|Year : 2015 | Volume
| Issue : 4 | Page : 559-564
Polarized light microscopic evaluation of remineralization by casein phosphopeptide-amorphous calcium phosphate paste of artificial caries-like lesion: An in vitro study
Shreyas Pradeepkumar Shah1, Praveen N Birur2
1 Department of Oral Medicine and Radiology, Bharati Vidyapeeth Dental College and Hospital, Navi Mumbai, Maharashtra, India
2 Department of Oral Medicine and Radiology, KLE's Institute of Dental Sciences, Bengaluru, Karnataka, India
|Date of Submission||29-May-2015|
|Date of Acceptance||07-Apr-2016|
|Date of Web Publication||19-Aug-2016|
Dr. Shreyas Pradeepkumar Shah
B-1003, Saiprasad Residency, Plot No. 252, Sector 10, Kharghar, Navi Mumbai 410 210, Maharashtra
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: Dental caries was considered to be an irreversible disease, but this concept of caries is changing because of the availability of many newer products for its treatment. One of such product is casein phosphopeptide-amorphous calcium phosphate (CPP-ACP). Aim: The present in vitro study was aimed to evaluate remineralization of artificial caries-like lesion by topical application of CPP-ACP paste. Materials and Methods: In this study, 40 caries-free premolar and molar teeth requiring extraction for orthodontic reasons and impactions were used. Artificial caries-like lesion was produced on sound enamel surface by immersing the samples in demineralizing solution for 96 h. Longitudinal sections were made through the white spot lesion with hard tissue microtome and were subjected to polarized light microscopic evaluation. The sections were divided into CPP-ACP group (Group I), positive control (Group II), and negative control (Group II). The Group I and Group II samples were subjected to 10 days of pH cycling, and all the samples were evaluated by polarized light microscopy. The lesion depth was calculated, and all the data was subjected to statistical analysis. Results: CPP-ACP group showed 27.306% decrease in lesion depth after 10 days of pH cycling. Conclusion: Based on the findings of this study, it can be concluded that the CPP-ACP paste can cause remineralization of white spot lesion and can be prescribed as topical application paste for the treatment of initial caries lesions.
Keywords: Casein phosphopeptide-amorphous calcium phosphate, demineralization, in vitro, pH cycling, polarized light microscopy
|How to cite this article:|
Shah SP, Birur PN. Polarized light microscopic evaluation of remineralization by casein phosphopeptide-amorphous calcium phosphate paste of artificial caries-like lesion: An in vitro study. J Indian Acad Oral Med Radiol 2015;27:559-64
|How to cite this URL:|
Shah SP, Birur PN. Polarized light microscopic evaluation of remineralization by casein phosphopeptide-amorphous calcium phosphate paste of artificial caries-like lesion: An in vitro study. J Indian Acad Oral Med Radiol [serial online] 2015 [cited 2022 Nov 28];27:559-64. Available from: http://www.jiaomr.in/text.asp?2015/27/4/559/188761
| Introduction|| |
The control of oral diseases presents one of the greatest challenges that must be met today by the dental profession. It has been a general failure of the dental profession that the treatment of disease has been overemphasized and the prevention minimized. Kauffman has stated that "the supreme ideal of dental profession should be to eliminate the necessity for its own existence." Dental caries is initiated by the demineralization of hard tissue of the teeth usually by organic acids produced from fermentation of dietary sugar by dental plaque odontopathogenic bacteria.  The very initial lesion appears chalky white so-called white spot lesion. At this stage, the dental caries is considered to be in a reversible stage. This process of demineralization may lead to white spot lesions and even cavitation in as little as 4 weeks if it is not treated, so early detection of these lesions is important to avoid cavitation and future loss of dental tissues by restorative procedures. Since many years, fluoride is the only material in its various forms proved to reduce dental caries in permanent and deciduous teeth. Recently, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) had shown anticariogenic property in various human and animal studies. It is claimed that this product when applied topically will remineralize the white spot lesions. This study was conducted with the aim to evaluate the remineralization of artificial caries-like lesion by CPP-ACP paste using polarized light microscopy.
| Materials and Methods|| |
The present study was carried out at the Department of Oral Medicine and Radiology, KLE Society's Institute of Dental Sciences, Bengaluru during June 2010 for 25 days. In this study, 40 caries-free premolar and molar teeth requiring extraction for orthodontic reasons and impactions were used. Teeth with visible or detectable caries, hypoplastic lesions, stains (extrinsic or intrinsic), and white spot lesions on any surface were excluded from the study. Artificial caries-like lesion was produced on sound enamel surface by immersing the samples with nail varnish with 1 × 1 mm exposed window in demineralizing solution for 96 h. The demineralizing solution used was containing 2.2 mM CaCl2, 2.2 mM KH2PO4, and 50 mM Acetic Acid. The remineralizing solution used was containing 1.5 mM CaCl2, 0.9 mM NaH2PO4, and 0.15 M KCl.
The longitudinal sections were made through the white spot lesion by mounting the samples on an acrylic block and using hard tissue microtome. They were then subjected to polarized light microscopic evaluation. The sections were divided into CPP-ACP group (Group 1), positive control (Group 2), and negative control (Group 3). The Group 1 and Group 2 samples were subjected to 10 days of pH cycling. pH cycling system was prepared using double organ bath, separating funnel, stand and rubber tubes [Figure 1]. The sections were placed in the pH cycling system for 10 days and each pH cycle involved 3 h of demineralization twice a day with in between remineralization. All the samples were evaluated by polarized light microscopy. The lesion depth was calculated using the software provided for polarized light microscope and all the data was subjected to statistical analysis [Figure 2].
|Figure 2: Representative pre-and post-treatment polarized light microscopic images of lesions in different groups|
Click here to view
| Results|| |
Lesions were found in samples of all groups with the polarized light microscope. Lesion depth was calculated for all three groups before and after the treatment. [Table 1] shows the average pretreatment lesion depth for the three experimental groups. In Group 1 (CPP-ACP), the average lesion depth was 619.127 μm with a range of 218.281 μm to 1265.688 μm. In Group 2 (positive control), the average lesion depth was 643.727 μm with a range of 350.000 μm to 950.406 μm and in Group 3 (negative control), the average lesion depth was 653.759 μm with a range of 500.344 μm to 816.313 μm. ANOVA found no significant differences among the three test groups (P = 0.895) [Table 2] and Graph 1.
[Table 3] shows the average post-treatment lesion depth for the three experimental groups. In Group 1 (CPP-ACP), the average lesion depth was 456.827 μm with a range of 150.0 μm to 853.094 μm. In Group 2 (positive control), the average lesion depth was 601.342 μm with a range of 369.313 μm to 884.141 μm and in Group 3 (negative control), the average lesion depth was 729.500 μm with a range of 503.125 μm to 875.000 μm. ANOVA found significant differences in lesion depths among the three test groups. Group 1 showed statistically significant difference in lesion depth when compared to that of Group 3 (P = 0.002) [Table 4] and Graph 1.
The paired t-test showed that Group 1 had a significant decrease in lesion depth after the specified treatment whereas Group 3 demonstrated a significant increase in lesion depths [Table 2] and Graph 2. [Table 5] and Graph 3 show the relative percentage of reduction of lesion depths. In Group 1, mean reduction in lesion depths was 27.306%; in Group 2, it was 5.187% and in contrast in Group 3 the lesion depths were increased by 11.774%. Even though the lesion depth decreased by 5.187% in the positive control, i.e., Group 2, it was less than in the group where CPP-ACP was used, i.e., Group 1 [Table 6].
| Discussion|| |
It is believed that CPP-ACP creates a large calcium and phosphate reservoir within the plaque to restrict mineral loss during a cariogenic episode. Certain animal studies have shown the remineralizing potential of CPP-ACP paste. ,, In many in vitro,,,,,,,,,,, as well as in vivo, studies the mechanism of its anticariogenic effect has been proposed. They have demonstrated that it is because of maintenance of supersaturation state of calcium around the tooth surface which reduces demineralization and promotes remineralization.  Hence, the use of CPP-ACP can be indicated in early caries/white spot lesions to prevent further caries progression. Many studies had demonstrated that higher the CPP-ACP concentration higher will be remineralization. ,, According to Schüpbach et al.,  CPP-ACP can be replaced with albumin in the pellicle so that it avoids the adherence of Streptococcus mutans and Streptococcus sobrinus. In our study, CPP-ACP demonstrated increase in remineralization.
In our study, Group 1 samples when subjected to 10 days of pH cycling with once daily topical application of CPP-ACP paste showed marked decrease in lesion depth. This could be because of subsurface remineralization of the white spot lesion. There was no increase in lesion depth in this group of samples, so it can be stated that the CPP-ACP paste has preventive role in lesion formation and lesion progression.
Group 2 samples were not treated with any medication and were subjected to 10 days of pH cycling and were kept in artificial saliva-like solution. This group also showed decrease in lesion depth but the decreased lesion depth was not statistically significant. Again this group did not show any increase in lesion depth. Hence, it can be stated that the saliva also has a protective role in lesion formation and lesion progression.
Group 3 samples were kept in normal saline during the test period. This group showed increase in lesion depth compared to the first readings. This could be because of progression of carious lesion as there was no protective or inhibitory effect from normal saline and no chance of remineralization of lesion. These results were expected since enamel with no protection will exhibit demineralization when exposed to an acidic challenge and the results were consistent with the findings of other studies. 
In our study, we have used pH cycling model. "pH cycling" refers to in vitro experimental protocols including exposure of substrates, enamel, or dentin, to combinations of remineralization and demineralization. These combination experiments are designed to simulate the dynamic variations in mineral saturation and pH associated with the natural caries process. As stated by ten Cate et al.,  modern pH cycling models have become methods of choice for many caries researchers applying in vitro techniques because they provide better simulation of the caries process for both mechanistic studies and for profile evaluations of toothpastes and mouthrinses. As in our study, we have used CPP-ACP paste as topical application paste and subjected to 10 days of pH cycling model, the results obtained from our study can be correlated clinically.
In most of the studies, polarized light microscope was used for qualitative assessment of lesion and microradiography as a quantitative measure of lesion mineralization as it shows more highly demineralized areas of the lesions as radiolucent areas and mineralized area as a radiopaque surface layer.  However, polarized light microscope can give high degree of differentiation between demineralized area and normal area of tooth sample. This differentiation is better than microradiography.  In our study, as we were concerned with lesion depth measurement, the polarized light microscope was supposed to be best method of choice.
In our study, freshly extracted, nondefective, noncarious premolars and third molars teeth were used. All the teeth were exposed to freshly prepared demineralizing solution with a standardized pH of 4.4. All teeth were exposed to freshly prepared remineralizing solution with a standardized pH of 7.00 and all surface treatments and data interpretation were performed by a single operator.
The limitations in our study are bacterial species that inhabit the oral cavity, and the formation of plaque on the tooth surface cannot be duplicated in an in vitro study. The varying pH levels in the mouth cannot be duplicated in vitro. Intraoral interactions with food, beverages, and salivary buffering effects cannot be duplicated in in vitro study.
This study has verified the effect of CPP-ACP paste on tooth mineralization. In our study, the CPP-ACP helped in reduction of lesion depth which correlates with the findings from previous studies. ,,,,,,,,,,, The present study has also shown the ease and efficacy of polarized light microscope in caries research.
| Conclusion|| |
In the present study, lesion depth of artificial caries-like lesion was measured with polarized light microscopy. The lesion depth was measured before and after the topical application of CPP-ACP paste in test group after 10 days of pH cycling. In this study, there were no significant differences among the groups and between the groups with regard to lesion depth. There is a significant difference between CPP-ACP group and negative control and between CPP-ACP group and positive control with respect to lesion depth.Based on the findings of this study, the CPP-ACP paste can be prescribed as topical application paste for the white spot lesions/initial caries lesion. The present study has also demonstrated the efficacy and ease of polarized light microscope in caries research studies.
This in vitro study used a small sample size in each group. It would be beneficial to perform an in vitro study similar to this one using more samples in all groups. Doing so, it might increase the level of significant differences among and between the groups. This study has not compared the tool used for lesion depth measurement; it would have been better if we use multiple evaluation methods for accurate results.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Moezizadeh M, Moayedi S. Anticariogenic effect of amorphous calcium phosphate stabilized by casein phosphopeptid: A review article. Res J Biol Sci 2009;4:132-6.
Adebayo OA, Burrow MF, Tyas MJ. Dentine bonding after CPP-ACP paste treatment with and without conditioning. J Dent 2008;36:1013-24.
Reynolds EC. The prevention of sub-surface demineralization of bovine enamel and change in plaque composition by casein in an intra-oral model. J Dent Res 1987;66:1120-7.
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.
Yamaguchi K, Miyazaki M, Takamizawa T, Inage H, Moore BK. Effect of CPP-ACP paste on mechanical properties of bovine enamel as determined by an ultrasonic device. J Dent 2006;34:230-6.
Devold TG, Rykke M, Isabey D, Sørensen ES, Christensen B, Langsrud T, et al
. In vitro
studies of adsorption of milk proteins onto tooth enamel. Int Dairy J 2006;16:1013-7.
Rahiotis C, Vougiouklakis G. Effect of a CPP-ACP agent on the demineralization and remineralization of dentine in vitro
. J Dent 2007;35:695-8.
Adebayo OA, Burrow MF, Tyas MJ. Effects of conditioners on microshear bond strength to enamel after carbamide peroxide bleaching and/or casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) treatment. J Dent 2007;35:862-70.
Oshiro M, Yamaguchi K, Takamizawa T, Inage H, Watanabe T, Irokawa A, et al.
Effect of CPP-ACP paste on tooth mineralization: An FE-SEM study. J Oral Sci 2007;49:115-20.
Sudjalim TR, Woods MG, Manton DJ, Reynolds EC. Prevention of demineralization around orthodontic brackets in vitro
. Am J Orthod Dentofacial Orthop 2007;131:705.e1-9.
Rees J, Loyn T, Chadwick B. Pronamel and tooth mousse: An initial assessment of erosion prevention in vitro
. J Dent 2007;35:355-7.
Shirahatti RV, Ankola AV, Nagesh L, Hallikerimath S. The effects of three different pastes on enamel caries formation and lesion depth progression - An in vitro
study. J Oral Health Community Dent 2007;1:1-6.
Lööf J, Svahn F, Jarmar T, Engqvist H, Pameijer CH. A comparative study of the bioactivity of three materials for dental applications. Dent Mater 2008;24:653-9.
Kumar VL, Itthagarun A, King NM. The effect of casein phosphopeptide-amorphous calcium phosphate on remineralization of artificial caries-like lesions: An in vitro
study. Aust Dent J 2008;53:34-40.
Piekarz C, Ranjitkar S, Hunt D, McIntyre J. An in vitro
assessment of the role of tooth mousse in preventing wine erosion. Aust Dent J 2008;53:22-5.
Ranjitkar S, Rodriguez JM, Kaidonis JA, Richards LC, Townsend GC, Bartlett DW. The effect of casein phosphopeptide-amorphous calcium phosphate on erosive enamel and dentine wear by toothbrush abrasion. J Dent 2009;37:250-4.
Manton DJ, Bhide R, Hopcraft MS, Reynolds EC. Effect of ozone and tooth mousse on the efficacy of peroxide bleaching. Aust Dent J 2008;53:128-32.
Sakr AK, Karargy AA, Sherif MM. A short-term clinical study on antimicrobial effects of Recaldent (CPP ACP). Dent Mater 2009;25:e5-46.
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.
Walker G, Cai F, Shen P, Reynolds C, Ward B, Fone C, et al.
Increased remineralization of tooth enamel by milk containing added casein phosphopeptide-amorphous calcium phosphate. J Dairy Res 2006;73:74-8.
Schüpbach P, Neeser JR, Golliard M, Rouvet M, Guggenheim B. Incorporation of caseinoglycomacropeptide and caseinophosphopeptide into the salivary pellicle inhibits adherence of mutans streptococci. J Dent Res 1996;75:1779-88.
ten Cate JM, Buijs MJ, Damen JJ. pH-cycling of enamel and dentin lesions in the presence of low concentrations of fluoride. Eur J Oral Sci 1995;103:362-7.
Wefel JS, Harless JD. Comparison of artificial white spots by microradiography and polarized light microscopy. J Dent Res 1984;63:1271-5.
Thylstrup A, Fejerskov O, Mosha HJ. A polarized light and microradiographic study of enamel in human primary teeth from a high fluoride area. Arch Oral Biol 1978;23:373-80.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]