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ORIGINAL ARTICLE
Year : 2017  |  Volume : 35  |  Issue : 4  |  Page : 312-318
 

An in vitro comparison of casein phosphopeptide-amorphous calcium phosphate paste, casein phosphopeptide-amorphous calcium phosphate paste with fluoride and casein phosphopeptide-amorphous calcium phosphate varnish on the inhibition of demineralization and promotion of remineralization of enamel


1 Department of Pedodontics and Preventive Dentistry, KLE VK Institute of Dental Sciences, Belagavi, Karnataka, India
2 Department of Oral Pathology and Microbiology, KLE VK Institute of Dental Sciences, Belagavi, Karnataka, India

Date of Web Publication15-Sep-2017

Correspondence Address:
Prachi Jayesh Thakkar
Department of Pedodontics and Preventive Dentistry, KLE VK Institute of Dental Sciences, KLE University, JNMC Campus, Nehru Nagar, Belagavi - 590 010, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JISPPD.JISPPD_308_16

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   Abstract 


Aim: This study aims to determine and compare the extent of inhibition of demineralization and promotion of remineralization of permanent molar enamel with and without application of three remineralizing agents. Materials and Methods: Forty extracted permanent molars were randomly divided into two groups 1 and 2, longitudinally sectioned into four and divided into subgroups A, B, C, and D. The sections were coated with nail varnish leaving a window of 3 mm × 3 mm. All sections of Group 1 were treated with their respective subgroup-specific agent: Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) paste for subgroup A, CPP-amorphous calcium phosphate fluoride (ACPF) paste for subgroup B, CPP-ACPF varnish for subgroup C and subgroup D served as a control. The sections were then subjected to demineralization for 12 days following which lesional depth was measured under the stereomicroscope. All the sections of Group 2 were subjected to demineralization for 12 days, examined for lesional depth, then treated with their respective subgroup specific agents and immersed in artificial saliva for 7 days. The sections were then examined again under the stereomicroscope to measure the lesional depth. Results: CPP-ACPF varnish caused significant inhibition of demineralization. All three agents showed significant remineralization of previously demineralized lesions. However, CPP-ACPF varnish showed the greatest remineralization, followed by CPP-ACPF paste and then CPP-ACP paste. Conclusion: This study shows that CPP-ACPF varnish is effective in preventing demineralization as well as promoting remineralization of enamel. Thus, it can be used as an effective preventive measure for pediatric patients where compliance with the use of tooth mousse may be questionable.


Keywords: Casein phosphopeptide, demineralization, fluoride, remineralization


How to cite this article:
Thakkar PJ, Badakar CM, Hugar SM, Hallikerimath S, Patel PM, Shah P. An in vitro comparison of casein phosphopeptide-amorphous calcium phosphate paste, casein phosphopeptide-amorphous calcium phosphate paste with fluoride and casein phosphopeptide-amorphous calcium phosphate varnish on the inhibition of demineralization and promotion of remineralization of enamel. J Indian Soc Pedod Prev Dent 2017;35:312-8

How to cite this URL:
Thakkar PJ, Badakar CM, Hugar SM, Hallikerimath S, Patel PM, Shah P. An in vitro comparison of casein phosphopeptide-amorphous calcium phosphate paste, casein phosphopeptide-amorphous calcium phosphate paste with fluoride and casein phosphopeptide-amorphous calcium phosphate varnish on the inhibition of demineralization and promotion of remineralization of enamel. J Indian Soc Pedod Prev Dent [serial online] 2017 [cited 2018 Jul 19];35:312-8. Available from: http://www.jisppd.com/text.asp?2017/35/4/312/214923





   Introduction Top


Dental caries is a highly prevalent disease, and although in most developed countries, its prevalence has declined, the disease remains a major public health problem. Signs of caries process cover a continuum from the first molecular changes in the apatite crystals of the tooth to a visible white spot lesion, through dentin involvement and eventual cavitation.[1]

Dental caries is initiated throgh demineralization of tooth hard tissue by organic acids produced from fermentable carbohydrates by cariogenic bacteria of the dental plaque. The presence of sufficient calcium and phosphate ions available in the immediate environment enables the rebuilding of partly dissolved apatite crystals which is called as remineralization. To restore the natural equilibrium, either remineralization must be enhanced, or demineralization must be retarded.

Fluoride is the most commonly used remineralizing agent. As the pH rises, new and larger crystals that contain more fluoride (fluorapatite) form, thereby reducing the enamel demineralization and enhancing remineralization.

To avoid the potential for adverse effects due to overexposure, high fluoride strategies cannot be followed. Also, for every two fluoride ions, ten calcium and six phosphate ions are required to form one unit cell of fluorapatite.(Ca10[PO4]6F2). Hence, on topical application of fluoride ions, the availability of calcium and phosphate ions can be a limiting factor for net enamel remineralization to occur.[2]

Casein phosphopeptide (CPP) can stabilize calcium phosphate in nanocomplexes in solutions such as amorphous calcium phosphate (ACP).[3] This CPP-ACP acts as reservoir of bio-available calcium and phosphate and maintains supersaturation of the solution thus facilitating remineralization.[4]

CPP-ACP is not only available as a paste for home use but also in the form of varnish in combination with fluoride (MI Varnish). This form becomes important for pediatric dentists particularly when the patient's level of cooperation is not ideal. Although CPP-ACP has been already shown to prevent enamel demineralization and to promote remineralization of subsurface enamel lesions in animal and human in situ caries models,[5],[6],[7] there is no published report to document whether there is any difference in the remineralizing potential of CPP-ACP in paste and varnish form.

Thus, the aim of this study was to determine and compare the extent of inhibition of demineralization and promotion of remineralization of sound permanent molar enamel with and without application of CPP-ACP paste, CPP-ACP paste with 900 ppm fluoride (CPP-amorphous calcium phosphate fluoride [ACPF] paste) and 5% sodium fluoride varnish containing CPP-ACP (CPP-ACPF varnish).


   Materials and Methods Top


An in vitro study was designed and conducted in the Department of Pedodontics and Preventive Dentistry with assistance from the Department of Oral Pathology. Ethical clearance for the study was obtained from the Institutional Review Board for the use of human extracted teeth. Freshly extracted permanent molars obtained from the Department of Oral and Maxillofacial Surgery were cleansed for debris and stored in 10% formalin until used for the study.

By Cohen's method, with type I error of 0.05 and power of 80%, to compare mean of four groups with eta [2] (eta square) of 0.15 the sample size estimated was: 40 extracted permanent molars.

Noncarious extracted permanent molars were included for the study. Attrided, abraded, hypoplastic, fractured, malformed, restored, endodontically treated teeth, and teeth extracted from patients with systemic diseases which could cause variation in salivary flow, tooth formation, and maturation were excluded from the study.

Methodology

Forty teeth selected according to the inclusion criteria and were equally divided randomly into two groups:

Group 1: To determine the inhibition of demineralization of enamel

Group 2: To determine the promotion of remineralization of enamel.

Each tooth from both groups was longitudinally sectioned, buccolingually, and mesiodistally into four sections using a diamond disc. The sections obtained were labelled and divided into subgroups A, B, C, and D, respectively. Thus, a total of 160 sections of teeth, i.e., 80 sections per group were made. Each of the Groups 1 and 2 had 20 sections each of A, B, C, and D.

An acid resistant nail varnish was applied on the tooth leaving a window of 3 mm × 3 mm on the middle third of the enamel at the edge of the section. [Figure 1] The sections were stored in 10% formalin until further use.
Figure 1: Windows prepared on each section

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The samples from Groups 1 and 2 were equally divided into subgroups depending on the remineralizing agent used:

Subgroup A: CPP-ACP paste-GC Tooth Mousse (GC Corporation Tokyo, Japan)

Subgroup B: CPP-ACP paste with 900 ppm fluoride-GC Tooth Mousse plus (GC Corporation Tokyo, Japan)

Subgroup C: 5% Sodium fluoride varnish with CPP-ACP-GC MI varnish (GC Corporation Tokyo, Japan)

Subgroup D: No agent was used. These sections served as control.

  1. To determine and compare the extent of inhibition of demineralization after application of a re-mineralizing agent:


  2. Sections obtained from each tooth of Group 1 were subjected to the following surface treatments:

    • Subgroups A and B: A layer of CPP-ACP paste and CPP-ACPF paste were applied respectively on Sections A and B and left undisturbed for 4 min
    • Subgroup C: A thin layer of CPP-ACPF varnish was applied; allowed being absorbed for 20 s and then air dried
    • Subgroup D: served as the control group where no surface treatment was performed
    • All specimens from Group 1 were immersed in the demineralizing solution for 12 days. [Figure 2] The composition of the demineralizing solution prepared was 2.2 mM CaCl2.2H2O, 2.2 mM KH2 PO4, 0.05 M acetic acid, and 10 M KOH. The pH was adjusted to 4.4.[3]
    Figure 2: Samples immersed in demineralizing solution

    Click here to view


    The acid resistant nail varnish was removed using acetone. The lesional depth was measured under stereomicroscope.

  3. To determine and compare the extent of promotion of remineralization following demineralization.


All specimens of Group 2 were immersed in demineralizing solution for 12 days, after which the depth of demineralization was measured under the stereomicroscope [Figure 3].
Figure 3: Demineralized section viewed under the stereomicroscope. Lesion depth measured from Point A to Point

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The samples were further subjected to the surface treatments as specified for Group 1. Finally, all specimens of Group 2 were immersed in artificial saliva with pH 7.2 for 7 days. The composition of artificial saliva prepared was 3.9 mM Na3 PO4, 4.29 mM NaCl, 17.98 mM KCl, 1.1 mM CaCl2 0.08 mM MgCl2,0.5 mM H2 SO4, 3.27 mM NaHCO3, and distilled water.[8] This artificial saliva solution was replaced each day.

After 7 days, each section was observed under the stereomicroscope to determine the depth of the lesion after remineralization [Figure 4]. The difference between the two depths gave the extent of remineralization.
Figure 4: Remineralized section viewed under the stereomicroscope

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The sections were observed under the stereomicroscope and the lesional depth measured as unit values which were then converted to micrometer. All the observations were made by a single observer who was blinded to the study groups.


   Results Top


All the data were tabulated and subjected to statistical analysis using IBM Statistical Package for Social Sciences software version 20.0 (Chicago, IL, USA.).

Comparison of four subgroups (A, B, C, D) was done by one-way analysis of variance (ANOVA) and pairwise comparisons of four subgroups (A, B, C, D) by Tukeys multiple post hoc procedures with respect to inhibition of depth of demineralization scores and percentage of change in-depth scores following remineralization.

[Table 1] and [Graph 1] explain the mean lesional depth, standard deviation and standard error for the samples from Group 1.
Table 1: Descriptive statistics for sections from Group 1

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The difference in the mean lesional depth of sections from subgroup C and subgroup D of Group 1 was found to be statistically significant (P < 0.05) as shown by one way ANOVA and Tukeys multiple post hoc procedures respectively for comparison of the four subgroups from Group 1 [Table 2].
Table 2: Pair wise comparisons of four subgroups (A, B, C, D) from Group 1 with respect to inhibition of depth of demineralization scores by Tukey's multiple post hoc procedures

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[Table 3] and [Graph 2] explain the mean lesional depth, standard deviation, and standard error for the samples from Group 2.
Table 3: Descriptive statistics for sections from Group 2

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Samples from subgroups A, B, and C showed significant remineralization when compared to control group (P < 0.05) as shown by one-way ANOVA and Tukeys multiple post hoc procedures, respectively for comparison of the four subgroups from Group 2 [Table 4].
Table 4: Pair wise comparisons of four subgroups (A, B, C, D) from Group 2 with respect to percentage of change in depth scores by Tukey's multiple post hoc procedures

Click here to view



   Discussion Top


The conventional treatment concept for all caries-attacked teeth included the removal of the affected tissues and the replacement with a restorative material. However, now a contemporary approach has been adopted which is the noninvasive intervention of the carious lesions which can be arrested if the cariogenic challenges of the microenvironment are sufficiently controlled or/and if therapeutic agents are applied for tissue healing.[9]

Fluoride is the most commonly used remineralizing agent. Featherstone has proved the efficacy of fluoride to increase the resistance of tooth mineral to demineralization by plaque acids as well as to promote remineralization of incipient lesions.[10]

Fluoride in the solution surrounding the apatite crystals of enamel is much more effective at inhibiting demineralization than fluoride that is incorporated into the crystals through fluoridated drinking water.[11] Primary caries preventive actions of fluoride are thus posteruptive through topical effects that can interfere with the dynamic equilibrium at the interface between mineral surface and oral fluid.

A study by Reynolds et al.[12] showed that although the fluoride uptake from a 1100 ppm fluoride containing dentifrice was considerable, not all the fluoride ions which diffused into the sub-surface were incorporated into the mineral phase. However, when used along with 2% CPP-ACP dentifrice, there was a significantly greater remineralization taking place suggesting that remineralization with fluoride dentifrice alone was calcium and phosphate limited.

Casein phosphopeptides are derived from milk protein casein that is complexed with calcium and phosphate, providing a reservoir of bioavailable calcium and phosphate in the saliva and on the surface of the tooth.[2]

The compound CPP-ACP has recently become available in varnish form in combination with fluoride. The varnish form is considered important over other topical forms as it is quick and easy to apply, less likely than gels to be swallowed by young children, useful alternative for caries control in special needs patients such as those with developmental disabilities, adheres to tooth surface for longer periods and can be an effective mode of prevention in patients where compliance is questionable.[13]

Experimental model based on the formation of lesions in vitro systems enable the researchers to selectively examine fundamental processes associated with mineral loss from the teeth and thus can be used to understand effects of various agents on carious processes.[14]

In vitro models face criticism as in the oral cavity, the pH alternates frequently, and the levels depend on the individual's dietary and oral hygiene habits and therefore it is difficult to exactly simulate the oral conditions that prevail in the mouth. Nevertheless, the in vitro model was chosen for this study because there is greater control over these variables which are extremely difficult to control in the mouth and artificial lesions are more homogenously reproducible than natural lesions.[15]

In the present study, permanent extracted molars were selected to enable obtaining four sections from each tooth with appropriate coding and systematic allocation to serve following purposes:[8]

  • One section from each tooth was allotted to each study group
  • During the measurement of lesion depth, the coding system helped to achieve blinding of the investigator
  • All the study groups received an equal number of mesiobuccal, distobuccal mesiolingual and distolingual tooth quarters.


A window was created to limit the area of paste/varnish application and to produce lesions only in the window area. This would help in lesion depth measurement compared to unaffected area covered by nail varnish.[16]

The artificial saliva composition used in this study [8] was a nonfluoride containing one, as the addition of fluoride could have interfered with the hypothesis being tested.

In group I, wherein inhibition of demineralization was determined, sections from subgroups A, B, and C showed a lesser mean lesion depth compared to the control group. The casein phosphopeptides (CPP) stabilizes amorphous calcium phosphate (ACP) by forming multiphosphoseryl sequences. CPP binds to ACP and prevents the dissolution of calcium and phosphate ions. The CPP-ACP maintains the solution supersaturated and acts as reservoir of bio-available calcium and phosphate, thus aiding in remineralization [2]. This is observed, as CPP-ACP would compete with calcium for plaque calcium binding sites. This is likely to restrict mineral loss during the cariogenic episode and maintain a state of supersaturation of the calcium and phosphate ions in close approximation with the tooth thus inhibiting demineralization and assisting in subsequent remineralization.[2]

In the present study, only CPP-ACPF varnish showed significant inhibition of demineralization when compared to the control group. This was similar to the results obtained by many other studies wherein varnishes have proved to be more effective than other forms of topical fluoride treatment.[17],[18] It could be attributed to the fact that the rosin base in fluoride varnishes allow them to stick to tooth surfaces and stay for up to 24 h whereby fluoride is gradually released from the varnish and is taken up by the tooth enamel and dentin. The strongly bound fluoride, incorporated onto the surface of the crystals of apatite, can reduce the solubility of the tooth mineral and hence inhibit demineralization due to acids generated by plaque bacteria.[19]

However, CPP-ACP paste and CPP-ACPF paste did not show a significant inhibition of demineralization. These results were in accordance with another in vitro study.[20] Contrasting results were reported by Roberts [21] when volunteers used a dentifrice containing 5% CPP and wore an in situ appliance with sound enamel. This could be attributed to the in situ design of the aforementioned study where the presence of plaque, bacteria and saliva could have significantly influenced the process. The literature points that CPP can bind to oral bacteria like Streptococcus mutans as reported by Rose [22] which could possibly be the reason why CPP-ACP group did not show significant resistance to lesion depth formation in vitro.

It is well established that CPP-ACP increases fluoride incorporation in subsurface enamel and substantially increases remineralization of subsurface lesions than with fluoride alone.[12] In Group II, the mean remineralization seen with CPP-ACP paste, CPP-ACPF paste and CPP-ACPF varnish was significantly greater when compared to the control group. The greater amount of remineralization in subgroups B and C may be attributed to the added benefit of fluoride as localization of Amorphous calcium phosphate fluoride at the tooth surface by the CPP which in effect would co-localize calcium, phosphate and fluoride thus forming a reservoir and for slow and prolong release of ions thus enhancing remineralization. However, the inter-subgroup comparison did not show a significant difference which was similar to the results obtained by Jayarajan et al.[23] Conflicting results have been found in some other studies [24],[25] which showed that CPP-ACP in combination with fluoride has increased remineralization potential when compared to CPP-ACP paste alone. This variation in results could be attributed to the higher standard deviation seen in subgroups A and B. This can probably be eliminated by including a larger sample size or limiting the age-group from which the extracted teeth were obtained to ensure similar susceptibility of all the sections to demineralization and remineralization thus reducing the wide range of values.

It was observed in the present study that remineralization had taken place throughout the body of the lesion in about 50% of the sections. Similar findings were shown by Reynolds et al.[12] wherein a more homogenous remineralization throughout the body of the lesion when 2%CPP-ACP was used along with fluoride dentifrice. This may be due to the fact that when fluoride ions come in contact with free calcium and phosphate ions, fluorapatite would rapidly form in the surface layer. However, in the presence of CPP, which prevents rapid transformation into calcium phosphate phases, the ions would be stabilized and maintained in a form that would drive diffusion down activity gradients into the subsurface lesion. CPP can localize, stabilize and deliver the ions at the tooth surface in the correct molar ratio [Ca: PO4:F = 5:3:1].[26]


   Conclusion Top


This study shows that:

  • The varnish form of casein phosphopeptide amorphous calcium phosphate with fluoride (CPP-ACPF varnish) caused significant inhibition of demineralization of permanent molar enamel
  • CPP-ACP paste, CPP-ACPF paste, and CPP-ACPF varnish are effective in remineralizing previously demineralised enamel. However, CPP-ACPF varnish showed greatest remineralization, followed by CPP-ACPF paste and then CPP-ACP paste.


Thus, we conclude that CPP-ACPF varnish is effective in preventing demineralization as well as promoting remineralization of enamel. It can be used as an effective preventive measure for pediatric patients where compliance with the use of tooth mousse may be questionable.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Reynolds EC. Casein phosphopeptide-amorphous calcium phosphate: The scientific evidence. Adv Dent Res 2009;21:25-9.  Back to cited text no. 2
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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. 3
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Reynolds EC, Cain CJ, Webber FL, Black CL, Riley PF, Johnson IH, et al. Anticariogenicity of calcium phosphate complexes of tryptic casein phosphopeptides in the rat. J Dent Res 1995;74:1272-9.  Back to cited text no. 6
    
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Reynolds EC, Black CL, Cai F, Cross KJ, Eakins D, Huq NL, et al. Advances in enamel remineralisation: Casein phosphopeptide-amorphous calcium phosphate. J Clin Dent 1999;10:86-8.  Back to cited text no. 7
    
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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. 8
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Marsh PD. The oral microflora and biofilm on the teeth. In: Fejerskov O, Kidd E, editors. Dental Caries: The Disease and its Clinical Management. Oxford (UK): Blackwell and Munksgaard; 2003. p. 29-47.  Back to cited text no. 9
    
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Featherstone JD. Prevention and reversal of dental caries: Role of low level fluoride. Community Dent Oral Epidemiol 1999;27:31-40.  Back to cited text no. 10
    
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Munshi AK, Reddy NN, Shetty V. A comparative evaluation of three fluoride varnishes: An in-vitro study. J Indian Soc Pedod Prev Dent 2001;19:92-102.  Back to cited text no. 13
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White DJ. The application of in vitro models to research on demineralization and remineralization of the teeth. Adv Dent Res 1995;9:175-93.  Back to cited text no. 14
    
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Thaveesangpanich P, Itthagarun A, King NM, Wefel JS. The effects of child formula toothpastes on enamel caries using two in vitro pH-cycling models. Int Dent J 2005;55:217-23.  Back to cited text no. 16
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

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



 

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