An in vitro evaluation of the demineralization inhibitory effect of F(-) varnish and casein phosphopeptide-amorphous calcium phosphate on enamel in young permanent teeth.

AIMS AND
OBJECTIVES: To determine the demineralization inhibitory potential of fluoride varnish and casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and to compare and evaluate the additive effect of fluoride varnish + CPP-ACP.
MATERIALS AND METHODS: Ten healthy premolar teeth that were extracted for orthodontic purposes were collected, and each tooth was longitudinally sectioned buccolingually and mesiodistally into four sections. The teeth were then assigned to four different treatment groups namely fluoride varnish, CPP-ACP, F(-) varnish followed by CPP-ACP and control. The prepared enamel samples were suspended in an artificial caries challenge for 10 days. The demineralizing inhibitory effects of the groups were recorded using polarized light microscopy. STATISTICAL ANALYSIS USED: Statistical analysis was carried out using analysis of variance and Duncan's multiple range tests.
RESULTS: The mean lesion depths of all the groups were Group 1 (fluoride varnish): 104.71, Group 2 (CPP-ACP): 127.09, Group 3: (F(-) varnish + CPP-ACP): 82.34, Group 4 (control): 146.93.
CONCLUSION: Demineralization inhibitory potential on the additive use of F(-) varnish and casein phosphopeptide was superior to fluoride varnish or CPP-ACP applied alone on the enamel of young permanent teeth.

Dental caries is a complex disease that afflicts a large proportion of the world regardless of age, gender, and ethnicity. During demineralization, the carbonate is lost and during remineralization it is excluded from the newly formed mineral. The calcium deficient, carbonate-rich regions of the crystal lattice are especially susceptible to attack by acid hydrogen ions during demineralization.[1]

The use of a topical fluoride (F− varnish) as a vehicle for topical application is intended to prolong the period of contact with the enamel surface. The amount of fluoride permanently retained in the enamel is increased, and this represents an important factor in reducing demineralization and enhancing remineralization.[2]

The casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) are multiphosphorylated peptides that stabilize calcium and phosphate ions in solution form and localize to tooth surfaces during acidogenic challenges. This maintains the super saturation of calcium and phosphate ions and ion pairs into the subsurface lesion thereby depressing enamel demineralization and thus affects high rates of remineralization.[1]

The mainstay in caries prevention and remineralization is due to the low frequent exposure to low levels of fluoride. This may be accompanied with fluoride toothpastes and mouth rinses, CPP-ACP and application of fluoride varnishes.[3] This study was taken up to evaluate the demineralization inhibitory effect of F− varnish and CPP-ACP.

Materials and Methods

Preparation of tooth sections

Ten healthy premolar teeth that were extracted for orthodontic purposes were collected for the study. All the teeth were examined under the magnifying lens to exclude the possibility of any developmental defect and then underwent fluoride free prophylaxis and soft tissue debridement using pumice slurry and polishing brush in a slowly rotating handpiece.[4] The teeth were then thoroughly washed under running water. The samples were stored in saline solution until further use.[5]

Each tooth was longitudinally sectioned buccolingually and mesiodistally into four sections using a high-speed diamond tipped disk under the constant water spray. A total of four enamel slabs were prepared from each tooth.[6] The sections were again rinsed under running water to clear them of debris and particles. They were then gently air-dried. An acid resistant coating of nail varnish was applied to caries free smooth enamel surface, leaving an exposed window of sound enamel (5 mm × 1 mm).[7]

The teeth were then assigned to four different treatment groups as follows:

Group 1: Fluoride varnish

Group 2: CPP-ACP

Group 3: F− varnish followed by CPP-ACP

Group 4: Control.

The fluoride varnish treatment and the CPP-ACP were carried out according to the manufacturers’ instructions.

Treatment of enamel sections

Group 1

In the windows prepared in the enamel sections, a thin layer of fluoride varnish (Bifluorid 12) was applied using Pele Tim foam pellet as per the manufacturer's instructions. The fluoride varnish was allowed to be adsorbed for 10–20 s and then dried with air.

Group 2

In the windows prepared in the enamel sections, a generous layer of CPP-ACP was applied using an application swab and left undisturbed for a minimum of 3 min as per the manufacturer's instructions.

Group 3

In the windows prepared in the enamel sections, a thin layer of fluoride varnish (Bifluorid 12) was applied using Pele Tim foam pellet as per the manufacturer's instructions. The fluoride varnish was allowed to be adsorbed for 10–20 s and then dried with air.

A generous layer of CPP-ACP was applied using an application swab and left undisturbed for a minimum of 3 min as per the manufacturer's instructions.

Group 4

One of the four sectioned specimens was kept as the control group and was not treated with either fluoride varnish or CPP-ACP.

Artificial caries challenge

The prepared enamel samples were then suspended in an artificial caries solution containing 2.2 mM Ca+2, 2.2 mM PO4−3, 50 mM acetic acid, 0.5 ppm F at a pH 4.0, 37°C for 10 days in an incubator until an artificial carious lesion was induced.[8]

After the lesions had been formed, a 100 μm mesiodistal longitudinal section was obtained from each tooth by hand grinding on 600 grit of silicon carbide paper.[9] The sections were then imbibed in water[10] and examined under × 10 magnification using a polarized light microscope.

Standardization of the micrometer

Before starting the measurement, the eyepiece micrometer was standardized as follows.

Eyepiece micrometer scale was fixed inside the eyepiece lens of the microscope

The stage micrometer with a 10X objective lens was focused

The number of divisions of eyepiece micrometer that coincided with that of the stage micrometer was found.

From this, the actual size of each division of the eyepiece micrometer was calculated as follows.

Seven divisions of eyepiece micrometer coincided with 100 μm

Therefore 1 division of eyepiece micrometer = 100/7 = 14.28 μm.

Photomicrographs of the lesions were projected on the microscope and measurements were made along the advancing front of the body of the lesion in three areas in order to determine the mean body of the lesion depth.[9] The three areas were calculated in units and later converted to μm, and the mean depth of these areas was obtained for the four groups, respectively [Figures 1–4].

Figure 1

Fluoride varnish

Figure 4

Control

Casein phosphopeptide-amorphous calcium phosphate

Fluoride varnish + casein phosphopeptide-amorphous calcium phosphate

Data were taken from each of the experimental and control groups, respectively, and were subjected to statistical analysis. Mean depth of the lesions was tabulated, and comparisons were made among the mean depths of each group using analysis of variance (ANOVA) [Table 1] and Duncan's multiple range test for a paired design with an alpha level of P < 0.001[79] [Tables 2–4].

Table 1

ANOVA

Table 2

Duncan's multiple range test between group 1 (F varnish) and group 2 (CPP-ACP), group 3 (F varnish + CPP-ACP), Group 4 (control)

Table 4

Duncan's multiple range test between group 3 (F varnish+CPP-ACP) and Group 4 (control)

Results

The mean lesion depths of all the groups were Group 1 (fluoride varnish): 104.71, Group 2 (CPP-ACP): 127.09, Group 3 (F− varnish + CPP-ACP): 82.34, Group 4 (control): 146.93.

The mean values of the experimental groups were compared with the control and the data obtained were subjected to ANOVA. The obtained P < 0.0001 from the ANOVA test results indicates that there is a highly significant difference among the groups [Table 1].

The mean depths for each treatment were compared with the control and among each other using ANOVA and Duncan's multiple range test [Tables 2–4] with P value being the level of significance and t-test of significance.

On comparing two groups, F− varnish (Group 1) and CPP-ACP (Group 2), the obtained P value was < 0.0001. This revealed a highly significant difference on the demineralization inhibitory effect between the two groups F− varnish over CPP-ACP [Table 2].

On comparing groups, F− varnish (Group 1) and control (Group 4), the P value obtained was < 0.0001. This revealed a highly significant demineralization inhibitory effect of F− varnish over control [Table 2].

On comparing fluoride varnish and F− varnish followed by CPP-ACP, the obtained P value was P < 0.0001. This revealed a highly significant difference on the demineralization inhibitory effect between the two groups, and F− varnish + CPP-ACP over F− varnish [Table 3].

Table 3

Duncan's multiple range test between group 2 (CPP-ACP) and group 3 (F varnish+ CPP-ACP), group 4 (control)

On comparing two groups CPP-ACP (Group 2) and F− varnish + CPP-ACP (Group 3), the P value obtained was <0.0001. This revealed a highly significant difference of F− varnish + CPP-ACP over CPP-ACP [Table 4].

Discussion

During demineralization, minerals primarily calcium and phosphate leak out of the hydroxyl apatite crystals as demineralization is outpaced by remineralization, and this leads to subsurface lesion development. Enamel is initially involved that results in white spot appearance as the dental hard tissues lose their optical properties. Saliva gets supersaturated with calcium and phosphate thereby preventing demineralization during a critical pH acid attack.[11]

Fluoride therapy has been the centerpiece of caries preventive strategies since the introduction of water fluoridation schemes over five decades. Systemic fluoride is considered to be important but has a lesser demineralization inhibitory effect and remineralization than topical forms of fluoride.[3] Topically applied fluoride products are used extensively as an operator applied caries preventive interventions. The most important anti-caries effect of fluoride is considered to result from its action on tooth/plaque interface by reducing tooth enamel solubility.[12]

The effectiveness of fluoride varnishes has been well-established in caries prevention studies involving permanent teeth. Varnishes are easy to apply, safe, and well-accepted by patients. They prolong the contact time between fluoride and dental enamel as they adhere to the tooth surface for longer periods (12 h or more) in a thin layer and prevent the immediate loss of fluoride after application by acting as a slow releasing reservoir of fluoride. Fluoride varnish hardens in contact with saliva. Fluoride ion can be incorporated into the hydroxylapatite structure of the tooth enamel by replacement of hydroxyl groups or by redeposition of dissolved hydroxylapatite as less soluble fluoridated forms. The physiochemical properties of teeth are thus modified by fluorapatite or fluor-hydroxylapatite thus inhibiting demineralization and enhancing remineralization.[1314]

Topical fluoride agents have shown to decrease enamel demineralization in vitro and in clinical studies.[15] Bifluorid 12 was seen to have the highest demineralization inhibitory effect among various fluoride varnishes evaluated. A significant elevation of fluoride levels in whole saliva occurred with Bifluorid 12 1 h after application,[16] and it exhibited a definite zone of inhibition (very clear and marked cellular zone).[17]

In this study, the enamel surfaces treated with Bifluorid12 varnish showed a significant reduction in lesion depth as compared with the untreated enamel surfaces and enamel surfaces treated with CPP-ACP.[16] Fluoride varnish treatment provided a significant decrease in the mean body of the lesion depths when compared with controls that are composed of pseudo isotropic areas of demineralized enamel indicative of lessened demineralization.[7]

Casein phosphopeptide is peptides that are derived from the milk protein casein that are complexed with calcium and phosphate. CPP binds to surfaces such as plaque, soft tissue, and dentin providing a reservoir of bio-available calcium and phosphate in the saliva and on the surface of the tooth. CPP contains phosphoryl cluster residues that stabilize and bind to spontaneously forming ACP nanoclusters, thus preventing the growth required for nucleation and precipitation.[18] The ACP is released from the CPP complex during oral acidic challenges. Under these conditions, the CPP bound to ACP would buffer plaque pH and in doing so would dissociate to calcium and phosphate ions. 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.[19]

In this study, the presence of CPP-ACP on surfaces provoked lower demineralization and higher remineralization in comparison with surfaces without CPP-ACP and is consistent with the studies reported by Reynolds et al. that CPP-ACP has protective influence on the enamel surfaces.[2021]

The use of CPP-ACP with fluoride and its synergistic effect on enamel remineralization have been attributed to the formation of CPP stabilized amorphous calcium fluoride phosphate resulting in the increase in corporation of fluoride ions into plaque together with the increase in concentration of bio-available calcium and phosphate ions. This synergistic effect of CPP-ACP with fluoride provoked lower demineralization and higher remineralization.[22]

A highly significant demineralization inhibitory effect was present in the group treated on the enamel surfaces with F− varnish + CPP than other experimental groups. The important facilitation effect of F− with CPP-ACP was demonstrated in a study by Shen et al.[23]

Fluoride requires a good source of calcium and phosphate for remineralization of tooth enamel with the more acid resistant fluorapatite; CPP-ACP provides this in an amorphous soluble form. The application of CPP-ACP or CPP-ACPF pastes to sound enamel surfaces resulted in inhibition of enamel demineralization, and a better effect was noted for latter paste.[24] Tooth mousse and high fluoride varnishes effectively inhibit demineralization under the experimental condition.[11] Regardless of the application sequence; the combination effect of CPP-ACP with fluoride provides an additional anti-cariogenic effect, thereby promoting a greater demineralization inhibitory effect on enamel in permanent teeth.

Conclusion

Demineralization inhibitory potential on the additive use of F− varnish + CPP was superior to fluoride varnish or CPP-ACP applied alone on the enamel of young permanent teeth. Thus, it can be said that although the fluoridated varnish offered a greater protective potential, samples treated with F− varnish + CPP-ACP paste were best able to resist the acidic challenge.

Financial support and sponsorship

Nil.

Conflict of Interest

There are no conflict of interest.