Effects of CPP-ACP with sodium fluoride on inhibition of bovine enamel demineralization: A quantitative assessment using micro-computed tomography

Abstract

Objectives

The present study evaluated the effects of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and CPP-ACP with 900 ppm fluoride (CPP-ACPF) pastes on inhibition of enamel demineralization over time, using polychromatic micro-computed tomography (micro-CT).

Methods

Enamel blocks were prepared from bovine teeth. The specimens were each treated by one of the following agents, 30 min daily for 7 days: deionized water (negative control); CPP-ACP paste; CPP-ACPF paste; and NaF solutions (positive controls) (90, 900, and 9000 ppm F). After treatment, the specimens were immersed in a demineralizing solution (pH 4.5) for 24, 72, and 120 h. Mean mineral loss (ML) and lesion depth (LD) after each period were determined from mineral density profiles obtained using micro-CT.

Results

ML values in all the treatment groups were significantly smaller than those in the control group after 72 and 120 h of demineralization (p < 0.05, two-way ANOVA and t-test with Bonferroni correction). ML values in CPP-ACPF and NaF solution groups were significantly smaller compared to CPP-ACP group after 72 h (p < 0.05). LD values in the CPP-ACPF and all the NaF solutions groups were significantly smaller compared to the control group after 120 h (p < 0.05). The 9000 ppm F group showed the lowest nominal ML and LD values.

Conclusions

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 the latter paste. Quantitative assessment using polychromatic micro-CT demonstrated to be useful for detecting mineral density changes.

Introduction

Enamel is the hardest substance in human body. Continuous balanced demineralization and remineralization are natural dynamic processes in enamel.¹ If the balance is interrupted and demineralization process dominates, it may eventually lead to the development of caries lesions in enamel and dentine.

In a neutral environment, the hydroxyapatite crystals of enamel are in a dynamic equilibrium with calcium and phosphate ions.² Demineralization is caused by acids which have intrinsic or extrinsic sources; some acids are produced when certain bacteria colonize on the tooth surface and metabolize carbohydrates, and some are introduced into the mouth as a part of food or drinks. The demineralization can be reverted if the pH is neutralized and there are sufficient bioavailable calcium and phosphate ions in the immediate environment.³

As the “Minimal Intervention” concept⁴ becomes widely spread, various products with tooth surface protection, anti-cariogenic or remineralizing effects attract an increasing attention. Casein phosphopeptides (CPP) containing the cluster sequence -Ser(P)-Ser(P)-Ser(P)- Glu-Glu- have been reported to bind amorphous calcium phosphate, forming small clusters of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), thereby stabilizing calcium phosphates in solution.5, 6 CPP-ACP is reported to promote remineralization through release of free calcium and phosphate ions by several studies; Reynolds et al.,5, 7 Poggio et al.,⁸ and Oshiro et al.⁹ reported that the paste was effective against enamel demineralization.

Historically, the effectiveness of topical sodium fluoride (NaF) as a cariostatic agent has been established well, and professional topical NaF applications are commonly used to arrest the progression of active caries. Reynolds et al. suggested that products containing CPP-ACP with NaF (CPP-ACPF) might be superior in reducing caries risk, when compared to CPP-ACP,⁷ whilst Pulido et al.¹⁰ and Kumar et al.¹¹ found a slight more demineralization with CPP-ACPF compared to fluoridated paste applied without CPP-ACP.

Non-destructive techniques for evaluation of mineral content enable long-term assessment of effects of the remineralizing agents on enamel. Micro-computed tomography (micro-CT) imaging provides a unique opportunity to capture three-dimensional (3D) architectural information from samples. Micro-CT has been frequently used in the experiments exploring mineral density and structure of mineral tissues, like bones and teeth.12, 13, 14 The technique is also a promising method to assess demineralization or remineralization in enamel or dentine.15, 16, 17, 18, 19 Micro-CT systems are mainly classified into two types; synchrotron radiation micro-CT that can produce parallel monochromatic X-ray beams,²⁰ and commercial or desktop micro-CT that can produce polychromatic X-rays.¹⁸ Where the polychromatic X-ray source is used, quantitative determination of mineral density requires correction of beam-hardening artifacts, especially in high mineral density tissues such as enamel.

Although micro-CT bears the advantage of obtaining 3D information non-destructively when compared to the destructive two-dimensional (2D) techniques such as transversal microradiography (TMR),21, 22, 23 few studies have to date employed this technique to quantitatively evaluate the effects of preventive agents on enamel.

Therefore, the aim of this study was to utilize polychromatic micro-CT for quantitative and non-destructive assessment of the effects of CPP-ACP and CPP-ACPF pastes on prevention of enamel demineralization over time. The null hypotheses were that: CPP-ACP would have no preventive effect on enamel demineralization, and that CPP-ACPF would have no additional benefits for prevention of demineralization compared to CPP-ACP.