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 Table of Contents  
ORIGINAL RESEARCH
Year : 2021  |  Volume : 13  |  Issue : 4  |  Page : 336-343

Effect of CPP-ACP pastes with/without fluoride on white spot lesion progression, salivary pH, and fluoride release in high caries risk patients: A randomized clinical trial


Conservative Dentistry Department, Faculty of Dentistry, Cairo University, Cairo, Egypt

Date of Submission26-Feb-2021
Date of Decision10-Apr-2021
Date of Acceptance27-Apr-2021
Date of Web Publication19-Aug-2021

Correspondence Address:
Dr. Doaa Gamal Ashour
263 8th District, El Sheikh Zayed, Giza.
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JIOH.JIOH_43_21

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  Abstract 

Aims: The aim of this article was to evaluate the efficacy of casein phosphopeptide amorphous calcium phosphate (CPP-ACP) with and without fluoride on white spot lesion (WSL) progression, salivary pH, and fluoride ion release compared with fluoridated toothpaste on high caries risk patients. Materials and Methods: A total number of 51 high caries risk participants with occlusal WSLs determined by VistaCam were randomly assessed into three equal groups (n = 17): group 1: CPP-ACP + Fluoridated toothpaste; group 2: CPP-ACFP + Fluoridated toothpaste; group 3: Fluoridated toothpaste only. WSL progression was assessed at baseline, 1 month, and 6 months. The pH values and fluoride ion release assessments were done at baseline, immediately after application, 1 day, 1 week, and 1 month. The pre-treatment and the post-intervention VistaCam scores, salivary pH, and fluoride ion release were tabulated and subjected to statistical analysis. Results: No statistically significant difference in VistaCam scores was found between the tested periods with Tooth Mousse, MI Paste Plus, and Fluoridated tooth paste. There was no statistically significant difference in salivary pH between different evaluation times with Tooth Mousse and control group. MI Paste Plus showed a statistically significant increase in pH value. Regarding fluoride ion release, there was a statistically significant difference between different evaluation periods with MI Paste Plus only. Also, intergroup comparison showed a statistically significant difference in each assessment period. Conclusion: In high caries risk patients, CPP-ACP-containing pastes have a slight remineralization effect like fluoridated toothpaste. The application of CPP-ACFP was effective in raising both salivary pH and fluoride ion release in saliva.

Keywords: CPP-ACP, Fluoride, pH, VistaCam, White Spot Lesion


How to cite this article:
Ashour DG, Farid MR, Mosallam RS, Abouauf EA. Effect of CPP-ACP pastes with/without fluoride on white spot lesion progression, salivary pH, and fluoride release in high caries risk patients: A randomized clinical trial. J Int Oral Health 2021;13:336-43

How to cite this URL:
Ashour DG, Farid MR, Mosallam RS, Abouauf EA. Effect of CPP-ACP pastes with/without fluoride on white spot lesion progression, salivary pH, and fluoride release in high caries risk patients: A randomized clinical trial. J Int Oral Health [serial online] 2021 [cited 2022 Jan 29];13:336-43. Available from: https://www.jioh.org/text.asp?2021/13/4/336/324145




  Introduction Top


Early enamel demineralization is noticed as white spot lesions (WSLs) which are opaque enamel areas produced by mineral loss from the subsurface layer of enamel and are capable of being reversible.[1] The use of fluoride toothpaste combined with other forms of topical fluoride treatment augments in the reduction in dental caries, compared with using fluoride toothpaste alone.[2],[3] However, an ideal remineralization system should include not only fluoride but also bioavailable calcium and phosphate to produce a more comprehensive sub-superficial remineralization.[4]

Casein phosphopeptide amorphous calcium phosphate (CPP-ACP) can stabilize calcium and phosphate in the amorphous state, increasing their levels in saliva.[5] CPP-ACP with 900 ppm sodium fluoride binds to the tooth surface and plaque which provides biocompatible calcium, phosphate, and fluoride needed to create fluorapatite crystals.[6] Most of in-vivo models, until then, were done for smooth surfaces after debonding of orthodontic brackets. Occlusal caries still remains a major challenge for research.

WSLs can be spotted by different methods; visual examination is a subjective method and has a low reproducibility in detecting occlusal caries.[7] Recently, a new florescence camera (VistaCam) has been developed. It is a fluorescence caries detection device that operates by illuminating a tooth with fluorescence light and then analyzes the emitted fluorescence light.[8]

Salivary pH is a quick chairside biochemical marker that provides useful diagnostic information. In addition, salivary molecules are also helpful in evaluating risk of dental caries. Thus, the purpose of this study was to evaluate and compare the effect of CPP-ACP on caries progression in high caries risk patients. On the basis of that, the null hypothesis tested was that the use of CPP-ACP with or without fluoride, in addition to fluoride toothpaste, does not have effect on remineralization of WSL, salivary pH, and fluoride ion release.


  Materials and Methods Top


Study setting

This randomized controlled clinical study was conducted in the Faculty of Dentistry, Cairo University, Egypt. The protocol of this study was registered in www.clinicaltrials.gov/ database, with unique identification number NCT03656432. All the procedures done in this study involve human participants, agreed with the ethical standards of the Research Ethics Committee (CREC) of Faculty of Dentistry, Cairo University with approval number 18-9-38.

Sample size calculation

Sample size calculation was done using G power 3.1 Software, University of Kiel, Germany. We calculated the pooled (within group) standard deviation which was 2.441 and the effect size was 0.504 fluorescence calibrating numerical value or unit (FCU). So, 42 subjects were divided into three equal groups to be able to reject the null hypothesis in which the means of the three studied groups are equal with power of 0.8 and type I error 38 probability of 0.05. To compensate for drops in the follow-up period, three subjects were added to each group resulting in a total sample size of 51 subjects.

Materials

Two types of remineralizing pastes were used in this study and were compared with fluoride toothpaste in [Table 1]:
Table 1: Material’s description, composition, lot number, and manufacturer

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  1. CPP-ACP (GC Tooth Mousse®);


  2. CPP-amorphous calcium phosphate with fluoride (ACFP) (GC MI Paste Plus®);


  3. Fluoridated toothpaste (Signal toothpaste®).


Armamentarium used in the study is as follows:

  1. VistaCam iX: light-induced fluorescence intraoral camera (LF) (Vista-Cam iX, DürrDental, Bietigheim-Bissingen, Germany);


  2. Digital pH meter: A calibrated digital pH meter (Adwa Hungary);


  3. Selective ion electrode for fluoride ion measurements.


Recruitment, study design, and grouping

Patients were recruited in the study that fulfill the eligibility criteria; the inclusion and exclusion criteria were given in [Table 2]. Screening was continued until the target population (51 participants) was achieved with high caries risk using the ADA caries risk assessment form. This study was a 3-month, double-blinded randomized three parallel arms clinical trial with 1:1 allocation ratio. Participants were randomly assigned into three groups (n = 17) according to the tested paste, in which A1group represents participants who were given CPP-ACP paste and A2 group represents participants who were given CPP-ACFP paste. Groups (A1) and (A2) were instructed to brush their teeth with standard fluoridated toothpaste, whereas (A3) represented participants given fluoridated toothpaste only. WSL progression, pH alteration, and fluoride release were evaluated at different assessment times: (T1) baseline before application, (T2) immediate after application, (T3) 1 day, (T4) 1 week, and (T5) 1 month. However, (T6) was supposed to be measured at 3 months but it was measured at 6 months due to the exceptional circumstances and the lockdown to prevent the spread of Covid-19 virus.
Table 2: Inclusion and exclusion criteria of teeth

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Randomization, sequence generation, and blinding

Simple randomization was done including the number of participants divided into three groups marked with letters A, B, and C. Randomization was generated using www.randomization.com. The subject and data analysts were blinded to the group assigned. The paste code was retained and given only after the analysis of the results. Participant were blinded to the group to which they have been assigned and received an unmarked tube corresponding to their allocated group.

Paste and regimen

First, the participants received a 3-month supply of the tested pastes at the start of the study and then they received additional 3 months supply as the research period was extended up to 6 months due to lockdown to prevent the spread of Covid-19. They were instructed to refine from any additional preventive protocols containing fluoride during the study period. Self-administration in groups A and B was standardized by specific stipulation to each participant. They were instructed to locally apply a standardized amount (pea-sized quantity) of the paste to the teeth twice daily after 3 min from brushing their teeth and for a period of 6 months. They were instructed to brush their teeth with pea-sized quantity of fluoridated toothpaste (Signal, 1450 ppm fluoride) twice per day in the morning and evening. The control group was instructed to use the fluoridated toothpaste only twice daily.

Outcome assessment

WSLs detection (primary outcome)

Following recruitment, baseline characteristics were assessed using a fluorescence-based camera following manufacturer’s instructions. A light-induced fluorescence intraoral camera (VistaCam iX) was placed on the tooth which was previously cleaned with a prophylactic paste (UltraproTM Tx Prophy Paste, Ultradent Company) and rinsed with water spray for 5 s and dried. The protective cover for the head was placed over the occlusal surface of the tooth perpendicularly and in contact with the enamel surface. Digital photographs in [Figure 1] of the occlusal surfaces were captured and analyzed by software which quantifies both the red and green components of fluorescence light into the caries scores from 0 to 3 in [Figure 2]. The same site is re-evaluated after 1 and 6 months after starting the treatment.
Figure 1: A. Tooth image capture. B. Image taken by VistaCam for initial caries lesions

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Figure 2: VistaCam scores and color scales

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Evaluation of salivary pH (secondary outcome)

Unstimulated saliva sample was done in the morning from 10 to 11 am. The participants were asked to refine from drinking or eating or any oral hygiene measures for at least 1 h before saliva collection. Saliva in the mouth was collected over a period of 2 min in a pre-labeled Falcon centrifuge tube. Then the pH was measured immediately to avoid any pH time-based changes due to CO2 loss using a handheld electronic pH meter.

Determination of fluoride ion release (tertiary outcome)

Stimulated saliva samples were collected for fluoride ion measurement. The patient was instructed to chew 1-g piece of unflavored paraffin wax for 3 min until 5 mL of saliva was collected.

Data collection methods

All data were entered electronically, and patient files were stored in numerical order and kept separately in sheet coded with the patient’s serial number for contact. The personal or outcome data were stored in locked cabinet and a soft copy was recorded at the Conservative Department.

Statistical analysis

Statistical analysis was performed using IBM SPSS Statistics Version 2.0 for Windows. Data were presented as mean, standard deviation (SD), frequencies (n), and percentages. To assess data normality of continuous data, the Kolmogorov–Smirnov and Shapiro–Wilk tests were used. The significance level was set at P ≤ 0.05. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed to compare salivary pH after using different pastes at each evaluation time. The Kruskal–Wallis test, followed by the Mann–Whitney test, was conducted to compare VistaCam readings and fluoride release of different pastes at each evaluation time. Repeated-measures ANOVA was used for comparisons of salivary pH between different evaluation times within each paste. The Friedman test followed by Wilcoxon’s signed-rank test was used for comparisons of VistaCam readings and fluoride release between different evaluation times within each paste.


  Results Top


Effect of tested pastes on WSL progression with respect to VistaCam scores

Regarding intragroup comparison, there was no statistically significant difference in VistaCam scores between different periods with Tooth Mousse (P = 0.529), MI Paste Plus (P = 0.193), and control group (P = 0.959). The highest VistaCam scores were recorded at baseline, while after 1 and 6 months there was a decrease in VistaCam scores, respectively. Regarding intergroup comparison, the Kruskal–Wallis test followed by the Mann–Whitney test revealed that there was a statistically significant difference in VistaCam scores between the tested pastes at 1 month (P = 0.006). MI Paste Plus group showed the highest significant mean scores, followed by Tooth Mousse whereas the control group showed the least significant readings. There was no statistically significant difference in VistaCam scores between different pastes at both baseline (P = 0.158) and 3 months (P = 0.221). Results are presented in [Table 3].
Table 3: Mean ± SD and P-value for the effect of pastes on WSL progression at different evaluation periods

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Effect of tested paste on salivary pH

Regarding intragroup comparison, there was no statistically significant difference in salivary pH between different evaluation times within Tooth Mousse group (P = 0.981) and control group (P = 0.971). There was a statistically significant difference in salivary pH in the MI Paste Plus group (P = 0.023). The significantly highest salivary pH was recorded at 1 month. Regarding intergroup comparison, one-way ANOVA followed by Tukey’s post hoc test showed that there was no statistically significant difference in salivary pH between different pastes at each assessment time (P = 0.928 at baseline, P = 0.767 immediately after application, P = 0.573 after 1 day, P = 0.201 after 1 week, and P = 0.074 after 1 month). Results are presented in [Table 4].
Table 4: Mean ± SD and P-value for the effect of pastes on salivary pH at different evaluation periods

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Effect of tested pastes on fluoride release

Regarding intragroup comparison, there was no statistically significant difference in fluoride ion release between different assessment times within Tooth Mousse (P = 0.721) and control group (P = 0.223). There was a statistically significant difference in fluoride ion release between different assessment times within MI Paste Plus (P = 0.023). The significantly highest mean fluoride release was recorded at 1 month of application of MI Paste Plus. Regarding intergroup comparison, the Kruskal–Wallis test followed by the Mann–Whitney showed that there was no statistically significant difference in fluoride ion release between different pastes at baseline (P = 0.115). There was a statistically significant difference in fluoride ion release between different pastes immediately after application (P = 0.018), after 1 day (P = 0.013), 1 week (P < 0.001), and 1 month (P < 0.001). At each assessment time, the MI Paste Plus group showed the significantly highest fluoride release when compared with that of Tooth Mousse and control groups, which were statistically similar. Results are presented in [Table 5].
Table 5: Mean ± SD and P-value for the effect of pastes on fluoride release at different evaluation periods

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  Discussion Top


Recently, dairy products have received attention for their anticariogenic effect in many studies. CPP-ACP products were selected in this study over fluoride products as they do not cause fluorosis of enamel and are ingestible in contrast to the topical fluoride products that may pose a risk if significant amount of fluoride was ingested.[9]

Also, they have the potential in enhancing remineralization. It ensures increased availability of calcium and phosphate in saliva and plaque at a supersaturated status and in close proximity to enamel lesion. The presence of CPP would inherently prevent rapid precipitation of calcium and phosphate on the superficial zones of the carious lesion, thus enhancing the remineralization process to occur inside the body of the lesion.[10] Interestingly, CPP-ACP products are also present containing fluoride such as CPP-ACFP in order to deliver fluoride to ensure efficient remineralization through increasing the degree of saturation of phosphate, calcium, and fluoride ions localized at the surface of tooth.[11] On the other side, the use of fluoride-containing toothpaste with all groups due to ethical aspect as the best clinical practice did not consider to totally exclude the use of regular fluoride toothpaste in the intervention groups and particularly in patients at high risk of caries.[12],[13]

Regarding the salivary pH values, unstimulated saliva was collected and pH measured using a portable pH meter due to its accuracy and convenience.[14],[15] Unstimulated saliva has lower pH values compared with stimulated saliva. The normal pH value of saliva is in the range of 6–7, which makes it slightly acidic; however when stimulated, it increases to about 7.8 due to higher concentration of both bicarbonate and phosphate concentration. In addition, unstimulated saliva is stable in pH compared with stimulated saliva which is sensitive to various stimuli such as diet, exercise, and innate metabolic difference.[16],[17] However, stimulated salivary samples were collected for fluoride level measurements, to ensure complete immersion of the electrodes for accurate readings of fluoride ion released into the saliva. Ion-selective electrode was used for rapid evaluation of the basic parameters such as concentration of different electrolytes.[18]

Regarding assessment methods, fluorescence-induced technology was selected in monitoring the progression of caries lesions. A fluorescence camera (VistaCam) was used in this study to detect WSLs with high reproducibility and good performance at various stages of the disease process.[1] In addition, this camera enables two-dimensional imaging of the tooth surface together with quantitative identification of caries score, which is impossible with conventional tools.[19] In addition, using blue light is more effective than the red light of the DIAGNOdent in detecting porphyrin, which is a product of bacterial metabolism.[20] WSLs indicate values in the range of 1–1.5. Therefore, a reduction in values represented is correlated with the increase in the tooth mineral content.[19]

For in any clinical trial, compliance is a crucial issue. In this study, seven participants dropped out at the last follow-up period which was not greater than planned. However, the final study participant numbers were actually more than planned as 17 participants were enrolled from the beginning. Regarding the results of WSL progression, there was a slight decrease in VistaCam scores after application of the tested pastes compared with baseline scores due to the formation of new hydroxyapatite crystals from the minerals existing in the paste by physical diffusion of ions to the destroyed hydroxyapatite.[21]

The slower and attenuated remineralization pattern observed with Tooth Mousse and MI Paste Plus may be explained by the presence of CPP in these used products, which will prevent precipitation of calcium and phosphate rapidly. In addition, the capability to deliver calcium, phosphate, and fluoride ions into the subsurface lesion in the correct molar ratio may be attributed to the capability of CPP to stabilize the ions and localize them in the correct molar ratio (Ca: PO4: F = 5:3:1) at the tooth surface.[22] These results were consistent with Jayarajan et al.,[23] Aykut-Yetkiner et al.,[21] and Ma et al.,[24] in which they reported that the slight decrease in VistaCam readings of the MI Paste Plus group might related to the changes in the porosity of enamel.

However, little evidence for a substantial effect of these products on pits and fissure caries was reported. The difficulty in penetrating deep into the anatomy of the occlusal surface may support the recommendations that caries prevention on the occlusal surface is best approached through the use of fissure sealants. As ACP-CPP is creamy in consistency, it could not properly wet the surface and diffuses properly into the lesion unlike other products with more flowy consistency as varnishes.[25] Most of the clinical studies are conducted to investigate the effect of these products on the smooth surface after orthodontic treatment, and their results are generally inconclusive. Moreover, the clearance time from the mouth may be a contributing factor for the reduced in vivo effect.

From the clinical point of view, the mechanism of action of CPP-ACP and fluoride toothpaste was not superior to fluoridated toothpaste alone within the limitations and duration of this study. The use of CPP-ACP-containing paste just after a regular fluoride-containing toothpaste delayed the remineralization due to the tendency of calcium in the CPP-ACP to bind with fluorides that was just being incorporated in the enamel surfaces after brushing with the fluoride toothpaste to form CaF2. Consequently, CaF2 was dissolved in saliva and will not be bioavailable during the following demineralization period.[26] These results were consistent with Meyer-Lueckel et al.,[2] who reported that CPP-ACP cream showed little beneficial effect in enhancing remineralization as it hampers the remineralization induced by the use of fluoride toothpaste.

Considering the results reported, it is logical to state that the addition of fluoride in the MI Paste Plus does not enhance remineralization. The additive anticariogenic effect of CPP-ACFP in MI Paste Plus was not statistically significant from CPP-ACP in Tooth Mousse. The results are in accordance to the study conducted by Lata et al.[27] Regarding the effect on salivary pH, the results of the current study showed that the significantly highest salivary pH change was recorded at 1 month, as changes in salivary parameters such as pH, salivary flow rate, plaque and levels of cariogenic micro-organism occur after 1 month.[6] In addition, applying MI Paste Plus caused the highest statistically significant increase in saliva pH when compared with Tooth Mousse and the control group. This could be attributed to pH-responsive feature of the material as it works effectively as a remineralizing agent at acidic pH, as well as in neutral and alkaline phosphate range.[28] The use of CPP-ACPF, plaque enzymes, as phosphatases and peptidases partially degrades CPP-based products and accordingly increases pH due to ammonia production.[29] The added fluoride to CPP restricts phosphatase action by extending the action of molecular complexes. This is probably due to the fact that the availability of mineral ions would offset any fall in pH. Furthermore, the neutral CaHPO4 which is formed takes the credit of consuming majority of the acid generated by the cariogenic bacteria.[30]

The significant increase in the fluoride ion concentration is related to the availability of high amount of calcium ions. The concentration of calcium in the plaque and saliva increased more than 2-folds, and the concentration of phosphate within plaque and saliva increased 3-folds after using CPP-ACPF.[31] The MI Paste Plus group showed statistically significant increase in fluoride ion release in saliva. Duckworth and Jones[32] stated that the fluoride retention in oral reservoirs was controlled by the concentration gradient through the oral fluid–reservoir interface while being used.

The retention period depends on the salivary flow rate which affects its clearance from the oral cavity.[32] This justifies the similarity in results of both Tooth Mousse and fluoridated toothpaste groups. After fluoride-containing toothpaste application, the fluoride bioavailability in saliva elevated dramatically for a short time period and then decreases till the baseline level after about 120 min.[33] Thierens et al.[25] reported that the clearance time from the oral cavity might be a contributing factor in the retention effects due to the fact that fluoride treatments show a rapid clearance from the oral cavity because when spitting out the excess saliva the major fraction of fluoride is lost.

This study represents that no favorable effect of CPP-ACP products on high caries risk patients could be detected over the time period of the study. The results suggest the rejection of the null hypothesis and confirm the in-vivo diverse effect of tested pastes on WSL progression, salivary pH, and fluoride ion release. Future research should focus on the long-term effect of these pastes to confirm its sustainability. Further researches are suggested to determine the longevity of pH changes following the use of amorphous calcium phosphate-containing products. Also, the use of supplementary diagnostic devices to measure the degree of remineralization effect of MI Paste and MI Paste Plus products is recommended. Longer periods of treatment are required to ensure the lack of significant difference in the performance of the tested agents, and clinical studies to explore the mechanism underlying the combined effect of different caries preventive products would also be meaningful.


  Conclusion Top


Under the conditions used in this clinical trial, the following conclusions could be highlighted: the findings of the study indicate that in patients with high caries risk, the effect of Tooth Mousse and MI Paste Plus is negligible. It also could be beneficial to use MI Paste Plus to increase the salivary pH compared with Tooth Mousse and the control group, whereas the fluoride ion content of saliva was improved with MI Paste Plus in comparison to Tooth Mousse and the control group.

Acknowledgement

Nil.

Financial support and sponsorship

This study was self-funded by the authors.

Conflicts of interest

Nil.

Authors’ contribution

All authors had contributed equally and finally approved the manuscript and given consent for publication.

Ethical policy and Institutional Review board statement

The Research Ethics Committee (CREC) of Faculty of Dentistry, Cairo University with approval number 18-9-38 (Date: 26 September 2018).

Patient declaration of consent

We have obtained all appropriate patient consent forms. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed; this randomized controlled clinical study was held in the Faculty of Dentistry, Cairo University, Egypt. The protocol of this study was registered in www.clinicaltrials.gov/database, with unique identification number NCT03656432. All procedures performed in this study, involving human participants, were in accordance with the ethical standards of the Research Ethics Committee (CREC) of Faculty of Dentistry, Cairo University with approval number 18-9-38 (date: 26 September 2018).

Data availability statement

Data are available upon reasonable request.



 
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