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.

AIM: The objective of this study was to quantitatively evaluate the remineralization potential of casein phosphopeptide-amor-phous calcium phosphate paste on enamel subsurface lesions using scanning electron microscopy with energy dispersive X-ray analysis (SEM-EDX).
MATERIALS AND METHODS: Ninety enamel specimens were prepared from extracted human molars. All specimens were evaluated for mineral content (% weight) using SEM-EDX. The specimens were placed in demineralizing solution for four days to produce artificial carious lesions. The mineral content (calcium/phosphorus ratios, Ca/P ratios) was remeasured using SEM-EDX. The specimens were then randomly assigned to five study groups and one control group of 15 specimens per group. Except for the control group, all group specimens were incubated in remineralizing paste (CPP-ACP paste) for 7, 14, 21, 28, and 35 days twice daily for three minutes. The control group received no treatment with remineralizing paste. All the 90 specimens were stored in artificial saliva at 37°C. After remineralization, the mineral content (% weight) of the samples was measured using SEM-EDX.
RESULTS: All the study groups showed very highly significant differences between Ca/P ratios of the demineralized and remineralized samples. There was no significant difference seen in the control group.
CONCLUSION: CPP-ACP paste could significantly remineralize the artificial enamel subsurface lesions in vitro: the remineralizing rates increasing with the time for which the samples were kept in the remineralizing paste. Energy dispersive X-ray analysis is an efficient way to quantitatively assess the changes in mineral content during demineralization and in vitro remineralization processes.

RCT Entities:   Population Interventions Outcomes

INTRODUCTION

Tooth structure undergoes continuous demineralization and remineralization in the oral environment. If this balance is disrupted, demineralization will progress, leading to a deterioration of the tooth structure.[1]

Caries initiation is associated with demineralization of the subsurface tooth enamel. Calcium and phosphate are lost from the subsurface enamel, resulting in the formation of a subsurface lesion. At this early stage, the caries lesion is reversible via a remineralization process involving the diffusion of calcium and phosphate ions into the subsurface lesion to restore the lost tooth structure. As several studies had demonstrated that milk-based products appeared to have anticariogenic properties in animal models, attention was focused on identifying the specific milk-based agents that were responsible for the ‘anticaries effect’[2] The concept of casein phosphopeptide-amorphous calcium phosphate as a remineralizing agent was first postulated in 1998. CPP-ACP nanocomplexes are derived from bovine milk protein, casein, and calcium and phosphate. A number of subsequent studies have demonstrated CPP-ACP to have anticariogenic activity in laboratory, animal and human in situ experiments.[2]

Modern prospective caries studies require the measurement of small changes in a tooth's mineral content, especially in a single caries lesion. One recent technique is scanning electron microscopy with an energy dispersive X-ray analysis attachment. It is a microanalytical technique that is employed to quantitatively estimate the amounts of mineral in a given tooth sample.[3]

The anticariogenic activity of CPP-ACP has led to its incorporation into food products and dental products as a new tool in the fight against dental caries.[2] Hence, the aim of this study was to assess the remineralization potential of casein phosphopeptide-amorphous calcium phosphate paste on enamel subsurface lesions.

MATERIALS AND METHODS

Ninety enamel specimens, 4 mm × 4 mm × 1 mm in size, were prepared from the buccal surfaces of extracted human molar teeth using a low-speed diamond disc. All specimens were evaluated for mineral content (% weight) using SEM-EDX [Figure 1]. The specimens were placed in the demineralizing solution containing 20 ml of acid buffer with 2 mmol/L Ca2+, 2 mmol/L PO43-, and 0.075 mol/L acetate at pH 4.3 for four days at 37°C to produce artificial carious lesions. All specimens were evaluated for any loss of mineral content (wt %) using SEM-EDX on the 5th day [Figure 2].

Figure 1

Elemental analysis of sound enamel sample by EDX

Figure 2

Elemental analysis of demineralized enamel sample by EDX

The specimens were randomly assigned to two groups: group 1 contained 15 specimens (control group) and group 2 contained 75 specimens (study group). The study group was subdivided into five groups of 15 specimens per group. Each subgroup was treated with remineralizing paste [10% CPP-ACP paste (GC-Tooth Mousse)] using a stainless steel spatula for seven days (subgroup 2a), 14 days (subgroup 2b), 21 days (subgroup 2c), 28 days (subgroup 2d), and 35 days (subgroup 2e), twice daily for three minutes followed by incubation in artificial saliva at 37°C. The specimens in the control group (group 1) were incubated in artificial saliva at 37°C after demineralization for a period of 35 days but received no treatment with remineralizing paste. SEM-EDX was done to measure mineral content after the remineralization process.

RESULTS

The present study evaluated the remineralization potential of CPP-ACP paste on artificial enamel subsurface lesions using SEM-EDX. Energy dispersive X-ray analysis was used to determine calcium and phosphorus content in % weight of sound, demineralized, and remineralized enamel in each group. The calcium and phosphorus content was then converted into Ca/P ratios for each group from the obtained data. Figures 3–7 demonstrates the elemental analysis of study groups [Sub groups 2a – 2e ] for different periods of treatment time with CPP-ACP. Table 1: Illustrates comparision of mean Ca/P ratios of Sound, Demineralized, Remineralized enamel samples.

Figure 3

Elemental analysis of enamel sample by EDX, after 7 days of remineralization

Figure 7

Elemental analysis of enamel sample by SEM, after 35 days of remineralization

Table 1

Comparision of mean Ca/P ratios of sound, demineralized, remineralized enamel samples using one - way ANOVA

Elemental analysis of enamel sample by EDX, after 14 days of remineralization

Elemental analysis of enamel sample by SEM, after 21 days of remineralization

Elemental analysis of enamel sample by SEM, after 28 days of remineralization

Statistical analysis was done using one-Way Anova, Tukey's HSD, and Student t-test. Comparison of Ca/P ratios of the sound enamel samples and Ca/P ratios of the demineralized enamel samples in all the groups using one-way Anova revealed that there was no statistically significant difference between the groups.

One-way Anova was applied to compare the mean Ca/P ratios of the study groups after remineralization, which was found to increase to 1.93 ± 0.02 on the 35th day. This increase in the mean Ca/P ratio from the seventh to the 35th days had P < 0.0005, implying a very high statistically significant difference in the remineralization potential over this period. Tukey HSD was done for intergroup comparison [Table 2] and P < 0.0005 for all the comparisons suggested very high significance.

Table 2

Intergroup comparison of the remineralization potential of study groups done using tukey Honestly significant difference post HOC multiple comparisons

The t-test was done to statistically analyze the mean Ca/P ratios of demineralized and remineralized specimens in each group [Table 3 and Figure 8]. The value of significance was set at P < 0.05 and it was seen that all the study groups revealed highly significant results between the Ca/P ratios of the demineralized and remineralized samples. Representative SEM images of the enamel specimens are shown in Figures 9–15. Enamel specimens treated with CPP-ACP paste revealed slight changes in their morphological features.

Table 3

T-test to analyze the mean Ca/P ratios of demineralized and remineralized specimens in study groups

Figure 8

Relationship between demineralization and the remineralization potential of study groups

Figure 9

Structural analysis of sound enamel sample by SEM

Figure 15

Structural analysis of enamel sample by SEM, after 35 days of remineralization

Structural analysis of demineralized enamel sample by SEM

Structural analysis of enamel sample by SEM, after 7 days of remineralization

Structural analysis of enamel sample by SEM, after 14 days of remineralization

Structural analysis of enamel sample by SEM, after 21 days of remineralization

Structural analysis of enamel sample by SEM, after 28 days of remineralization

DISCUSSION

In the present study, the remineralization potential of CPP-ACP for enamel subsurface lesions was evaluated using SEM-EDX. GC Tooth Mousse is a water-based, lactose-free creme containing 10% w/w Recaldent CPP-ACP. When CPP-ACP is applied in the oral environment, it will bind to biofilms, plaque, bacteria, hydroxyapatite, and soft tissue, localizing bioavailable calcium localizes and phosphate.

CPP-ACP thus, localizes ACP on the tooth surface, and buffers the free calcium and phosphate ion activities, helping to maintain a state of supersaturation with respect to the enamel by suppressing demineralization and enhancing remineralization.[1] The CPP has a substantial ability to stabilize calcium phosphate in solution. The peptide was found to bind 21 calcium and 14 phosphorus ions per molecule.

ACP nuclei spontaneously form in neutral and alkaline supersaturated calcium phosphate solutions. It is proposed that the peptide binds to the forming ACP nanoclusters, producing a metastable solution and preventing ACP growth to the critical size required for nucleation and phase transformation.[3] A 1.0% w/v CPP solution can stabilize 60 mmol/L CaCl2 and 36 mmol/L sodium phosphate at pH 7.0 to form colloidal amorphous calcium phosphate-CPP nanocomplexes.[4]

The remineralization process involves diffusion of calcium and phosphate ions through the protein/water-filled pores of the carious surface enamel into the body of the enamel lesion. Once in the body of the enamel lesion, these calcium and phosphate species increase the activities of Ca2+ and PO43-, thereby increasing the degree of saturation with respect to hydroxyapatite.

The formation of hydroxyapatite in the lesion would lead to the generation of acid and phosphate, which would diffuse out of the lesion along a concentration gradient. By stabilizing calcium phosphate in a metastable solution, the CPP facilitates high concentrations of calcium and phosphate ions, which can then diffuse into the enamel subsurface lesion. The CPP will also maintain high activities of the free calcium and phosphate ions during remineralization through the reservoir of the bound ACP. By being in dynamic equilibrium with free calcium and phosphate ions, the bound ACP will maintain the concentrations of the species involved in diffusion into the lesion. Furthermore, dissociation of the CPP-bound ACP will be facilitated by the acid generated during enamel remineralization. This would explain why CPP-supported, metastable calcium phosphate solutions are such efficient remineralizing solutions, as they would consume the acid generated during enamel lesion remineralization by generating more calcium and phosphate ions, thus maintaining their high concentration gradients into the lesion.[4]

In the present study, a paste type formulation of CPP-ACP was used. Patients can use this kind of oral hygiene paste just like tooth paste with tooth brushes and also apply the paste with cotton slabs.[1]

EDX has been used for elemental analysis at the ultrastructural level. It is a microanalytical technique that is used in conjunction with SEM wherein SEM does the structural analysis and the elemental analysis is done by EDX. The principle is based on the energy emitted in the form of X-ray photons when electrons from external sources collide with the atoms in a material, thus generating characteristic X-rays of that element. When the sample is bombarded by the electron beam of the SEM, electrons are ejected from the atoms on the specimen's surface (secondary electrons). A resulting electron vacancy is filled by an electron from a higher shell, and an X-ray is emitted (characteristic X-rays) to balance the energy difference between the two electrons. The EDX X-ray detector measures the number of emitted X-rays vs. their energy. The energy of the X-ray is characteristic of the element from which the X-ray was emitted. A spectrum of the energy vs. relative counts of the detected X-rays is obtained and evaluated for qualitative and quantitative determinations of the elements present in the specimen using a computer-based program.[3]

The results of this in vitro study showed that 10% CPP-ACP paste remineralized subsurface lesions in human enamel. The remineralization was maximal in the samples kept for 35 days, thus, it could be said that the remineralization was dose-dependent. The results of this study were consistent with the proposed remineralization mechanism of CPP-ACP and are in accordance with the one obtained by Reynolds who demonstrated that CPP-stabilized calcium phosphate solutions remineralized subsurface lesions in human enamel in vitro.[4]

According to Yamaguchi et al . Hegde et al .[5] and Oshiro et al .[6] the inorganic components contained in high concentrations in CPP-ACP acted to enhance remineralization of the enamel. This is consistent with the results of the present study.

Enamel specimens treated with CPP-ACP paste revealed slight changes in their morphological features. The surface morphologies of the specimens in the study groups showed no apparent differences among the different storage periods. This is in accordance with the study conducted by Oshiro et al.[6]

CONCLUSION

Within the limitations of this study, the following conclusions can be drawn:

10% CPP-ACP paste significantly remineralized the artificial enamel subsurface lesions in vitro.

The remineralization achieved was dose-dependent as the remineralizing rate increased with the time for which the enamel was exposed to the CPP-APP paste.

EDX was found to be an efficient way to quantitatively assess the changes in mineral content during in vitro caries studies.