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Table of Contents   
ORIGINAL RESEARCH  
Year : 2013  |  Volume : 24  |  Issue : 6  |  Page : 681-689
Comparative evaluation of remineralization potential of casein phosphopeptide-amorphous calcium phosphate and casein phosphopeptide-amorphous calcium phosphate fluoride on artificial enamel white spot lesion: An in vitro light fluorescence study


Department of Pedodontics and Preventive Dentistry, JSS Dental College and Hospital, JSS University, Mysore, Karnataka, India

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Date of Submission02-Apr-2013
Date of Decision19-Apr-2013
Date of Acceptance13-Jun-2013
Date of Web Publication20-Feb-2014
 

   Abstract 

Background: World-wide, the contribution of dental caries to the burden of oral diseases is about 10 times higher than that of periodontal disease, the other common oral condition. Owing to its globally high prevalence, dental caries is a "pandemic" disease characterized by a high percentage of untreated carious cavities causing pain, discomfort and functional limitations. Untreated carious cavities; furthermore, have a significant impact on the general health of children and on the social and economic well-being of communities. A surgical approach to the elimination of carious lesion was developed a century ago; this approach was necessary at that time, because there was no valid alternative. The focus in caries has recently shifted to the development of methodologies for the detection of the early stages of caries lesions and the non-invasive treatment of these lesions. The non-invasive treatment of early lesions by remineralization has the potential to be a major advance in the clinical management of the disease. Remineralization of white-spot lesions may be possible with a variety of currently available agents containing fluoride, bioavailable calcium and phosphate and phosphate. This concept bridges the traditional gap between prevention and surgical procedures, which is just what dentistry needs today.
Aims and Objectives: The aim of this in vitro study was to evaluate and to compare the remineralization potential of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and casein phosphopeptide-amorphous calcium fluoride phosphate (CPP-ACFP) on artificial white spot enamel lesions using the quantitative light fluorescence (QLF).
Materials and Methods: A total of 45 caries-free extracted maxillary first premolars were embedded in acrylic resin. The samples were randomly divided into three groups namely control group, CPP-ACP group and CPP-ACFP group with 15 samples in each group. The samples of each group were subjected to demineralization process for a period of 96 h. The samples were then mounted in the artificial mouth model and subjected to remineralization and pH cycling for a period of 21 days. QLF readings were recorded at the end of demineralization (1 st , 7 th , 14 th and 21 st day) and were statistically analyzed.
Results: As compared with artificial saliva both CPP-ACP and CPP-ACFP produced significant amount of remineralization of the artificial enamel white spot lesion (P < 0.001), however when the remineralizing effect of CPP-ACP was compared with the remineralizing effect of CPP-ACFP there was no significant difference. Significant amount of remineralization was produced by CPP-ACP and CPP-ACFP only after the 7 th day. After the 14 th day, the remineralization produced by both CPP-ACP and CPP-ACFP as compared to artificial saliva was non-significant.

Keywords: Artificial mouth model, casein phosphopeptide amorphous calcium phosphate, casein phosphopeptide amorphous calcium fluoride phosphate, demineralization, remineralization, quantitative light fluorescence

How to cite this article:
Mehta R, Nandlal B, Prashanth S. Comparative evaluation of remineralization potential of casein phosphopeptide-amorphous calcium phosphate and casein phosphopeptide-amorphous calcium phosphate fluoride on artificial enamel white spot lesion: An in vitro light fluorescence study. Indian J Dent Res 2013;24:681-9

How to cite this URL:
Mehta R, Nandlal B, Prashanth S. Comparative evaluation of remineralization potential of casein phosphopeptide-amorphous calcium phosphate and casein phosphopeptide-amorphous calcium phosphate fluoride on artificial enamel white spot lesion: An in vitro light fluorescence study. Indian J Dent Res [serial online] 2013 [cited 2018 Dec 10];24:681-9. Available from: http://www.ijdr.in/text.asp?2013/24/6/681/127610
Diagnostic methods to detect early demineralization in vivo include visual inspection, tactile examination using dental probe, radiography, electronic monitoring and FOTI Fibreoptic transillumination. All these methods have limitations affecting their diagnostic ability or their practicability in the clinical setting. Several of these methods such as radiography cannot detect the carious process until it is significantly advanced while other produces arbitrary data that cannot be correlated with actual mineral loss. Methods have been developed that permit this quantifiable detection, i.e., transverse microradiography of very early lesions but are destructive and cannot be used in vivo. [1]

These traditional visual, tactile and radiographic methods of detecting dental caries can only detect lesions that are well advanced involving 300-500 μm of the enamel. During the past 10 years, optical methods have been developed for the quantification of enamel caries. The common principle for these methods is enamel fluorescence. Quantitative light fluorescence (QLF) and DIAGNOdent are the devices, which make use of this optical property to detect early caries and there progression and their regression. This technique has high sensitivity and specificity, especially for carious lesions on occlusal surfaces. However, the device supplies the information as an arbitrary value and has to be calibrated frequently for longitudinal comparisons. The probe must also be rotated in all directions to detect the highest reading, which is very technique sensitive. QLF presents a solution to the problem by combining the sensitivity and specificity of the destructive in vitro techniques with clinical usefulness of those employed in vivo. [1]

A number of methods and agents are available to mineralize early enamel lesions. Fluoride is the most commonly used remineralizing agent. The topical application of mineral rich concentrates can also be used for remineralization of incipient caries lesions. These provide calcium and phosphate ions that can easily diffuse through porous enamel in order to remineralize incipient enamel lesion. [2]

The influence of milk and milk products on caries is known since 1950's, when cheese was considered to have a substantial cariostatic effect. It was attributed to the physical nature of cheese and the presence of casein, calcium and phosphate contents. [3] Latter investigations on the role of casein and calcium phosphate concentrates derived from milk on remineralization concluded that casein phosphopeptide (CPP) complexes stabilized the calcium phosphate and remineralize the incipient carious lesions. [3],[4],[5] The combination of CPP-amorphous calcium phosphate (ACP) and fluoride, i.e., casein phosphopeptide-amorphous calcium fluoride phosphate (CPP-ACFP) also brings about the remineralization of the incipient caries lesions. [5] When such agents are used clinically, the practitioner should evaluate the remineralization using non-invasive techniques. Therefore, the aim of this in vitro study was to evaluate the remineralization of artificial white spot enamel lesions using CPP-ACP and CPP-ACFP using QLF.


   Materials and Methods Top


The study was conducted in the Department of Pedodontics and Preventive Dentistry, JSS Dental College and Hospital and Water and Health Department of JSS College of Pharmacy, JSS University, Mysore, Karnataka, India.

Maxillary first premolars teeth, freshly extracted for Orthodontic reasons from young adults were obtained for the present study from the Department of Oral and Maxillofacial Surgery at JSS Dental College and Hospital, Mysore. The samples were selected based on the following inclusion and exclusion criteria.

Inclusion criteria

  • Caries free teeth
  • Teeth extracted for orthodontic reasons only.


Exclusion criteria

  • Teeth with fracture of either crown or root detected using FOTI
  • Teeth with caries lesions including white spot lesions detected using QLF and FOTI
  • Teeth with QLF score >1
  • Teeth with hypoplastic lesions detected visually as well as on QLF
  • Teeth with intrinsic stains
  • Teeth with any wasting diseases like attrition, abrasion, erosion
  • Teeth with developmental anomalies
  • Teeth with any restoration.


In order to detect a clinically relevant difference of 20% at 5% level of significance with 80% power, the required sample size was arrived at 15 samples in each group.

A total of 45 caries-free premolars without any detectable caries, hypoplastic lesions, stains (intrinsic or extrinsic and white spot lesions) were included in the study.

Sample preparation

These teeth samples were embedded in acrylic blocks such that only their crown portion was exposed using a standardized jig. Using this customized jig 45 samples of 3.2 × 1.2 × 1.2 mm each, were prepared. The samples were subsequently examined using QLF and FOTI for the detection of any subclinical lesions or hypoplastic areas and cracks on the buccal surface of the samples. Any defective samples as identified by QLF and FOTI were replaced by the new ones meeting the norms.

A polyvinyl stencil of 4 × 4 mm dimension was placed on the buccal surface of the samples. The samples were then coated with a transparent acid resistant nail varnish (REVLON, NY, USA) except for the 4 × 4 mm area of the polyvinyl stencil. After setting of the varnish, the stencil was removed leaving a window of 4 × 4 mm on the buccal surface. This 4 × 4 mm area is the area of interest (AOI) as this is the only area, which was analyzed for the change in fluorescence values following demineralization and remineralization.

The samples were then randomly divided into three groups with fifteen each. The groups being:

  • Control group
  • CPP-ACP group (remineralized using GC Tooth Mousse cream, RECALDENT TM )
  • CPP-ACFP group (remineralized using GC Tooth Mousse plus cream, RECALDENT TM ).


Artificial enamel white spot lesion production

The demineralizing solution was prepared by adding 2.2 mM calcium chloride, 2.2 mM potassium dihydrogen orthophosphate dehydrate to 0.05 M Acetic acid. The pH was adjusted to 4.4 with 1 M potassium hydroxide. The samples were stored individually in 25 ml plastic bottles containing 10 ml of demineralization solution for a period of 96 h to produce lesions of 120-200 μm deep. [6] The bottles were placed in an orbital incubator at 37°C to provide gentle agitation.

At the end of 96 h, the samples were removed from the demineralization solution. Each sample was air dried for 5 s using a chip blower to inspect visually the AOI for the detection of white spot lesion using the fluorescence camera (FC).

Remineralization procedure

The test samples were then remineralized by once daily application of GC Tooth Mousse, RECALDENT TM for CPP-ACP group, GC Tooth Mousse Plus, RECALDENT TM for CPP-ACFP group. The paste was applied onto the tooth surface window (AOI) with a brush and left in place for 3 min. During the demineralization procedure the test samples and the control samples were mounted in the artificial mouth models.

Preparation of artificial saliva: The artificial saliva was prepared according to method given by Sato et al. [7]

  • Sodium phosphate Na 3 PO 4 - 3.90 mM
  • Sodium chloride NaCl - 4.29 mM
  • Potassium chloride KCl - 17.98 mM
  • Calcium chloride CaCl 2 - 1.1 mM
  • Magnesium chloride MgCl 2 - 0.08 mM
  • Sulphuric acid H 2 SO 4 - 0.05 mM
  • Sodium carbonate NaHCO 3 - 3.27 mM
  • Distilled water.


The pH was set at a level of 7.2.

The inorganic component of the artificial saliva is similar to that of natural saliva.

Artificial mouth model

Three artificial mouth models were made, one for each group [Figure 1] and [Figure 2].
Figure 1: Artificial mouth model

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Figure 2: Arrangement of the artificial mouth models in hot air oven

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Artificial mouth model consist of a rectangular polyvinyl box (Tupperware) of dimension 25 × 15 mm with the multiple compartments (20) within. Each compartment measured 5 × 5 cm in dimension.

Each sample was mounted in the base of the individual compartment such that their buccal surface faced upward. Double-sided adhesive tape was used to mount the samples. Once the samples were mounted a circular opening of 5 mm in diameter was made in the walls of the compartment at a distance of 5 mm from the base for draining of the solutions. A circular exit hole of 15 mm in diameter was made in the last compartment for the exit pipe, which finally drained into a waste collector. Waste collector was a polyvinyl container of dimension 36 × 45 × 17 mm. For each compartment, a controlled dropper was fixed on the lid of the polyvinyl box such that the drop of solutions would fall exactly on the exposed window surface of each sample. The controlled dropper was adjusted such that it delivered 5 drops of solution on the AOI of the buccal surface of the tooth. The controlled droppers of each compartment were interconnected using polyvinyl tubes of 0.5 mm diameter with the help of two-way plastic connectors. Each row was then connected individually to the drip set with a controller for the flow of solutions. The controller was so adjusted that the flow of solutions was 5 drops/min. The drip set of each row was connected to the polyvinyl pipe of 1 cm diameter, which had its one end connected to the storage tank for the solutions and other end closed with a washer. In the similar way, other two artificial mouth models were prepared for the other subgroups with individual inlet for the solutions and individual outlet, which finally drained into the waste collector. A water pump was fixed inside the waste collector to pump out the waste solutions collected. A polyvinyl pipe of 1 cm diameter was connected to the pump to drain the waste solutions out of the hot air oven. The pipe came out through the exit opening of the hot air oven. The whole set up except for the storage tank for the solutions was placed in the hot air oven, the temperature of which was maintained at 37°C simulating oral temperature.

Thus, in this artificial mouth models there was a continuous flow of saliva with intermittent flow of 5 pH buffer solution simulating oral conditions.

pH cycling

pH cycling consisted of continuous exposure of the samples to artificial saliva with intermittent exposure to buffer solution of 5 pH, 3 times daily for a period of 15 min each for 21 days to resemble the pH changes occurring in mouth.

Light fluorescence analysis

Light fluorescence analysis of the lesions was conducted by a single blinder examiner using Light fluorescence device (FC, Duerr Dental, Vistaproof, Germany), which uses the DD view software to analyze the carious lesions.

This recently devised FC device emits blue light at 405 nm and records fluorescence from the teeth as digital images. The FC (VistaProof, Durr Dental, Germany) is a system that has been modified by exchanging the white light emitting diodes (LEDs) of the camera with six blue gallium nitride LEDs emitting at 405 nm. An optical long pass filter has been introduced into the beam path in front of the charged coupled deviceensor to cut down the excitation light below 495 nm. DD view software is used to digitize the video signal to create the images of 720 × 576 pixels with 3 × 8 bit intensities of red, green and blue channels and resolution of 72 pixels/inch. These images are analyzed with the software, which quantifies the red and green components of fluorescence. This software shows the region of the teeth that emits fluorescence ranging from 0 to 3 corresponding to the lesion severity and calculated as the intensity ratio of the red and green fluorescence. Caries lesions are identified when the red/green ratio was higher than that of sound tissue. The fluorescence ratio of caries lesions was taken as the maximum red/green ratio recorded. [8]

Criteria for caries detection using FC

  • 0-1: Normal (green)
  • 1-1.5: Beginning of enamel caries (blue)
  • 1.5-2: Deep enamel caries (red)
  • 2-2.5: Dentinal caries (brown)
  • 2.5-3: Deep dentinal caries (yellow).


FC reading indicates the change in the value of fluorescence. Increase in the FC reading indicates loss of fluorescence, which corresponds to demineralization while decrease in the Fluorescence reading corresponds to remineralization.

Light fluorescence examination was done during the following procedures:

  • Selection of the samples
  • After varnish application
  • After demineralization cycle (1 st day)
  • At the end of 7 th day, 14 th day and 21 st day of pH cycling to check the amount of remineralization.


For the light fluorescence analysis only the AOI was analyzed for the change in fluorescence value after demineralization and during remineralization process.

For light fluorescence analysis the samples were mounted in a customized positioner, which was colored matted black to avoid ambient light interfering with the analysis.

The FC was held at a fixed distance from the samples using a customized positioner to avoid magnification errors during the successive imaging procedures [Figure 3].
Figure 3: Quantitative light fluorescence analysis procedure using standardized positioner for the samples and the fluorescence camera

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The light fluorescence reading corresponding to loss of fluorescence was recorded:

  • Highest score point: The point in the AOI with highest light fluorescence reading as identified at the end of demineralization (day 1) as generated by the FC. The same point was followed up on 7 th , 14 th and 21 st day by mapping.


Relocation of the highest score point

The X axis and Y axis coordinates of the highest score point were noted down as given by the image software analyzer (DDview software Durr Dental software on day 1 and during the subsequent QLF examinations the point with the same values of the X axis and Y axis coordinates was recorded [Figure 4]. During the QLF examination, standardized imaging procedure was followed using the customized positioner to avoid any angulation errors of the FC.

The FC image of the sample was compared with the previous FC image taken on day 1 to avoid any magnification error before recording the highest score point.
Figure 4: Relocation of the highest score point using the X and Y coordinates

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The other two light fluorescence readings recorded were:

  • Highest tooth score: The point in the AOI, which showed the highest QLF reading at 1 st , 7 th , 14 th and 21 st day as identified by manual mapping of the tooth
  • Lowest tooth score: The point in the AOI, which showed the lowest QLF reading at 1 st , 7 th , 14 th and 21 st day identified by manual mapping of the tooth.


Average tooth score: It is the average of the highest tooth score and lowest tooth score for each sample.

The digital readings of Light FC obtained after demineralization and remineralization of all the samples of the test groups were statistically analyzed using Paired samples t-test, independent t-test. All statistical operations were done through Statistical Presentation System Software (SPSS) (version 16.0, Chicago, USA).

Means of the highest score point readings for different groups at 1 st , 7 th , 14 th , 21 st day

Mean highest score point readings of the control group at different time interval was 1.55 ± 0.19, 1.13 ± 0.96, 1.09 ± 007 and 1.01 ± 0.04 at 1 st , 7 th 14 th and 21 st day respectively [Table 1].
Table 1: Means of the highest score point for different groups at 1st, 7th, 14th and 21st day


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Mean highest score point readings of the CPP-ACP group at different time interval was 1.47 ± 0.17, 1.27 ± 0.10, 1.09 ± 0.07, 1.05 ± 0.06, at 1 st , 7 th 14 th and 21 st day respectively.

Mean highest score point readings of the CPP-ACFP group at different time interval was 1.49 ± 0.53, 1.20 ± 0.1.77, 1.02 ± 0.04, 0.95 ± 0.06 at 1 st , 7 th 14 th and 21 st day respectively.

Intragroup comparison of the mean difference in the highest score point reading for the control group, CPP-ACP group and CPP-ACFP group

When intragroup comparison was made, all three groups showed statistically significant difference (P < 0.001) in mean highest score point reading at the day intervals of 1 st day-7 th day, 1 st day-14 th day, 1 st day-21 st day, 7 th day-21 st day and 14 th day-21 st day [Table 2]. The mean highest score point reading decreased from day 1 to day 21. This change in the highest score point reading suggest that a significant amount of remineralization has taken place in the samples of each group. The samples of control group also showed remineralization because of artificial saliva, which was used in the pH cycling in the artificial mouth model.
Table 2: Intragroup comparison between control, CPP-ACP and CPP-ACFP groups


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Intergroup comparison of mean difference in highest score point reading for the Control group and CPP-ACP group

When the mean difference in highest score point reading for the control group from day 1 and day 7 and the mean difference in the highest score point reading for the CPP-ACP group from day 1 to day 7 were compared [Table 3] the difference was highly significant (P < 0.01).
Table 3: Intergroup comparison between control, CPP-ACP and CPP-ACFP groups


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The mean difference in the highest score point reading for the control group from day 7 to day 14 and the mean difference in the highest score point reading for the CPP-ACP group were compared, the difference was highly significant (P < 0.01) suggesting that CPP-ACP group performed better than the control group from 7 th day to 14 th day.

When the mean difference in the highest score point reading for the control group from day 7 to day 21 and the mean difference in the highest score point reading for the CPP-ACP group were compared the difference was significant (P < 0.05).

Intergroup comparison of the mean difference in the highest score point reading for the control group and CPP-ACFP group

The mean difference in the highest score point reading for the control group from day 7 to day 14 and the mean difference in the highest score point reading for the CPP-ACFP group were compared, the difference was highly significant (P < 0.01) suggesting that CPP-ACFP group performed better than the control group from 7 th day to 14 th day [Table 3].

When the mean difference in the highest score point reading for the control group from day 7 to day 21 and the mean difference in the highest score point reading for the CPP-ACP group were compared the difference was significant (P < 0.05).

Intergroup comparison of the mean difference in the highest score point reading for the CPP-ACP group and CPP-ACFP group

When the mean difference [Table 3] in highest score point reading for CPP-ACP group and the CPP-ACFP group among 1 st , 7 th , 14 th and 21 st day were compared the difference was not significant.


   Discussion Top


In our study, we use teeth with natural surface curvature, without cutting or grinding the enamel surface flat along with the light fluorescence technology to estimate the changes of fluorescence. The purpose of this in vitro project was to simulate the clinical intraoral situation and investigate the correlation between the fluorescence loss and demineralization depth, so that the absolute lesion depth could be estimated to evaluate the stage of early demineralization.

In the present in vitro study, the samples were subjected to pH cycling in an artificial mouth model were there was a continuous flow of artificial saliva with intermittent flow of 5 pH buffer solution for 15 min thrice, once in the morning, in afternoon and once at night to resemble minimum three time acid attack in a day for a period of 21 days.

In previous in vitro studies carried out by Kumar et al. and Lata et al., the pH cycling was carried out for a period of 10 days and 5 days respectively to evaluate the effect of CPP-ACP on remineralization of artificial caries like lesions. [9],[10] However in our study, the pH cycling was carried out for a period of 21 days with daily once application of the CPP-ACP and CPP-ACFP for the test groups in order to evaluate whether the extended use of CPP-ACP and CPP-ACFP have any additional remineralization effect on the artificial enamel white spot lesions.

In the previous studies carried out by Von der Fehr et al. and Rana et al., the demineralization solution of pH 4.4 was used. [11],[12] The demineralization solution used in these studies contained 2.2 mM CaCl 2, which would by itself have remineralizing effect and would enhance the remineralizing effect of CPP-ACP and CPP-ACFP. Hence, when such pH cycling methods are applied to the evaluation of caries preventive agents, the investigator should realize the limitations of the method. Hence to avoid any bias a 5 pH buffer solution was used. The artificial mouth model used in the present study almost simulated the pH changes occurring in the oral cavity.

In the previous studies carried out by Von der Fehr et al. and Rana et al., the pH cycle involved 3 h of demineralization twice daily and 2 h of remineralization in between which does not completely simulate the pH changes taking place in the oral cavity. [11],[12] Hence, in the present study to simulate the dynamics of pH changes occurring in the oral cavity, 3 times exposure to 5 pH buffer solution for 15 min resembling acid attacks during breakfast, lunch and dinner with continuous exposure to artificial saliva was done.

In the previous studies, [6],[11],[12],[13] the samples were just stored in the stagnant artificial saliva; however in the present study, there was a continuous flow of fresh artificial saliva over the samples thus avoiding the pooling of the saliva simulating salivary flow in the oral cavity.

In a previous study carried out by Pulido et al., CPP-ACP was applied for 3 min and the surfaces were rinsed and the remnants of the products were removed immediately after the treatment application. [13]

In the present study, the CPP-ACP and CPP-ACFP were in contact with the tooth surface and it was washed out by the continuous flowing artificial saliva simulating the washout of the CPP-ACP and CPP-ACFP paste in oral cavity.

For the QLF analysis, three readings were recorded, the highest score point, highest tooth score and lowest tooth score. A change in the fluorescence of the test samples from demineralization to remineralization indicates that a significant amount of remineralization had taken place in these samples.

The highest score point is the point on the AOI with highest QLF reading as identified by the QLF device at the end of demineralization. As this same point was followed-up on 7 th , 14 th and 21 st day, the discussion of the results is based on the statistical analysis of this QLF reading [Figure 5], [Figure 6], [Figure 7], [Figure 8].
Figure 5: Quantitative light fluorescence analysis at the end of demineralization

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Figure 6: Quantitative light fluorescence analysis at the end of 7th day of remineralization

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Figure 7: Quantitative light fluorescence analysis at the end of 14th day of remineralization

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Figure 8: Quantitative light fluorescence analysis at the end of 21st day of remineralization

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When the mean difference in the highest score point reading for control group and the CPP-ACP group from day 1 to day 7 were compared the difference was very highly significant (P < 0.001) suggesting that the control group performed better than the CPP-ACP group for the first 7 days indicating that artificial saliva produced better remineralization than CPP-ACP for the first 7 days.

Reynolds and Walsh, suggested that CPP-ACP molecules need an acid challenge to be activated and it should separate ACP from the casein. In the present study, the samples were subjected to demineralization cycle immediately after the application of the CPP-ACP paste following the suggestions of Reynolds and Walsh. This result of the current study in the first seven days may be because CPP-ACP might have been incorporated into the lesion, but not activated when it was necessary or even washed away in the demineralizing solution. This may be due to a different time between the release of ACP from CPP during the acid challenge and the time of a gradient necessary to deposit calcium and phosphate into the lesion during remineralization. [14] Other possible reason for the result obtained may be due to the short duration of treatment application. Hence, it is necessary to have a longer application time to be able to detect some deposition of calcium and phosphate in a remineralized lesion. Hence, when the mean difference in the highest score point readings for the CPP-ACP group and control group from 7 th day to 14 th day and 7 th day to 21 st day were compared there was a significant reduction indicating significant remineralization produced by the CPP-ACP paste as compared to artificial saliva for the control group.

Thus from the results obtained in the present study, it can be interpreted that though artificial saliva produced better remineralization initially in the first week, the remineralization obtained using CPP-ACP paste gradually increased in the subsequent weeks as evident by the significant difference in the mean difference in the highest score point of CPP-ACP and control group among the day's interval of 7 th -14 th and 7 th -21 st .

However, when the mean difference in the highest score point readings among the day's interval of 14 th -21 st was compared there was no significant difference inferring that the lesions were remineralized by the end of 14 th day following which there was not much influx. Similarly for the CPP-ACFP group, though artificial saliva produced better remineralization initially in the first week, the remineralization obtained using CPP-ACFP paste gradually increased in the subsequent weeks as evident from the significant difference in the mean difference of the highest score point of CPP-ACFP group and control group at the days interval of 7 th -14 th and 7 th -21 st .

However, when the mean difference in the highest score point readings among the day's interval of 14 th -21 st was compared there was no significant difference inferring that the lesions were remineralized by the end of 14 th day following which there was not much influx of calcium, phosphate and fluoride ions into the artificial white spot lesions.

The presence of 0.2% NaF (900 ppm) with CPP-ACP in the CPP-ACFP paste would have co-localized calcium, phosphate and fluoride ions at the tooth surface presumably as CPP-ACP/F nanocomplexes as explained by Cross et al. [15] At the enamel surface, when fluoride ions come into contact with free calcium and phosphate ions, fluorapitite would rapidly form in the surface layer. However in the presence of CPP, which prevents rapid transformation of the calcium phosphate phases, the ions would be stabilized and maintained in the form that would drive diffusion down activity gradient into the subsurface lesion producing higher activity of ions in the subsurface lesion fluid, resulting in higher level of remineralization and fluoride incorporation. Hence, the ability to deliver calcium phosphate and fluoride ions in the correct molar ratio into the subsurface lesion may be attributed to the ability of the CPP to localize and stabilize the ions at the tooth surface in the correct molar ratio (Ca: PO 4 :F = 5:3:1). [16]

When the mean difference in highest score point reading for CPP-ACP group and the CPP-ACFP group among 1 st , 7 th , 14 th and 21 st day were compared the difference was not significant. From these results it can be interpreted that the remineralizing effect of CPP-ACP and CPP-ACFP was almost the same in between the days intervals from day 1 to day 21.

Thus in our study, the remineralization effect of CPP-ACP and CPP-ACFP on the artificial enamel lesions was greater than that observed with artificial saliva. However, when the remineralization effect of CPP-ACP was compared with the remineralization effect of CPP-ACFP, there was no significant difference.

These results are in accordance to the study conducted by (Lata et al., 2010), which concluded that combination of fluoride and CPP-ACP does not provide any additive remineralization potential compared to fluoride alone. [10]

Another in vitro study by Pulido et al. also concluded that there was no significant difference in percentage of reduction in lesion size between artificial saliva and MI paste neither there was any significant difference between the NaF 1100 ppm and combination application of MI paste and NaF 1100 ppm. [12]

The present study was conducted simulating a few intraoral conditions under an in vitro artificial mouth model to demonstrate the remineralization of simulated carious lesions. The replication of the dynamics of the caries process and the complexity of the oral environment in these in vitro models is limited. The remineralization effect of natural saliva and the effect of bacterial assaults in a clinical situation were not determined.

Studies that can equate to the amount of mineral loss or gain quantitatively to change in FC readings, in order to establish appropriate cut-off points to monitor the progress of demineralization and remineralizaition are required. This can make it more convenient to set clinical standards for the use of the FC as a chair side method. Taking into consideration, the limitations of the present in vitro study it can be concluded that:

  • As compared to artificial saliva both CPP-ACP and CPP-ACFP produced significant amount of remineralization of the artificial enamel caries lesion, however when the remineralizing effect of CPP-ACP was compared with the remineralizing effect of CPP-ACFP, there was no significant difference
  • Significant amount of remineralization was produced by CPP-ACP and CPP-ACFP only after the 7 th day. After the 14 th day the remineralization produced by both CPP-ACP and CPP-ACFP as compared with artificial saliva was non-significant.


 
   References Top

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4.Exterkate RA, Damen JJ, ten Cate JM. A single-section model for enamel de- and remineralization studies. 1. The effects of different Ca/P ratios in remineralization solutions. J Dent Res 1993;72:1599-603.  Back to cited text no. 4
    
5.Reynolds EC, Cai F, Cochrane NJ, Shen P, Walker GD, Morgan MV, et al. Fluoride and casein phosphopeptide-amorphous calcium phosphate. J Dent Res 2008;87:344-8.  Back to cited text no. 5
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6.Itthagarun A, Wei SH, Wefel JS. Morphology of initial lesions of enamel treated with different commercial dentifrices using a pH cycling model: Scanning electron microscopy observations. Int Dent J 1999;49:352-60.  Back to cited text no. 6
    
7.Sato Y, Sato T, Niwa M, Aoki H. Precipitation of octacalcium phosphates on artificial enamel in artificial saliva. J Mater Sci Mater Med 2006;17:1173-7.  Back to cited text no. 7
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8.Rodrigues JA, Hug I, Diniz MB, Lussi A. Performance of fluorescence methods, radiographic examination and ICDAS II on occlusal surfaces in vitro. Caries Res 2008;42:297-304.  Back to cited text no. 8
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9.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. 9
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10.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. 10
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11.Von der Fehr FR, Löe H, Theilade E. Experimental caries in man. Caries Res 1970;4:131-48.  Back to cited text no. 11
    
12.Rana R, Itthagarun A, King NM. Effects of dentifrices on artificial caries like lesions: An in vitro pH cycling study. Int Dent J 2007;57:243-8.  Back to cited text no. 12
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13.Pulido MT, Wefel JS, Hernandez MM, Denehy GE, Guzman-Armstrong S, Chalmers JM, et al. The inhibitory effect of MI paste, fluoride and a combination of both on the progression of artificial caries-like lesions in enamel. Oper Dent 2008;33:550-5.  Back to cited text no. 13
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14.Reynolds EC, Walsh LJ. Additional aids to remineralization of tooth structure. In: Mount GJ, Hume WR, editors. Preservation and Restoration of Tooth Structure. Sandgate: Knowledge Books and Software; 2005. p. 111-8.  Back to cited text no. 14
    
15.Cross KJ, Huq NL, Stanton DP, Sum M, Reynolds EC. NMR studies of a novel calcium, phosphate and fluoride delivery vehicle-alpha (S1)-casein (59-79) by stabilized amorphous calcium fluoride phosphate nanocomplexes. Biomaterials 2004;25:5061-9.  Back to cited text no. 15
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16.Cross KJ, Huq NL, Palamara JE, Perich JW, Reynolds EC. Physicochemical characterization of casein phosphopeptide-amorphous calcium phosphate nanocomplexes. J Biol Chem 2005;280:15362-9.  Back to cited text no. 16
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Correspondence Address:
R Mehta
Department of Pedodontics and Preventive Dentistry, JSS Dental College and Hospital, JSS University, Mysore, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.127610

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