|Year : 2015 | Volume
| Issue : 4 | Page : 269-273
Effect of casein phosphopeptide-amorphous calcium phosphate (cpp-acp) on caries-like lesions in terms of time and nano-hardness: An in vitro study
Shanthan Mettu1, Namineni Srinivas2, CH Reddy Sampath2, Nallanchakrava Srinivas1
1 Department of Pediatric Dentistry, Panineeya Institute of Dental Sciences, Kamalanagar, Dilsukhnagar, Hyderabad, India
2 Department of Pediatric Dentistry, Sri Sai College of Dental Surgery and Research Center, Vikarabad, Andhra Pradesh, India
|Date of Web Publication||18-Sep-2015|
Dr. Shanthan Mettu
Senior Lecturer, Department of Pediatric Dentistry, Panineeya Institute of Dental Sciences, Kamalanagar, Dilsukhnagar, Hyderabad - 500 060, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context: Time bound increase in the nanohardness of the enamel after remineralization with casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) in a regular interval of 1 h has not been explored in the literature to a greater extent. Aims: To determine and compare the maximum hardness of the remineralized caries-like lesions, in terms of nanohardness and the rate of achieving maximum hardness at 1-h interval, after treatment with artificial saliva and CPP-ACP, over 12 h. Materials and Methods: Fifty longitudinal sections of extracted sound permanent maxillary central, lateral incisors were immersed in demineralizing solution for 4 days. The samples were then randomly divided into three groups, consisting of 12 sections each for soaking them in three different media-isotonic saline, artificial saliva, and CPP-ACP for 12 h. The nanohardness was measured on the labial surface, at baseline, after erosion, and after remineralization at 1-h interval. Statistical Analysis Used: The data was analyzed with paired t-test, one-way analysis of variance (ANOVA) and post-hoc analysis. Results: CPP-ACP increased the enamel hardness significantly (P < 0.001), at an increased rate, than artificial saliva. Conclusions: This study has provided an insight into the frequency of use of CPP-ACP, once per day, as the nanohardness of enamel samples increased within 1 h of application and remained within the normal limits after 12 h.
Keywords: CPP-ACP, nanohardness, remineralization
|How to cite this article:|
Mettu S, Srinivas N, Reddy Sampath C H, 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 Pedod Prev Dent 2015;33:269-73
|How to cite this URL:|
Mettu S, Srinivas N, Reddy Sampath C H, 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 Pedod Prev Dent [serial online] 2015 [cited 2019 Dec 10];33:269-73. Available from: http://www.jisppd.com/text.asp?2015/33/4/269/165657
| Introduction|| |
The current concept regarding cariogenesis is that a carious lesion, either clinically invisible or detectable, is the accumulation of numerous episodes of de- and remineralization, rather than a unidirectional demineralization process. Studies  have shown certain areas of the incipient caries lesions can be demineralizing, whereas other areas can be remineralizing, implying that remineralization could occur in clinically detectable incipient lesions. Incipient carious lesion appears white because the loss of mineral changes the refractive index, when compared to that of surrounding normal enamel. 
Since almost a decade, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) derived from cow's milk has been reported to reduce the demineralization process and enhance the remineralization process of the tooth structure.  In-depth analysis have shown that a particular part of casien protein, CPP, is responsible for tooth protective activity. CPP plays an important role as ACP carrier, thereby localizing the highly soluble calcium phosphate phase at the tooth surface. This localization maintains high concentration gradients of calcium and phosphate ions in the subsurface enamel, thereby enabling remineralization. 
In various enamel hardness studies ,,,,,,, on correlation between hardness and chemical composition of the human teeth, it was revealed that the hardness depended on the degree of mineralization of the enamel and the baseline enamel hardness values were reported to be ranging from 330.06 to 430.53 Vickers hardness number (VHN). Even minor changes in the surface of enamel during demineralization, remineralization, and storage are important consideration for accurate determination of nano-mechanical properties, which can be appreciated by nanoindentations. As nanoindentation can examine a surface layer as thin as 1μm with a load as low as 10nN, for the changes, its importance as an accurate technique for determination of mechanical properties of biological hard tissues has increased during the recent years.
Hence, an in vitro study was conducted to determine and compare the maximum hardness of the remineralized caries-like lesions, in terms of nanohardness, achieved after treatment with artificial saliva and CPP-ACP. Also the rate of achieving maximum hardness at 1-h intervals each was examined after remineralization of the demineralized enamel samples in artificial saliva and CPP-ACP, over 12 h.
| Materials and Methods|| |
This study was approved by the Ethical Committee of the Institutional Review Board and was carried out in collaboration with Defence Metallurgical Research Laboratory (DMRL), Hyderabad, Andhra Pradesh, India.
The sample size consisted of 25 permanent maxillary incisors, extracted due to periodontal compromise. The extracted teeth were washed with deionized water, and the debris covering the tooth surface was removed using scalers and sterilized using gamma radiation of 25 kGy.  Evaluation of the teeth was done under stereomicroscope to include the teeth free of caries, developmental defects, enamel fractures, discoloration, and teeth with no attempted pulp therapy. In order to maintain the physical properties, the specimens were stored in Hanks' Balanced Salts Solution (HBSS). 
The selected 25 teeth were longitudinally sectioned into 50 halves and the roots were cutoff with 400 μm thick diamond cutoff wheel at 200 rpm, using Minitome  and were reevaluated for microfractures. The cut-sections were then stored in HBSS at room temperature till the experiment started. Four of these longitudinal sections were randomly selected and mounted for nanoindentation, to determine the baseline values for normal enamel.
The remaining 46 longitudinal sections of enamel were mounted in self-cured acrylic resin, with the cut surface leveled on top and lying flat and parallel to the horizontal plane. Caries-like lesions were prepared in each using carbopol method of White as modified byReynolds at pHof4.8.  The enamel sections were incubated in the demineralizing solution for 4 days at 37°C with a change of solution after 2 days. Four of these demineralized samples were nanoindented for the baseline values of the demineralized enamel.
The artificial saliva based on xanthan gum  was prepared (pH 7.0) and stored in an amber-colored plastic bottle, at room temperature, in a cool dark place.
Out of the remaining 44 demineralized samples, 36 were randomly divided into three groups:
- Group A (negative control)-12 longitudinal teeth sections, soaked in isotonic saline.
- Group B (positive control group)-12 longitudinal teeth sections, soaked in artificial saliva.
- Group C (experimental group)-12 longitudinal teeth sections, soaked in CPP-ACP.
The samples from these three groups were soaked in their respective solutions, in 36 different specimen bottles, labeled as A-I to A-XII, B-I to B-XII, and C-I to C-XII, respectively, for each group. The samples of the Group A were soaked in isotonic saline solution in different specimen bottles from 1 to 12 h, taking away one sample in an interval of every hour; that is, sample from the specimen bottle A-I was removed after soaking it in isotonic saline for 1 h and A-II after 2 h, so on and so forth till 12 h. The similar procedure as above was performed for the samples of Group B, which were soaked in artificial saliva. The samples of Group C were applied with a 0.5-mm layer of CPP-ACP (GC Tooth Mousse™ ) on their surfaces for 3 min.  Manufacturer's instructions regarding the procedure and post-application care, which advice avoidance of rinsing after application were followed. Later each sample was stored in artificial saliva in different specimen bottles from 1 to 12 h, taking away one sample at an interval of every hour, similar to the Groups A and B.
These samples were washed in deionized water, blotted dry, and mounted on the indentation table of the nanohardness tester. Area of the enamel to be indented was evaluated under a light microscope with camera, which was attached to the CSM TM nanohardness tester. An automated reference indentation was carried out and the positions of the indenations to be made were programmed. These samples were subjected to nanoindentations with electromagnetically controlled load of 10 nN and the values were tabulated.
Statistical analysis was done using Statistical Package for Social Sciences (SPSS) software (version 12.0). P-value <0.05 was considered statistically significant. The recorded values were analyzed statistically using the independent samples' Student's t-test for two group comparison and one-way analysis of variance (ANOVA) for multiple group comparison with post-hoc analysis using least significant difference (LSD) method for pair-wise comparisons. The recorded values are presented as mean ± standard deviation, range, and significance.
| Results|| |
When mean hardness was measured in relation to time, no change in hardness was observed in enamel samples of Group A, which were treated with isotonic saline solution, for a period of 12 h. On the other hand, mean hardness of samples in artificial saliva (Group B), showed an increase to normal limits at 3 rd h, reached the maximum (445.80 VHN) at 6 th h, and gradually reduced from the 7 th h itself. In Group C (samples treated with CPP-ACP), increase in mean hardness (394.47 VHN) was noticed within 1 h, reaching the peak (447.79 VHN) at 6 th h, to remain well within normal limits till 12 h [Figure 1].
|Figure 1: No change in mean hardness was observed in enamel samples of Group A treated with isotonic saline solution for 12 h. Mean hardness of samples in artificial saliva, that is, Group B, showed an increase to normal limits at 3 rd h, reached the maximum at 6 th h, and gradually reduced from the 7 th h. For enamel samples treated with CPP-ACP, that is, Group C, increase in mean hardness was noticed within 1 h, reaching the peak at 6 th h and remained well within normal limits till 12 h. CPP-ACP = Casein phosphopeptide-amorphous calcium phosphate|
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The mean nanohardness of the normal enamel samples (395.01 ± 5.97 VHN) was found to be significantly higher (P < 0.001) when compared to that of demineralized enamel samples (276.62 ± 2.21 VHN), using Student's t-test [Table 1]. The overall mean hardness was highest for Group C (429.54 ± 17.60 VHN) was significantly higher as compared to Groups A (272.64 ± 2.85 VHN) and B (381.50 ± 54.83 VHN). Likewise, Group B showed a significantly higher mean hardness as compared to Group A. The difference of nano-hardness values between the three groups and also within them were statistically significant (P < 0.001) using ANOVA and post-hoc analysis using LSD [Table 2] and [Table 3].
|Table 1: Comparison of the mean, standard deviation, and significance of baseline hardness values of enamel; before and after demineralization|
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|Table 2: Comparison of the mean, standard deviation, and range of the hardness of the demineralized enamel after remineralization among the control and experimental groups|
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|Table 3: Descriptive analysis shows the intergroup comparison of the mean difference and significance (P) value of nanohardness of the enamel samples among the control and experimental groups|
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| Discussion|| |
The cross-sections of permanent maxillary central and lateral incisors were chosen in this study for induction of artificial caries and for nanoindentation because these are flat on surface and straight in profiles that are mandatory for proper contact of the indenter tip. Teeth were sterilized using gamma radiation of 25 kGy as it does not change either enamel hardness or its resistance to demineralization.  A minitome was used in this study to achieve longitudinal sections for nanoindentations. 
Featherstone  et al., compared the artificial caries-like lesions by quantitative microradiography and microhardness profiles and concluded that mineral gain or loss in enamel as a result of remineralization and demineralization can be measured as hardness change. In this study, the mean nanohardness of the longitudinal sections of the teeth, before being subjected to demineralization was 395.02 ± 31.84 VHN. These values are similar to those in studies by Ryu et al., and Collys et al., , but they were found to be higher than the values reported by Wongkhantee et al.,  and Sukasame et al.  It was reported that the enamel hardness varies depending on the degree of mineralization of the enamel and it decreases from the outer enamel surface towards the dentinoenamel junction, which explains the range of baseline values. ,
The complexity of the development of caries lesion has led to the development of model systems to improve our understanding of the mechanism of de- and remineralization processes. In vitro chemical models provide information about the effects of caries preventive agents on the demineralization and remineralization dynamics at the surface and in the subsurface of the teeth.  Chemical caries model, similar to carbopol method of White as modified by Reynolds at pH of 4.8,  for the production of artificial caries lesion was used, which consisted 0.1 mol/l lactic acid, 500 mg/l hydroxyapatite, and 20 g/l carbopol C907 as this demineralization procedure produced consistent subsurface lesions of 80-110 μm depth with intact surface layers. In our study after the longitudinal sections of tooth samples were demineralized, the mean nanohardness was measured and recorded as 276.63 ± 16.04 VHN. These baseline nanohardness values for demineralized sample were similar to those in studies by Wongkhantee and coworkers,  Sukasame and colleagues,  and Wilson and Love.  In the present study, the mean nanohardness reduction, after the demineralization was 118.39 VHN.
In this present study, nanohardness of the enamel samples in Group A, those immersed in isotonic solution, did not increase significantly, which is at par with the results reported by Eisenburger and colleagues. 
The artificial saliva used in the study contained xanthan gum, potassium chloride, sodium chloride, magnesium chloride, calcium chloride, dipotassium hydrogen orthophosphate, and methyl para-hydroxybenzoate, which helps to remineralize the enamel.  The nanohardness of the teeth specimen in Group B, increased significantly after remineralization by artificial saliva. The mean enamel nanohardness increased by 104.87 VHN in the artificial saliva group. The maximum hardness, similar to the baseline values for this group was attained at the 6 th h. This finding is consistent with results of the in vitro studies done by Eisenburger and colleagues. 
In this study, a 0.5 mm layer of CPP-ACP was applied on the enamel surfaces of the samples in Group C, for 3 min, as per manufacturer's instructions and each sample was immersed in artificial saliva as recommended by Muratha and colleagues  to simulateoral environment. The increase in the nanohardness resulting from remineralization by CPP-ACP had started during the 1 st h itself, after the exposure of enamel sample. The initial mean nanohardness (394.47 VHN) achieved in this group during the 1 st h of exposure was well within the baseline values for the normal enamel resulting in the mean increase by 152.91 VHN.
Adding calcium and phosphate together will result in the formation of insoluble calcium phosphate crystals, but in the presence of CPP this does not happen, and the calcium and phosphate stay in a form that can penetrate into the tooth enamel and repair the areas that have been damaged by the activity of caries bacteria. CPP has an ability to stabilize ACP in solution as nanocomplexes and substantially increase the level of calcium phosphate in dental plaque. The proposed mechanism of anticariogenicity for the CPP-ACP is that they localize ACP in dental plaque which buffers the free calcium and phosphate ion activities, thereby helping to maintain a state of supersaturation with respect to tooth enamel and reducing demineralization and enhancing remineralization.  Rose  investigated the proposed mechanism of action of CPP-ACP and demonstrated that CPP-ACP and calcium competed for the same binding sites on Streptococcus mutans.
During the in vitro remineralization process of the demineralized enamel using either artificial saliva or CPP-ACP, there is an initial surge in the absorption of calcium and phosphate. The time taken for this initial surge when treated with CPP-ACP is less when compared to artificial saliva as the calcium and phosphate in CPP-ACP are in amorphous soluble state. After this initial surge in absorption of calcium and phosphate, the maximum hardness is attained. Because of the Guar gum, a binder and retainer present in CPP-ACP, the calcium and phosphate bind more to the tooth surface, which is responsible for the sustainability of the remineralization effect of CPP-ACP for longer duration when compared to artificial saliva. ,
Minor changes in the surface of enamel during demineralization, remineralization, and storage are important consideration for accurate determination of nanomechanical properties.  Nanoindentation can examine a surface layer as thin as 1μm with a load as low as 10nN. The loading control system can be powered by electromagnetic force and the load ranges from 10 to 100nN. The position of the nanoindentation could be programmed and the indents observed with a charge-coupled device (CCD) camera attached to the tester. An automated reference indentation is mandatory before the indentations are carried out at the programmed positions, as it ensures that the indenter tip is perpendicular to the surface to be indented.
The incipient caries lesions are the surface carious lesions and any changes in mechanical properties in these layers can be best detected by nanoindentation technique. The added advantage of using nanoindentation is that it checks the breakage of the enamel rods without causing any further damage to the enamel surface. Even the forces within the physiological limits such as, the normal tooth brushing force can bring about change in the mechanical properties of the remineralizing incipient lesions. Hence in the present study, the nanoindentation technique with a minimum load of 10nN was considered. ,
| Conclusions|| |
The following conclusions can be made from the present study:
- The maximum mean hardness of the demineralized enamel samples increased from 276.63 ± 16.04 VHN to 381.50 ± 54.83 VHN when remineralized with artificial saliva and to 429.54 ± 17.60 VHN when remineralized with CPP-ACP. No change in the hardness was observed in enamel samples treated with isotonic saline solution.
- The application of CPP-ACP paste with continuous replenishment of saliva like solution significantly hardened the demineralized enamel, within 1 h of application and the hardness remained within the normal limits even till 12 h.
- The CPP-ACP increased the hardness of the demineralized enamel and had a greater effect on enamel in terms of time and hardness than did artificial saliva.
| Acknowledgement|| |
I offer my sincere thanks and acknowledge the support given by Dr. B. Venkatraman, Scientist and Head, and Dr. Gokul Lakshmi, Ph. d, scientist C, Surface Engineering Group, Defence Metallurgical Research Laboratory, Kanchanbagh, Hyderabad for their enthusiastic help during the Nanoindentation.
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[Table 1], [Table 2], [Table 3]