Ó 2013 Eur J Oral Sci
Eur J Oral Sci 2014; 122: 78–83 DOI: 10.1111/eos.12107 Printed in Singapore. All rights reserved
European Journal of Oral Sciences
Inhibition of enamel demineralization by buffering effect of S-PRG fillercontaining dental sealant Kaga M, Kakuda S, Ida Y, Toshima H, Hashimoto M, Endo K, Sano H. Inhibition of enamel demineralization by buffering effect of S-PRG filler-containing dental sealant. Eur J Oral Sci 2014; 122: 78–83. © 2013 Eur J Oral Sci The buffering capacity and inhibitory effects on enamel demineralization of two commercially available dental sealants were evaluated in this study. The effects of filler particles were also examined. Disks of enamel and cured sealant materials of BeautiSealant (silica or S-PRG filler) or Teethmate F-1 were incubated in lactic acid solutions (pH 4.0) for 1–6 d. The pH changes and amounts of ions released in the solutions were assessed, and enamel surfaces were observed using a scanning electron microscope. The pH of the solution with BeautiSealant (S-PRG filler) was neutralized from pH 4.0 to pH 6.1 (after incubation for 1 d) and from pH 4.0 to pH 6.7 (after incubation for 6 d). In addition, no release of calcium ions was detected and the enamel surface was morphologically intact in scanning electron microscopy images. However, the pH of the solution with Teethmate F-1 remained below pH 4.0 during incubation from days 1 to 6. Calcium release was increased in solutions up to and after 6 d of incubation. Scanning electron microscopy images showed that the structures of hydroxyapatite rods were exposed at the specimen surfaces as a result of demineralization. Ions released from S-PRG filler-containing dental sealant rapidly buffered the lactic acid solution and inhibited enamel demineralization.
The clinical usage of dental sealants requires bonding techniques for penetration into deep fissures (1). Complicated pit and fissure morphologies provide ideal sites for collection of food debris, bacterial growth, and biofilm formation. Deep sites in fissures are inaccessible hidden portions and are not cleaned perfectly by mechanical or chemical cleaning in dental practice. Moreover, sealant does not reach the bottom of a fissure and hence a space remains under the cured sealant material (2, 3). In such cases, attempts to improve sealant application using a mechanical technique are limited. To resolve these problems, introduction of a chemical operating agent or a new material is needed for fissure sealing or treatment of hidden caries. It has been reported that glass-ionomer cement releases several types of ions, resulting in pH neutralization (4, 5), and that it is effective for controlling bacterial growth (6). A fissure sealant with fluoride-releasing properties is effective for protecting adjacent enamel from acid demineralization (7–9). Therefore, a bioactive material with an antibacterial effect that is effective for enamel remineralization and can eliminate initial caries is needed. A surface reactiontype glass-ionomer (S-PRG) filler has recently been developed: it can be incorporated into resinous materials and has been used clinically as a resin bonding agent, a sealant material, or a resin composite (10). The S-PRG
Masayuki Kaga1, Shinichi Kakuda2, Yusuke Ida1, Hirokazu Toshima1, Masanori Hashimoto1, Kazuhiko Endo1, Hidehiko Sano2 1
Division of Biomaterials and Bioengineering, Department of Oral Rehabilitation, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido; 2 Department of Restorative Dentistry, Division of Oral Health Science, Graduate School of Dental Medicine, Hokkaido University, Sapporo, Hokkaido, Japan
Visiting Prof. Masayuki Kaga, Division of Biomaterials and Bioengineering, Department of Oral Rehabilitation, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 0610293, Japan E-mail: [email protected] Key words: buffering capacity; demineralization; dental sealants; ion release; S-PRG filler Accepted for publication November 2013
filler is prepared with fluoroboroaliminosilicate glass and polyacrylic acid solution through an acid-based reaction (10). Interestingly, multiple ions are released in neutral and lactic acid conditions by S-PRG-containing resinous materials (11). Fluoride and strontium form an acid-resistant layer and reinforce tooth structure by acting on hydroxylapatite to convert it to a fluoride-apatite (12, 13) and strontium-apatite (14) complexes (14). In addition, a sealant releasing fluoride has shown inhibitory activity against mutans streptococci (15). Based on those findings, sealants that have the ability to release multiple ions should have more potential for long-term durability and self-repair ability against demineralization at the sealant–enamel interface. Therefore, the purpose of this study was to evaluate the buffering effects of dental sealants containing S-PRG filler on enamel decalcification. The null hypothesis is that ions released from sealant materials neutralize lactic acid and inhibit enamel decalcification.
Material and methods Test materials Two commercially available fissure sealants – BeautiSealant (BS) (Shofu, Kyoto, Japan) and Teethmate F-1
Buffering effect of S-PRG filler Table 1 Chemical formulations of the test materials Materials (manufacturers)
Chemical formulations
BeautiSealant (S-PRG) (Shofu, Kyoto, Japan) Experimental sealant (silica filler) (Shofu, Kyoto, Japan) Teethmate F-1 (Kuraray Noritake Dental, Tokyo, Japan)
UDMA, TEGDMA, S-PRG filler UDMA, TEGDMA, silica filler MDP, MF-MMA, TEGDMA, HEMA, hydrophobic aromatic dimethacrylate
HEMA, 2-hydroxyethyl methacrylate; MDP, 10-methacryloyloxydecyl dihydrogen phosphate; MF-MMA, methacryloyl fluoride methacrylate; TEGDMA, triethylene glycol dimethacrylate; UDMA, urethane dimethacrylate.
(Kuraray-Noritake, Tokyo, Japan) – and one experimental sealant were used in this study. To compare the effects of filler particles, we used the following two BS groups: one group with 40 mass% of S-PRG filler (particle size: approximately 1 lm) contained in the resin matrix (S-PRG group) and one group in which the S-PRG filler was replaced with silica filler (particle size: approximately 1 lm) to have the same viscosity as BS (experimental group). The formulations and compositions of test materials used in this study are shown in Table 1. Initial ion release from test materials Uncured liquids of sealants were placed in silicone molds (13 mm in diameter and 1 mm in thickness), clamped between cover glasses, and then immediately light-cured using a light curing unit (Blue shot; Shofu, Kyoto, Japan) for 60 s on each side. The disk surface was gently polished with 600-grit silicon carbide paper under running water to remove the uncured layer on the surface (n = 6 for each group). A lactic acid solution of pH 4.0 (5 ml) was pipetted into a 50-ml plastic tube, and a cured sealant disk was placed in the solution and incubated at 37°C for 1 d. The concentrations of silicon (Si), strontium (Sr), aluminum (Al), boron (B), and sodium (Na) were measured using inductively coupled plasma atomic emission spectroscopy (ICP-AES) (ICPS-8000; Shimadzu, Kyoto, Japan). Fluoride (F) release was analyzed using a fluoride ion-selective electrode (Orion 9609 BNWP; Thermo Fisher Scientific, Waltham, MA, USA) connected to an ion analyzer (Orion 2115010 Dual Star pH/ion-meter; Thermo Fisher Scientific). TISAB III buffer was added to the sample solution to provide a constant background of ionic strength to the fluoride sample in 10% volume and was stirred with a magnetic stirrer for 20 s. The fluoride electrode was immediately placed in the sample solution. pH measurement Four crown parts of a bovine upper central incisor were used in this study. A flattened enamel surface on the buccal aspect of the crown was longitudinally exposed using a low-speed diamond saw (Isomet; Buehler, Lake Bluff, IL, USA) and polished with 600-, 1,200-, and 2,000-grit silicon carbide papers under running water. Twenty-four enamel specimens were cut into sections measuring approximately 4 mm 9 4 mm and 1 mm in thickness, and were cleaned
79
with an ultrasonic cleaner for 15 s in distilled water. The following four groups (n = 6 for each group) were used for tests: (i) enamel disk (control); (ii) enamel disk with a cured Teethmate F-1 (TM) disk; (iii) enamel disk with a cured BS disk (S-PRG filler); and (iv) enamel disk with a cured experimental sealant disk (silica filler). Cured sealant disks were prepared as previously described. The test solution (5 ml of lactic acid solution adjusted to pH 4.0) was pipetted into a 50-ml conical tube (BD Falcon, Franklin Lakes, NJ, USA). The cured sealant and an enamel disk were then immersed in the test solution. A pH electrode (Orion 8102BNUWP; Thermo Fisher Scientific) connected to a pH/ion-meter (Orion 2115010 Dual Star pH/ion-meter; Thermo Fisher Scientific) was immediately inserted in the center of the plastic tube and maintained at room temperature. The pH was measured after 1, 2, 3, 4, 5, and 6 d. Sample tubes were incubated at 37°C for the duration of the experiment. Calcium release and scanning electron micriscopy observation of the enamel surface The amounts of calcium released from enamel in 5 ml of lactic acid were measured using an atomic absorption spectrometer (AAnalyst 200; Perkin-Elmer, Norwalk, CT, USA). The instrument was calibrated using standards containing 0, 0.1, 1, and 5 lg ml 1 of calcium ion in 0.1 mol l-1 HNO3-containing lanthanum chloride solutions. Two-hundred microliters of the lactic acid solution containing calcium released from enamel was transferred to a new tube and was diluted by adding 19.8 ml of 0.1 M HNO3-containing lanthanum chloride solution for analysis after incubation for 6 d. After incubation for 6 d, enamel disks were removed from the lactic solution and then rinsed with distilled water. The enamel disks were dried at room temperature for 48 h. The surfaces of enamel disks were sputter-coated with gold and observed using a scanning electron microscope (S-4000; Hitachi, Tokyo, Japan) with an acceleration voltage of 25 kV in a vacuum. Statistics The data (pH and ion release) were analyzed using twoway ANOVA followed by the Turkey-HSD post-hoc test. Significance was set at P < 0.05 (n = 6 for each group).
Results Initial ion release
Table 2 shows the amount of each ion released from sealants. Significantly larger amounts of Si, Sr, Al, B, and Na ions were released in the BS (S-PRG) group than in the other groups (P < 0.05). In the solution with experimental sealant (silica filler), only Si (0.21 lg ml 1), which was released from silica oxide, and a very low level of F were detected in the solution. There was no significant difference between the BS (S-PRG) and the TM groups (P < 0.05) in the amount of F ions released. pH measurement
Figure 1 shows changes in pH values in the solutions containing specimens. The pH of the solution with an
80
Kaga et al. Table 2 Concentration of ions
Material BS (S-PRG) Experimental sealant (silica filler) TF
Si
Sr
Al
B
Na
F
1.79 (0.05)a 0.21 (0.01)c 0.42 (0.03)b
10.80 (0.33) None 0.09 (0.05)
2.06 (0.07) None 0.08 (0.01)
5.16 (0.32) None 0.06 (0.01)
13.03 (0.58) None 0.81 (0.06)
7.53 (0.80)d 0.03 (0.01)e 7.85 (0.78)d
Values are given as mean (SD). Groups with different superscript letters are statistically different (P < 0.05, n = 6). The ion-release profile of tested materials (lg ml 1) was analyzed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) [for detecting the concentrations of silicon (Si), strontium (Sr), aluminum (Al), boron (B) and sodium (Na)] and a pH/ion-meter [for detecting the concentration of fluoride (F)] following 1 d of incubation at 37°C in lactic acid solution (pH 4.0). BS (S-PRG), BeautiSealant (S-PRG filler); TF, Teethmate F-1.
enamel disk (control) changed to 4.7 after 1 d and to 5.4 after 2 d. The pH of the solution with BS (S-PRG) showed a rapid increase from pH 4.0 to pH 6.1 at 1 d and a further, gradual, increase over the duration of the study to pH 6.7 at 6 d. The pH of the solution with experimental sealant (silica filler) was 5.5 at 3 d. However, the pH of the solution with TM showed a gradual decrease, to pH 3.6 at 1 d, and remained below pH 4.0 from 2 to 6 d. The pH of the solution with BS (S-PRG) was higher than the pH values in the other three groups from 1 to 3 d (P < 0.05). The pH of solution with TM was significantly lower on all days (P < 0.05). Calcium release and scanning electron microscopy observation
Table 3 shows Ca ion release from test materials and enamel disks after storage in lactic acid solution for 6 d. The lowest concentration of Ca ion release was observed in the BS (S-PRG) group, and was significantly smaller than that observed in all other groups (P < 0.05). No significant difference in Ca ion release was found between the solution with experimental sealant (silica filler) and the control group (enamel disk). The concentration of Ca ion released in the solution with TM was significantly higher than the concentration released in the other three groups (P < 0.05). Microstructure profiles of the enamel surfaces determined by scanning electron microscopy at 6 d are shown in Fig. 2. The enamel surface was smooth and polishing lines were observed in specimens in the control group (Fig. 2A). The groups of BS (Fig. 2B, S-PRG filler; and Fig. 2C, silica filler) showed fine and smooth surfaces with no sign of prism outlines. Enamel surfaces in the TM group exhibited a typical etched pattern with the removal of enamel prism peripheries, revealing rigid enamel rods with hydroxylapatite prism cores remaining (Fig. 2D).
Discussion Dental plaque and dental biofilm contain numerous acidogenic bacteria that ferment carbohydrates and produce various organic acids, which dissolve hard tissue and shift the calcium phosphate balance (16). Low pH facilitates caries formation. STEPHAN found that
Fig. 1. Changes in pH values in the different groups – enamel disk (control), enamel disk with BeautiSealant (S-PRG filler) [BS (S-PRG)], enamel disk with experimental sealant (silica filler), and enamel disk with Teethmate F-1 (TM) – when incubated for 1–6 d in lactic acid solution (pH 4.0). Different letters denote a statistically significant difference compared with the other groups for days 1–3 (P < 0.05, n = 6). The pH in the TM group was significantly lower than the pH in the other three groups on all days of measurement (P < 0.05).
enamel dissolution leading to caries formation occurred after prolonged exposure to a pH of pH 6.3 at 3 d (Fig. 1). In contrast, in the BS (S-PRG) group, the initial pH of the lactic acid solution (pH 4.0) rapidly increased to pH 6.2 after 1 d of incubation. However, no buffering effect of TM was observed during the 6-d experimental period in this study. Therefore, the null hypothesis of this study was proved for BS but rejected for TM. The buffering capacity of BS (S-PRG) and its protective effect on enamel demineralization were clearly
Buffering effect of S-PRG filler Table 3 Concentration of Ca ions Material
[Ca] (lg ml 1)
Control (enamel disk) BS (S-PRG) Experimental sealant (silica filler) TM
6.4 2.0 5.5 27.1
(4.3)b (2.5)a (4.5)b (9.0)c
Values are given as mean (SD) concentration of Ca ions released from enamel disks after storage in lactic acid solution for 6 d. Groups with different superscript letters are statistically significant (P < 0.05, n = 6). BS, BeautiSealant (S-PRG filler); TM, Teethmate F-1.
proved by the modest quantities of Si, Sr, Al, B, Na, and F ions released. However, the solution with TM showed a low pH (below pH 4.0), and 27.1 lg ml 1 of Ca was detected at 6 d. The results for demineralized Ca release coincide with the consistent pH curve below pH 4.0 and the scanning electron microscopy images showing a typical etched pattern of the enamel surface. The sealant TM consists of a methacryloyl fluoridemethylmethacrylate (MF-MMA) copolymer resin (Table 1), which contains acidic fluoride covalently bonded to carbonyl groups and releases fluoride ions slowly by hydrolysis in an aqueous environment (20, 21). During the process of hydrolysis, it formed either carboxylic acid or carboxylate groups (20). Another reasonable speculation is that the monomers of 2-hydroxyethyl methacrylate (HEMA) or 10-methacryloyloxydecyl dihydrogenphosphate (MDP) might be related to the low pH value observed during the experimental period.
81
The constant low pH, of
Eur J Oral Sci 2014; 122: 78–83 DOI: 10.1111/eos.12107 Printed in Singapore. All rights reserved
European Journal of Oral Sciences
Inhibition of enamel demineralization by buffering effect of S-PRG fillercontaining dental sealant Kaga M, Kakuda S, Ida Y, Toshima H, Hashimoto M, Endo K, Sano H. Inhibition of enamel demineralization by buffering effect of S-PRG filler-containing dental sealant. Eur J Oral Sci 2014; 122: 78–83. © 2013 Eur J Oral Sci The buffering capacity and inhibitory effects on enamel demineralization of two commercially available dental sealants were evaluated in this study. The effects of filler particles were also examined. Disks of enamel and cured sealant materials of BeautiSealant (silica or S-PRG filler) or Teethmate F-1 were incubated in lactic acid solutions (pH 4.0) for 1–6 d. The pH changes and amounts of ions released in the solutions were assessed, and enamel surfaces were observed using a scanning electron microscope. The pH of the solution with BeautiSealant (S-PRG filler) was neutralized from pH 4.0 to pH 6.1 (after incubation for 1 d) and from pH 4.0 to pH 6.7 (after incubation for 6 d). In addition, no release of calcium ions was detected and the enamel surface was morphologically intact in scanning electron microscopy images. However, the pH of the solution with Teethmate F-1 remained below pH 4.0 during incubation from days 1 to 6. Calcium release was increased in solutions up to and after 6 d of incubation. Scanning electron microscopy images showed that the structures of hydroxyapatite rods were exposed at the specimen surfaces as a result of demineralization. Ions released from S-PRG filler-containing dental sealant rapidly buffered the lactic acid solution and inhibited enamel demineralization.
The clinical usage of dental sealants requires bonding techniques for penetration into deep fissures (1). Complicated pit and fissure morphologies provide ideal sites for collection of food debris, bacterial growth, and biofilm formation. Deep sites in fissures are inaccessible hidden portions and are not cleaned perfectly by mechanical or chemical cleaning in dental practice. Moreover, sealant does not reach the bottom of a fissure and hence a space remains under the cured sealant material (2, 3). In such cases, attempts to improve sealant application using a mechanical technique are limited. To resolve these problems, introduction of a chemical operating agent or a new material is needed for fissure sealing or treatment of hidden caries. It has been reported that glass-ionomer cement releases several types of ions, resulting in pH neutralization (4, 5), and that it is effective for controlling bacterial growth (6). A fissure sealant with fluoride-releasing properties is effective for protecting adjacent enamel from acid demineralization (7–9). Therefore, a bioactive material with an antibacterial effect that is effective for enamel remineralization and can eliminate initial caries is needed. A surface reactiontype glass-ionomer (S-PRG) filler has recently been developed: it can be incorporated into resinous materials and has been used clinically as a resin bonding agent, a sealant material, or a resin composite (10). The S-PRG
Masayuki Kaga1, Shinichi Kakuda2, Yusuke Ida1, Hirokazu Toshima1, Masanori Hashimoto1, Kazuhiko Endo1, Hidehiko Sano2 1
Division of Biomaterials and Bioengineering, Department of Oral Rehabilitation, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido; 2 Department of Restorative Dentistry, Division of Oral Health Science, Graduate School of Dental Medicine, Hokkaido University, Sapporo, Hokkaido, Japan
Visiting Prof. Masayuki Kaga, Division of Biomaterials and Bioengineering, Department of Oral Rehabilitation, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 0610293, Japan E-mail: [email protected] Key words: buffering capacity; demineralization; dental sealants; ion release; S-PRG filler Accepted for publication November 2013
filler is prepared with fluoroboroaliminosilicate glass and polyacrylic acid solution through an acid-based reaction (10). Interestingly, multiple ions are released in neutral and lactic acid conditions by S-PRG-containing resinous materials (11). Fluoride and strontium form an acid-resistant layer and reinforce tooth structure by acting on hydroxylapatite to convert it to a fluoride-apatite (12, 13) and strontium-apatite (14) complexes (14). In addition, a sealant releasing fluoride has shown inhibitory activity against mutans streptococci (15). Based on those findings, sealants that have the ability to release multiple ions should have more potential for long-term durability and self-repair ability against demineralization at the sealant–enamel interface. Therefore, the purpose of this study was to evaluate the buffering effects of dental sealants containing S-PRG filler on enamel decalcification. The null hypothesis is that ions released from sealant materials neutralize lactic acid and inhibit enamel decalcification.
Material and methods Test materials Two commercially available fissure sealants – BeautiSealant (BS) (Shofu, Kyoto, Japan) and Teethmate F-1
Buffering effect of S-PRG filler Table 1 Chemical formulations of the test materials Materials (manufacturers)
Chemical formulations
BeautiSealant (S-PRG) (Shofu, Kyoto, Japan) Experimental sealant (silica filler) (Shofu, Kyoto, Japan) Teethmate F-1 (Kuraray Noritake Dental, Tokyo, Japan)
UDMA, TEGDMA, S-PRG filler UDMA, TEGDMA, silica filler MDP, MF-MMA, TEGDMA, HEMA, hydrophobic aromatic dimethacrylate
HEMA, 2-hydroxyethyl methacrylate; MDP, 10-methacryloyloxydecyl dihydrogen phosphate; MF-MMA, methacryloyl fluoride methacrylate; TEGDMA, triethylene glycol dimethacrylate; UDMA, urethane dimethacrylate.
(Kuraray-Noritake, Tokyo, Japan) – and one experimental sealant were used in this study. To compare the effects of filler particles, we used the following two BS groups: one group with 40 mass% of S-PRG filler (particle size: approximately 1 lm) contained in the resin matrix (S-PRG group) and one group in which the S-PRG filler was replaced with silica filler (particle size: approximately 1 lm) to have the same viscosity as BS (experimental group). The formulations and compositions of test materials used in this study are shown in Table 1. Initial ion release from test materials Uncured liquids of sealants were placed in silicone molds (13 mm in diameter and 1 mm in thickness), clamped between cover glasses, and then immediately light-cured using a light curing unit (Blue shot; Shofu, Kyoto, Japan) for 60 s on each side. The disk surface was gently polished with 600-grit silicon carbide paper under running water to remove the uncured layer on the surface (n = 6 for each group). A lactic acid solution of pH 4.0 (5 ml) was pipetted into a 50-ml plastic tube, and a cured sealant disk was placed in the solution and incubated at 37°C for 1 d. The concentrations of silicon (Si), strontium (Sr), aluminum (Al), boron (B), and sodium (Na) were measured using inductively coupled plasma atomic emission spectroscopy (ICP-AES) (ICPS-8000; Shimadzu, Kyoto, Japan). Fluoride (F) release was analyzed using a fluoride ion-selective electrode (Orion 9609 BNWP; Thermo Fisher Scientific, Waltham, MA, USA) connected to an ion analyzer (Orion 2115010 Dual Star pH/ion-meter; Thermo Fisher Scientific). TISAB III buffer was added to the sample solution to provide a constant background of ionic strength to the fluoride sample in 10% volume and was stirred with a magnetic stirrer for 20 s. The fluoride electrode was immediately placed in the sample solution. pH measurement Four crown parts of a bovine upper central incisor were used in this study. A flattened enamel surface on the buccal aspect of the crown was longitudinally exposed using a low-speed diamond saw (Isomet; Buehler, Lake Bluff, IL, USA) and polished with 600-, 1,200-, and 2,000-grit silicon carbide papers under running water. Twenty-four enamel specimens were cut into sections measuring approximately 4 mm 9 4 mm and 1 mm in thickness, and were cleaned
79
with an ultrasonic cleaner for 15 s in distilled water. The following four groups (n = 6 for each group) were used for tests: (i) enamel disk (control); (ii) enamel disk with a cured Teethmate F-1 (TM) disk; (iii) enamel disk with a cured BS disk (S-PRG filler); and (iv) enamel disk with a cured experimental sealant disk (silica filler). Cured sealant disks were prepared as previously described. The test solution (5 ml of lactic acid solution adjusted to pH 4.0) was pipetted into a 50-ml conical tube (BD Falcon, Franklin Lakes, NJ, USA). The cured sealant and an enamel disk were then immersed in the test solution. A pH electrode (Orion 8102BNUWP; Thermo Fisher Scientific) connected to a pH/ion-meter (Orion 2115010 Dual Star pH/ion-meter; Thermo Fisher Scientific) was immediately inserted in the center of the plastic tube and maintained at room temperature. The pH was measured after 1, 2, 3, 4, 5, and 6 d. Sample tubes were incubated at 37°C for the duration of the experiment. Calcium release and scanning electron micriscopy observation of the enamel surface The amounts of calcium released from enamel in 5 ml of lactic acid were measured using an atomic absorption spectrometer (AAnalyst 200; Perkin-Elmer, Norwalk, CT, USA). The instrument was calibrated using standards containing 0, 0.1, 1, and 5 lg ml 1 of calcium ion in 0.1 mol l-1 HNO3-containing lanthanum chloride solutions. Two-hundred microliters of the lactic acid solution containing calcium released from enamel was transferred to a new tube and was diluted by adding 19.8 ml of 0.1 M HNO3-containing lanthanum chloride solution for analysis after incubation for 6 d. After incubation for 6 d, enamel disks were removed from the lactic solution and then rinsed with distilled water. The enamel disks were dried at room temperature for 48 h. The surfaces of enamel disks were sputter-coated with gold and observed using a scanning electron microscope (S-4000; Hitachi, Tokyo, Japan) with an acceleration voltage of 25 kV in a vacuum. Statistics The data (pH and ion release) were analyzed using twoway ANOVA followed by the Turkey-HSD post-hoc test. Significance was set at P < 0.05 (n = 6 for each group).
Results Initial ion release
Table 2 shows the amount of each ion released from sealants. Significantly larger amounts of Si, Sr, Al, B, and Na ions were released in the BS (S-PRG) group than in the other groups (P < 0.05). In the solution with experimental sealant (silica filler), only Si (0.21 lg ml 1), which was released from silica oxide, and a very low level of F were detected in the solution. There was no significant difference between the BS (S-PRG) and the TM groups (P < 0.05) in the amount of F ions released. pH measurement
Figure 1 shows changes in pH values in the solutions containing specimens. The pH of the solution with an
80
Kaga et al. Table 2 Concentration of ions
Material BS (S-PRG) Experimental sealant (silica filler) TF
Si
Sr
Al
B
Na
F
1.79 (0.05)a 0.21 (0.01)c 0.42 (0.03)b
10.80 (0.33) None 0.09 (0.05)
2.06 (0.07) None 0.08 (0.01)
5.16 (0.32) None 0.06 (0.01)
13.03 (0.58) None 0.81 (0.06)
7.53 (0.80)d 0.03 (0.01)e 7.85 (0.78)d
Values are given as mean (SD). Groups with different superscript letters are statistically different (P < 0.05, n = 6). The ion-release profile of tested materials (lg ml 1) was analyzed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) [for detecting the concentrations of silicon (Si), strontium (Sr), aluminum (Al), boron (B) and sodium (Na)] and a pH/ion-meter [for detecting the concentration of fluoride (F)] following 1 d of incubation at 37°C in lactic acid solution (pH 4.0). BS (S-PRG), BeautiSealant (S-PRG filler); TF, Teethmate F-1.
enamel disk (control) changed to 4.7 after 1 d and to 5.4 after 2 d. The pH of the solution with BS (S-PRG) showed a rapid increase from pH 4.0 to pH 6.1 at 1 d and a further, gradual, increase over the duration of the study to pH 6.7 at 6 d. The pH of the solution with experimental sealant (silica filler) was 5.5 at 3 d. However, the pH of the solution with TM showed a gradual decrease, to pH 3.6 at 1 d, and remained below pH 4.0 from 2 to 6 d. The pH of the solution with BS (S-PRG) was higher than the pH values in the other three groups from 1 to 3 d (P < 0.05). The pH of solution with TM was significantly lower on all days (P < 0.05). Calcium release and scanning electron microscopy observation
Table 3 shows Ca ion release from test materials and enamel disks after storage in lactic acid solution for 6 d. The lowest concentration of Ca ion release was observed in the BS (S-PRG) group, and was significantly smaller than that observed in all other groups (P < 0.05). No significant difference in Ca ion release was found between the solution with experimental sealant (silica filler) and the control group (enamel disk). The concentration of Ca ion released in the solution with TM was significantly higher than the concentration released in the other three groups (P < 0.05). Microstructure profiles of the enamel surfaces determined by scanning electron microscopy at 6 d are shown in Fig. 2. The enamel surface was smooth and polishing lines were observed in specimens in the control group (Fig. 2A). The groups of BS (Fig. 2B, S-PRG filler; and Fig. 2C, silica filler) showed fine and smooth surfaces with no sign of prism outlines. Enamel surfaces in the TM group exhibited a typical etched pattern with the removal of enamel prism peripheries, revealing rigid enamel rods with hydroxylapatite prism cores remaining (Fig. 2D).
Discussion Dental plaque and dental biofilm contain numerous acidogenic bacteria that ferment carbohydrates and produce various organic acids, which dissolve hard tissue and shift the calcium phosphate balance (16). Low pH facilitates caries formation. STEPHAN found that
Fig. 1. Changes in pH values in the different groups – enamel disk (control), enamel disk with BeautiSealant (S-PRG filler) [BS (S-PRG)], enamel disk with experimental sealant (silica filler), and enamel disk with Teethmate F-1 (TM) – when incubated for 1–6 d in lactic acid solution (pH 4.0). Different letters denote a statistically significant difference compared with the other groups for days 1–3 (P < 0.05, n = 6). The pH in the TM group was significantly lower than the pH in the other three groups on all days of measurement (P < 0.05).
enamel dissolution leading to caries formation occurred after prolonged exposure to a pH of pH 6.3 at 3 d (Fig. 1). In contrast, in the BS (S-PRG) group, the initial pH of the lactic acid solution (pH 4.0) rapidly increased to pH 6.2 after 1 d of incubation. However, no buffering effect of TM was observed during the 6-d experimental period in this study. Therefore, the null hypothesis of this study was proved for BS but rejected for TM. The buffering capacity of BS (S-PRG) and its protective effect on enamel demineralization were clearly
Buffering effect of S-PRG filler Table 3 Concentration of Ca ions Material
[Ca] (lg ml 1)
Control (enamel disk) BS (S-PRG) Experimental sealant (silica filler) TM
6.4 2.0 5.5 27.1
(4.3)b (2.5)a (4.5)b (9.0)c
Values are given as mean (SD) concentration of Ca ions released from enamel disks after storage in lactic acid solution for 6 d. Groups with different superscript letters are statistically significant (P < 0.05, n = 6). BS, BeautiSealant (S-PRG filler); TM, Teethmate F-1.
proved by the modest quantities of Si, Sr, Al, B, Na, and F ions released. However, the solution with TM showed a low pH (below pH 4.0), and 27.1 lg ml 1 of Ca was detected at 6 d. The results for demineralized Ca release coincide with the consistent pH curve below pH 4.0 and the scanning electron microscopy images showing a typical etched pattern of the enamel surface. The sealant TM consists of a methacryloyl fluoridemethylmethacrylate (MF-MMA) copolymer resin (Table 1), which contains acidic fluoride covalently bonded to carbonyl groups and releases fluoride ions slowly by hydrolysis in an aqueous environment (20, 21). During the process of hydrolysis, it formed either carboxylic acid or carboxylate groups (20). Another reasonable speculation is that the monomers of 2-hydroxyethyl methacrylate (HEMA) or 10-methacryloyloxydecyl dihydrogenphosphate (MDP) might be related to the low pH value observed during the experimental period.
81
The constant low pH, of
