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The in vitro remineralizing effect of CPP-ACP and CPP-ACPF after 6 and 12 weeks on initial caries lesion

Abstract Objective: The aim of this in vitro study was to determine the effects of remineralization promoting agents containing casein phosphopeptide-stabilized amorphous calcium phosphate (CPP-ACP), or CPP-ACP in combination with fluoride (CPP-ACPF) on artificial white spot lesions (WSLs) after 6 and 12 weeks. Methodology: White spot lesions were created on 123 sectioned premolars (246 specimens) with a demineralization solution during a 96 hours pH-cycling regime. Two experimental groups were created: a CPP-ACP group (Tooth Mousse™), and a CPP-ACPF group (Mi Paste Plus™). Additionally, two control groups were created, one using only a conventional toothpaste (1450 ppm fluoride) and another one without any working agents. All teeth were also daily brushed with the conventional toothpaste except the second control group. Tooth Mousse™ and Mi Paste Plus™ were applied for 180 seconds every day. The volume of demineralization was measured with transverse microradiography. Six lesion characteristics regarding the lesion depth and mineral content of WSLs were also determined. Results: The application of CPP-ACP and CPP-ACPF had a significant regenerative effect on the WSLs. Compared to Control group 1 and 2 the volume of demineralization after 6 weeks decreased significantly for CPP-ACP (respectively p<0.001 and p<0.001) and CPP-ACPF (respectively p=0.001 and p=0.003). The same trend was observed after 12 weeks. For the CPP-ACPF group, WSL dimensions decreased significantly between 6 and 12 weeks follow-up (p=0.012). The lesion depth reduced significantly after application of CPP-ACP and CPP-ACPF but increased significantly in the Control groups. Mineral content increased for CPP-ACP and CPP-ACPF after an application period of 12 weeks, but this was only significant for CPP-ACP. Conclusions: Long-term use of CPP-ACP and CPP-ACPF in combination with a conventional tooth paste shows beneficial effects in the recovery of in vitro subsurface caries lesions.


Introduction
The white spot lesion (WSL) is the first clinical sign of dental caries. 1 It is defined as a subsurface enamel porosity caused by carious demineralization and is clinically presented as a milky white opacity when located on smooth surfaces. 2 The outermost layer of enamel, covering the lesion, remains relatively intact and appears radiopaque in contact radiographs. 3 In general, orthodontic patients have significantly more white spot lesions than non-orthodontic patients. [2][3][4][5] The prevalence of WSLs prior to an orthodontic treatment ranges from 15% to 40%, whereas the incidence of WSLs occurring during orthodontic treatment ranges between 30% and 70%. 6,7 WSL assessment by means of quantitative light-induced fluorescence (QLF) even reported a prevalence of 96%. 8 An adequate oral hygiene regime and a diet with low carbohydrate intake are important factors reducing the risk for decalcification and WSL formation. 6,9,10 There are many possibilities to intervene in the process of demineralization of enamel to arrest or even reverse the progress of the lesion. 1 The influence of dairy products (milk, milk concentrates, and cheeses) on caries is known ions deep into the lesion enables substantial crystal growth (remineralization) throughout the body of the lesion. 13,15,16 However, several in vitro studies detected only limited effects or were even unable to detect any significant clinical effects of CPP-ACP(F). Such discrepancy could be explained by the fact that sufficient time is needed for an effective diffusion of CPP-ACP(F) into the body of a subsurface lesion and a subsequent remineralization. Consequently, the study duration must be long enough so that CPP-ACP(F) can act as a saliva biomimetic. Therefore, the objectives of this study were to determine the remineralizing effect of CPP-ACP and CPP-ACPF compared to a conventional toothpaste (1450 ppm fluoride) and a negative control group, and to evaluate the characteristics of white spot lesions in each group after 6 and 12 weeks.

Methodology Preparation of enamel blocks
One hundred and twenty-three extracted premolars were collected, washed in tap-water, and stored in 0.1% thymol solution at 4°C. Only premolars with a healthy enamel surface were selected for this study. Teeth with

Study and control groups
The premolar parts were divided in four groups.
The prophylactic protocol and the application of remineralisation promoting agents as described by the manufacturer are shown in Table 1. Each of the four groups was divided into 2 subgroups with 6 or 12 weeks follow-up, respectively.
Volume quantification of demineralization, mineral content, and caries lesion depth  these parameters are visually presented in a chart with the mineral content (volume fraction of the mineral) plotted against the depth of the lesion. The average of three measurements for each white spot lesion was used for each parameter.

Volume of demineralization
An univariate 2-way ANOVA was used to compare the mean differences of the volume of demineralization between the experimental and control groups, and between 6 and 12 weeks follow-up. In order to avoid a type-1 error, the significance value was adjusted by the Bonferroni correction. Post-hoc testing was conducted by using an independent samples t-test to evaluate the single effect of time and treatment. Normality and homogeneity of variances were checked.

Lesion characteristics
An independent, non-parametric sample test

Descriptive statistics
By sectioning the premolars, some of the samples became useless due to fracturing of the outer surface of the white spot lesion. The final study sample for 6 and 12 weeks follow-up is shown in Table 1.

Volume of demineralization
The assumptions for homogeneity (p>0.05) and normality after log-transformation (p>0.05) are met for every combination. Table 2   clearly illustrated in Figure 2. Table 2 shows the means and standard deviations of the lesion characteristics of the four study groups.

Significant differences between groups are indicated
with the corresponding p-value. After 6 weeks, significant differences occur for MBL (p<0.001), DL (p<0.001), and BMC (p<0.001). Post-hoc testing of these significant lesion characteristics shows significant differences, primarily between the experimental groups CPP-ACP and/or CPP-ACPF at one side, and control groups 1 and/or 2 at the other side.
A significant difference is also determined between control groups 1 and 2 for BMC (p=0.039).
After 12 weeks, significant differences occur for DSL (p=0.001), DBL (p<0.001), MBL (p<0.001), DL (p<0.001), and BMC (p=0.039). Post-hoc testing of these significant lesion characteristics shows similar results compared to 6 weeks follow-up. Significant differences are detected between the experimental groups CPP-ACP and/or CPP-ACPF at one side, and control groups 1 and/or 2 at the other side.
The differences within each study group between 6 and 12 weeks follow-up for the lesion characteristics are shown in Table 3

Discussion
Long-term application of CPP-ACP(F) combined with a conventional tooth paste reduced the amount of demineralization of artificial white spot lesions ( Table   2). The amount of demineralization was comparable application of CPP-ACP and CPP-ACPF was mainly due to an increase in the mineral content in the body of the lesion (MBL, Table 2) and a concomitant decrease in lesion depth especially after 12 weeks when compared to the control groups (DL, Table 2).
On the other hand, the mineral content of the enamel layer covering the lesion remained unchanged (MSL, Table 2). These results clearly indicate that the long- The duration of similar in vitro studies varies, going from 10 days 11,13,27 up to 21 days 23,28 . This is in contrast with the 6 and 12 weeks used in the current study.
In vitro studies with a duration of 12 weeks are labor intensive, but this approach also yielded new insights.
Both volume and mineral content showed significant improvement going from 6 to 12 weeks of follow-up.
The remineralizing potential of CPP-ACP and CPP-ACPF as observed in the current study has also been observed in other in vitro studies, 11,25,29 in situ studies, 15,30 and in vivo studies. 31  The latter emphasizes the importance of prolonged use in order to reduce the white spot lesion.