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Table of Contents   
ORIGINAL RESEARCH  
Year : 2011  |  Volume : 22  |  Issue : 1  |  Page : 77-82
Efficacy of CPP-ACP and CPP-ACPF on enamel remineralization - An in vitro study using scanning electron microscope and DIAGNOdent®


Department of Orthodontics, Meenakshi Ammal Dental College, Chennai, Tamil Nadu, India

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Date of Submission09-Sep-2009
Date of Decision01-Jun-2010
Date of Acceptance09-Aug-2010
Date of Web Publication25-Apr-2011
 

   Abstract 

Introduction: Remineralization as a treatment procedure has received a lot of attention both from clinicians as well researchers. The objective of this in vitro study was to find out the efficacy of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and casein phosphopeptide-amorphous calcium phosphate fluoride (CPP-ACPF) in remineralizing enamel surface on which artificial caries lesion had been created. The changes were analyzed using DIAGNOdent® (KaVo) and scanning electron microscope (SEM).
Materials and Methods: Ninety maxillary premolars were selected and divided into three groups of 30 teeth each: A (artificial saliva), B (CPP-ACP), and C (CPP-ACPF). All the samples were assessed using DIAGNOdent® at the baseline and after demineralization and remineralization. Three samples were randomly selected from each group after remineralization for surface evaluation using SEM.
Results: Statistical analysis showed that group B {CPP-ACP (4.1±1.8)} and group C {CPP-ACPF (4.8±1.2)} had a significantly higher amount of remineralization than group A (1.7±0.7).
Conclusion: All the three groups showed a statistically significant amount of remineralization. However, because of the added benefit of fluoride (NaF 0.2%), CPP-ACPF (Tooth Mousse-Plus®) showed marginally more amount of remineralization than CPP-ACP (Tooth Mousse®).

Keywords: CPP-ACP fluoride, DIAGNOdent® , remineralization, SEM

How to cite this article:
Jayarajan J, Janardhanam P, Jayakumar P, Deepika. Efficacy of CPP-ACP and CPP-ACPF on enamel remineralization - An in vitro study using scanning electron microscope and DIAGNOdent®. Indian J Dent Res 2011;22:77-82

How to cite this URL:
Jayarajan J, Janardhanam P, Jayakumar P, Deepika. Efficacy of CPP-ACP and CPP-ACPF on enamel remineralization - An in vitro study using scanning electron microscope and DIAGNOdent®. Indian J Dent Res [serial online] 2011 [cited 2014 Sep 1];22:77-82. Available from: http://www.ijdr.in/text.asp?2011/22/1/77/80001
An opaque white enamel lesion is frequently a sequel of orthodontic fixed appliance therapy. These lesions are due to either surface or subsurface demineralization. [1] Clinical evidence has shown that the white spot lesion can be reversed completely or at least in part. More information is necessary before an optimal remineralization program for orthodontic patients can be established.

A number of remineralization techniques have been tried out, among which the use of fluoride is well known. A newer concept for remineralization is the use of milk and milk products, which appear to have a protective effect against the development of dental caries. It has been suggested that the anticariogenic properties of milk is due to the presence of casein, calcium, and phosphate, [2],[3] which are responsible for resistance to acid dissolution. When casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) or casein phosphopeptide-amorphous calcium phosphate fluoride (CPP-ACPF) is applied to the oral environment, the sticky CPP part of the CPP-ACP/ACPF complex binds readily to the enamel, biofilm, and soft tissues, delivering the calcium and phosphate ions exactly where it is needed. The free calcium and phosphate ions move out of the CPP, enter the enamel rods, and reform the apatite crystals

This study was undertaken to evaluate the ability of topically applied CPP­-ACP and CPP­-ACPF in bringing about remineralization changes on enamel surface that has been exposed to an artificial caries challenge in a simulated oral environment.


   Materials and Methods Top


One hundred and twenty human maxillary first premolars that had been extracted for orthodontic procedures were collected and stored in 0.1% thymol solution to prevent any fungal or bacterial growth. The teeth were thoroughly cleaned of its debris, calculus, and soft tissues. All the teeth were sliced mesiodistally into buccal and lingual halves using a diamond disk burr. The buccal halves of the teeth were used for the study, taking into consideration the technique sensitivity of DIAGNOdent® (KaVo) and the ease of mounting the sample on a scanning electron microscope (SEM).

The samples that showed no evidence of white spot lesion, enamel cracks, or caries on visual inspection were taken for evaluation. A white stick-on paper of 4×4 mm dimension was stuck on each tooth before applying an acid-resistant nail varnish; this was done to limit the area of study.

All the samples were examined using DIAGNOdent® (KaVo) [4],[5] to assess for any surface changes present on the labial window [Table 1]. As recommended by the manufacturer, prior to every measurement session the instrument was calibrated against its own ceramic standards. Two types of probes are available: type A used in pit and fissures and type B used for smooth caries. In this study, the type B probe was used. The labial window area was carefully scanned using the type B probe by holding the tip in close contact with the tooth surface and tilting the tip around the measuring site in order to collect the fluorescence from all directions.
Table 1: DIAGNOdent® interpretation

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Samples showing a moment value between 3 and 7 on the digital display were selected. These values suggest that the enamel surface is intact. [5] Samples showing a value greater than 7 were discarded. The teeth were randomly divided into three groups containing 30 samples each and color-coded. The baseline values of the three groups were recorded.

Each tooth was then immersed individually into separate plastic containers numbered from 1 to 90, each containing 4 ml of demineralizing solution [6] (2.2 mM KH 2 PO 4 , 50 mM acetic acid, and 2.2 mM CaCl 2 ), for a period of 5 h. This demineralizing procedure was intended to produce a consistent subsurface lesion. After 5 h in the demineralizing solution the teeth were taken out, washed with deionized water, dried, and placed back in their respective clean containers.

The teeth were evaluated with DIAGNOdent® and the samples showing a moment value of 9 and above on the digital display were taken for further evaluation. This value indicated the presence of a subsurface lesion on the tooth surface. The readings were recorded for statistical analysis.

The samples in each group were treated with the remineralizing agent, after which they were rinsed with deionised water, dried, and stored in artificial saliva, [7] consisting of 20 mmol/l NaHCO 3 , 3 mmol/l NaH 2 PO 4 , and 1 mmol/l CaCl 2 at room temperature, neutral pH, and constant circulation for 1 week [Figure 1]. Each sample in group A (brown) was washed with deionized water and placed back in a clean container containing artificial saliva. Each sample in group B (pink) was rubbed with CPP-ACP paste for 4 min using a gloved finger, following which they were washed with deionized water and placed back in artificial saliva. Each sample in group C (red) was rubbed with CPP-ACPF paste for 4 min, washed with deionized water, and placed back in artificial saliva.
Figure 1: Randomly selected samples

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Following this, the surface was assessed using DIAGNOdent® to record the values after the remineralization procedure. The values obtained were tabulated and statistically analyzed using Student's 't' test followed by one-way ANOVA; the comparison of means was conducted using Tukey's multiple comparison tests. P≤.05 was considered to be significant.

To assess the remineralized surface changes on the enamel, three samples from each group were randomly selected and compared with each other using an SEM (Quanta 200). Pictures were taken at 1000× and 2000× magnification for all the three groups.


   Results Top


Inference from [Table 1]: Moment values between 3 and 7 indicate normal tooth structure. Moment values between 7 and 9 indicate white spot lesion. Moment values more than 9 indicate demineralization of the tooth structure.

Inference from [Table 2]: Mean value at baseline was highest for group C (4.2±1.6), followed by group B (4.1±1.2) and then group A (3.7±0.8). However, there was no statistically significant difference between the three groups.
Table 2: Statistical values

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Mean value at demineralization was highest for group C (11.0±1.3), followed by group A (11.0±1.2) and then group B (10.9±1.4). There was no statistically significant difference in the mean values between the three groups. This shows that the demineralization solution that was used for the study produced uniform artificial carious lesions. The difference between the baseline value and the demineralization value was statistically significant (P<.0001) in all the three groups.

The maximum remineralization was seen in group C (6.2±1.6), followed by group B (6.8±1.3) and then group A (9.4±1.3) (the lower the mean value the higher the amount of remineralization). One-way ANOVA showed that there was significant difference among the three study groups (P<.0001). Further, the multiple range test by the Tukey HSD procedure showed that group C had significantly better remineralization than group B and group A. Thus, saliva (group A) by itself could not increase the levels of calcium and phosphate to the same extent as CPP-ACP (group B) and CPP-ACPF (group C).

Inference from [Table 3]: The mean values in group B (4.1±1.8) and group C (4.8±1.2) were significantly higher than the mean value in group A (1.7±0.7) (P<.05). However, there was no significant difference between group B and group C (P<.05).
Table 3: Change in mean values from demineralization to remineralization

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SEM evaluation

In group A (control; artificial saliva), the configuration of sound enamel topography is apparent with certain porous defects [[Figure 2]a; 1000× magnification], but the enamel rods are barely discernable at this magnification. In [Figure 2]b, at a higher magnification of 2000×, the porosities are more evident and faint lines of mineralization can be seen in and around the porosities.
Figure 2: (a) Group A at 1000× magnification (b) Group A at 2000× magnification

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In group B (CPP-ACP), the interprismatic substances are evident, with porosities and areas of remineralization also seen [[Figure 3]a; 1000×]. [Figure 3]b (2000×) of the same group displays thick and more frequent lines of remineralization along the prismatic borders; certain areas of calcifications are evident along the porosities (indicated by arrow marks).
Figure 3: (a) Group B at 1000× magnification (b) Group B at 2000× magnification

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In group C (CPP-ACPF), enamel rods and prismatic substance are not discernable [[Figure 4]a; 1000×], but the areas of calcified deposits are more evident and are seen concentrated along the porous defects. In [Figure 4]b, at a higher magnification of 2000×, areas of mineralized deposits are discernble and are seen profusely scattered along the porous defects (indicated by arrow marks).
Figure 4: (a) Group C at 1000× magnification (b) Group C at 2000× magnification

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   Discussion Top


Though remineralization has been a major area of investigation, it is still difficult to exactly define the efficacy of various remineralization methods. Until today, fluoride therapy has remained the most popular of all caries prevention and remineralization methods. [8] Recently, dairy products (milk, milk concentrates, and cheeses) have received a lot of attentionfor their anticariogenic effect in animal and human in situ caries models. [2] The protective effect seen with dairy products is possibly best attributed to the phosphoprotein casein and calcium phosphate contents. Casein is rapidly degraded by intraoral bacterial proteolytic activity, [9] with the released phosphopeptides being relatively stable to further degradation. The recently proposed remineralization and anticariogenic mechanism of CPP-ACP involves the incorporation of nanocomplexes into dental plaque and onto the tooth surface, which thereby acts as a calcium and phosphate reservoir. [3]

Demineralization is an unwanted but inevitable adverse effect of fixed orthodontic therapy, a problem faced by all clinicians. Demineralization occurs when the pH in the oral environment falls below 5.5, allowing calcium and phosphate ions to diffuse from the enamel surface. The duration and frequency of the acid challenge can influence the level of damage to the tooth substance, which can range from small white lesions to frank cavitations. [10] It should be emphasized that when the surface remains partially intact, there is a possibility of arresting or even reversing the lesion. This challenge can be arrested or reversed by the combined action of salivary minerals and fluoride therapy. [11]

Demineralization can be detected by two methods: (1) invasive and (2) noninvasive. [1] White spot lesions are not detectable visually until they have progressed 200-300 μm into the enamel. Some of the noninvasive diagnostic methods available are quantitative light fluorescence (QLF), electrical resistance caries monitoring device, fiberoptic transillumination, optical coherence tomography, laser fluorescence (DIAGNOdent® ), [4] and SEM. [12]

DIAGNOdent® (KaVo), a non-invasive method, uses laser fluorescence to measure early demineralization. Various literature reports have shown the value of DIAGNOdent® as a reliable non-invasive caries-detecting device. The organic and inorganic materials present on the tooth surface absorb the laser light and emit fluorescence in the infrared region of the spectrum. The presence of a demineralized area increases the fluorescence, with an audible sound indicating the fluorescence increase. Higher number of sounds and higher-pitched sounds indicate more demineralization.

This study was undertaken to determine the remineralization potential of CPP-ACP and CPP-ACPF and to compare it to the inherent remineralization potential of saliva. The CPP molecules contain a cluster of phosphoseryl residues that markedly increase the apparent solubility of calcium phosphate by stabilizing amorphous calcium phosphate under neutral and alkaline conditions. [3] CPP can stabilize over 100 times more calcium phosphate than is normally possible in aqueous solution at neutral or alkaline pH before spontaneous precipitation occurs. [13] CPP-ACPF has, in addition, fluoride. Studies have shown that remineraliszation of small lesions with low-dose fluoride therapy is more efficient. [14]

All the samples were observed using DIAGNOdent® (KaVo), [4] and the baseline value, the amount of demineralization, and the remineralization values were tabulated. A SEM (Quanta 200) was used to assess the surface changes seen on the enamel.

The mean and standard deviation were calculated and tests of significance were done for each group. Comparison of the mean remineralization value of group A (artificial saliva; 9.4±1.3) with its mean demineralization value (11.0±1.2) shows that there is a certain amount of remineralization; this indicates that saliva by itself has some remineralization potential. On comparing the remineralization value of group B (CPP-ACP; 6.8±1.3) to its demineralization value (10.9±1.4) it is evident that a significant amount of remineralization has occurred. On comparing group B and group A, the former showed a significantly higher amount of remineralization. Though saliva has some remineralization potential, [11] it cannot by itself increase the levels of calcium and phosphate release. For mineral deposition to occur within the body of the lesion, calcium and phosphate ions must first penetrate the surface layer of enamel. This explains why the CPP-supported metastable calcium phosphate solutions are such efficient remineralizing solutions: they are able to consume the acid generated during enamel lesion remineralization by generating more calcium and phosphate ions, including CaHPO4, thus maintaining the high concentration gradient into the lesion. [15]

On comparing the change in mean values from demineralization to remineralization between group C (CPP-ACPF; 4.8±1.2) and group B (CPP-ACP; 4.1±1.8), the former showed a much higher amount of remineralization. This can be attributed to the synergistic anticariogenic effects of CPP-ACP and fluoride. The fluoride ions are adsorbed onto the surface of enamel crystals, inhibiting dissolution and increasing remineralization. The softened surface lesions remineralize faster and more completely than subsurface lesions. Ogaard and coworkers [16] warned against treating visible white spots that develop during orthodontic therapy with concentrated fluoride agents immediately after debonding, because this procedure will arrest the lesions and prevent complete repair by surface hypermineralization. Hence, the use of fluoride alone as a remineralizing agent in our clinical situation is not beneficial. Treating visible white spots that develop during orthodontic therapy with concentrated fluoride agents will arrest the lesions and prevent complete repair. With the use of low fluoride concentration as is present in CPP-ACPF (0.2% or 900 ppm of NaF), there is a complex localization of free calcium phosphate and fluoride ion activities, which helps in maintaining a state of supersaturation by suppressing demineralization. [17] Thus CPP-ACPF (Tooth Mousse Plus ) is an excellent local slow-delivery system to treat the white spot lesion. In this study we have limited the application to a week. If the same had been extended to a scheduled routine protocol, the amount of remineralization may probably have approached the baseline value. The SEM pictures of the three groups suggest remineralization in the same order, with the CPP-ACPF showing the greatest amount of mineral deposits, followed by CPP-ACP and then artificial saliva.


   Conclusion Top


CPP-ACP (Tooth Mousse™) and CPP-ACPF (Tooth Mousse Plus™) are excellent delivery vehicles available in a slow-release amorphous form to localize calcium, phosphate, and fluoride at the tooth surface.

CPP-ACPF can be included in the routine hygiene and maintenance instructions for reversing or arresting white spot lesions in orthodontic patients.

 
   References Top

1.Anusavice KJ. Caries risk assessment. Oper Dent Suppl 2001;6;1-5.  Back to cited text no. 1
    
2.Genkins GN, Ferguson DB. Milk and dental caries. Br Dent J 1966;144:40-5.  Back to cited text no. 2
    
3.Rose RK. Effects of anticariogenic casein phosphopeptide on calcium diffusion in streptococcal model dental plaque. Arch Oral Biol 2000;45:569-75.  Back to cited text no. 3
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4.Al-Khateeb S, Forsberg C M, de Josselin de Jong E, Angmar-Mansson B. A longitudinal laser fluorescence study of white spot lesions in orthodontic patients. Am J Orthod Dentofacial Orthop 1998;113:595-602.  Back to cited text no. 4
    
5.Ross RG. Product profile, Pit and Fissure Caries Diagnosis: Clinical guidelines. Oral Health journal April 2007.  Back to cited text no. 5
    
6.Corry A, Millett DT, Creanor SL, Foye RH, Gilmour WH. Effect of fluoride exposure on cariostatic potential of orthodontic bonding agents: An in vitro evaluation. J Orthod 2003;30:323-9.  Back to cited text no. 6
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7.Todd MA, Staley RN, Kanellis MJ, Donly KJ, Wefel JS. Effects of a fluoride varnish on demineralization adjacent to orthodontic brackets. Am J Orthod Dentofacial Orthop 1999;116:159-67.  Back to cited text no. 7
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8.Geiger AM, Gorelick L, Gwinnett AJ, Benson BJ. Reducing white spot lesion in orthodontic populations with fluoride rinsing. Am J Orthod Dentofacial Orthop 1992;101;404-7.  Back to cited text no. 8
    
9.Reynolds EC, Riley PF. Protein dissimilation by human salivary sediment bacteria. J Dent Res 1989;68:124-9.  Back to cited text no. 9
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10.Ogaard B, Rolla G, Arends J. Orthodontic appliances and enamel demineralization. Part 1 Lesion development. Am J Orthod Dentofacial Orthop 1988;94:68-73.  Back to cited text no. 10
    
11.Amaechi BT, Higham SM. In vitro remineralisation of eroded enamel lesions by saliva. J Dent Res 2001;29:371-6.  Back to cited text no. 11
    
12.Oshiro M, Yamaguchi K, Takamizawa T, Inage H, Watanabe T, Irokawa A, et al. Effects of CCP-ACP paste on tooth mineralization: An FE-SEM study. J Oral Sci 2007;49:115-20.  Back to cited text no. 12
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13.Holt C,Van Kemenade M.The interaction of phophoprotiens with calcium phosphate. Calcified tissue. In: Hukins DW, editor. Boca Raton, Fl: CRC Press; 1989. p. 175-213.  Back to cited text no. 13
    
14.Zahradnik RT. Effect of Fluoride rinses upon in vitro enamel remineralization. J Dent Res 1980;59:105-6.  Back to cited text no. 14
    
15.Reynolds EC. Remineralization of enamel subsurface lesion by Caesin phosphopeptide- stabilized calcium phosphate solution. J Dent Res 1997;76:1587-95.  Back to cited text no. 15
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16.Ogaard B, Rolla G, Arends J, Tencate JM. Orthodontic appliances and enamel demineralization: Part 2- Prevention and treatment of lesions. Am J Orthod Dentofacial Orthop 1988;94:123-8.  Back to cited text no. 16
    
17.Holler BE, Friedl KH, Jung H, Hiller KA, Schmalz G. Fluoride uptake and distribution in enamel and dentin after application of different fluoride solutions. Clin Oral Investig 2002;6:137-44.  Back to cited text no. 17
[PUBMED]  [FULLTEXT]  

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Correspondence Address:
Jayanth Jayarajan
Department of Orthodontics, Meenakshi Ammal Dental College, Chennai, Tamil Nadu
India
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DOI: 10.4103/0970-9290.80001

PMID: 21525682

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    Figures

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    Tables

  [Table 1], [Table 2], [Table 3]

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