- Email: [email protected]
44Ca doped remineralization study on dentin by isotope microscopy
d e n t a l m a t e r i a l s 3 4 ( 2 0 1 8 ) e57–e62
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ScienceDirect journal homepage: www.intl.elsevierhealth.com/journals/dema
44
Ca doped remineralization study on dentin by isotope microscopy
N. Hiraishi a,∗ , S. Kobayashi b , H. Yurimoto b,c , J. Tagami a a
Cariology and Operative Dentistry, Department of Oral Health Sciences, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan b Isotope Imaging Laboratory, Creative Research Institute, Hokkaido University, Kita 8, Nishi 5, Kita-ku, Sapporo 001-0021, Japan c Department of Natural History Sciences, Hokkaido University, Kita 8, Nishi 5, Kita-ku, Sapporo 001-0021, Japan
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objective. The dental caries is developed as a result of an alternative course of mineral gain
Received 3 June 2017
and loss. In order to distinguish between intrinsic Ca (tooth-derived mineral) and extrinsic
Received in revised form
Ca (solution-derived mineral) uptakes, a
1 January 2018
(a stable calcium isotope) remineralization solution.
Accepted 16 January 2018
Methods. The natural abundance of
40
44
Ca doped pH-cycling was performed using
Ca and
remineralization solution was prepared using
44
44
Ca
Ca is 96.9% and 2.1%, respectively. The
44
Ca to contain 1.5 mmol/L CaCl2 (44 Ca),
0.9 mmol/L KH2 PO4, 130 mmol/L KCl, 20 mmol/L HEPES at pH 7.0. The pH-cycling was conKeywords:
ducted on bovine root dentin daily by demineralization (pH 5.0) for 2 h, incubation in 0%
Isotope microscope
(control) and 0.2% NaF (900 ppm fluoride) for 2 h and
Calcium
After 14 days pH-cycling, the specimens were sectioned longitudinally. On the sectioned 40
44
44
Ca doped remineralization for 20 h.
Secondary ion mass spectrometry
surface, isotope imaging of
Remineralization
high mass-resolution stigmatic secondary ion 77 (Camera IMS 1270, Gennevilliers Cedex,
Fluoride
France).
Dentin
Results. Uptake of 44 Ca was greater in intensity for the 0.2% fluoride group than the control,
Ca and
Ca labeled mineral distribution was observed by a
especially in the superficial lesions. The control group showed 40 Ca (intrinsic) distribution in the subsurface lesions and in the superficial lesions, meanwhile the fluoride group showed 40
Ca distribution limited in subsurface lesions. The total Ca (44 Ca + 40 Ca) image revealed
more homogeneously for the control than the fluoride group. Significance. Since the fluoride-treated surface is more acid-resistant than intrinsic dentin, alternative minerals were dissolved from the intact intrinsic lesion in the demineralization cycle. © 2018 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
∗
Corresponding author. E-mail addresses: [email protected] (N. Hiraishi), [email protected] (S. Kobayashi), [email protected] (H. Yurimoto), [email protected] (J. Tagami). https://doi.org/10.1016/j.dental.2018.01.022 0109-5641/© 2018 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
e58
1.
d e n t a l m a t e r i a l s 3 4 ( 2 0 1 8 ) e57–e62
Introduction
Dental caries is caused by an action of acids on an enamel surface and developed into dentin lesion. Unlike bone, toothregeneration 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 toothderived mineral (intrinsic Ca) or solution-derived mineral uptake (extrinsic Ca). To overcome this limitation, we used 44 Ca (a stable isotope) doped remineralization solution while a pH-cycling. Since the natural abundance of calcium isotopes, 40 Ca, 42 Ca and 44 Ca, is 96.941%, 0.647% and 2.086%, respectively, 40 Ca represents intrinsic Ca, which is derived from tooth, meanwhile 44 Ca 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 44 Ca 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 40 Ca and 44 Ca 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.
2.
Materials and methods
2.1.
Preparation of bovine root dentin specimens
Root dentin blocks were obtained from root surfaces of six bovine incisor teeth. This 44 Ca 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 dentine surface was painted with a nail vanish, leaving a 5 mm diameter window.
2.2.
Methods of pH-cycling
A stable Ca isotope, 44 Ca, was purchased from ISOFLEX (San Francisco, Ca, USA), and other chemical ingredients were from Sigma-Aldrich (St. Louis, MN, USA). The remineralization solution was prepared using 44 Ca to contain 1.5 mmol/L CaCl2 (44 Ca), 0.9 mmol/L KH2 PO4, 130 mmol/L KCl, 20 mmol/L HEPES buffer at pH 7.0. The demineralizing solution was prepared using a conventional calcium compound, and contained 50 mmol/L acetic acid, 1.5 mmol/L CaCl2 and 0.9 mmol/L KH2 PO4 adjusted to pH 5.0. The dentin samples were soaked in the demineralization solution (pH 5.0) for 2 days to make pre-lesion (about 150 m). The pH-cycling was employed by demineralization (pH 5.0) for 2 h, treatment with 0% (control) or 0.2% NaF (900 ppm fluoride) at pH 7 for 2 h and 44 Ca doped remineralization for 20 h. The period of the cycling was 14 days. Since we aimed to observe calcium intakes from the remineralization solution, we soaked the samples in a prolonged remineralization cycle.
2.3. Microradiography observation and isotope imaging of Ca distribution The samples were sectioned into halves, each for TMR and isotope imaging. For TMR, the sectioned samples were further cut longitudinally into 220 ± 20 m thickness. The cut sections were put in a solution containing 80% of glycerin and 20% of water to prevent shrinkage. Photo plates (HY2, Konica Minolta Holdings, Inc., Tokyo, Japan) were exposed at 4 mA and 25 kV for 5 min using an X-ray generator (Type SRO-M50, Sofron Company Lit., Tokyo, Japan). After the plates were developed and fixed, the microradiographs were taken under a microscope (BX 41, Olympus Co., Tokyo, Japan) with CCD camera (DP70, Olympus Co., Tokyo, Japan). For isotopic microscopy, the sectioned surface was embedded and polished using a series of silicon carbide papers up to 4000-glit. Isotope imaging of 40 Ca+ and 44 Ca+ mineral distribution was observed by a high mass-resolution stigmatic SIMS instrument (Camera IMS 1270, Gennevilliers Cedex, France) [4,5]. The 130 m diameter on sample surface was irradiated by O — primary beam (23 KeV, 10 nA). Secondary ions 40 Ca+
d e n t a l m a t e r i a l s 3 4 ( 2 0 1 8 ) e57–e62
e59
Fig 1 – TMR and isotope microscopy images of three sections: (I) the control group and (II) the 0.2% fluoride treatment group. Lesions encircled with in bold line in TMR images were observed with isotope microscope. Brightness for 40 Ca, 44 Ca and 40 Ca + 44 Ca images are adjusted to distinguish each distribution. For the control group (Ia, Ib and Ic), the 40 Ca (intrinsic Ca) is present in their superficial layers, and 44 Ca (extrinsic Ca) shows moderate uptake in their surface layers. The total 40 Ca + 44 Ca indicates a homogeneous distribution in the lesions. For the 0.2% fluoride treatment group (IIa, IIb and IIc), the 40 Ca is less in their surface lesion (arrows in 40 Ca images) especially in 0–10 m depth for (IIa); 0–30 m depth for (IIb); 0–20 m depth for (IIc). The 44 Ca images shows greater uptake especially in superficial layer (arrows in 44 Ca images). The total 40 Ca + 44 Ca exhibits a heterogeneous distribution, lesser in their subsurface lesions (arrows in 40 Ca + 44 Ca images). The squares in 40 Ca isotope images are profiled to determine intrinsic mineral loss. Each 190 × 190 pixel in 40 Ca isotope image in a dotted line square is used to calculate the intrinsic mineral loss.
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d e n t a l m a t e r i a l s 3 4 ( 2 0 1 8 ) e57–e62
Fig. 2 – Gray values of the 190 × 190 pixel in 40 Ca isotope images as shown in Fig. 1 (the squares in dotted line). The integrated area of the 190 × 190 pixel was assigned as Ca(40). The straight line with G(high) indicates the highest gray value. The integrated area between the G(high) and gray value was assigned as the loss of 40 Ca, Ca(loss). Intrinsic mineral loss was calculated as Ca(loss)/Ca(40). Ca(40)
Ca(loss).
and 44 Ca+ were obtained with exposure time of 10 and 25 s, respectively. These irons were constructed as a mass-filtered ion image using stigmatic ion optics of IMS 1270 and visualized when directly projected on a pixel matrix of stacked CMOS-type active pixel sensor. The obtained 40 Ca data was processed into a gray-scale image by the use of a customized image processing software (ImageJ, version1.42q, Wayne Rasband, NIH, USA). Representative 190 × 190 pixels in 40 Ca data was selected from the intrinsic dentin toward the boundary surface where extrinsic 44 Ca was detected. The integrated area of the 190 × 190 pixel was assigned as Ca(40) and the highest gray value was recorded as G(high). In the 40 Ca profile, the integrated area between G(high) and gray value was assigned as Ca(loss), loss of 40 Ca. Then, the intrinsic mineral loss was calculated as Ca(loss)/Ca(40), which was statistically analyzed using Ttest (p < 0.05) between the control and fluoride groups using a statistical software package (SigmaStat Version 16.0, SPSS, Chicago, IL, USA).
3.
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 40 Ca (intrinsic) distribution in their subsurface lesion and superficial lesion as well. Meanwhile the fluoride group shows that
40 Ca (intrinsic) distribution is present in the subsurface lesions
for all samples, but lesser toward the surface lesions, especially at 0–10 m depth for (IIa), 0–30 m depth for (IIb) and 0–20 m depth for (IIc). Uptake of 44 Ca (extrinsic) was greater in intensity for the superficial layer of the fluoride group than the control. The total Ca (44 Ca + 40 Ca) image reveals more homogeneously for the control than the fluoride group. For the fluoride group, the total Ca (44 Ca + 40 Ca) is intensified on the superficial lesion, but less in the subsurface lesion, at 15 m depth for (IIa), 40 m depth for (IIb) and 30 m depth for (IIc). These total Ca (44 Ca + 40 Ca) images are in consistence with corresponding TMR images, which show deficiency in mineral in the subsurface. As shown in Fig. 2, the intrinsic mineral loss was 0.107% ± 0.021% and 0.412% ± 0.072% for the control and fluoride group, respectively. T-test showed that there was a significant difference (p = 0.012).
4.
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
d e n t a l m a t e r i a l s 3 4 ( 2 0 1 8 ) e57–e62
is, on the other hand, determined as a total mineral gain of tooth-derived mineral (intrinsic Ca) and solution-derived mineral uptake (extrinsic Ca). The conventional pH-cycling and subsequent TMR measurement provides an apparent mineral gain, in which the authentic mineral gain cannot be validated. As shown in Fig. 1, the isotope microscopic measurement exhibited the distribution of intrinsic Ca and extrinsic Ca. The control (0% fluoride treatment) showed a homogeneous mineral distribution in the surface and subsurface lesion, in which a relatively intact subsurface layer was remained. Interestingly, for the fluoride group, there was a different distribution of mineral (40 Ca and 44 Ca). Dissolved intrinsic minerals (40 Ca) have not been compensated by the mineral gain in the subsurface lesion, in which the total mineral was less than the surface lesion. The effect of fluoride has been addressed in the WHO Global Oral Health Programme, which has focused its effect in dental caries prevention and in improvement communitybased public oral health [9]. The action of fluoride on mineral has been well documented in previous studies [10–16]. At a low fluoride concertation (i.e. less than 100 ppm), fluoride incorporation in apatite structure takes place, due to an ionic exchange of F− for OH− in apatite or epitaxial crystal growth of fluorapatite when solution is supersaturated for the crystal growth [14]. At a higher fluoride concertation than 100 ppm, CaF2 -like formation is initiated [10,14–16]. At this concentration, fluoride ions may be loosely bound or non-specifically adsorbed on minerals, where calcium and other ions such as phosphate are utilized to form phosphate-containing CaF2 like minerals [14–16]. This phosphate-containing CaF2 is slightly more soluble than pure CaF2, thus acting as a fluoride reservoir during the pH-cycling [17]. In the remineralization pH-cycling, the crystals in remaining minerals serve as nuclei for an accumulation of new mineral which fluoride is incorporated into, resulting in epitaxial crystal growth of fluorapatite [14]. In this event, fluorhydroxyapatite and/or fluorapatite is formed and precipitated in the surface layer [11]. The fluorhydroxyapatite and fluorapatite remain stable if not at lower than a pH 4.7, meanwhile mineral in dentin are easily dissolved at critical pH values for dentin at pH 6.3 [11]. In our study, the dentin samples were treated with 900 ppm fluoride for 2 h between remineralization and demineralization cycling. When the tooth samples were treated with 900 ppm fluoride, fluoride ions were bound on dentin surface, and CaF2 -like minerals was initially formed, acting as fluoride ions reservoir. In the following remineralizing cycling, fluoride ions were reacted with the extrinsic 44 Ca ions to promote epitaxial crystal growth of fluorapatite and/or fluorhydroxyapatite. This fluoride action rendered the surface lesion more acid-resistant than the subsurface lesion. Another noteworthy fact is that native intrinsic apatite in hard tissue in tooth and bone are formed in corporation with carbonated hydroxyapatite mineral, which is more soluble than pure hydroxyapatite mineral and fluorapatite-like mineral [13]. In the demineralizing cycle at pH 5, the aforementioned fluorhydroxyapatite and/or fluorapatite were hardly dissolved. Meanwhile, the mineral of subsurface lesion was alternatively dissolved because intrinsic dentin in the subsurface lesion was less acid-resistant. This alternative mineral loss has been continuously occurred from the subsurface
e61
intrinsic lesion. As a results, the intrinsic mineral loss that was calculated as Ca(loss)/Ca(40) was statistically higher in the fluoride group than the control group. The gradual depletion of intrinsic dentin was contributed to the less total mineral (40 Ca + 44 Ca) as shown in the subsurface of the fluoride lesion when compared with the control group. In other word, when the solubility on the surface was modified by fluoride to be acid-resistant, alternative minerals were dissolved at the expense of intact intrinsic mineral. Within limitations, it may be concluded that the extrinsic Ca uptake was increased at the superficial lesion by the use of fluoride; however, the intrinsic Ca loss was predominant, resulting that total Ca was less in that lesion. In further studies, the effect of fluoride must be investigated in vivo condition and clinical approach. Furthermore, it would be interesting to perform isotope microscopic observation on enamel specimens to investigate early caries progression, clinically shown as a white opaque spot.
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).
references
[1] ten Cate JM, Duijsters PP. Alternating demineralization and remineralization of artificial enamel lesions. Caries Res 1982;16:201–10. [2] Featherstone JD, Ten Cate JM, Shariati M, Arends J. Comparison of artificial caries-like lesions by quantitative microradiography and microhardness profiles. Caries Res 1983;17:385–91. [3] Dowker SE, Elliott JC, Davis GR, Wilson RM, Cloetens P. Synchrotron X-ray microtomographic investigation of mineral concentrations at micrometre scale in sound and carious enamel. Caries Res 2004;38:514–22. [4] Yurimoto H, Abe K, Abe M, Ebihara M, Fujimura A, Hashiguchi M, et al. Oxygen isotopic compositions of asteroidal materials returned from Itokawa by the Hayabusa mission. Science 2011;333:1116–9. [5] Yurimoto H, Nagashima K, Kunihiro T. High precision isotope micro-imaging of materials. Appl Surf Sci 2003;20(203–204):793–7. [6] Buzalaf MA, Pessan JP, Honório HM, ten Cate JM. Mechanisms of action of fluoride for caries control. Monogr Oral Sci 2011;22:97–114. [7] Buzalaf MA, Hannas AR, Magalhães AC, Rios D, Honório HM, Delbem AC. pH-cycling models for in vitro evaluation of the efficacy of fluoridated dentifrices for caries control: strengths and limitations. J Appl Oral Sci 2010;18:316–34. [8] Teruel Jde D, Alcolea A, Hernández A, Ruiz AJ. Comparison of chemical composition of enamel and dentine in human, bovine, porcine and ovine teeth. Arch Oral Biol 2015;60:768–75. [9] Petersen PE, Lennon MA. Effective use of fluorides for the prevention of dental caries in the 21st century: the WHO approach. Community Dent Oral Epidemiol 2004;32:319–21. [10] Larsen MJ, Jensen SJ. Experiments on the initiation of calcium fluoride formation with reference to the solubility or dental enamel and brushite. Arch Oral Biol 1994;9:7–23.
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[11] Lussi A, Hellwig E, Klimek J. Fluorides-mode of action and recommendations for use. Schweiz Monatsschr Zahnmed 2012;122:1030–42. [12] Featherstone JD, Glena R, Shariati M, Shields CP. Dependence of in vitro demineralization of apatite and remineralization of dental enamel on fluoride concentration. J Dent Res 1990;69:620–5. [13] Featherstone JD. The science and practice of caries prevention. J Am Dent Assoc 2000;131:887–99. [14] Roˇsin-Grget K, Peroˇs K, Sutej I, Baˇsic´ K. The cariostatic mechanisms of fluoride. Acta Med Acad 2013;42:179–88.
[15] Rølla G, Saxegaard E. Critical evaluation of the composition and use of topical fluorides, with emphasis on the role of calcium fluoride in caries inhibition. J Dent Res 1990;69:780–5. [16] Ogaard B. CaF2 formation: cariostatic properties and factors of enhancing the effect. Caries Res 2001;35:40–4. [17] Christoffersen J, Christoffersen MR, Kibalczyc W, Perdok WG. Kinetics of dissolution and growth of calcium fluoride and effects of phosphate. Acta Odontol Scand 1988;46:325–36.
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ScienceDirect journal homepage: www.intl.elsevierhealth.com/journals/dema
44
Ca doped remineralization study on dentin by isotope microscopy
N. Hiraishi a,∗ , S. Kobayashi b , H. Yurimoto b,c , J. Tagami a a
Cariology and Operative Dentistry, Department of Oral Health Sciences, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan b Isotope Imaging Laboratory, Creative Research Institute, Hokkaido University, Kita 8, Nishi 5, Kita-ku, Sapporo 001-0021, Japan c Department of Natural History Sciences, Hokkaido University, Kita 8, Nishi 5, Kita-ku, Sapporo 001-0021, Japan
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objective. The dental caries is developed as a result of an alternative course of mineral gain
Received 3 June 2017
and loss. In order to distinguish between intrinsic Ca (tooth-derived mineral) and extrinsic
Received in revised form
Ca (solution-derived mineral) uptakes, a
1 January 2018
(a stable calcium isotope) remineralization solution.
Accepted 16 January 2018
Methods. The natural abundance of
40
44
Ca doped pH-cycling was performed using
Ca and
remineralization solution was prepared using
44
44
Ca
Ca is 96.9% and 2.1%, respectively. The
44
Ca to contain 1.5 mmol/L CaCl2 (44 Ca),
0.9 mmol/L KH2 PO4, 130 mmol/L KCl, 20 mmol/L HEPES at pH 7.0. The pH-cycling was conKeywords:
ducted on bovine root dentin daily by demineralization (pH 5.0) for 2 h, incubation in 0%
Isotope microscope
(control) and 0.2% NaF (900 ppm fluoride) for 2 h and
Calcium
After 14 days pH-cycling, the specimens were sectioned longitudinally. On the sectioned 40
44
44
Ca doped remineralization for 20 h.
Secondary ion mass spectrometry
surface, isotope imaging of
Remineralization
high mass-resolution stigmatic secondary ion 77 (Camera IMS 1270, Gennevilliers Cedex,
Fluoride
France).
Dentin
Results. Uptake of 44 Ca was greater in intensity for the 0.2% fluoride group than the control,
Ca and
Ca labeled mineral distribution was observed by a
especially in the superficial lesions. The control group showed 40 Ca (intrinsic) distribution in the subsurface lesions and in the superficial lesions, meanwhile the fluoride group showed 40
Ca distribution limited in subsurface lesions. The total Ca (44 Ca + 40 Ca) image revealed
more homogeneously for the control than the fluoride group. Significance. Since the fluoride-treated surface is more acid-resistant than intrinsic dentin, alternative minerals were dissolved from the intact intrinsic lesion in the demineralization cycle. © 2018 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
∗
Corresponding author. E-mail addresses: [email protected] (N. Hiraishi), [email protected] (S. Kobayashi), [email protected] (H. Yurimoto), [email protected] (J. Tagami). https://doi.org/10.1016/j.dental.2018.01.022 0109-5641/© 2018 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
e58
1.
d e n t a l m a t e r i a l s 3 4 ( 2 0 1 8 ) e57–e62
Introduction
Dental caries is caused by an action of acids on an enamel surface and developed into dentin lesion. Unlike bone, toothregeneration 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 toothderived mineral (intrinsic Ca) or solution-derived mineral uptake (extrinsic Ca). To overcome this limitation, we used 44 Ca (a stable isotope) doped remineralization solution while a pH-cycling. Since the natural abundance of calcium isotopes, 40 Ca, 42 Ca and 44 Ca, is 96.941%, 0.647% and 2.086%, respectively, 40 Ca represents intrinsic Ca, which is derived from tooth, meanwhile 44 Ca 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 44 Ca 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 40 Ca and 44 Ca 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.
2.
Materials and methods
2.1.
Preparation of bovine root dentin specimens
Root dentin blocks were obtained from root surfaces of six bovine incisor teeth. This 44 Ca 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 dentine surface was painted with a nail vanish, leaving a 5 mm diameter window.
2.2.
Methods of pH-cycling
A stable Ca isotope, 44 Ca, was purchased from ISOFLEX (San Francisco, Ca, USA), and other chemical ingredients were from Sigma-Aldrich (St. Louis, MN, USA). The remineralization solution was prepared using 44 Ca to contain 1.5 mmol/L CaCl2 (44 Ca), 0.9 mmol/L KH2 PO4, 130 mmol/L KCl, 20 mmol/L HEPES buffer at pH 7.0. The demineralizing solution was prepared using a conventional calcium compound, and contained 50 mmol/L acetic acid, 1.5 mmol/L CaCl2 and 0.9 mmol/L KH2 PO4 adjusted to pH 5.0. The dentin samples were soaked in the demineralization solution (pH 5.0) for 2 days to make pre-lesion (about 150 m). The pH-cycling was employed by demineralization (pH 5.0) for 2 h, treatment with 0% (control) or 0.2% NaF (900 ppm fluoride) at pH 7 for 2 h and 44 Ca doped remineralization for 20 h. The period of the cycling was 14 days. Since we aimed to observe calcium intakes from the remineralization solution, we soaked the samples in a prolonged remineralization cycle.
2.3. Microradiography observation and isotope imaging of Ca distribution The samples were sectioned into halves, each for TMR and isotope imaging. For TMR, the sectioned samples were further cut longitudinally into 220 ± 20 m thickness. The cut sections were put in a solution containing 80% of glycerin and 20% of water to prevent shrinkage. Photo plates (HY2, Konica Minolta Holdings, Inc., Tokyo, Japan) were exposed at 4 mA and 25 kV for 5 min using an X-ray generator (Type SRO-M50, Sofron Company Lit., Tokyo, Japan). After the plates were developed and fixed, the microradiographs were taken under a microscope (BX 41, Olympus Co., Tokyo, Japan) with CCD camera (DP70, Olympus Co., Tokyo, Japan). For isotopic microscopy, the sectioned surface was embedded and polished using a series of silicon carbide papers up to 4000-glit. Isotope imaging of 40 Ca+ and 44 Ca+ mineral distribution was observed by a high mass-resolution stigmatic SIMS instrument (Camera IMS 1270, Gennevilliers Cedex, France) [4,5]. The 130 m diameter on sample surface was irradiated by O — primary beam (23 KeV, 10 nA). Secondary ions 40 Ca+
d e n t a l m a t e r i a l s 3 4 ( 2 0 1 8 ) e57–e62
e59
Fig 1 – TMR and isotope microscopy images of three sections: (I) the control group and (II) the 0.2% fluoride treatment group. Lesions encircled with in bold line in TMR images were observed with isotope microscope. Brightness for 40 Ca, 44 Ca and 40 Ca + 44 Ca images are adjusted to distinguish each distribution. For the control group (Ia, Ib and Ic), the 40 Ca (intrinsic Ca) is present in their superficial layers, and 44 Ca (extrinsic Ca) shows moderate uptake in their surface layers. The total 40 Ca + 44 Ca indicates a homogeneous distribution in the lesions. For the 0.2% fluoride treatment group (IIa, IIb and IIc), the 40 Ca is less in their surface lesion (arrows in 40 Ca images) especially in 0–10 m depth for (IIa); 0–30 m depth for (IIb); 0–20 m depth for (IIc). The 44 Ca images shows greater uptake especially in superficial layer (arrows in 44 Ca images). The total 40 Ca + 44 Ca exhibits a heterogeneous distribution, lesser in their subsurface lesions (arrows in 40 Ca + 44 Ca images). The squares in 40 Ca isotope images are profiled to determine intrinsic mineral loss. Each 190 × 190 pixel in 40 Ca isotope image in a dotted line square is used to calculate the intrinsic mineral loss.
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Fig. 2 – Gray values of the 190 × 190 pixel in 40 Ca isotope images as shown in Fig. 1 (the squares in dotted line). The integrated area of the 190 × 190 pixel was assigned as Ca(40). The straight line with G(high) indicates the highest gray value. The integrated area between the G(high) and gray value was assigned as the loss of 40 Ca, Ca(loss). Intrinsic mineral loss was calculated as Ca(loss)/Ca(40). Ca(40)
Ca(loss).
and 44 Ca+ were obtained with exposure time of 10 and 25 s, respectively. These irons were constructed as a mass-filtered ion image using stigmatic ion optics of IMS 1270 and visualized when directly projected on a pixel matrix of stacked CMOS-type active pixel sensor. The obtained 40 Ca data was processed into a gray-scale image by the use of a customized image processing software (ImageJ, version1.42q, Wayne Rasband, NIH, USA). Representative 190 × 190 pixels in 40 Ca data was selected from the intrinsic dentin toward the boundary surface where extrinsic 44 Ca was detected. The integrated area of the 190 × 190 pixel was assigned as Ca(40) and the highest gray value was recorded as G(high). In the 40 Ca profile, the integrated area between G(high) and gray value was assigned as Ca(loss), loss of 40 Ca. Then, the intrinsic mineral loss was calculated as Ca(loss)/Ca(40), which was statistically analyzed using Ttest (p < 0.05) between the control and fluoride groups using a statistical software package (SigmaStat Version 16.0, SPSS, Chicago, IL, USA).
3.
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 40 Ca (intrinsic) distribution in their subsurface lesion and superficial lesion as well. Meanwhile the fluoride group shows that
40 Ca (intrinsic) distribution is present in the subsurface lesions
for all samples, but lesser toward the surface lesions, especially at 0–10 m depth for (IIa), 0–30 m depth for (IIb) and 0–20 m depth for (IIc). Uptake of 44 Ca (extrinsic) was greater in intensity for the superficial layer of the fluoride group than the control. The total Ca (44 Ca + 40 Ca) image reveals more homogeneously for the control than the fluoride group. For the fluoride group, the total Ca (44 Ca + 40 Ca) is intensified on the superficial lesion, but less in the subsurface lesion, at 15 m depth for (IIa), 40 m depth for (IIb) and 30 m depth for (IIc). These total Ca (44 Ca + 40 Ca) images are in consistence with corresponding TMR images, which show deficiency in mineral in the subsurface. As shown in Fig. 2, the intrinsic mineral loss was 0.107% ± 0.021% and 0.412% ± 0.072% for the control and fluoride group, respectively. T-test showed that there was a significant difference (p = 0.012).
4.
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
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is, on the other hand, determined as a total mineral gain of tooth-derived mineral (intrinsic Ca) and solution-derived mineral uptake (extrinsic Ca). The conventional pH-cycling and subsequent TMR measurement provides an apparent mineral gain, in which the authentic mineral gain cannot be validated. As shown in Fig. 1, the isotope microscopic measurement exhibited the distribution of intrinsic Ca and extrinsic Ca. The control (0% fluoride treatment) showed a homogeneous mineral distribution in the surface and subsurface lesion, in which a relatively intact subsurface layer was remained. Interestingly, for the fluoride group, there was a different distribution of mineral (40 Ca and 44 Ca). Dissolved intrinsic minerals (40 Ca) have not been compensated by the mineral gain in the subsurface lesion, in which the total mineral was less than the surface lesion. The effect of fluoride has been addressed in the WHO Global Oral Health Programme, which has focused its effect in dental caries prevention and in improvement communitybased public oral health [9]. The action of fluoride on mineral has been well documented in previous studies [10–16]. At a low fluoride concertation (i.e. less than 100 ppm), fluoride incorporation in apatite structure takes place, due to an ionic exchange of F− for OH− in apatite or epitaxial crystal growth of fluorapatite when solution is supersaturated for the crystal growth [14]. At a higher fluoride concertation than 100 ppm, CaF2 -like formation is initiated [10,14–16]. At this concentration, fluoride ions may be loosely bound or non-specifically adsorbed on minerals, where calcium and other ions such as phosphate are utilized to form phosphate-containing CaF2 like minerals [14–16]. This phosphate-containing CaF2 is slightly more soluble than pure CaF2, thus acting as a fluoride reservoir during the pH-cycling [17]. In the remineralization pH-cycling, the crystals in remaining minerals serve as nuclei for an accumulation of new mineral which fluoride is incorporated into, resulting in epitaxial crystal growth of fluorapatite [14]. In this event, fluorhydroxyapatite and/or fluorapatite is formed and precipitated in the surface layer [11]. The fluorhydroxyapatite and fluorapatite remain stable if not at lower than a pH 4.7, meanwhile mineral in dentin are easily dissolved at critical pH values for dentin at pH 6.3 [11]. In our study, the dentin samples were treated with 900 ppm fluoride for 2 h between remineralization and demineralization cycling. When the tooth samples were treated with 900 ppm fluoride, fluoride ions were bound on dentin surface, and CaF2 -like minerals was initially formed, acting as fluoride ions reservoir. In the following remineralizing cycling, fluoride ions were reacted with the extrinsic 44 Ca ions to promote epitaxial crystal growth of fluorapatite and/or fluorhydroxyapatite. This fluoride action rendered the surface lesion more acid-resistant than the subsurface lesion. Another noteworthy fact is that native intrinsic apatite in hard tissue in tooth and bone are formed in corporation with carbonated hydroxyapatite mineral, which is more soluble than pure hydroxyapatite mineral and fluorapatite-like mineral [13]. In the demineralizing cycle at pH 5, the aforementioned fluorhydroxyapatite and/or fluorapatite were hardly dissolved. Meanwhile, the mineral of subsurface lesion was alternatively dissolved because intrinsic dentin in the subsurface lesion was less acid-resistant. This alternative mineral loss has been continuously occurred from the subsurface
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intrinsic lesion. As a results, the intrinsic mineral loss that was calculated as Ca(loss)/Ca(40) was statistically higher in the fluoride group than the control group. The gradual depletion of intrinsic dentin was contributed to the less total mineral (40 Ca + 44 Ca) as shown in the subsurface of the fluoride lesion when compared with the control group. In other word, when the solubility on the surface was modified by fluoride to be acid-resistant, alternative minerals were dissolved at the expense of intact intrinsic mineral. Within limitations, it may be concluded that the extrinsic Ca uptake was increased at the superficial lesion by the use of fluoride; however, the intrinsic Ca loss was predominant, resulting that total Ca was less in that lesion. In further studies, the effect of fluoride must be investigated in vivo condition and clinical approach. Furthermore, it would be interesting to perform isotope microscopic observation on enamel specimens to investigate early caries progression, clinically shown as a white opaque spot.
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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