Ability of Barrier Coat S-PRG coating to arrest artificial enamel lesions in primary teeth

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1 Research Article Ability of Barrier Coat S-PRG coating to arrest artificial enamel lesions in primary teeth YUMIKO HOSOYA, DDS, PHD, SUSUMU ANDO, DDS, PHD, HIDEJI OTANI, DDS, TETSUHIRO YUKINARI, DDS, MASASHI MIYAZAKI, DDS, PHD & FRANKLIN GARCIA-GODOY, DDS, MS, PHD, PHD ABSTRACT: Purpose: To evaluate the effects of a surface pre-reacted glass-ionomer (S-PRG) filled coating material to arrest artificial enamel lesions in primary teeth. Methods: Buccal and lingual enamel was demineralized in 0.1 M lactic acid buffer solution (ph 4.75) for 5 days and then divided in the PRG-applied and non-prg areas. Proximal surfaces were used as a control area without demineralization and coating application. Teeth were divided into three groups (n= 4) according to the 1-week immersion in different solutions: Group 1 (distilled water), Group 2 (demineralizing solution) and Group 3 (artificial saliva). Hardness and Young s modulus by nano-indentation test, and elemental contents and ultrastructure by SEM/EDX analysis were obtained. Data were statistically analyzed using ANOVA and Fisher s PLSD at = Results: Only for the non-prg area in Group 1, the hardness and Young s modulus of the demineralized surface enamel were significantly lower than those of the enamel μm beneath the surface. Demineralized enamel of non-prg and PRG-applied areas showed similar SEM views. Only for the non-prg area in Group 2 and control area in Group 3, the Ca/P of the surface enamel was significantly higher than that of the enamel 5-10 μm beneath the surface. There was no significant difference of the Ca/P among the measuring points from the surface to 10 μm depth of enamel for the PRG applied area in Group 2. (Am J Dent 2013;26: ). CLINICAL SIGNIFICANCE: After 1-week immersion in demineralizing solution in Group 2, the Ca/P on the demineralized surface enamel area without PRG application was significantly higher than that of the enamel beneath the surface. On the PRG applied area, progression of demineralization on the surface enamel was not observed. S-PRG filled coating material might be effective to arrest caries and remineralize the incipient enamel caries of primary teeth. : Dr. Yumiko Hosoya, Department of Pediatric Dentistry, Unit of Translational Medicine, Course of Medical and Dental Science, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1, Sakamoto, Nagasaki , Japan. E- : hosoya@nagasaki-u.ac.jp Introduction Incipient enamel caries lesions can be arrested or even remineralized. 1,2 The common treatment strategy comprises application of fluorides, oral hygiene education and proper diet. 3-6 Recently, sealing or infiltration of incipient enamel caries lesions with low viscosity light curing resins was also used. 7,8 Surface pre-reacted glass-ionomer (S-PRG) fillers are a new type of particles that can be incorporated into resin materials. They are prepared by an acid-base reaction between fluoroboroaluminosilicate glass and a polyacrylic acid aqueous solution. 8 A ligand exchange mechanism within the pre-reacted hydrogel endows S-PRG fillers with the ability to release and recharge fluoride. 9 In addition to fluoride, S-PRG fillers released Al, B, Na, Si and Sr ions. 8,10 Silicate and fluoride strongly induce remineralization of the dentin matrix. 11 Strontium and fluoride also improve the acid resistance of teeth by converting hydroxyapatite to strontium apatite and fluoroapatite. 12 S-PRG fillers alter the ph of the surrounding environment within a weakly alkaline range when they come into contact with water or acidic solutions. 13 Recently, a resin coating material containing S-PRG fillers became available for use in caries prevention where it is hoped that it would enhance mineralization and reduce acidic attack by oral cariogenic bacteria. 14 Treatment to arrest caries of primary teeth is useful in the pediatric dental field especially for managing caries in uncooperative young child patients and in disabled patients. Treatment to arrest caries is also useful on incipient caries lesions in teeth not fully erupted and with immature mineralization. Coating materials make the enamel surface smooth and protect against bacterial retention in the incipient caries lesion. The esthetic advantage of coating materials is also preferable. However, little information has been reported on the efficacy of S-PRG filled coating material on treatment to arrest caries for primary tooth enamel. This study examined the effects of Barrier Coat, a a S-PRG filled coating material, on changes in structure of artificiallydemineralized primary tooth enamel. The null hypothesis tested was that Barrier Coat application does not influence the physiccal qualities and elemental contents of artificially-demineralized primary tooth enamel. Materials and Methods Twelve healthy primary teeth (six primary canines and six primary molars) that were extracted to expedite eruption of the permanent teeth, or for orthodontic reasons, were used as substrates for the present study. Informed consent for tooth collection was obtained from parents and subjects, according to the regulations of Nagasaki University Dental School (Permission No. 26). The teeth were frozen in physiologic saline within 10 minutes after extraction. The dental pulp was removed and each tooth was ultrasonically cleaned in distilled water. The pulp chamber of each tooth was filled with white-colored wax. a The enamel surfaces of the teeth except for the labial surfaces of primary canines and buccal and lingual surfaces of primary molars were coated

2 American Journal of Dentistry, Vol. 26, No. 5, October, 2013 with nail varnish. Proximal surfaces were set for the control (Control area) without demineralization or PRG Barrier Coat application. Teeth were demineralized in 0.1 M lactic-acid buffered solution (ph 4.75 demineralization solution; 0.75 mm CaCl 2 /2H 2 O and 0.45 mm KH 2 PO 4 ) for 5 days. The buccal, lingual or labial areas of each tooth were divided into a PRG Barrier Coat applied area (PRG) and a non-applied area (non- PRG) at the center of the tooth (Fig. 1). In the PRG applied area, a thin film coating was applied to the demineralized enamel and light cured for 10 seconds with a 2000 mw/cm 2 highpower LDA light-curing unit (G-Light Prima- b ). The composition of PRG Barrier Coat Base are: S-PRG fillers, polymeric fillers and water, and of the active: carboxylic acid monomer, phosphoric acid monomer, 2,2'-bis (4-methacryloxy polyethoxyphenyl) propane (Bis-MPEPP), tetraethyleneglycol dimethacrylate (TEGDMA), polymeric monomer and photo initiator. The teeth were divided into three groups (n= 4) according to the soaking solutions: Group 1 (distilled water), Group 2 (demineralizing solution) and Group 3 (artificial saliva). Artificial saliva soaking was set to simulate oral environment, and soaking in demineralizing solution to replicate the acidic oral condition with high caries activity. Distilled water soaking was thought not to directly influence the components of the coating material. Artificial saliva, with an electrolyte composition similar to that of human saliva, was prepared from 1.09 mmol/l CaCl 2, 0.68 mmol/l KH 2 PO 4, 30 mmol/l KCl and 2.6 mmol/l NaF. The artificial saliva was buffered to ph 7.0 with 50 mmol/l N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Each tooth was immersed in a glass bottle filled with 10 ml of solution at 24 C for 7 days. The solutions were changed every 48 hours. After 1-week immersion, the S-PRG coating material was removed with a dental explorer. The specimens were sectioned perpendicular to the longitudinal axis. Sectioning was performed using a low-speed diamond saw (Isomet c ) under copious water-cooling. Two to three sectioned specimens were obtained from each tooth. After sectioning, the specimens were polished with wet 1,200-grit silicon carbide papers d with final polish with 0.3 μm aluminum oxide lapping films. d The polished specimens were stored in the containers at 4 C with 100% wet condition until the nano-indentation testing. One of the polished specimens from each tooth was used for nano-indentation evaluation using a nano-indentation tester (ENT-1100a e ) with a chamber temperature of 26 C. Hardness (H) and Young s modulus (Y) were calculated following previously reported procedures. 15,16 Indentations were made at 10 μm intervals from the enamel surface to the pulp chamber direction, using a load of 300 mgf for 10 seconds. Data obtained from the first seven indentations of each line were analyzed. Three lines of indentations were made for each specimen. The indentations were observed using a microscope with a CCD camera attached to the tester at x700 magnification, to ensure symmetrical indents and adequate spacing between the indentations. Scanning electron microscopy (SEM) was used after argon ion etching (ERA-8800 FE e ) to examine the quality of the indentations and its morphology. Data derived from the two biomechanical parameters of H and Y were found to be normally distributed (Kolmogorov- Smirnoff test) and homoscedastic (Levene test). They were then analyzed separately using two-way ANOVA to examine the Barrier coat to arrest artificial enamel lesion 287 Fig. 1. Areas and measuring lines for nano-indentation test and SEM/EDX analysis. A. (PRG applied area), B. (non-prg area), and C. (control area). effect of nanoindentation locations (i.e. distances away from the interface) and (i.e. control, non-prg and PRG areas for each group) and the interaction of these two factors on the two biomechanical parameters. For each parameter, post-hoc comparisons were performed using Fisher s PLSD test with = For SEM/EDX analysis, the elemental contents of enamel were analyzed at 20 kv and x2000 magnification. Analysis was conducted perpendicular to the tooth surface, in 5 μm intervals toward the pulp chamber, thereby creating measuring points 0 (tooth-surface), 1 (just beneath the surface), 2 (5 μm beneath the surface) and 3 (10 μm beneath the surface). Three measuring lines were established for each test surface. (Fig. 1) Analysis was performed with ZAF correction (atomic number, absorption and fluorescence) based on standardless correction. The surfaces were carbon-coated for SEM/EDX analysis (GENESIS 2000 f ), and Au coated for SEM observation (JSM- 6340F g ). Both of the measuring points, one for SEM/EDX analysis and another for the nanoindentation test were set at the same level and locations from the surface. The data was statistically analyzed using a one-way ANOVA to examine the effects in locations (i.e. measuring points 0-3.) for each area (control, PRG and non-prg) in each of Groups 1, 2 and 3. Post-hoc comparisons were performed using Fisher s PLSD test with = Results Table 1 shows the mean hardness (H) values. Hardness was compared among different measuring points for control, non- PRG and PRG areas in each group: for the control area in all groups, there was no significant difference for the H among all measuring points. A significant difference was clearly observed only for non-prg area and PRG area in Group 1. Table 2 shows the mean Young s modulus. When comparing the Y among different measuring points for each of control, non-prg and PRG areas in each group, for all areas in Groups 2 and 3, there was no significant difference of the Y among all measuring points. For the non-prg area in Group 1, the Y of measuring points 1 and 2 were significantly lower than that of measuring points 4, 5, 6 and 7.

3 288 Hosoya et al American Journal of Dentistry, Vol. 26, No. 5, October, 2013 Table 1. Means and standard deviations of hardness values (unit: GPa) ± 2.61 a, ± 0.45 a, ± 0.92 a, ± 0.98 a, ± 2.30 a, ± 1.48 a, ± 0.82 a, ± 0.94 a,1, ± 1.48 a, ± 2.00 a, ± 1.19 a,c, ± 0.90 a,c, ± 0.47 a, ± 1.34 a, ± 1.23 a, ± 1.18 a, ± 0.73 a,b, ± 0.76 a, ± 1.34 a, ± 0.81 a,d,e, ± 1.24 a, ± 0.81 a, ± 0.72 a,1, ± 1.37 b, ± 0.81 a, ± 0.62 a,b, ± 0.81 a, ± 0.71 a, ± 2.06 b, ± 0.74 a,d, ± 0.90 a, ± 0.86 a, ± 0.76 a,b, ± 0.36 a, ± 0.86 a,b, ± 1.27 a, ± 0.53 a, ± 1.17 b,c,e, ± 0.90 a,d, ± 2.03 a, ± 0.63 a, ± 1.02 a,b, ± 0.34 a, ± 0.89 b, ± 1.19 a, ± 1.28 a, ± 0.63 b,d,e, ± 0.84 b,d, ± 0.88 a, ± 0.32 a, ± 0.40 a,b, ± 0.92 a, ± 0.50 a,b, ± 0.68 a, ± 1.35 a, ± 1.35 b, ± 0.95 b,c, ± 0.73 a, ± 0.84 a, ± 1.22 a,b, ± 1.16 a, ± 0.25 a,b, ± 1.13 a,2 A = Measuring point. B = Distance from surface (μm). For each of the control, non-prg and PRG areas for each group (Group 1, Group 2, Group 3), different letters (a, b, c, d, e) among different measuring points represent statistical significance (P< 0.05). For each of the measuring points for each group (Group 1, Group 2, Group 3), different numerical designators (1, 2) among control, non-prg, and PRG areas represent statistical significance (P< 0.05). Table 2. Means and standard deviations of Young s modulus (unit: GPa) ± 9.48 a, ± 1.55 a, ± 2.97 a, ± 2.08 a, ± a, ± 3.53 a, ± 4.92 a, ± 2.29 a, ± 3.88 a, ± 4.71 b, ± 2.50 a, ± 2.57 a, ± 1.50 a, ± 3.48 a, ± 3.13 a, ± 4.68 a, ± 2.07 a, ± 2.64 a, ± 2.79 a,b, ± 2.65 a,c, ± 4.59 a,b,1, ± 2.57 a, ± 1.94 a, ± 2.96 a, ± 2.84 a, ± 1.92 a, ± 2.21 a, ± 1.25 a,b, ± 5.10 b, ± 1.97 a,b, ± 4.30 a, ± 2.72 a, ± 1.72 a, ± 3.88 a, ± 2.53 a, ± 3.71 a, ± 1.21 a,b, ± 3.96 b,c, ± 2.51 a,b,1, ± 5.35 a, ± 2.12 a, ± 2.42 a, ± 2.40 a, ± 2.74 a,1, ± 3.13 a, ± 2.26 a,b, ± 3.12 b,c, ± 1.89 a,b, ± 4.86 a, ± 1.14 a,1, ± 1.26 a, ± 3.79 a, ± 1.40 a, ± 2.31 a, ± 1.60 a,b, ± 4.33 b,c, ± 2.38 b, ± 2.23 a, ± 1.68 a, ± 2.55 a, ± 5.92 a, ± 1.44 a, ± 2.86 a,2 A = Measuring point. B = Distance from surface (μm). For each the control, non-prg and PRG areas of Group 1, Group 2, and Group 3, different alphabetical letters (a, b, c) among different measuring points represent statistical significance (P< 0.05). For each of the measuring points for each group (Group 1, Group 2, Group 3), different numerical designators (1, 2) among control, non-prg and PRG areas represent statistical significance (P< 0.05). Table 3. Means and standard deviations of Ca/P ratio (0.1) a 1.4 (0.2) a 3.8 (3.8) a 1.6 (0.1) a 2.0 (0.4) a 1.7 (0.3) a 2.3 (0.0) a 1.5 (0.0) a 2.5 (0.0) a (0.1) a 2.0 (0.5) a 1.8 (0.2) b 1.9 (0.3) a 1.6 (0.3) a,b 1.6 (0.2) a 2.1 (0.0) a 1.8 (0.0) a 1.9 (0.5) a (0.2) a 1.7 (0.2) a 2.0 (0.4) a,b 1.8 (0.4) a 1.5 (0.3) b 1.5 (0.4) a 1.9 (0.1) b 1.9 (0.0) a 2.1 (0.0) a (0.0) a 2.2 (1.2) a 1.9 (0.2) a,b 1.7 (0.5) a 1.5 (0.2) b 1.6 (0.1) a 1.8 (0.0) b 1.8 (0.0) a 1.7 (0.3) a A= Measuring point. B=Distance from surface ( m). For each of the control, non-prg and PRG areas for each of the Group 1, Group 2 and Group 3, different alphabetic letters (a, b) among different measuring points represent statistical significance (P< 0.05). Figure 2 shows representative SEM views for different areas of Group 1. The images of a, b and c are expanded views for each of A, B and C. In the control area (A and a), the enamel surface was smooth. Compared to the non-prg area, deeper and stronger demineralization was observed in PRG area. Table 3 shows the mean Ca/P ratio. For the non-prg area in Group 2 and control area in Group 3, the Ca/P of the measuring point 0 was significantly higher than that of measuring points 2 and 3. Discussion In the present study, depth of artificially demineralized enamel showed a large variation among the teeth and locations, and the depth ranged from less than 1 μm to over 10 μm (Fig. 2). Tooth to tooth differences influenced the results of both the nano-indentation test and EDX analysis (Tables 1-3). A previous study reported that both nanohardness and elastic modulus gradually decreased from the enamel surface toward the dentin-enamel junction, and such a variation correlates well with the decreasing trend of calcium composition. 17 This phenomenon indicates that mineral content has a strong influence on nanohardness. A study of nanoindentation mapping of molar tooth enamel reported that the range of the hardness (H) and Young s modulus (Y) observed over an individual tooth showed great variations in different areas. 18 These variations corresponded to the changes in chemistry, microstructure, and prism alignment, and showed the strongest correlations with changes in the average chemistry of enamel. 18 The highest CaO or P 2 O 5 were observed with the highest H and Y at the enamel surface, and the lowest CaO and P 2 O 5 were observed with the lowest H and Y at the inner enamel region. 18 In this study, when comparing the H and Y among different measuring points for each of area in each group, only the non- PRG area and PRG area in Group 1 (1-week immersion in distilled water), for H, and non-prg area in Group 1 for Y showed significant differences (Tables 1 and 2). The H and Y of demineralized surface area of measuring point 1 was significantly lower than that of the measuring points μm beneath the surface. These results showed that regardless of PRG application, 1 week immersion in distilled water could not recover the hardness of demineralized surface enamel. The H and Y of measuring point 1 on demineralized surface of Group

4 American Journal of Dentistry, Vol. 26, No. 5, October, 2013 Barrier coat to arrest artificial enamel lesion 289 Fig. 2. Representative SEM views on different areas in Group 1 (soaked in distilled water). A. ( 500) and a. ( 3000) in control area, the enamel surface was smooth. B. ( 500) and b. ( 3000) in non-prg area, 1-2 μm thick slight demineralization at the enamel surface was seen. C. (x500) and c. ( 3000) in PRG applied area, demineralized surface enamel and amorphous materials on the enamel surface were observed. 2 (1-week immersion in demineralized solution) and Group 3 (1-week immersion in artificial saliva) did not show significant lower values compared to the values of enamel μm beneath the surface (Tables 1 and 2). The influence of the PRG coating material and /or artificial saliva for remineralization and/or arresting demineralization might be considered. In Group 2, the PRG coating material might have been dissolved by the demineralizing solution and the efficacy of PRG might have been influenced not only by the PRG-applied area, but also by the non-prg area. In Group 3, fluoride and other ions from PRG coating material 8,9 to PRG area, and calcium and fluoride from artificial saliva to non-prg area might induce remineralization. Relatively large standard deviations caused by tooth to tooth and region to region differences 18 were also one of the reasons for no significant difference in other areas, especially for control areas in all the groups. The results of this study showed that 1-week immersion in demineralized solution did not cause additional demineralization with significantly lower H and Y on the demineralized surface enamel. This result may suggest that the S-PRG coating material used in this study may arrest caries on primary tooth enamel. The crystalline qualities of calcium phosphate (apatite) are thought to be improved by increases in Ca and the Ca/P ratio. 19 In this study, increased Ca/P ratio in the enamel surface (Table 3) was observed only at the non-prg area in Group 2 (immersed in demineralizing solution) and control area in Group 3 (immersed in artificial saliva), however, neither significant increase of Ca% nor significant decrease of P% was observed. In this study, the depth of demineralization of many specimens was less than 5 μm beneath the surface, however, in some specimens, the depth of demineralization was more than 10 μm beneath the surface. Although the depth of demineralization exceeded 10 μm beneath the surface in some specimens (Fig. 2), EDX analysis was tested for the enamel less than 10 μm beneath the surface. It might be considered that in some areas, all measuring points were set at the demineralized enamel, thus it is hard to obtain a statistically significant difference among the measuring points (Table 3). In future studies, SEM/EDX analysis should also evaluate the enamel more than 10 μm beneath the surface including sound and not demineralized enamel in all the specimens. The Ca/P ratio on the demineralized enamel surface (measuring point 0) was not significantly lower than that 10 μm beneath the surface of all groups including the Group 2 (immersed in demineralizing solution) (Table 3). Influence of demineralizing function to S-PRG coating material should be observed in a future study. Remineralization efficacy 9 and modulation effect on the acidic conditions 14 of S-PRG may be expected to arrest caries on primary enamel. Release of B, Na, Si, and Sr ions were not detected on the S-PRG applied surface after 7-day immersion in the three different solutions tested. Small amounts of these ions might have dissolved in the solution especially in the demineralizing solution, however, the level of recharge into the demineralized enamel lesion might be below the level of detection. In the present study, the possibility of remineralization in the demineralized enamel was uncertain; however, enamel demineralization may be arrested by the S-PRG coating. It is not certain that 7-day S-PRG coating with Barrier-Coat and/or 7-day immersion in artificial saliva is adequate to arrest the demineralization. Further studies are required to evaluate the effect of repeated PRG application and longer time soaking in artificial saliva to demonstrate the remineralization and arresting caries potential of the S-PRG coating. Within the limitations of the present study, the null hypothesis that S-PRG coating did not influence the physical qualities and elemental contents of artificially demineralized primary tooth enamel was rejected. The effect of Barrier Coat S-PRG coating was only partially validated in this study using demineralized primary tooth enamel. To demonstrate the efficacy of Barrier Coat in arresting caries for primary enamel, further studies should evaluate the stability of this coating material in the oral environment and the clinical efficacy of this material in arresting caries. a. Shofu Inc., Kyoto, Japan. b. GC Co., Tokyo Japan. c. Buehler, Lake Bluff, IL, USA. d. Maruto Co., Tokyo, Japan.

5 290 Hosoya et al American Journal of Dentistry, Vol. 26, No. 5, October, 2013 e. Elionix Co., Tokyo, Japan. f. EDAX Inc., Tokyo, Japan. g. JEOL, Tokyo, Japan. Acknowledgements: To Mr. Takuji Ito and Yukiko Omata (Elionix Co., Tokyo, Japan) for technical assistance in operating the nano-indentation tester and SEM/EDX analysis. Disclosure statement: The authors declared no conflict of interest. Dr. Hosoya is Associate Professor, Department of Pediatric Dentistry, Unit of Translational Medicine, Course of Medical and Dental Science, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan. Dr. Ando is Associate Professor and Dr. Miyazaki is Professor, Department of Operative Dentistry, Nihon University School of Dentistry, Tokyo, Japan. Dr. Otani is Pediatric Dental Practitioner, Otani Dental Clinic, Nayoro, Japan. Dr. Yukinari is a pediatric and orthodontic privatel practitioner, Yukinari Pediatric and Orthodontic Dental Clinic, Nagasaki, Japan. Dr. Garcia-Godoy is Professor, Senior Executive Associate Dean for Research and Director, Bioscience Research Center, College of Dentistry, University of Tennessee, Memphis, and Senior Clinical Investigator, The Forsyth Center, Cambridge, Massachusetts, USA. References 1. Featherstone JD. The science and practice of caries prevention. J Am Dent Assoc 2000;131: Featherstone JD. Prevention and reversal of dental caries: Role of low level fluoride. Community Dent Oral Epidemiol 1999;27: Zantner C, Martus P, Kielbassa AM. Clinical monitoring of the effect of fluoride on long-existing white spot lesions. Acta Odontol Scand 2006; 64: Lagerweij MD, Ten Cate JM. Remineralization of enamel lesion with daily application of high-concentration fluoride gel and fluoridated toothpaste: An in situ study. Caries Res 2002;36: Koivusilta L, Honkala S, Honkala E, Rimpela A. Tooth-brushing as part of the adolescent lifestyle products education level. J Dent Res 2003; 82: Burt BA, Ismail AI. Diet, nutrition, and food cariogenicity. J Dent Res 1986;65: El-Kalla IH, Sandi HIA, El-Agany RAI. Effect of adhesive resin application on the progression of cavitated and non-cavitated incipient carious lesions. Am J Dent 2012;25: Ito S, Iijima M, Hashimoto M, Tsukamoto N, Mizoguchi I, Saito T. Effects of surface pre-reacted glass-ionomer fillers on mineral induction by phosphoprotein. J Dent 2011;39: Kamijyo K, Mukai Y, Tominaga T, Iwaya I, Fujino F, Hirata Y, Terasaka T. Fluoride release and recharge characteristics of denture base resins containing surface pre-reacted glass-ionomer filler. Dent Mater J 2009;28: Fujimoto Y, Iwasa M, Murayama R, Miyazaki M, Nagafuji A, Nakatsuka T. Detection of ions released from S-PRG fillers and their modulation effect. Dent Mater J 2010;29: Saito T, Toyooka H, Ito S, Crenshaw MA. In vitro study of remineralization of dentin: Effects of ions on mineral induction by decalcified dentin matrix. Caries Res 2003;37: Thuy TT, Nakagaki H, Kato K, Hung PA, Mukai J, Tsuboi S, Hirose MN, Igarashi S, Robinson C. Effect of strontium in combination with fluoride on enamel remineralization in vitro. Arch Oral Biol 2008;53: Murayama R, Furuichi T, Yokokawa M, Takahashi F, Kawamoto R, Takamizawa T, Kurokawa H, Miyazaki M. Ultrasonic investigation of the effect of S-PRG filler-containing coating material on bovine tooth demineralization. Dent Mater J 2012;31: Ma S, Imazato S, Chen J, Mayanagi G, Takahashi N, Ishimoto T, Nakano T. Effects of a coating resin containing S-PRG filler to prevent demineralization of root surfaces. Dent Mater J 2012;31: Hosoya Y, Marshall Jr GW. The nano-hardness and elastic modulus of carious and sound primary canine dentin. Oper Dent 2004;29: Hosoya Y, Marshall Jr GW. The nano-hardness and elastic modulus of sound deciduous canine dentin and young premolar dentin. Preliminary study. J Mater Sci Mater Med 2005;16: Jeng YR, Lin TT, Hsu HM, Chang HJ, Shieh DB. Human enamel rod presents anisotropic nanotribological properties. J Mech Behav Biomed Mater 2011;4: Cuy JL, Mann AB, Livi KJ, Teaford MF, Weihs TP. Nanoindentation mapping of the mechanical properties of human molar teeth enamel. Arch Oral Biol 2002;47: Eanes ED, Termine JD, Nylem MU. An electron microscopic study of the formation of amorphous calcium phosphate and its transformation to crystalline apatite. Calcif Tissue Res 1973;12: