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Table of Contents   
ORIGINAL RESEARCH  
Year : 2019  |  Volume : 30  |  Issue : 6  |  Page : 820-825
Effect of casein phosphopeptide–amorphous calcium phosphate as a remineralizing agent – An In Vivo study


1 Department of Pedodontics and Preventive Dentistry, Rungta College of Dental Sciences and Research, Bhilai, Chhattisgarh, India
2 Department of Orthodontics, Saraswati Dental College, Lucknow, Uttar Pradesh, India

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Date of Submission28-Dec-2017
Date of Decision14-Jul-2018
Date of Acceptance11-Sep-2018
Date of Web Publication14-Jan-2020
 

   Abstract 


Introduction: Demineralization and remineralization have a crucial impact on the hardness and strength of teeth. Casein phosphopeptide–amorphous calcium phosphate (CPP-ACP) Trident white chewing gum has anticariogenic property and also stimulates saliva, which buffers the oral cavity and promotes remineralization. Trident sugar-free gum, therefore, is an excellent delivery vehicle for promoting enamel remineralization. Aims and Objectives: The aim of this study is to show that CPP-ACP-containing chewing gum would increase the level of calcium concentration of saliva, thereby supplying calcium and phosphorus to whole dentition for a prolonged period and aid in remineralization of tooth surfaces. Materials and Methods: An in vivo nonrandomized clinical trial study was carried among 60 children. Unstimulated saliva from each 60 selected participants was collected. Then each participant was given two pellets of chewing gum containing CPP-ACP and asked to chew for a period of 20 min, after which saliva samples were again collected from each participant. The study was carried out for 15 days, and at three intervals, calcium and phosphorus levels were assessed using affiliated reagent kits and spectrophotometer. Results: Significant difference was found in the calcium and phosphorus concentration of saliva before and after chewing CPP-ACP-containing chewing gum. When post calcium and phosphate levels were analyzed among different time intervals, a highly statistically significant difference was observed (P = 0.000). Conclusion: Clinical trial study shows that chewing sugar-free gum containing CPP-ACP can be regarded as an additional caries prevention tool.

Keywords: Amorphous calcium phosphate, casein phosphopeptide, enamel demineralization, enamel remineralization, nano hydroxyapatite

How to cite this article:
Dewani N, Kashyap N, Avinash A, Kumar B, Singh M, Pawar P. Effect of casein phosphopeptide–amorphous calcium phosphate as a remineralizing agent – An In Vivo study. Indian J Dent Res 2019;30:820-5

How to cite this URL:
Dewani N, Kashyap N, Avinash A, Kumar B, Singh M, Pawar P. Effect of casein phosphopeptide–amorphous calcium phosphate as a remineralizing agent – An In Vivo study. Indian J Dent Res [serial online] 2019 [cited 2020 Feb 20];30:820-5. Available from: http://www.ijdr.in/text.asp?2019/30/6/820/275882



   Introduction Top


Teeth are tools that have evolved to aid in the survival of the species. Nature has fashioned the hard enamel covering to serve this function. It is a paradox that though enamel is the hardest biological substance, its acellular and avascular nature makes it incapable of any natural defence mechanism based on cellularity. When tooth erupts into the oral cavity, the hypomineralized enamel encounters a highly complex ecology. Imbalance in this complex environment holds the clue to the origin of this unique disease called dental caries. The onset of dental caries requires the establishment of necessary conditions for mineral dissolution.[1]

Tooth enamel is the highest mineralized tissue of the body. It is mainly composed of 96% inorganic material, 4% organic material and water. Oral environment is a combat zone for activities such as demineralization and remineralization. Therefore, it is necessary to maintain equilibrium between these two processes.[2] Nowadays, newer noninvasive interventions for noncarious lesions are implicated as a preventive treatment.[3] Casein, a bovine milk phosphorus protein, is a natural food component found in cow's milk, which interacts with calcium and phosphate ions. Technically, it is known as casein phosphopeptide–amorphous calcium phosphate (CPP-ACP). It contains cluster sequence of [-Ser(P)-Ser(P)-Ser(P)-Glu-Glu] which stabilizes calcium and phosphate in solution and increases the level of calcium and phosphate in dental plaque and tooth. A pioneer of CPP-ACP in dentistry is Professor Eric Reynolds from the School of Dental Science at the University of Melbourne in Australia.[4]

Remineralization can be enhanced by enriching tissues with hydroxyapatite which is the same inorganic component as enamel.[5] ACPs are highly soluble and can be readily converted to hydroxyapatite which makes it a suitable remineralizing agent.[2]

The most common mode of using CPP-ACP is through sugar-free sorbitol or xylitol-based chewing gums. Apart from chewing gum, the other vehicles are milk, mouth rinses, lozenges, and dental cream.[6] Trident is a commercially available (Trident Total; Kraft Foods/Cadbury Adams SP, Brazil) chewing gum containing CPP-ACP and patented as Recaldent.[7]

Therefore, this nonrandomized clinical trial study was done to assess the efficacy of CPP-ACP as a remineralizing agent.


   Materials and Methods Top


An in vivo nonrandomized clinical trial study was conducted. For this proposed study, 60 children age between 8 and 14 years were selected. This study was reviewed by the institutional ethical committee and ethical approval was obtained. Informed consent was then obtained from the caretakers of the participants. The inclusion criteria were as follows: children with no active carious lesion, no pathological changes in oral mucosa or periodontal tissue, sample collection done in morning hours, and in those who had breakfast 2 h before (children not consumed food or drink between this period). Exclusion criteria were as follows, children who were allergic to milk protein, children who were on antibiotics as it may hamper salivary flow, and children with fluorosis.

The following armamentarium was used for the study: CPP-ACP-containing chewing gum (Trident Cadbury Adams USA) watermelon flavor serving two pellets – 3 g, serum calcium and serum phosphorus reagent kit (Accucare Labcare Diagnostic Pvt., India), spectrophotometer, sputum bottle, and micropipette [Figure 1].
Figure 1: Armamentarium

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The protocol of the study was explained to participants in detail, and it was informed that the study will be carried out in three phase – baseline, first week, and second week. Before the study, 1.5 mL of saliva sample was collected and labeled as BEFORE SAMPLE [Figure 2]. Analysis of saliva sample was started to detect presalivary calcium and pre-salivary phosphorus level with spectrophotometer [Figure 3]. Meanwhile, the subjects were given two pellets of CPP-ACP “Trident chewing gum” (Cadbury Adams USA) and asked to chew for 20 min, and again 1.5 mL of saliva sample was collected and labeled as AFTER SAMPLE. Immediately after collection, salivary calcium and phosphorus levels were detected with spectrophotometer.
Figure 2: Spectrophotometer

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Figure 3: Collection of saliva

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Then for the next six consecutive days, the subjects were given two pellets of chewing gum [Figure 4], and on the seventh day again pre- and post-salivary calcium and phosphorus levels were detected with the help of spectrophotometer and the same procedure was again repeated for the next six days, and then on 15th day again pre- and post-salivary calcium and phosphorus levels were detected. The data were statistically analyzed using SPSS version 19.0. Student's paired t-test and analysis of variance (ANOVA) were performed to know the effect of variables and to reveal statistical significance.
Figure 4: Two pellets of chewing gum given

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   Results Top


In this study, a total of 60 participants with a mean age of 9.35 ± 1.02 years were selected. Before chewing trident gum, the mean pre-calcium level in baseline was found to be 9.68 ± 1.13 mg/dL and pre-phosphorus level in baseline was 4.67 ± 0.25 mg/dL. After chewing the gum containing calcium phosphate, it resulted in an increase in the mean salivary calcium level to 10.48 ± 0.98 mg/dL and phosphorus level to 4.80 ± 0.11 mg/dL [Table 1]. Similar results were obtained in the first and second consecutive weeks with a mean pre-salivary calcium level of 9.80 ± 1.14 and 9.31 ± 1.37 mg/dL, respectively, and salivary phosphorus level of 4.76 ± 0.18 and 4.73 ± 0.22 mg/dL, respectively. The chewing gum in the consecutive week resulted in increased post-salivary calcium levels to 10.78 ± 1.10 and 11.47 ± 1.04 mg/dL, respectively, and post-salivary phosphorus levels to 5.32 ± 0.36 and 5.69 ± 0.22 mg/dL.
Table 1: Calcium and phosphorus levels at baseline using paired t-test

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From the results observed, it is stipulated that the mean salivary calcium and phosphate levels after chewing the gum containing calcium phosphate were numerically greater at all points of time when compared with levels before chewing the gum, with a mean increase in calcium levels at baseline, first week, and second week to be 0.80 ± 0.63, 0.974 ± 0.65, and 2.16 ± 1.79 mg/dL, respectively. This shows that chewing gum containing calcium phosphate is effective in increasing salivary calcium and phosphate levels in vivo as highly statistically significant increase in salivary calcium was achieved at all the three time intervals (P = 0.000). Similar results observed with phosphate levels were also numerically greater after chewing the gum at all points of time when compared with levels before chewing the gum, with a mean increase in phosphate levels at baseline, first week and second week observed to be 0.12 ± 0.23, 0.57 ± 0.37 and 0.96 ± 0.29 mg/dL respectively. This result shows highly statistically significant increase in salivary phosphate levels achieved at all the three time intervals (P = 0.000).

When we compared the baseline calcium and phosphate levels, the test chewing gum resulted in a significant increase in calcium and phosphate levels of 800 ± 0.63 and 0.12 ± 0.23 mg/dL (P = 0.000) [Table 1]. After application of ANOVA, there was no statistically significant difference among pre-calcium at baseline, first week, and second week (P = 0.076) [Table 2]. When we analyzed post-calcium and phosphate levels among different time intervals, a highly statistically significant difference was observed (P = 0.000). It was also observed that statistically significant increase in mean salivary calcium levels was found to be between baseline and second week, that is, 0.992 mg/dL (p = 0.000) and between first and second weeks, that is, 0.695 mg/dL (P = 0.001) [Table 3]. Similarly, when ANOVA was applied, it showed that there was no statistically significant difference among pre-phosphate levels at baseline, first week and second week (P = 0.117) [Table 4], while we analyzed post-phosphate levels among different time intervals a highly statistically significant difference was observed (P = 0.000). However the levels of salivary phosphate increased significantly at all the three points of time (P = 0.000) [Table 5].
Table 2: Comparison of pre-chewing Cal levels at baseline, 1st week and 2nd week scores using ANOVA

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Table 3: Comparison of post-chewing Cal levels at baseline, 1st week and 2nd week scores using ANOVA

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Table 4: Comparison of pre-chewing P-levels at baseline, 1st week and 2nd week scores using ANOVA

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Table 5: Comparison of post-chewing P-levels at baseline, 1st week and 2nd week scores using ANOVA

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   Discussion Top


Dental caries progressively lead to demineralization of tooth enamel that occurs when microorganisms metabolize dietary sugars into acids that release calcium and phosphate ions from hydroxyapatite. Dental caries is a highly prevalent disease and it is a major public health problem. For maximum remineralization to occur, calcium, phosphorus, and fluoride must all be available at sufficient levels in the oral environment.[8]

Nowadays, three strategies are used for managing dental caries, that is, prevention, control and treatment, and it is based on appropriate methods of disease detection from the earliest stage as the lesion starts forming.[9]

Recent concept uses the CPP-ACP molecule (Recaldent) which is a protein found in milk called casein which acts as a demineralizing agent and is remarkably capable of stabilizing calcium and phosphate. It is one of the promising noninvasive procedures for management of early carious lesions.

CPP-ACP possesses anticariogenicity properties as they act as a calcium phosphate reservoir, buffering the activities of free calcium and phosphate ions in plaque fluid and they maintain a state of supersaturation with respect to enamel minerals, and therefore they enhance remineralization and suppress demineralization.[4]

Rose and Grychtol et al. demonstrated that CPP-ACP and calcium competed for the same binding sites on Streptococcus mutans.[9],[10] CPP-ACP has been increasingly accepted as an adjunct to oral hygiene procedures. Chewing gum stimulates saliva and helps maintain buffer activity of saliva. It has been demonstrated evidently that CPP-ACP increases the level of calcium and phosphorus ion in plaque fivefold in humans in in situ studies and short-term mouthwash studies.[4]

However, Grychtol et al. found no significant differences between chewing gum containing calcium and calcium-free chewing gums with regard to change in mineral loss and lesion depth of enamel samples.[11]

This study stipulated that the mean salivary calcium and phosphate levels after chewing gum containing calcium phosphate were numerically greater at all time points when compared with levels before chewing the gum, with a mean increase in calcium levels at baseline, first week, and second week to be 0.80 ± 0.63, 0.974 ± 0.65, and 2.16 ± 1.79 mg/dL, respectively. This shows that chewing gum containing calcium phosphate is effective in increasing salivary calcium and phosphate levels in vivo.

Similar findings have been reported by various researchers in different parts of the world. Santhosh BP et al. reported 70.6% increase in salivary calcium concentration after chewing CPP-ACP Trident chewing gum. However, they found 28.3% decrease in phosphorus concentration after chewing CPP-ACP chewing gum.[12]

Ruchi Vashisht et al. applied GC Tooth Mousse 3 min thrice daily after meals for 14 days and they showed remineralization of the lesions and significant alterations seen in surface morphologies of enamel after 14 days of experiment.[13]

Similarly, Shen et al. found chewing sugar-free gum containing CPP-ACP produced 4-7 fold increase in salivary flow rate over a 2-min period. Preliminary studies show that no CPP-ACP could be detected in gum bolus after 10 min of chewing, hence in in situ studies they tested for 20 min asking to chew the chewing gum and found enamel remineralization of 100%. On the basis of this study, the time chosen in this study is for 20 min, so that active ingredient of CPP-ACP gets properly dissolved into the saliva.[14]

Shen and Reynolds in their study revealed that CPP-ACP in a sugar-free chewing gum enhanced remineralization of enamel subsurface lesions by 100%–150%. They revealed a dose-related increase in enamel remineralization independent of chewing gum weight and type.[14] Similar results were obtained by D Gurunathan et al. and Carmen llena et al.[2],[15]

Reynolds EC had combined CPP-ACP nanocomplexes and fluoride in a toothpaste and found a greater increase in enamel remineralization.[16],[17] Similar to our study, Catarina Ribeiro assessed remineralizing potential of CPP-ACP chewing gum. They found no active carious lesion at two levels, that is, at two and 24 h and suggested that chewing gum with CPP-ACP showed higher hardness recovery at both levels, that is, two and 24 h.[7]

In this study, two pellets of Trident chewing gum were given for 20 min for 15 days once daily and it was stipulated that the mean salivary calcium and phosphate levels after chewing the gum containing calcium phosphate were numerically greater at all time points when compared with levels before chewing the gum. But when pre-calcium and phosphorus levels were assessed at first and second weeks, no significant difference was seen in calcium and phosphorus levels; this may be due to removal of softened enamel (remineralized) which was produced by CPP-ACP trident chewing gum by tongue abrasion.[14] Similar reports were observed by E.C. Reynolds in 1997.[18]

F Cai incorporated CPP-ACP into the lozenges and found significantly increasing remineralization by 78% to 176%, respectively, after 14 days of consumption and they also found no significant relation between individual stimulated or unstimulated salivary flow rates. Dawes and Macpherson compared salivary stimulation between lozenges and sugar-free chewing gum and reported that consumption of sugar-free chewing gum produces significantly greater increase in salivary flow (3–4 mL/min) which was also reported by Shen et al.[14],[19]

Reynolds E.C. in 1998 used 1.0% of CPP-ACP solution twice daily which resulted in a 144% increase in calcium level and a 160% increase in inorganic phosphate level which was similar to our study.[4],[20]

Limitations and implications

This in vivo study investigated the effect of periodic CPP-ACP chewing gum as a remineralizing agent. But in every day situation, the enamel surfaces are bathed in saliva between the demineralization periods when dental plaque metabolizes sugar into acids. The pH stays low for some time but returns back to normal. In oral cavity, the lesion formed is covered by plaque and debris. This can make the situation altogether different from in vivo condition.


   Conclusion Top


The introduction of CPP-ACP in preventive dentistry has provided a novel way of remineralizing dental tissues. The incorporation of CPP-ACP in chewing gum, lozenges, mouthwashes, dentifrices, and so on provides children with a new way to fight dental caries. Hence, the use of CPP-ACP as a preventive adjunct should be encouraged.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. 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.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Margolis HC, Zhang YP, Lee CY, Kent RLJ, Moreno EC. Kinetics of enamel demineralization in vitro. J Dent Res 1999;78:1326-35.  Back to cited text no. 1
    
2.
Gurunathan D, Somasundaram S, Kumar SA. Casein phosphopeptide-amorphous calcium phosphate: Are mineralizing agent of enamel. Aust Dent J 2012;57:404-8.  Back to cited text no. 2
    
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ElSayad I, Sakr A, Badr Y. Combining casein phosphopeptide-amorphous calcium phosphate with fluoride: Synergistic remineralization potential of artificially demineralized enamel or not? J Biomed Opt 2009;14:1-6.  Back to cited text no. 3
    
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Al-Batayneh OB. The clinical applications of Tooth Mousse™ and other CPP-ACP products in caries prevention: Evidence-based recommendations. Smile Dental J 2009;4:8-12.  Back to cited text no. 4
    
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Divyapriya GK, Yavagal PC, Veeresh DJ. Casein phosphopeptide–amorphous calcium phosphate in dentistry: An update. Int J Oral Health Sci 2016;6:18-25.  Back to cited text no. 5
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Yengopal V, Mickenautsch S. Caries preventive effect of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP): a meta-analysis. Acta Odontol Scand 2009;67:321-32.  Back to cited text no. 6
    
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De Alencar CRB, Magalha AC, Moreira MA, De Oliveira TM, Hono Rio HM, Rios. In situ effect of a commercial CPP-ACP chewing gum on the human enamel initial erosion. J Dent 2014;42:1502-7.  Back to cited text no. 7
    
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C. Llena AM, Leyda L. Forner. CPP-ACP and CPP-ACFP versus fluoride varnishing remineralisation of early caries lesions. A prospective study. Eur J Paediat Dent 2015;16:181-6.  Back to cited text no. 8
    
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Mettu S, Srinivas N, Reddy S, Srinivas N. Effect of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) on caries-like lesions in terms of time and nano-hardness: An in vitro study. J Indian Soc Pedodont Prev Dent 2015;33:269-74.  Back to cited text no. 9
    
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Rose RK. Binding characteristics of Streptococcus mutans for calcium and casein phosphopeptides. Caries Res 2000;34:427-31.  Back to cited text no. 10
    
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Grychtol S, Basche S, Hannig M, Hannig C. Effect of CPP/ACP on initial bioadhesion to enamel and dentin in situ. Sci World J 2014;3:1-9.  Back to cited text no. 11
    
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Santhosh BP, Jethmalani, Shashibhushan KK, Reddy S. Effect of casein phosphopeptide-amorphous calcium phosphate containing chewing gum on salivary concentration of calcium and phosphorus. J Indian Soc Pedodont Prev Dent 2012;30:146-50.  Back to cited text no. 12
    
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Vashisht R, Kumar AR, Indira MR, Srinivasan S, Ramachandran. Remineralization of early enamel lesions using casein phosphopeptide amorphous calcium phosphate: An ex-vivo study. Contemp Clin Dent 2010;1:210-4.  Back to cited text no. 13
[PUBMED]  [Full text]  
14.
Shen R, Cai F, Nowicki A, Vincent J, Reynolds EC. Remineralization of enamel subsurface lesions by sugar-free chewing gum containing casein phosphopeptide-amorphous calcium phosphate. J Dent Res 2001;80:2066-70.  Back to cited text no. 14
    
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Llena C, Forner L, Baca P. Anticariogenicity of casein phosphopeptide-amorphous calcium phosphate: A review of the literature. J Contemp Dent Pract 2009;10:1-9.  Back to cited text no. 15
    
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Reynolds EC, Cai F, Shen P, walker GD. Retention in plaque and remineralization of enamel lesions by various forms of calcium in a mouthrinse or sugar-free chewing gum. J Dent Res 2003;82:206-11.  Back to cited text no. 16
    
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Reynolds EC, Cai F, Cochrane NJ, Shen P, Walker GD, Morgan MV, Reynolds C. Fluoride and casein phosphopeptide-amorphous calcium phosphate. J Dent Res 2008;87:344-8.  Back to cited text no. 17
    
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Reynolds EC. Remineralization of enamel subsurface lesions by casein phosphopeptide-stabilized calcium phosphate solutions. J Dent Res 1997;6:1587-95.  Back to cited text no. 18
    
19.
Cai F, Shen P, Morgan MV, Reynolds EC. Remineralization of enamel subsurface lesions in situ by sugar-free lozenges containing casein phosphopeptide-amorphous calcium phosphate. Aust Dent J 2003;48:240-3.  Back to cited text no. 19
    
20.
Reynolds EC. Anticariogenic complexes of amorphous calcium phosphate stabilized by casein phosphopeptides: A review. Spec Care Dent 1998;18:9:8-16.  Back to cited text no. 20
    

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Correspondence Address:
Dr. Brij Kumar
Department of Pedodontics and Preventive Dentistry, Rungta College of Dental, Sciences and Research, Bhilai, Chhattisgarh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.IJDR_779_17

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