44Ca doped remineralization study on dentin by isotope microscopy
Graphical abstract
Introduction
Dental caries is caused by an action of acids on an enamel surface and developed into dentin lesion. Unlike bone, tooth-regeneration is not a feasible physiological process. We have to consider prevention of caries, detection incident of caries at an early stage and strategies of remineralization for decayed lesions. Cariology researchers have worked on in vitro models to investigate caries progress, remineralization possibility of decalcified lesions and effects of anticariogenic agents such as fluoride. Among in vitro protocols, the pH-cycling method is most commonly employed, which involves an exposure of dental substrates (enamel and dentin) alternatively to remineralizing and demineralizing solutions [1]. The pH-cycling yields experimental lesions as a result of mineral loss at an acidic pH and mineral gain at a neutral pH. After the pH-cycling is performed, the mineral content in the lesion is evaluated to represent de- and remineralization progress. Transverse microradiography (TMR) is used as a gold standard method for the evaluation of mineral content [2]. More recently, microcomputed tomography (micro-CT) has been introduced as a non-destructive evaluation in Cariology research [3]. These methods allow measurements for mineral content from intact lesions to extensive demineralized lesions, including longitudinal observation of lesions development. For effective caries preventions, anticariogenic approaches have been attempted to decrease mineral loss and increase mineral gain. However, TMR and micro-CT have their limitations when it comes to distinguish between tooth-derived mineral (intrinsic Ca) or solution-derived mineral uptake (extrinsic Ca).
To overcome this limitation, we used 44Ca (a stable isotope) doped remineralization solution while a pH-cycling. Since the natural abundance of calcium isotopes, 40Ca, 42Ca and 44Ca, is 96.941%, 0.647% and 2.086%, respectively, 40Ca represents intrinsic Ca, which is derived from tooth, meanwhile 44Ca does extrinsic Ca, which is derived from the remineralization solution. Each Ca distribution was detected using isotope microscopy. This technique employs secondary ion mass spectrometry (SIMS), which has been developed as a method to identify isotope ratios within micro-areas of materials and to allow high-precision isotope imaging in cosmo- and geochemistry [4], [5].
In our study, imaging by SIMS was applied for bovine dentin lesions that underwent the pH-cycling using 44Ca doped remineralization solution. We performed the pH-cycling with or without fluoride treatment between de- and remineralization cycles to investigate an effect of fluoride on Ca distribution. Topical fluoride such as toothpastes, mouthrinses, gels or varnishes is widely used for preventing dental caries. It is thought that fluoride is absorbed to the surface of the apatite crystals and further at high concentration is incorporated into fluorhydroxyapatite, which are resistant to demienarlization [6], [7]. The imaging by SIMS was attempted to gain insight into the action of fluoride on dentin in pH-cycling model.
The aim of this preliminary Ca-isotope study was to distinguish 40Ca and 44Ca distributions which have been derived from tooth substrates and from the remineralization solution, respectively, and to discuss the fluoride effect on formation of remineralized lesions.
Section snippets
Preparation of bovine root dentin specimens
Root dentin blocks were obtained from root surfaces of six bovine incisor teeth. This 44Ca doped pH-cycling study required consistency in dentin samples in terms of compositional and morphological property. Therefore, we used bovine dentin as a substitute for human dentin because bovine and human dentins show a great similarity in chemical composition [8]. The blocks embedded in acrylic resin were polished to expose the root dentine using a series of silicon carbide papers up to 2000-glit. Each
Results
The TMR and Ca-distribution images of the control (0% fluoride treatment) and the 0.2% fluoride group are shown in Fig. 1(I) and (II), respectively. The control group showed 40Ca (intrinsic) distribution in their subsurface lesion and superficial lesion as well. Meanwhile the fluoride group shows that 40Ca (intrinsic) distribution is present in the subsurface lesions for all samples, but lesser toward the surface lesions, especially at 0–10 μm depth for (IIa), 0–30 μm depth for (IIb) and 0–20 μm
Discussion
In a number of previous studies, TMR observations have given us an insight of the dynamics of mineral loss and gain as a consequence of pH-cycling, and provided a quantitative measurement of the amount of mineral and lesion depth [1], [2]. In light of mineral gain, there might be authentic and apparent mineral gain. Authentic mineral gain is defined as additional mineral source that is derived from external remineralization solution (extrinsic Ca). Apparent mineral gain is, on the other hand,
Acknowledgments
All authors declared no conflict of interest associated with this work. This work was supported by Grant-in-Aid for the Japan Society for the Promotion of Science Fellows (15J40094).
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