 4] and evaluated for qualitative and quantitative determinations of the elements present in the specimen using a computer based program [14] .

The results of this study were consistent with the proposed remineralization mechanism of CPP-ACP and are in accordance with the one obtained by EC Reynolds [11] .


   Conclusion Top


Under the parameters of this study following conclusions were made­

1. 10 % CPP-ACP paste significantly remineralized the artificial enamel sub-surface lesion in vitro.

2.The remineralization achieved was dose dependent since the remineralizing rate increased with the experiment time.

3. Energy dispersive X-ray analysis (EDAX) was found to be an efficient way to quantitatively access the changes in mineral content during in vitro caries studies.

 
   References Top

1.Arends J and Bosch JJT. Demineralization and remineralization evaluation techniques. Journal of Dental Research 1992; 71 (special issue): 924-928.  Back to cited text no. 1    
2.Bosch JJT& Mansson A. A review of quantitative methods for studies of mineral content of intra-oral incipient caries lesions. Journal of Dental Research 1991; 70 (1): 2-14  Back to cited text no. 2    
3.Harper DS, Osborn JC, Hefferren JJ, Clayton R. Cariostatic evaluation of cheeses with diverse physical and compositional characteristics. Caries Res 1986; 20: 123-130.  Back to cited text no. 3    
4.Kaczmarek E, Surdacka A, Matthews T, Miskowiak B. Digital image analysis and visualization of early caries changes in human teeth. Mat Sci-Poland2005; 23(2): 551-558  Back to cited text no. 4    
5.Krobicka A, Bowen WH, Pearson S, Young DA. The effect of cheese snacks on caries in desalivated rats. J Dent Res 1987; 66: 1116-1119.  Back to cited text no. 5    
6.Mishima H, Kozawa K. SEM and EDS analysis of calcospherites in human teeth. Eur J Oral Sci 1998; 106(S l ):392-396.  Back to cited text no. 6    
7.Reynolds EC, Johnson 1. Effect of milk on caries incidence and bacterial composition of dental plaque in the rat. Arch Oral Biol 1981; 26: 445-451.  Back to cited text no. 7    
8.Reynolds EC. An Effect of casein and whey­protein solutions on caries experience and feeding patterns of the rat. Arch Oral Biol 1984; 29:927-933.  Back to cited text no. 8    
9.Reynolds EC. The prevention of sub-surface demineralization of bovine enamel and changes in plaque composition by casein in an intraoral model. J Dent Res 1987: 66:1120-1127.  Back to cited text no. 9    
10.Reynolds EC, Cain CJ, Webber FL, Black CL, Riley PF, Johnson IH. Anticariogenicity of tryptic casein and synthetic-phosphopeptides in the rat. J Dent Res 1995; 74:1272-1279.  Back to cited text no. 10    
11.Reynolds EC. Remineralization of enamel subsurface lesions by casein phosphopeptide stabilized calcium phosphate solutions. J Dent Res 1997; 76: 1587-1595.  Back to cited text no. 11    
12.Reynolds EC. Anticariogenic complexes of amorphous calcium phosphate stabilized by casein phosphopeptides. A review. Spec Care Dent 1998; 18: 8-16.  Back to cited text no. 12    
13.Reynolds EC, Cai F, Shen P, Walker GD. Retention in plaque and remineralization of enamel lesions by various forms of calcium in a mouthrinse or sugar-free chewing gum. J Dent Res 2003; 82: 206-211.  Back to cited text no. 13    
14.Russ JC. Electron probe x-ray microanalysis­principles. In: Erasmus DA, ed. Electron probe microanalysis in biology. London: Chapman & Hall, 1978: 5-36.  Back to cited text no. 14    
15.Sakoolnamarka R, Burrow MF, Swain M, Tyas MJ. Microhardness and Ca:P ratio of carious and CarisolyTM treated caries-affected dentine using an ultra-micro-indentation system and energy dispersive analysis of x-rays A pilot study.Aus Dent J 2005; 50(4): 246-250.  Back to cited text no. 15    
16.Shen P, Cai F, Nowicki A, Vincent J, Reynolds EC. Remineralization of enamel subsurface lesions by sugar-free chewing gum containing casein phosphopeptide-amorphous calcium phosphate. J Dent Res 2001; 80: 2066-2070.  Back to cited text no. 16    
17.White DJ and Featherstone JBD. A longitudinal microhardness analysis of fluoride dentifrice effects n lesion progression in vitro. Caries research 1987- 1 21: 502-512  Back to cited text no. 17    

Top
Correspondence Address:
Mithra N Hegde
Department of Conservative Dentistry & Endodontics, A.B Shetty Memorial Institute of Dental Sciences, Deralakatte, Mangalore- 574160
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-0707.42276

Rights and Permissions


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]

This article has been cited by
1 Evaluation to determine the caries remineralization potential of three dentifrices: An in vitro study
Arun Balakrishnan,R Jonathan,P Benin,Arvind Kuumar
Journal of Conservative Dentistry. 2013; 16(4): 375
[Pubmed] | [DOI]



 

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


    Abstract
    Introduction
    Materials and Me...
    Results
    Discussion
    Conclusion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed4345    
    Printed333    
    Emailed3    
    PDF Downloaded0    
    Comments [Add]    
    Cited by others 1    

Recommend this journal