Journal of Conservative Dentistry
Home About us Editorial Board Instructions Submission Subscribe Advertise Contact e-Alerts Login 
Users Online: 336
Print this page  Email this page Bookmark this page Small font sizeDefault font sizeIncrease font size
 


 
Table of Contents   
ORIGINAL ARTICLE  
Year : 2013  |  Volume : 16  |  Issue : 4  |  Page : 342-346
Role of casein phosphopeptide amorphous calcium phosphate in remineralization of white spot lesions and inhibition of Streptococcus mutans?


1 Department of Conservative Dentistry and Endodontics, National Dental College, Dera Bassi, Punjab, India
2 Department of Conservative Dentistry and Endodontics, Ragas Dental College and Hospital, Chennai, India
3 Department of Conservative Dentistry and Endodontics, Sir Venkateswara Dental College and Hospital, Chennai, India

Click here for correspondence address and email

Date of Submission11-Dec-2012
Date of Decision05-Mar-2013
Date of Acceptance01-Apr-2013
Date of Web Publication2-Jul-2013
 

   Abstract 

Introduction: To promote the remineralization by ionic exchange mechanism instead of invasive techniques many remineralizing agents can be used.
Objective: To evaluate the remineralization effects of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) on white spot lesions (WSLs) and its inhibitory effect on Streptococcus mutans colonization.
Materials and Methods: The study group consisted of 60 subjects exhibiting at least 1-WSL. Subjects were randomly divided into 2 groups: A test group using CPP-ACP cream (GC-Tooth Mousse, Leuven, Belgium) and a control group using only fluoride containing toothpaste for a period of 3-month. Baseline WSLs were scored using DIAGNOdent device (KaVo Germany) and the saliva samples were collected to measure S. mutans counts. After the 3-month period the WSLs were again recorded and the saliva collection was repeated.
Result: DIAGNOdent measurements were increased by time (P = 0.002) in the control group and no statistically significant difference (P = 0.217) was found in the test group by the 3-month period. In both groups, the mutans counts were decreased in the 3-month experimental period.
Conclusion: These clinical and laboratory results suggested that CPP-ACP containing cream had a slight remineralization effect on the WSL in the 3-month evaluation period however, longer observation is recommended to confirm whether the greater change in WSLs is maintained.

Keywords: Casein phosphopeptide-amorphous calcium phosphate; enamel; remineralization; white-spotlesions

How to cite this article:
Vashisht R, Indira R, Ramachandran S, Kumar A, Srinivasan MR. Role of casein phosphopeptide amorphous calcium phosphate in remineralization of white spot lesions and inhibition of Streptococcus mutans?. J Conserv Dent 2013;16:342-6

How to cite this URL:
Vashisht R, Indira R, Ramachandran S, Kumar A, Srinivasan MR. Role of casein phosphopeptide amorphous calcium phosphate in remineralization of white spot lesions and inhibition of Streptococcus mutans?. J Conserv Dent [serial online] 2013 [cited 2018 Dec 10];16:342-6. Available from: http://www.jcd.org.in/text.asp?2013/16/4/342/114370

   Introduction Top


Dental caries is one of the most common and preventable diseases of childhood. The process of caries formation is a cycle of remineralization and demineralization with various stages being either reversible or irreversible. White spot lesions (WSLs) are manifestations of the earliest stage of caries progression and are capable of being reversible. [1] These lesions are characterized by a white, chalky, opaque appearance and are commonly located in pits, fissures, and smooth surfaces of teeth. The most frequently detected localization of WSLs is on the cervical third of the tooth.

The WSLs can be spotted by many methods using a range of relatively new detection systems, including laser fluorescence, digital imaging fiber-optic trans illumination, and quantitative light-induced fluorescence. These techniques are considered as possible supplemental techniques for detecting incipient carious lesions. [2] The DIAGNOdent (KaVo, Germany) is a laser fluorescence caries detection device that operates by illuminating a tooth surface with pulses of red laser light and then analyses the emitted fluorescence. Changes in the mineral content result in changes in patterns of fluorescence. [3],[4] A numerical value is assigned to the degree of fluorescence, which is used to indicate the extent of caries.

The non-invasive treatment of early caries lesions by remineralization is a major advantage in clinical management and researchers have investigated the low cariogenic potential and possible cariostatic activity of dairy products such as milk, casein, caseinates, and cheeses. [5] Recently, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) derived from milk protein casein has been reported to reduce demineralization of the tooth structure and enhance remineralization. The anticariogenic potential and remineralizing effects have been shown in in vitro and in situ studies. [6],[7],[8],[9],[10],[11],[12],[13],[14],[15] CPP contains the cluster sequence of Ser (P)-Ser (P)-Ser (P)-Glu-Glu from casein [10],[12] and CPP-ACP is reported to have topical anticariogenic effects due to its ability to stabilize calcium and phosphate in an amorphous state. In acidic environment, ACP will separate from CPP, thereby increasing salivary calcium and phosphate levels. [7],[16] Moreover, CPP can stabilize the level of ACP in saliva by preventing precipitation of calcium and phosphate, and stabilize the level of calcium. It can be anticipated that routine use of CPP-ACP cream would prevent WSL formation and remineralize present lesions.

Therefore, the aim of this clinical study was to evaluate the remineralizing effect of a topical remineralizing cream containing CPP-ACP on WSL and its inhibitory effect on Strepteccocus mutans colonization and the tested hypothesis was that the use of CPP-ACP would have a remineralizing effect on WSLs and inhibiting effect on S. mutans.


   Materials and Methods Top


Study population

Approval for the clinical trial was obtained from the Ethics Committee. Sixty healthy subjects of both sexes with no complicating medical history exhibiting orthodontic treatment and having at least one WSL were invited to participate in the study. The patients who had taken antibiotics within the last month and are allergic to milk protein were excluded. The mean age of was 18 years ± 0.68 years and the mean DMFS (Decayed missing filled score) was 1.95 ± 2.22.

Treatment procedures

The subjects were randomly divided into 2 groups: A test group of 29 patients using Tooth Mousse, (GC Europe N.V., Leuven, Belgium) (Test group) and a control group with 31 patients using routine fluoride toothpastes (Colgate, 1450 ppm F). Oral health education was provided to both groups at baseline and 3-month. The test group was instructed to apply a standardized amount (approx. 1 g) of CPP-ACP containing cream. They were advised to use the cream on the teeth twice a day after brushing his/her teeth with fluoride toothpaste for a period of 3-month. The control group were informed and encouraged to use the same fluoride toothpaste 2 times a day during 3-month. The products were distributed to the subjects at baseline and they were asked to suffer from additional preventive measures based on fluoride during the experimental period.

DIAGNOdent measurements and visual examination

The teeth were isolated with sterile cotton rolls and the surfaces of the teeth were cleaned with sterile gauze pads and the baseline WSLs were scored by using two methods: Quantitatively measured using DIAGNOdent device, which was calibrated according to the manufacturer's instructions before every use. The same examiner made all measurements for 3 times to eliminate the operator effect for each lesion and was calibrated before the study in the use of DIAGNOdent in line with the manufacturer's directions.

The second method was the visual examination with the International Caries Detection and Assessment System II (ICDASII) index and only the subjects with scores 1 or 2 were included in the study. After the 3-month experimental period the WSLs were again recorded using the DIAGNOdent device and the changes in the amount of the values and scores were recorded.

Saliva collection and microbiological analysis

The unstimulated saliva samples were collected for 5 min from each participant at baseline and salivary flow rate and pH of the saliva was measured. The salivary samples were then transferred to the microbiology laboratory for S. mutans counts and the sample collections were repeated at the end of the 3-month experimental period. The samples were vortex-mixed to disperse bacterial aggregates and 0.1 ml of the samples were inoculated onto Mitis Salivarius Agar (- Mitis salivarius agar Salivarius Agar), Difco, Detroit, Mich, U.S.A.) supplemented with 15% sucrose and bacitracin (0.2 U/ml) (Sigma Chemical Co., St. Louis, Mo., U.S.A.) for the selective isolation of S. mutans.[17] Two to three isolates representing colonial types of S. mutans were subcultured for each sample, and biochemically differentiated by colony morphology, the presence of catalase, [18] fermentation of inulin, mannitol, melibiose, raffinose, sorbitol, production of acetoin and dextran, hydrolysis of arginin and esculin. [19] Counts below 10 colony-forming units were beyond detectable limits and recorded as 0 (not detectable).


   Statistical Methods Top


All data were processed by SPSS software, (version 19.0, SPSS Inc., Chicago, IL, USA). The differences in the groups at baseline and 3-month interval were calculated by Wilcoxon Signed Ranks Test. Mann Whitney U-test was used to determine statistically significant differences between both the groups at baseline and 3-month interval. The level of significance for all the tests was set at 5%.


   Results Top


The baseline salivary flow rate, salivary pH, DIAGNOdent measurements and salivary mutans values were not significantly different between the groups and were shown in [Table 1].
Table 1: The baseline salivary flow rate, salivary pH, DIAGNOdent measurements and salivary mutans values of both groups (mean±SD)

Click here to view


DIAGNOdent measurements

DIAGNOdent readings increased in the control group after 3-month period and were shown in [Figure 1] and this increase was found to be statistically significant (P = 0.002). There was a decrease between the baseline and 3-month results of the DIAGNOdent measurements in the test group, but this was not statistically significant (P = 0.217). Photographs of two patients who were in the test group were presented in [Figure 2] and [Figure 3].
Figure 1: DIAGNOdent measurements, (*) presents statistically significant difference

Click here to view
Figure 2: Intraoral photographs of a patient (a) white spot lesions were seen at baseline; (b and c) at 3-month evaluation period after the application of tooth mousse

Click here to view
Figure 3: Intraoral photographs of a patient (a) at baseline; (b) after the 3-month evaluation period after using tooth mousse 161 mm × 164 mm

Click here to view


Microbiological analysis

The S. mutans levels in the saliva at the baseline and 3-month periods were shown in [Figure 4]. In the both the control and the test groups, the mutans counts in the saliva were decreased in 3-month experimental period, which was found to be statistically significant (P = 0.000 for both groups).
Figure 4: Streptococcus mutans levels in the saliva at the baseline and 3-month periods, (*, ^) presents statistically significant

Click here to view



   Discussion Top


This study evaluated the remineralization efficiency of a 3-month home application of CPP-ACP on WSL and the inhibitory effect on S. mutans in comparison with a control group. WSL, which are the earliest phase of the caries process and which are reversible1 can be treated by conventional approaches involving the disadvantage of being invasive. [20] Therefore, many remineralization agents can be used to promote remineralization by ionic exchange mechanism instead of invasive techniques. In the current study, CPP-ACP containing product is used, which has been reported to have the potential in promoting remineralization [7],[8],[9],[10],[11],[12] and which maintains calcium and phosphate at a super saturated status compared to calcium and saliva and preserves them in close proximity to the enamel lesion, thereby decreasing demineralization and enhancing remineralization of enamel lesions. [6],[21]

In this study, laser fluorescence system was used to detect the WSLs in our study. The patented laser fluorescence method of DIAGNOdent works on the basis of the differing fluorescence between healthy dental substance and diseased dental substance and it detects even the smallest lesions; using a number scale and an audible alert. It has an acceptable sensitivity and specificity, which was reported before as compared with quantitative light-induced fluorescence. [22] Similarly, high sensitivity and acceptable specificity were also observed in other studies, such as those by Rocha et al. [23] and Anttonen et al. [24]

DIAGNOdent is considered to be useful and reproducible method that could be used to quantify WSLs and test the effectiveness of the preventive strategies. In the present study 3-month after the application of CPP-ACP in vivo, DIAGNOdent measurements exhibited a slight decrease in the demineralization process in comparison with the control group. The slight decrease in DIAGNOdent readings for the WSLs in TM group compared to that of the control group may be taken to indicate a reversal of the early caries process due to the usage of CPP-ACP might have taken place showing that new hydroxyapatite crystals were formed from the minerals existing in the paste by physical diffusion of the ions to the destroyed hydroxyapatite. The slight decrease in DIAGNOdent readings of TM group might also be explained by the changes in the surface properties of TM applied WSL, like the changings in the porosity of enamel because the main function of CPPs is to modulate bioavailability of calcium phosphate levels by maintaining ionic phosphate and calcium super saturation to increase remineralization however, longer observation is recommended to confirm whether the greater change in WSLs is maintained. Andersson et al. [25] reported a significant reduction in WSLs after the application of CPP-ACP at 12-month follow-up as we expected. In a systematic review, Yengopal and Mickenautsch [26] reviewed the short-term remineralization effect of CPP-ACP in clinical in situ trials and long-term caries-preventing effect for CPP-ACP in vivo in a randomized control trial. According to our results, CPPs could be suggested as an alternative preventive system against demineralization of early enamel lesions as published before. [13],[14],[27]

Our results indicated that the numbers of S. mutans decreased after the 3-month evaluation period in TM group. CPP also is believed to have an antibacterial and buffering effect on plaque and interfere in the growth and adherence of Streptococcus species.

In the current study, we cannot correlate the reduction in S. mutans values only to the inhibiting effect of CPP-ACP because there was a significant decrease in the control group, too. In an in vitro study of Erdem et al., it was reported that there was a reduction in bacterial viability of S. mutans in biofilm after the application of CPP-ACP but this reduction was not statistically significant. [28] These reductions in both groups might also be related to the changes in oral hygiene behaviors of the children, because all of the children received oral health education at the beginning of the study individually. It could be suggested that patients in both groups got more aware of their oral health-care and followed the oral hygiene instructions more and consumed less cariogenic food in comparison with before.

The subjects used the CPP-ACP containing paste just after a routine fluoride containing toothpaste because of the ethical reasons and it is reported that fluoride also has a tendency to interact with the ACP component of the casein complex and may precipitate out as calcium fluoride, leading both inorganic components ineffective. [29] But this might happen only if massive excess of fluoride exists, because there is an also fluoride containing CPP-ACP product, which contains 900 ppm of fluoride and Reynolds observed that a dentifrice containing 2% CPP-ACP plus 1100 ppm F was superior to all other formulations of mouth rinses and dentifrices containing CPP-ACP and fluoride. [13],[27] Controversially in a study of Beerens et al. [30] who evaluated the effects of CPP-ACPF (Amorphous calcium phosphate fluoride) paste on dental plaque and on the remineralization of enamel WSLs after the removal of fixed orthodontic appliances during a 3-month time period, it was reported that there were no differences between the commercially available CPP-ACPF (Amorphous calcium phosphate fluoride) paste and the fluoride free control paste on the remineralization of enamel WSLs and plaque composition.

It can be concluded that within the limitations of this study CPP-ACP containing remineralizing paste had a slight remineralization effect in the 3-month evaluation period and longer observation period with an enlarged sample size is recommended to confirm whether the greater remineralization in early caries lesions is maintained.

 
   References Top

1.Sudjalim TR, Woods MG, Manton DJ. Prevention of white spot lesions in orthodontic practice: A contemporary review. Aust Dent J 2006;51:284-9.  Back to cited text no. 1
[PUBMED]    
2.Yoon RK, Best JM. Advances in pediatric dentistry. Dent Clin North Am 2011;55:419-32.  Back to cited text no. 2
[PUBMED]    
3.Pinheiro IV, Medeiros MC, Ferreira MA, Lima K. Use of laser fluorescence (DIAGNOdent®) for in vivo diagnosis of occlusal caries: A systematic review. w 2008;1:45-51.  Back to cited text no. 3
    
4.Jayarajan J, Janardhanam P, Jayakumar P, Deepika. Efficacy of CPP-ACP and CPP-ACPF on enamel remineralization: An in vitro study using scanning electron microscope and DIAGNOdent. Indian J Dent Res 2011;22:77-82.  Back to cited text no. 4
[PUBMED]  Medknow Journal  
5.Carounanidy U, Sathyanarayanan R. Dental caries: A complete changeover, Part III: Changeover in the treatment decisions and treatments. J Conserv Dent 2010;13:209-17.  Back to cited text no. 5
[PUBMED]  Medknow Journal  
6.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
[PUBMED]    
7.Reynolds EC. Remineralization of enamel subsurface lesions by casein phosphopeptide-stabilized calcium phosphate solutions. J Dent Res 1997;76:1587-95.  Back to cited text no. 7
[PUBMED]    
8.Reynolds EC. Anticariogenic complexes of amorphous calcium phosphate stabilized by casein phosphopeptides: A review. Spec Care Dentist 1998;18:8-16.  Back to cited text no. 8
[PUBMED]    
9.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. 9
[PUBMED]    
10.Shen P, 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. 10
[PUBMED]    
11.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. 11
[PUBMED]    
12.Iijima Y, Cai F, Shen P, Walker G, Reynolds C, Reynolds EC. Acid resistance of enamel subsurface lesions remineralized by a sugar-free chewing gum containing casein phosphopeptide-amorphous calcium phosphate. Caries Res 2004;38:551-6.  Back to cited text no. 12
[PUBMED]    
13.Reynolds EC. Calcium phosphate-based remineralization systems: Scientific evidence? Aust Dent J 2008;53:268-73.  Back to cited text no. 13
[PUBMED]    
14.Reynolds EC. Casein phosphopeptide-amorphous calcium phosphate: The scientific evidence. Adv Dent Res 2009;21:25-9.  Back to cited text no. 14
[PUBMED]    
15.Zero DT. Recaldent: Evidence for clinical activity. Adv Dent Res 2009;21:30-4.  Back to cited text no. 15
[PUBMED]    
16.Hegde MN, Shetty S, Pardal D. Remineralization of enamel sub-surface lesion using casein phosphopeptide amorphous calcium phosphate: A quantitative energy dispersive X-ray analysis (EDAX). J Conserv Dent 2007;10:19-25.  Back to cited text no. 16
  Medknow Journal  
17.Gold OG, Jordan HV, Van Houte J. A selective medium for Streptococcus mutans. Arch Oral Biol 1973;18:1357-64.  Back to cited text no. 17
[PUBMED]    
18.Coykendall AL, Specht PA, Samol HH. Streptococcus mutans in a wild, sucrose-eating rat population. Infect Immun 1974;10:216-9.  Back to cited text no. 18
[PUBMED]    
19.Perch B, Kjems E, Ravn T. Biochemical and serological properties of Streptococcus mutans from various human and animal sources. Acta Pathol Microbiol Scand B Microbiol Immunol 1974;82:357-70.  Back to cited text no. 19
[PUBMED]    
20.Melrose CA, Appleton J, Lovius BB. A scanning electron microscopic study of early enamel caries formed in vivo beneath orthodontic bands. Br J Orthod 1996;23:43-7.  Back to cited text no. 20
[PUBMED]    
21.Hegde MN, Moany A. Remineralization of enamel subsurface lesions with casein phosphopeptide-amorphous calcium phosphate: A quantitative energy dispersive X-ray analysis using scanning electron microscopy: An in vitro study. J Conserv Dent 2012;15:61-7.  Back to cited text no. 21
[PUBMED]  Medknow Journal  
22.Shi XQ, Tranaeus S, Angmar-Månsson B. Comparison of QLF and DIAGNOdent for quantification of smooth surface caries. Caries Res 2001;35:21-6.  Back to cited text no. 22
    
23.Rocha RO, Ardenghi TM, Oliveira LB, Rodrigues CR, Ciamponi AL. In vivo effectiveness of laser fluorescence compared to visual inspection and radiography for the detection of occlusal caries in primary teeth. Caries Res 2003;37:437-41.  Back to cited text no. 23
[PUBMED]    
24.Anttonen V, Seppä L, Hausen H. Clinical study of the use of the laser fluorescence device DIAGNOdent for detection of occlusal caries in children. Caries Res 2003;37:17-23.  Back to cited text no. 24
    
25.Andersson A, Sköld-Larsson K, Hallgren A, Petersson LG, Twetman S. Effect of a dental cream containing amorphous cream phosphate complexes on white spot lesion regression assessed by laser fluorescence. Oral Health Prev Dent 2007;5:229-33.  Back to cited text no. 25
    
26.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. 26
[PUBMED]    
27.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. 27
[PUBMED]  Medknow Journal  
28.Erdem AP, Sepet E, Avshalom T, Gutkin V, Steinberg D. Effect of CPP-ACP and APF on Streptococcus mutans biofilm: A laboratory study. Am J Dent 2011;24:119-23.  Back to cited text no. 28
[PUBMED]    
29.Azarpazhooh A, Limeback H. Clinical efficacy of casein derivatives: A systematic review of the literature. J Am Dent Assoc 2008;139:915-24.  Back to cited text no. 29
[PUBMED]    
30.Beerens MW, van der Veen MH, van Beek H, ten Cate JM. Effects of casein phosphopeptide amorphous calcium fluoride phosphate paste on white spot lesions and dental plaque after orthodontic treatment: A 3-month follow-up. Eur J Oral Sci 2010;118:610-7.  Back to cited text no. 30
[PUBMED]    

Top
Correspondence Address:
Ruchi Vashisht
National Dental College, DERA Bassi, Punjab, House Number-1101, Sector-21B, Chandigarh, Union-Territory
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-0707.114370

Rights and Permissions


    Figures

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

  [Table 1]

This article has been cited by
1 enamel remineralization assessment after treatment with three different remineralizing agents using surface microhardness: an in vitro study
shetty, s. and hegde, m. and bopanna, t.
journal of conservative dentistry. 2014; 17(1): 49-52
[Pubmed]
2 Welk bewijs is er over het gebruik van Tooth Mousse bij het bevorderen van remineralisatie van witte vlek cariŽs?
Marit Kuijer,Merahl Philips
Mondhygienisten vademecum. 2014; 12(3): 1
[Pubmed] | [DOI]



 

Top
 
 
 
  Search
 
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  
 


    Abstract
   Introduction
    Materials and Me...
   Statistical Methods
   Results
   Discussion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed4902    
    Printed90    
    Emailed2    
    PDF Downloaded266    
    Comments [Add]    
    Cited by others 2    

Recommend this journal