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ORIGINAL ARTICLE
Year : 2019  |  Volume : 22  |  Issue : 11  |  Page : 1546-1552

Comparative evaluation of combined remineralization agents on demineralized tooth surface


1 Department of Pediatric Dentistry, Faculty of Dentistry, Harran University, Sanliurfa, Turkey
2 Department of Pediatric Dentistry, Faculty of Dentistry, Dicle University, Diyarbakir, Turkey

Date of Submission04-Apr-2019
Date of Acceptance24-May-2019
Date of Web Publication13-Nov-2019

Correspondence Address:
Dr. A Aras
Department of Pediatric Dentistry, Faculty of Dentistry, Harran University, Sanliurfa - 63300
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njcp.njcp_188_19

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   Abstract 


Objectives: The aim of this in vitro study was to evaluate the effects of casein phosphopeptides (CPP)-ACPF, NovaMin+ fluoride-containing toothpaste and Xylitol+ fluoride containing cream on demineralized areas on the enamel surface. Materials and Methods: A total of 100 enamel slab samples was prepared to investigate in the laboratory experiments. For this purpose, a total of 50 freshly extracted third molar teeth which completed root formation split into two portions in the mesiodistal direction. Enamel surfaces were immersed in a pH cycling protocol as described in the literature to simulate oral conditions for 9 days in order to evaluate the effect of test materials on the artificial enamel lesions. Then the remineralization agents were applied on the enamel surfaces, and we analyzed their effects. Results: We used Vickers Microhardness with the purpose of calculating the amount of lost or acquisition of minerals on the enamel surface qualitatively; inductively coupled plasma atomic emission spectroscopy (ICP-AES) to define the calcium and phosphorus ions that dissolved in acid. One-way ANOVA and Tukey's T Post-Hoc tests were performed to distinguish significant differences among groups (P < 0.05). Conclusions: Remineralization was provided in all treated groups, according to the data obtained from all tests. NovaMin was more effective in increasing acid resistance. It was also found that all three experimental groups were effective in increasing the surface hardness, but CPP-ACPF and NovaMin are more effective than Xylitol. However, there was no statistically significant difference between the experimental groups.

Keywords: Casein phosphopeptide, demineralization, fluoride, NovaMin, remineralization, xylitol


How to cite this article:
Aras A, Celenk S, Dogan M S, Bardakci E. Comparative evaluation of combined remineralization agents on demineralized tooth surface. Niger J Clin Pract 2019;22:1546-52

How to cite this URL:
Aras A, Celenk S, Dogan M S, Bardakci E. Comparative evaluation of combined remineralization agents on demineralized tooth surface. Niger J Clin Pract [serial online] 2019 [cited 2022 Jan 25];22:1546-52. Available from: https://www.njcponline.com/text.asp?2019/22/11/1546/270854




   Introduction Top


Today there is no standard protective program to prevent dental caries. In addition to some traditional methods such as regular dentist control, brushing teeth with fluoride toothpaste, topical fluoride applications and regulation of diet, in some occasions more effective applications are needed.[1]

The leading classic prophylaxis application is brushing teeth with fluoride toothpaste and cleaning all the tooth interfaces with dental floss. However, an examination of prophylaxis programs shows that, in addition to the previous applications, using several types of mouthwash and chewing gums whose caries preventing effectiveness are proved with studies are recommended to individuals at an increasing rate. As a result, caries observation frequency has declined today and changed in terms of type and progress rate.[2],[3]

In addition to fluoride-based materials, such materials as calcium, phosphate, casein phosphopeptide, casein glycopeptide, chlorhexidine, xylitol, and ozone, all of which have antimicrobial additives, are being effective in remineralization.[4],[5],[6]

Phosphopeptides, which are obtained from milk casein, are used as a contemporary remineralization technology.[7] These casein phosphopeptides (CPP) which include multiphosphoryl stabilize calcium phosphate in a nanocomplex structure in amorph calcium phosphate (ACP) solution. As a result of this multiphosphoryl structure, CPP is tied to ACP in stable solution and growth in the molecular structure is prevented as well as calcium-phosphate formation in low crystal form. These soluble calcium-phosphate ions create a form which is resistant against acid attacks and accelerate remineralization. Also, it is reported that CPP-ACP and flour combination (ACFP) create a synergic effect in preventing caries.[8],[9]

Bioactive glasses produced in the form of toothpaste recently in the treatment of dentin sensitivity and enamel remineralization were first developed as bone regeneration material.[10] These materials interact with body heat and ensure hydroxycarbonate apatite accumulation. NovaMin which has calcium sodium phosphosilicate (CSPS) as an active content is similar to natural dental minerals in chemical terms. It has been observed that therapeutic doses of fluoride increased remineralization in caries lesions when combined with NovaMin.[11],[12]

Another caries preventive method is the consumption of food with sweetening content; xylitol is the most frequently used sugar variable in this area. As regards oral and dental health, it is essential to use this sugar alcohol which minimizes dental caries risk in candies, beverages, and gums to replace sucrose.[5],[13] Xylitol which cannot be fermented by S. mutans shows antiacidogenic and anticariogenic features. Another role of xylitol in oral health is that it reduces the amount of plaque accumulation in the mouth. It shows a synergic impact with fluoride and increases the effectiveness of oral hygiene products.[14]

In our study, the purpose is to evaluate the effectiveness of toothpaste with NovaMin and fluoride content, CPP-ACFP content cream and Xylitol-HA-fluoride content cream on artificially prepared incipient enamel lesions by using ınductively coupled plasma-atomic emission spectrometry (ICP-AES) and Vicker's Microhardness analysis methods.


   Materials and Methods Top


In this study, 50 extracted embedded human wisdom teeth were used in order to obtain standard affected enamel surfaces. The roots of the teeth were separated using ISOMET (Buehler, USA) water-cooled microtome device, double-sided cutting thin diamond blade; then, crown parts were divided into two in occlusocervical direction and mesiodistal direction. In the buccal and lingual surfaces of the obtained 100 samples, 3 × 3 mm tags were stuck to prepare 3 × 3 mm windows on 1-2 mm upper side of the enamel-cement border and the natural tooth surfaces which are not covered by the tag were covered with double-layer durable nail polish. Finally, enamel samples were separated into five random groups.

Groups:

Group 1: Positive (+) control group, the group which was exposed only to the demineralization cycle.

Group 2: Negative (−) control group, the sound tooth group which was not exposed to any application.

Group 3: The group which was exposed to CPP-ACFP cream form; in our study MI Paste Plus (GC, Japan) containing 0.2% NaF (900 ppm) in addition to 10% CPP-ACP was used as CPP-ACFP.

Group 4: The group which was exposed to NovaMin-fluoride content toothpaste; in our study, Sensodyne repair and protection toothpaste (GlaxoSmithKline, UK) was preferred as NovaMin-fluoride content toothpaste.

Group 5: The group which was exposed to Ksilitol-HA-fluoride content cream; in our study, ReminPro (Voco, DE) was preferred as Ksilitol-HA-fluoride content cream.

Formation of artificial incipient enamel caries on framed enamel surfaces of the samples

With the exception of Group 2, deremineralization cycle (pH cycle) was applied to the other four groups which are similar to the clinical conditions and incipient enamel caries were formed. pH cycle model imitates these phases and is used in the evaluation of materials which play an essential role in the caries mechanism under laboratory conditions. This model changes in periods of 5, 9, or 14 days. pH cycle used in our study was applied throughout 9 days in the form of 24-h periods.

Preparation of demineralization and remineralization cycle

  1. First, samples were left in 20 ml glass flasks in demineralization solution arranged with 4.3 pH which included 1,5 mM CaCl2, 0,9 mM KH2 PO4, 50 mM acetic acid at 37.5° C.
  2. Then these samples were extracted from this solution and washed with distilled water.
  3. Samples were later left in 20 ml glass flasks in remineralization solution with 7.0 pH value which consisted of 1.5 mM CaCl2, 5 mM KH2 PO4, 100 mM acetic acid for the next 17.5 h at 37.5°C.
  4. Then, the samples were taken out of this solution and washed with distilled water and placed again in demineralization water. This pH cycle process continued from 9 days. These solutions were changed in every 3 days, and the formation of any saturation was prevented.


Application of treatments after the formation of artificial incipient caries on the framed enamel surfaces of samples

After the samples were separated into groups and artificial incipient enamel caries were formed, no remineralization agent was applied to the groups 1 and 2. In the CPP-ACFP cream application to group 3, manufacturer company instructions were followed and the teeth were slightly dried; then, the cream was applied for 3 min. In group 4, toothpaste with NovaMin-fluoride content was applied for 2 min considering the average rushing period. In group 5, Ksilitol-HA-fluoride content cream was applied for 3 min after slightly drying the teeth surfaces as per manufacturer company instructions.

Then, all samples were washed in distilled water and, in order to imitate the clinical conditions in 20 ml glass flask, they were left in artificial saliva for 30 min at 37°C. Artificial saliva solution was prepared using %(wt) 0.08NaCl, %(wt) 0.12 KCl, %(wt) 0.01 MgCl2.6H2O, %(wt) 0.03 KH2 PO4, %(wt) 0,01 CaCl2.2H2O, %(wt) 0.10 CMC-Na, %(wt) 99.6 deionized water in 1 l of solution. The pH of the solution was adjusted at 7.0 with NaOH.

Examination of enamel surfaces

With the purpose of determining the demineralization and remineralization in the enamel, two different tests were applied. Vicker's microhardness test was used in order to determine the changes in surface hardness depending on mineral loss or gain created on the enamel surface by the applied treatments. ICP-AES was preferred in order to identify the amount of calcium (Ca) and phosphorus (P) ions solved from hard tissues treated with acid.

Examination of samples with ICP-AES

The enamels inside the buccal or lingual frame of 50 teeth samples in total were preserved; all the remaining parts were removed using a diamond separator. Then, the weights of the obtained enamel samples were measured using a precision scale. Each sample was shaken for 30 min using a mini mixer inside 20 ml glass flasks containing 2.5 ml of 0.5 M perchloric acid (pH = 0.53). 10 ml distilled water was added on the solution and the total volume was completed to 12.5 ml.

Once the samples were prepared, they were taken to Selcuk University Faculty of Agriculture ICP-AES laboratory (Konya, Turkey). In the meantime, the enamel which remained without being solved in the solution was taken to oven for drying. After measuring its final dry weight, the last dry weight was subtracted from the first dry weight, and the number of materials solved from enamel tissue was found in mg, whereas the dilution coefficient of the values taken from ICP-AES was calculated and found in mg/1 (ppm).

Performing the micro-hardness measurements of samples

In our study, the microhardness analysis was conducted in Afyon Kocatepe University Technology Application and Research Centre. First, in order to ensure parallelism of framed enamel surfaces in the samples to the ground, the samples were placed in acrylic blocks and fixed. Then, the microhardness analysis was conducted using Buehler® Micromet 5114 (Lake Bluff, Illinois, USA) microhardness analysis device which had one Vicker's diamond indentor.

Fifty tooth samples in total which were placed to the plate in microhardness measurement device so that their lower and upper surfaces would be parallel to each other facing upwards. The diagonal length of the groove created by the end of Vicker's diamond indentor was identified using the measurement system in the microscope placed on the device. Vicker's hardness value was automatically calculated using the calculation device on the instrument. Measurements were performed at four different points applying 200 g force for 5 s. The means of microhardness values were taken.

Statistical analyses

Groups were compared using one-way ANOVA at a significance level 0.05, followed by a Tukey's T post-hoc test.


   Results Top


Evaluation findings with Icp-Aes method

The mean solved calcium amounts obtained as a result of the statistical evaluation revealed that the lowest mean values were witnessed in Group 2 followed by Group 4, Group 3, and Group 5. The highest calcium loss was seen in Group 1. The statistical analysis conducted found the only significant difference between Group 4 and Group 1 [Table 1]. No statistically significant difference was found between other groups (P > 0.05). [Table 2] provides the mean solved calcium amounts and standard deviation values according to groups.
Table 1: Pairwise comparison of all groups whose calcium findings are evaluated with ICP-AES method

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Table 2: Mean solved Ca ratios measured with ICP-AES method

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An examination of the solved phosphor amounts showed similar results. According to statistical analysis, the lowest mean phosphor loss was witnessed in Group 2 which was followed by Group 4, Group 3, and Group 5. The highest phosphor loss was seen in Group 1. Pairwise comparisons showed that there was a significant difference between Group 4 and Group 1, which were experiment groups, and Group 3 and Group 5 with Group 2 [Table 3] (P > 0,05). [Table 4] gives the solved average phosphor amounts and standard deviation scores per groups.
Table 3: Pairwise comparisons of all groups whose phosphor findings are evaluated with ICP-AES method

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Table 4: Mean solved P ratios measured with ICP-AES method

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Evaluation findings with Vicker's Microhardness analysis

An examination of mean hardness values of experiment and control groups shows that the highest hardness was determined in Group 2 where no process was applied. This group was followed by Group 3, Group 4, and Group 5. The lowest hardness value was found in Group 1 which was subjected to pH cycle and demineralized and for which no treatment was applied [Table 5].
Table 5: Mean micro-hardness values of groups

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As a result of pairwise comparisons, a significant difference was found between Group 1 and other experiment groups (Group 3, 4, 5) (P < 0.05). A significant difference was found between Group 5 and both Group 1 and Group 2 (P < 0.05), whereas no significant difference was found between Groups 3, 4, and 5 (P > 0.05) [Table 6].
Table 6: Pairwise comparisons of all groups whose findings are evaluated with micro-hardness method

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   Discussion Top


Active enamel decay lesions at an incipient status which do not require a restorative treatment are exposed to the oral environment, and affected by acids is still one of the frequently witnessed controversial topics in the literature in terms of both prognosis and treatment. During the last four decades, remineralization of incipient enamel lesions has been the topic of several studies.[15],[16]

In this study, we applied such remineralization agents as CPP-ACFP, NovaMin-fluoride, and Xylitol-HA-fluoride on artificially prepared incipient enamel lesions and aimed at evaluating their effectiveness using ICP-AES and microhardness analysis.

Deremineralization phase in the oral environment occurs together; as a result, clinical conditions cannot be completely ensured with the only demineralization. Featherstone et al. developed a pH cycle model for this kind of studies which covered a 9-day deremineralization period.[17] Particularly in areas limited to enamel, pH cycle model has been preferred in several scientific studies to create artificial caries which displayed incipient enamel caries features called subsurface lesion.[18]

Although the literature indicates a few different pH-cycle techniques, the changing parameters are the time intervals during when the samples are subjected to demineralization or remineralization, undersaturation or oversaturation level with fluoro-apatite or other phases and whether or not human saliva is used.[19]

Studies conducted using pH cycle model are used in different studies by being modified in periods of 5, 9, or 14 days.[17],[20],[21],[22] In our study, considering the previous researches, a 9-day remineralization period was preferred. Experiment samples prepared for pH cycle were left in demineralization solution for 6 h and remineralization solution for 17.5 h. The 6 h demineralization period was used to imitate the average acid attack period after eating. The 17.5 h remineralization period was used to imitate the buffering period of saliva. The content of deremineralization solutions that we used is identical to the solutions used by Gilgrass et al.[23] These solutions were preferred in our study as they imitated deremineralization cycles in the oral environment in a similar form and due to their simple and clear content.

Studies conducted on deremineralization employed different test methods in order to determine the changes on the dental surface. Surface microhardness test is one of these tests which is reported as a simple, reliable, nondestructive and fast method which facilitates obtaining information on the mineral changes on dental hard tissues.[24] Featherstone et al.[17] conducted a study and identified significant relation between the microhardness values of enamel and the mineral ratio in caries lesions and reported that microhardness measurement was sensitive enough to identify early demineralization of enamel. For this reason, in our study microhardness test was preferred in order to be able to measure the change in hardness which occurs on enamel surface after deremineralization.

ICP-AES is another popular method used in studies aiming remineralization of incipient enamel caries. This method is used to examine the calcium, phosphor, and other minor elements which are separated from the tissue under examination at ppm level. Obtained data allow us to provide interpretations on the resistance of enamel surface against acid.[25] For this reason, in the study we conducted, the ICP-AES method was preferred to measure the number of elements solved when treated with acid.

Fluoride application is one of the most effective methods in the prevention and control of dental caries.[26] Fluoride agents are mostly compared with CPP-ACP which has been subject to several studies until today which employed solution, sugarless gum, mouthwash, lozenge, and paste forms and examined the prevention of deremineralization of incipient caries.[27],[28],[29],[30] Some of the studies which used fluoride and CPP-ACP tested how the combined usage of both agents would react, and it was found out that such combined usage created a synergic effect.[28],[31] In their study, El Sayad et al.[9] reported that the combined usage of CPP-ACP with fluoride had a synergetic effect.

Srinivasan et al.[32] left 45 enamel samples in coke for 8 min and created erosion effect; then they placed them into the mouths of five volunteers using intra-oral apparatuses. Following deremineralization cycle, the teeth were subjected to Viker's microhardness test. At the end of the study, they found out that 46.24% increase was observed in the group which used CPP-ACP, whereas 64.25% increase was witnessed in the group which used CPP-ACP + 900 ppm fluoride (Tooth mousse Plus). In our study, it has been identified that CPP-ACPF which had the highest mean surface hardness among test groups provided remineralization and that it increased surface hardness at a statistically significant level compared with control groups. However, its impact on increasing resistance towards acid was not found to be statistically significant.

Another agent which is found to have a remineralizing effect by being added to the content of toothpaste is NovaMin. In the study conducted by Golpayageni et al.[33] on NovaMin, 40 extracted solid teeth were subjected to pH cycle first. The teeth were separated into two groups and applied with NovaMin and 1.1% neutral fluoride gel; then, their microhardness levels were compared. It was found out that the toothpaste with NovaMin content had more advanced caries preventing features. Also, it was reported that NovaMin increased surface microhardness significantly and that combined use of these two pastes could make a synergetic effect in terms of remineralization. Neto et al.[34] conducted a study which compared a toothpaste with 1,100 ppm F, Novamin content, CCP-ACP, and a cream consisting of CPP-ACP+900 ppm F content with a microhardness method and found out that Novamin, 1,100 ppm F, CCP-ACFP showed the least mineral loss.

Wang et al.[35] performed a study on dental deremineralization and identified that kinds of toothpaste with NovaMin content, GC Tooth mousse and MI Paste Plus did not show any statistically significant difference in terms of remineralization. One of the different results seen in the conducted studies is that NovaMin is an agent whose remineralization effect is revealed with several studies in general.[12],[33],[34],[36] We believe that diversity in application methods causes these differences. In our study, NovaMin appeared as the most resistant group against acid according to Ca and P amounts, and it increased surface hardness and acid resistance at a statistically significant level. However, no statistically significant difference has been found between NovaMin and other experimental groups.

One study on xylitol, another famous agent used in the remineralization of incipient enamel caries, Miake et al.[37] reported that xylitol (facilitating the movement and passage of Ca ions) induced the remineralization of the demineralized enamel in deeper layers. Another study revealed that when xylitol was used with fluoride, it inhibited demineralization and accelerated remineralization.[38]

The study conducted by Sano et al.[39] mixed three kinds of toothpaste with different contents (500 ppm F, 500 ppmF+xylitol, silica-based 500 ppm F content); the researchers concluded that the toothpaste with 500 ppm F and 5% xylitol content was significantly superior compared with the others. As a result, it was reported that adding xylitol to fluoride toothpaste could improve remineralization.

The inductive effects of xylitol and fluoride on remineralization are combined in a single product and launched to the market under the name ReminPro. Kamath et al.[40] conducted a study on this new agent and found out that Mc Innes solution reduced hardness value, whereas using ReminPro increased hardness value. In another study, 37% carbamide peroxide whitening agent was applied on 30 dental surfaces; then the teeth were subjected to CPP-ACFP (MI Paste Plus), ReminPro. The measurements showed that MI Paste Plus and ReminPro reduced roughness but that there was no statistically significant difference between them.[41]

In our study, it was found out that the xylitol group had the lowest mean surface roughness among experiment groups and that it did not lead to a statistically significant increase in improving acid resistance.


   Conclusions Top


In accordance with the data we obtained in our study:

  1. It has been observed that the 9-days pH cycle model used in the study was an effective and sufficient method in creating artificial incipient caries.
  2. It has been seen that ICP-AES and Vicker's microhardness tests are successful and easily-applicable tests in remineralization studies.
  3. According to the data obtained from all the tests used, it was found out that remineralization could be achieved in all experimental groups.
  4. It was found out that NovaMin was more effective compared with other groups in increasing acid resistance.
  5. It was also found out that in terms of increasing surface hardness, all three experiment groups were effective. However, CPP-ACPF and NovaMin showed more effectiveness.


We believe that it would be beneficial to conduct further studies to see whether these materials have any negative effect on other tissues and organs of the body.

Ethical approval

All procedures performed were in accordance with the ethical standards of the institutional and national research committee. The study protocol was submitted to and approved by the local institutional review board of the University of Dicle, Diyarbakır, Turkey (approval number 2013-1:7).

Financial support and sponsorship

This study has been funded by departmental funds only. No external funding has been received.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

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


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