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Buffering or non-buffering; an action of pit-and-fissure sealants
Accepted Manuscript Title: Buffering or non-buffering; an action of pit-and-fissure sealants Author: Shinichi Kakuda Sharanbir K. Sidhu Hidehiko Sano PII: DOI: Reference:
S0300-5712(15)30007-5 http://dx.doi.org/doi:10.1016/j.jdent.2015.06.013 JJOD 2487
To appear in:
Journal of Dentistry
Received date: Revised date: Accepted date:
28-12-2014 29-6-2015 30-6-2015
Please cite this article as: Kakuda Shinichi, Sidhu Sharanbir K, Sano Hidehiko.Buffering or non-buffering; an action of pit-and-fissure sealants.Journal of Dentistry http://dx.doi.org/10.1016/j.jdent.2015.06.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Buffering or non-buffering; an action of pit-and-fissure sealants Authors Shinichi Kakuda a*, Sharanbir K Sidhu b and Hidehiko Sano a Department a Department of Restorative Dentistry, Division of Oral Health Science, Graduate School of Dental Medicine, Hokkaido University, Sapporo, Japan b Institute of Dentistry, Barts & The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom
* Corresponding author: Shinichi Kakuda: Department of Restorative Dentistry, Division of Oral Health Science, Graduate School of Dental Medicine, Hokkaido University, Sapporo, 060-8586, JAPAN. Tel: +81 11 706 4261 Fax: +81 11 706 4878 E-mail address: [email protected]
Abstract Objectives The aim of this study was to evaluate the buffering capacity of glass-ionomer material in vitro. The null hypothesis tested was that there is no effect of cured glass-ionomer pit-and-fissure sealant (PFS) pastes on the environmental acidity as well as the tooth substrate. Method For each material, a cured PFS disk and a section of human enamel were simultaneously soaked in lactic acid solution in a conical tube, and the pH of the solution was measured daily for one week. Subsequently, the total amount of calcium leached out in solution was also measured with ICP-AES. Results The results showed that the acidity of the solutions changed over time. Significant differences of calcium ion concentration in solution were observed as a result of decalcification. As the PFS products tested did not include calcium, the concentration of calcium ion released indicated acidic erosion of the tooth enamel. Conclusions The glass-ionomer countered the acid of the solution rapidly and preserved the structure of human tooth enamel.
1.
Introduction Dental caries is initiated by demineralization of the outer surface of the tooth due to organic acids
produced locally by bacteria that ferment deposits of dietary carbohydrates.1,2 Acid of aged plaque 1
falls to approximately pH 4.0,
3
as any glucose rinse results in lactic acid production in supra-
gingival plaque.4 The current restorative approach is that caries should be detected and monitored in the earliest stages.5 One of the most effective ways to prevent caries in pits and fissures, and to arrest undiagnosed caries in these areas, is to place sealants.6 There are predominantly two types of pit and fissure sealants (PFSs); resin-based sealants and glass-ionomer cements.6,7 However, the caries preventive effects of glass-ionomer PFSs have been previously shown to be superior to those of resin-based PFSs.6,8,9 Glass-ionomer cements have the potential to neutralize the acidity of their environment10,11 ; however, previous research in this area did not utilize teeth or tooth fragments. Moreover, relatively few reports have evaluated the effect of acid and dental materials on tooth substrate; there is no information regarding changes in acidity, efficacy of sealants and the effect on tooth tissue. Hence, this study was proposed to determine both the demineralization of teeth in aqueous solution as well as the role of glass-ionomer in acidic circumstances. The main objective was to investigate the effect of cured glass-ionomer PFS pastes on the acidity of the environment and tooth substrate. The null hypothesis tested was that there is no effect of cured glass-ionomer PFS pastes on the environmental acidity as well as the tooth substrate.
2.
Materials and methods
Tooth fragments Twenty-four human third molars collected under approval by the Hokkaido University Ethics Committee (reference number #2010-2) were stored in 0.5% Chloramine-T solution at 4°C. Disks of tooth tissue of 1mm thickness were made by slicing the buccal surface of each tooth using a sectioning machine (Isomet low speed saw; Buehler, Lake Bluff, IL, USA). The slabs were then polished with ascending numbers of water-resistant SiC paper (#600, #1200 and #2000 grits) under running tap water, and finished with deerskin under water. The polished slabs were squared off to form cuboids of dimensions 3x3x1mm using a dental portable unit (OPU-7; OSADA Electric Co., Nagoya, Japan) and diamond-points (M104; Morita, Osaka, Japan). Cured PFS disks BeautiSealant (BS), Fuji III LC (IIILC) and TEETHMATE F-12.0 Clear (TM) were used in this study (Table 1). The IIILC product required mixing of the powder and liquid material before use. The primer of BS, etchant of TM and conditioner of IILC, although supplied by the manufacturers for the relevant products, were not used in this study. In order to prepare six disk-shaped specimens of each product, pastes of IIILC as well as pastes of BS and TM were each packed into moulds (13mm diameter, 1mm thickness), and then subjected to a dental blue light (BlueShot, Shofu Inc., Kyoto, Japan) for 40sec per exposed side (top and bottom). Each side of the specimen was gently polished with #600-grit SiC paper under running water, after which the disk was dried with
2
absorbent paper (Kimwipe, Nippon Paper Crecia Co., Tokyo, Japan). Acid solution Distilled water was used to prepare pH4.0 solutions with lactic acid (Wako Pure Chemical industries, Ltd., Osaka, Japan) under room temperature (20°C) using a pH electrode (# 8102BNUWP, Thermo Fisher SCIENTIFIC, Beverly, MA, USA) and a desk-top pH/ion meter (Orion Dual Star, Thermo Fisher SCIENTIFIC, Waltham, MA, USA). Following this, 5ml of the solution was measured and placed in 50ml polypropylene conical tubes (BD Falcon, Franklin Lakes, NJ, USA). Experimental method (with tooth fragments) Each tooth fragment was soaked together with one of the PFS disks in the prepared solution (Figure 1). As controls, lactic acid solutions with tooth fragments but without any sealant disk (LA) were also used. There were six separate conical tubes with a tooth fragment and a PFS disk each, as well as six with the acid solution tested altogether (BS, IIILC, TM and LA). The acidities of the solutions were measured daily with the pH/ion meter kit which was calibrated immediately before use every day. The lids of the conical tubes were securely closed at all times (apart from the measurement procedures) and they were stored in a thermostat chamber at 37°C. After 7 days, the tooth and sealant disks were removed from the individual solutions for analysis. Analysis of tooth fragments and ions released in solution The surfaces of the tooth specimens were sputter-coated with Pt-Pd ion for 120 seconds at an accelerating voltage of 0.4kV (E-1030 Ion Sputter; HITACHI, Tokyo, Japan) and then observed using a field-emission scanning electron microscope (FE-SEM: S-4000; HITACHI, Tokyo, Japan) at an accelerating voltage of 10kV. The concentration of fluoride ion in solution was measured using a fluoride ion electrode (# 9609BNWP, Thermo Fisher SCIENTIFIC, Beverly, MA, USA) and the pH/ion meter. Each solution was diluted to obtain a fluoride ion level of less than 5 ppm and 0.1ml of ionic strength adjuster (TISAB III, Orion Research Inc., Jacksonville, FL, USA) was added to 1ml of each diluted solution. Measurement of released ions of B, Al, Si, P, Ca, Sr and Na was conducted using an Inductively Coupled Plasma Atomic Emission Spectrometer (ICP-AES; ICPS-8000, Shimadzu, Kyoto, Japan). Four standard solutions of ascending concentrations were prepared for each ion (B: 0, 2, 5, 10 ppm; Al: 0, 2, 5, 10 ppm; Si: 0, 4, 10, 20 ppm; P: 0, 2, 5, 10 ppm; Ca: 0, 2, 5, 10 ppm; Sr: 0, 2, 5, 10 ppm; Na; 0, 4, 10, 20 ppm). A calibration curve for each tested solution was obtained, and the densities of these ions were measured. The tested solutions were eluted within the range of the calibration line of several ions. Experimental measurement for releasing ions of exclusively PSF (without tooth fragments) Conical tubes with the acid solution were prepared as previously described, prior to insertion of PFS disks (1 disk per tube, 6 tubes per PFS material); these tubes had no tooth fragments (BS, IILC,
3
TM). The volume of solution and the cured PFS paste specimens were as described in the previous section. The tubes were stored at 37°C for 7 days after which the disks were removed for measurement of ions released (Na, B, Al, Si, P, Ca, Sr and F). Data analysis The data obtained were compared for significant differences using a statistical analysis program (SPSS Ver.17; IBM Japan, Tokyo, Japan). The concentrations of calcium and fluoride in the 7-day solutions were analyzed with the Games-Howell test.
3.
Results
pH changes Daily changes in pH of the solutions are shown in Figure 2 as pH-curves. The pH of solutions with tooth tissue, but LA which did not include any PFS showed a gradual rise in pH. The pH of solutions with tooth fragments together with cured BS or cured IIILC pastes showed rapid initial rises in pH followed by positive or negative plateaus. The solutions with tooth fragments and TM were fairly constant in terms of pH throughout the 7-days period. Concentration of calcium and other ions by ICP-AES ICP-AES was used to determine the concentration of Ca2+ and other non-gas ions in solution after the 7-days period. The data were shown in Figures 3.1 and 3.2. Figure3.1 showed ion solutes in the tube with both PFS and tooth. Significant differences of Ca2+ concentration in solutions with tooth fragments were described by the Games-Howell test, which were collated in ascending order (ppm: average ±SD): IIILC (0.03 ±0.02)
4
result of the grinding process using SiC paper were observed. The edges of the marks appeared sharp. Figure4b, 4c, 4d and 4e showed enamel surfaces after 7 days of storage in lactic acid together with sealant fragments. Figure 4b was an enamel surface soaked with a BS specimen while Figure 4c was one with an IIILC specimen. In both, debris powder covered the surface giving a smooth appearance while grinding marks with sharp edges were observed. Figure 4d showed an enamel surface after 7 days of storage in lactic acid without any sealant disk (LA). The surface consists of debris which appeared rough. Grinding marks were barely observed, and the edges of the marks were not distinct. Figure 4e was an enamel surface of a tooth fragment soaked in lactic acid with a specimen of TM. Neither debris nor grinding marks were observed. Structures of enamel prisms were clearly observed.
4.
Discussion In the oral cavity, it was thought that tooth substrate keeps its form due to the buffering capacity
of saliva.12 The pH curve of LA showed that the tooth tissue results were the only way to counter acid challenge in the present study. The erosion of tooth tissue by lactic acid was not extremely rapid, but was gradual. On the other hand, the rate of pH change with BS or IIILC was more rapid than with LA. It took less than 24 hours for the pH to rise above pH 6 with BS and IIILC. IIILC is a resin-modified glass-ionomer, while BS includes S-PRG fillers which have surface-layers of prereacted glass-ionomer around the core fillers.13,14 Glass-ionomer has been reported to neutralize acidic solution11 by releasing multiple ions,15,16 but it has not been assessed with tooth substrate. Previous studies only extrapolated a potential capacity of the material to neutralize the environment without using teeth in the vicinity. The results of the present study which included tooth substrate showed that both BS and IIILC could neutralize solution more rapidly than teeth. The calcium-ion concentration of solutions with BS and IIILC were significantly lower than soaking teeth alone (LA). During the neutralization with BS or IIILC, multiple ions were released into solution. Hence, glass-ionomer was a factor in contributing to the neutralization of acidic conditions, and the phenomenon of multiple ion release would have been the same as that with the materials encountering acid. Because the dissolving action of glass-ionomer is its neutralizing effect, the neutralization effect would sustain as far as the structure remain. In this study, we could measure Ca in case of PFS with tooth (Figure3.1), although PFS did not include Ca (Figure3.2). Therefore, it was thought that specimens of teeth were demineralized by acidic solution of pH4.0. SEM observations revealed that BS and IIILC preserved enamel debris and its form on a nonaffected enamel surface. In the current study, the pH curve of solutions with TM was lower in acidity than others due to the acid supplied. Although the teeth were demineralized and countered
5
with released H+, dissolution of tooth substrates did not keep up the proton release from metacryloyl fluoride with methyl methacrylate (MF-MMA); MF-MMA polymer in the paste of TM releases both ionic fluoride and H+ by hydrolysis in aqueous solution.17,18 As a result, calcium solute with TM showed a significantly higher concentration than with the others, and significant demineralization was observed by SEM. A former report showed that TM used in permanent teeth clinically sealed not only sound fissures but also cavitated fissures.19 The authors reported that incomplete sealant penetration was observed.19 If water exists in the enclosed area which they called a “black area” in their images, it is possible that cured TM continues to decalcify. Ionic fluoride has been the main means of preventing caries for a long time. Previously, “alkaline-earth” factors in the soil have been a prime focus in caries prevention.1 Subsequently, multiple ions have been targeted for research on caries prevention. Although dental caries research is often divided into hard tissue and microbiological evaluation, comprehensive caries prevention protocols should encompass fluoride and other agents affecting the de-/re-mineralization balance as well as antimicrobial strategies.5 Fluoride has been the focus of mainstream caries prevention procedures for a long time. However, the concentration of fluoride and pH of solution in relation to fluoride has recently been of interest in caries prevention.11 In clinical research reports, the effectiveness of treatment using glass-ionomer was superior to that of resin.6,8,20 These studies, however, assumed that this was due to the effect of fluoride only. Fluoride has a possibility to prevent dental caries. Ionized fluoride does not counter acid directly. Salts affect the acidic balance due to their dissolution. Hence, glass-ionomer controlled acidity from a harmful level to a neutral one.21 The acidity control for local areas which have a predilection for dental caries is a different approach from fluoridation for caries prevention. The function of materials is both neutralization due to dissolution as well as bioactivity towards microbes and teeth by the ions released. Neutralization is related to the process of ionization, but the function of neutralization is different from the function of ions. Tooth activity by neutralization is neither an action of anti-microbial nor a morphological modification of tooth construction. This study suggested that neutralization could occur prior to the effect of the ions. Neutralization is one factor that helps conserve tooth substrate. Neutralization by glass-ionomer maintained the homeostasis of tooth structure in this study. In the oral environment, saliva and its contents act in a buffering capacity against environmental acidity responsible for tooth erosion. The physical function of saliva is neutralization from acid to a neutral situation in the oral cavity. The function of neutralization of glass-ionomer is in an artificial buffering capacity. It is proposed that glass-ionomer cements have two caries preventive properties. One is in terms of an artificial buffering capacity which alters to a given buffering capacity of saliva. The other is a function of the ions released, such as fluoride. These two properties are not independent of each other but correlate with each other as the ions released are due to the dissolution of the glassionomer. The curing system of BS is more resin-like; however, BS includes glass-ionomer
6
ingredients in its formulation. Therefore, it is not surprising that the acidity change with BS was similar to IIILC. In conclusion, the behaviour of various PFSs in acidic solutions differs. Hence, the null hypothesis was rejected. An artificial buffering capacity due to neutralization by a cured paste of PFS may have the potential to conserve tooth substrate. On the other hands, although this study showed that a resin modified glass-ionomer cement (IIILC) and a resin composing glass-ionomer filler material (BS) had a potential of neutralization, this study did not explain that the specific component or process contributed to neutralization. Future research is expected to solve that unexplained mechanism.
Acknowledgements This work was supported by JSPS KAKENHI Grant Number 26861587. The authors would like to thank the generous donations of materials by the relevant manufacturers, as well as to Yusuke Ida, Hirokazu Toshima, Masanori Hashimoto, Kazuhiko Endo and Masayuki Kaga who worked with us in an early stage of this study.
Reference 1. Scherp HW. Dental caries: prospects for prevention. Science 1971;173:1199-205. 2. Martins C, Castro GF, Siqueira MF, Xiao Y, Yamaguti PM, Siqueira WL. Effect of Dialyzed Saliva on Human Enamel Demineralization. Caries Research 2012;47:56-62. 3. Clarke NG, Fanning EA. Plaque pH and calcium sucrose phosphate: a telemetric study. Australian Dental Journal 1971;16:13-6. 4. Takahashi N, Washio J. Metabolomic effects of xylitol and fluoride on plaque biofilm in vivo. Journal of Dental Research 2011;90:1463-8. 5. Ten Cate JM. Novel anticaries and remineralizing agents: prospects for the future. Journal of Dental Research 2012;91:813-5. 6. Antonson SA, Antonson DE, Brener S, Crutchfield J, Larumbe J, Michaud C, Yazici AR, Hardigan PC, Alempour S, Evans D, Ocanto R. Twenty-four month clinical evaluation of fissure sealants on partially erupted permanent first molars: glass-ionomer versus resin-based sealant. Journal of the American Dental Association 2012;143:115-22. 7. Beauchamp J , Caufield PW, Crall JJ, Donly KJ, Feigal R, Gooch B, Ismail A, Kohn W, Siegal M, Simonsen R. Evidence-based clinical recommendations for the use of pit-and-fissure sealants: a report of the American Dental Association Council on Scientific Affairs. Journal of the American Dental Association 2009;139:257-68. 8. Trairatvorakul C, Kladkaew S, Songsiripradabboon S. Active management of incipient caries and choice of materials. Journal of Dental Research 2008;87:228-32. 9. Frencken JE, Wolke J. Clinical and SEM assessment of ART high-viscosity glass-ionomer sealants after 8-13 years in 4 teeth. Journal of Dentistry 2010;38:59-64. 10. Nicholson JW, Aggarwal A, Czarnecka B, Limanowska-Shaw H. The rate of change of pH of lactic acid exposed to glass-ionomer dental cements. Biomaterials 2000;21:1989-93. 11. Nakajo K, Imazato S, Takahashi Y, Kiba W, Ebisu S, Takahashi N. Fluoride released from glass-ionomer cement is responsible to inhibit the acid production of caries-related oral streptococci. Dental Materials 2009;25:703-8. 12. Stephan RM. Intra-oral hydrogen-ion concentrations associated with dental caries activity. Journal of Dental Research 1944;23:257-66. 13. Fujimoto Y, Iwasa M, Murayama R, Miyazaki M, Nagafuji A, Nakatsuka T. Detection of ions released from S-PRG fillers and their modulation effect. Dental Materials Journal 2010;29:392-7. 14. Ito S, Iijima M, Hashimoto M, Tsukamoto N, Mizoguchi I, Saito T. Effects of surface pre7
reacted glass-ionomer fillers on mineral induction by phosphoprotein. Journal of Dentistry 2011;39:72-9. 15. Czarnecka B, Limanowska-Shaw H, Nicholson JW. Buffering and ion-release by a glassionomer cement under near-neutral and acidic conditions. Biomaterials 2002;23:2783-8. 16. Czarnecka B, Nicholson JW. Ion release by resin-modified glass-ionomer cements into water and lactic acid solutions. Journal of Dentistry 2006;34:539-43. 17. Kadoma Y, Kojima K, Masuhara E. Studies on dental fluoride-releasing polymers. IV: Fluoridation of human enamel by fluoride-containing sealant. Biomaterials 1983;4:89-93. 18. Tanaka M, Ono H, Kadoma Y, Imai Y. Journal of Dental Research 1987;66:1591-3. 19. Hevinga MA, Opdam NJ, Frencken JE, Bronkhorst EM, Truin GJ. Can caries fissures be sealed as adequately as sound fissures? Journal of Dental Research 2008;87:495-8. 20. Miller BH, Komatsu H, Nakajima H, Okabe T. Effect of glass-ionomer manipulation on early fluoride release. American Journal of Dentistry 1995;8:182-6. 21. Imazato S. Bio-active restorative materials with antibacterial effects: new dimension of innovation in restorative dentistry. Dental Materials Journal 2009;28:11-9.
Figure legends Table 1 Pit and fissure sealant materials used Figure 1 Schematic description for method of present study: Teeth fragments were cut and polished. Sealant pastes were put into a frame, were cured with dental curing-light and were polished. Distilled water was conditioned for certain acidity with lactic acid at room temperature. After that, 5ml of conditioned aqueous was into a tube. And, fragments of both teeth and sealant were put into the tube at the same time. Figure 2 Daily changes of acidity: Abbreviations; BS: acidity of solution with BeautiSealant and fragment of human enamel, IIILC: acidity of solution with Fuji IIILC and fragment of human enamel, LA: acidity of exclusively lactic acid solution without any sealant fragments, TM: acidity of solution with TEETHMATE F-12.0 and fragment of human enamel Figure 3 Measurement of respective released ions in aqueous after 7days storage: Figure 3.1 showed concentrations of various ions (Na, B, Al, Si, P, Ca and Sr) in solution after 7 days as determined with ICP-AES. The tested solution included a tooth fragment. Figure 3.2 showed concentrations of the ions in the solutions without tooth fragments as determined with ICP-AES. Figure 3.3 showed the concentration of fluoride (F) in the solution with tooth fragments, as determined by the Ion-Electrode. Figure 3.4 showed the concentration of fluoride (F) in the solutions without tooth fragments, as determined by the Ion-Electrode. Abbreviations; BS: BeautiSealant; IIILC: Fuji III LC; LA: lactic acid without any sealant fragments; TM: TEETHMATE F-12.0 Figure 4 Field emission scanning electron microscopic images enamel surfaces: All of them were observed at 10,000x magnification. “a” was enamel surface just after preparation. “b”, “c”, “d” and “e” were enamel surfaces of “after 7-days’ soaking” with/without PFS cured-disk 8
(b; with BeautiSealant, c; with Fuji III LC, d; exclusively lactic acid, e; with TEETHMATE F-12.0)
Fig. 1
9
Fig. 2
Fig. 3
10
Fig. 4
Material / Manufacturer
Main composition
FUJI III LC / GC CORPORATION, Japan
Powder: Alumino-fluoro-silicate glass (amorphous)
Liquid: Methacrylic acid ester、Polyacrylic acid、Distilled water
BeautiSealant (Paste) / SHOFU INC., Japan
S-PRG filler particles,UDMA,TEGDMA
TEETHMATE F-12.0 / Kuraray Noritake Dental Inc., Japan
TEGDMA, HEMA, 10-MDP, MF-MMA
Abbreviation: S-PRG; Surface pre-reacted glass, UDMA; Urethane dimethacrylate, TEGDMA; Triethylene glycol dimethacrylate, HEMA; 2-hydroxyethyl methacrylate, 10-MDP; 10-Methacryloyloxydecyl dihydrogen phosphate, MF-MMA; Methacryloylfluoride-methyl methacrylate copolymer
11
S0300-5712(15)30007-5 http://dx.doi.org/doi:10.1016/j.jdent.2015.06.013 JJOD 2487
To appear in:
Journal of Dentistry
Received date: Revised date: Accepted date:
28-12-2014 29-6-2015 30-6-2015
Please cite this article as: Kakuda Shinichi, Sidhu Sharanbir K, Sano Hidehiko.Buffering or non-buffering; an action of pit-and-fissure sealants.Journal of Dentistry http://dx.doi.org/10.1016/j.jdent.2015.06.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Buffering or non-buffering; an action of pit-and-fissure sealants Authors Shinichi Kakuda a*, Sharanbir K Sidhu b and Hidehiko Sano a Department a Department of Restorative Dentistry, Division of Oral Health Science, Graduate School of Dental Medicine, Hokkaido University, Sapporo, Japan b Institute of Dentistry, Barts & The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom
* Corresponding author: Shinichi Kakuda: Department of Restorative Dentistry, Division of Oral Health Science, Graduate School of Dental Medicine, Hokkaido University, Sapporo, 060-8586, JAPAN. Tel: +81 11 706 4261 Fax: +81 11 706 4878 E-mail address: [email protected]
Abstract Objectives The aim of this study was to evaluate the buffering capacity of glass-ionomer material in vitro. The null hypothesis tested was that there is no effect of cured glass-ionomer pit-and-fissure sealant (PFS) pastes on the environmental acidity as well as the tooth substrate. Method For each material, a cured PFS disk and a section of human enamel were simultaneously soaked in lactic acid solution in a conical tube, and the pH of the solution was measured daily for one week. Subsequently, the total amount of calcium leached out in solution was also measured with ICP-AES. Results The results showed that the acidity of the solutions changed over time. Significant differences of calcium ion concentration in solution were observed as a result of decalcification. As the PFS products tested did not include calcium, the concentration of calcium ion released indicated acidic erosion of the tooth enamel. Conclusions The glass-ionomer countered the acid of the solution rapidly and preserved the structure of human tooth enamel.
1.
Introduction Dental caries is initiated by demineralization of the outer surface of the tooth due to organic acids
produced locally by bacteria that ferment deposits of dietary carbohydrates.1,2 Acid of aged plaque 1
falls to approximately pH 4.0,
3
as any glucose rinse results in lactic acid production in supra-
gingival plaque.4 The current restorative approach is that caries should be detected and monitored in the earliest stages.5 One of the most effective ways to prevent caries in pits and fissures, and to arrest undiagnosed caries in these areas, is to place sealants.6 There are predominantly two types of pit and fissure sealants (PFSs); resin-based sealants and glass-ionomer cements.6,7 However, the caries preventive effects of glass-ionomer PFSs have been previously shown to be superior to those of resin-based PFSs.6,8,9 Glass-ionomer cements have the potential to neutralize the acidity of their environment10,11 ; however, previous research in this area did not utilize teeth or tooth fragments. Moreover, relatively few reports have evaluated the effect of acid and dental materials on tooth substrate; there is no information regarding changes in acidity, efficacy of sealants and the effect on tooth tissue. Hence, this study was proposed to determine both the demineralization of teeth in aqueous solution as well as the role of glass-ionomer in acidic circumstances. The main objective was to investigate the effect of cured glass-ionomer PFS pastes on the acidity of the environment and tooth substrate. The null hypothesis tested was that there is no effect of cured glass-ionomer PFS pastes on the environmental acidity as well as the tooth substrate.
2.
Materials and methods
Tooth fragments Twenty-four human third molars collected under approval by the Hokkaido University Ethics Committee (reference number #2010-2) were stored in 0.5% Chloramine-T solution at 4°C. Disks of tooth tissue of 1mm thickness were made by slicing the buccal surface of each tooth using a sectioning machine (Isomet low speed saw; Buehler, Lake Bluff, IL, USA). The slabs were then polished with ascending numbers of water-resistant SiC paper (#600, #1200 and #2000 grits) under running tap water, and finished with deerskin under water. The polished slabs were squared off to form cuboids of dimensions 3x3x1mm using a dental portable unit (OPU-7; OSADA Electric Co., Nagoya, Japan) and diamond-points (M104; Morita, Osaka, Japan). Cured PFS disks BeautiSealant (BS), Fuji III LC (IIILC) and TEETHMATE F-12.0 Clear (TM) were used in this study (Table 1). The IIILC product required mixing of the powder and liquid material before use. The primer of BS, etchant of TM and conditioner of IILC, although supplied by the manufacturers for the relevant products, were not used in this study. In order to prepare six disk-shaped specimens of each product, pastes of IIILC as well as pastes of BS and TM were each packed into moulds (13mm diameter, 1mm thickness), and then subjected to a dental blue light (BlueShot, Shofu Inc., Kyoto, Japan) for 40sec per exposed side (top and bottom). Each side of the specimen was gently polished with #600-grit SiC paper under running water, after which the disk was dried with
2
absorbent paper (Kimwipe, Nippon Paper Crecia Co., Tokyo, Japan). Acid solution Distilled water was used to prepare pH4.0 solutions with lactic acid (Wako Pure Chemical industries, Ltd., Osaka, Japan) under room temperature (20°C) using a pH electrode (# 8102BNUWP, Thermo Fisher SCIENTIFIC, Beverly, MA, USA) and a desk-top pH/ion meter (Orion Dual Star, Thermo Fisher SCIENTIFIC, Waltham, MA, USA). Following this, 5ml of the solution was measured and placed in 50ml polypropylene conical tubes (BD Falcon, Franklin Lakes, NJ, USA). Experimental method (with tooth fragments) Each tooth fragment was soaked together with one of the PFS disks in the prepared solution (Figure 1). As controls, lactic acid solutions with tooth fragments but without any sealant disk (LA) were also used. There were six separate conical tubes with a tooth fragment and a PFS disk each, as well as six with the acid solution tested altogether (BS, IIILC, TM and LA). The acidities of the solutions were measured daily with the pH/ion meter kit which was calibrated immediately before use every day. The lids of the conical tubes were securely closed at all times (apart from the measurement procedures) and they were stored in a thermostat chamber at 37°C. After 7 days, the tooth and sealant disks were removed from the individual solutions for analysis. Analysis of tooth fragments and ions released in solution The surfaces of the tooth specimens were sputter-coated with Pt-Pd ion for 120 seconds at an accelerating voltage of 0.4kV (E-1030 Ion Sputter; HITACHI, Tokyo, Japan) and then observed using a field-emission scanning electron microscope (FE-SEM: S-4000; HITACHI, Tokyo, Japan) at an accelerating voltage of 10kV. The concentration of fluoride ion in solution was measured using a fluoride ion electrode (# 9609BNWP, Thermo Fisher SCIENTIFIC, Beverly, MA, USA) and the pH/ion meter. Each solution was diluted to obtain a fluoride ion level of less than 5 ppm and 0.1ml of ionic strength adjuster (TISAB III, Orion Research Inc., Jacksonville, FL, USA) was added to 1ml of each diluted solution. Measurement of released ions of B, Al, Si, P, Ca, Sr and Na was conducted using an Inductively Coupled Plasma Atomic Emission Spectrometer (ICP-AES; ICPS-8000, Shimadzu, Kyoto, Japan). Four standard solutions of ascending concentrations were prepared for each ion (B: 0, 2, 5, 10 ppm; Al: 0, 2, 5, 10 ppm; Si: 0, 4, 10, 20 ppm; P: 0, 2, 5, 10 ppm; Ca: 0, 2, 5, 10 ppm; Sr: 0, 2, 5, 10 ppm; Na; 0, 4, 10, 20 ppm). A calibration curve for each tested solution was obtained, and the densities of these ions were measured. The tested solutions were eluted within the range of the calibration line of several ions. Experimental measurement for releasing ions of exclusively PSF (without tooth fragments) Conical tubes with the acid solution were prepared as previously described, prior to insertion of PFS disks (1 disk per tube, 6 tubes per PFS material); these tubes had no tooth fragments (BS, IILC,
3
TM). The volume of solution and the cured PFS paste specimens were as described in the previous section. The tubes were stored at 37°C for 7 days after which the disks were removed for measurement of ions released (Na, B, Al, Si, P, Ca, Sr and F). Data analysis The data obtained were compared for significant differences using a statistical analysis program (SPSS Ver.17; IBM Japan, Tokyo, Japan). The concentrations of calcium and fluoride in the 7-day solutions were analyzed with the Games-Howell test.
3.
Results
pH changes Daily changes in pH of the solutions are shown in Figure 2 as pH-curves. The pH of solutions with tooth tissue, but LA which did not include any PFS showed a gradual rise in pH. The pH of solutions with tooth fragments together with cured BS or cured IIILC pastes showed rapid initial rises in pH followed by positive or negative plateaus. The solutions with tooth fragments and TM were fairly constant in terms of pH throughout the 7-days period. Concentration of calcium and other ions by ICP-AES ICP-AES was used to determine the concentration of Ca2+ and other non-gas ions in solution after the 7-days period. The data were shown in Figures 3.1 and 3.2. Figure3.1 showed ion solutes in the tube with both PFS and tooth. Significant differences of Ca2+ concentration in solutions with tooth fragments were described by the Games-Howell test, which were collated in ascending order (ppm: average ±SD): IIILC (0.03 ±0.02)
4
result of the grinding process using SiC paper were observed. The edges of the marks appeared sharp. Figure4b, 4c, 4d and 4e showed enamel surfaces after 7 days of storage in lactic acid together with sealant fragments. Figure 4b was an enamel surface soaked with a BS specimen while Figure 4c was one with an IIILC specimen. In both, debris powder covered the surface giving a smooth appearance while grinding marks with sharp edges were observed. Figure 4d showed an enamel surface after 7 days of storage in lactic acid without any sealant disk (LA). The surface consists of debris which appeared rough. Grinding marks were barely observed, and the edges of the marks were not distinct. Figure 4e was an enamel surface of a tooth fragment soaked in lactic acid with a specimen of TM. Neither debris nor grinding marks were observed. Structures of enamel prisms were clearly observed.
4.
Discussion In the oral cavity, it was thought that tooth substrate keeps its form due to the buffering capacity
of saliva.12 The pH curve of LA showed that the tooth tissue results were the only way to counter acid challenge in the present study. The erosion of tooth tissue by lactic acid was not extremely rapid, but was gradual. On the other hand, the rate of pH change with BS or IIILC was more rapid than with LA. It took less than 24 hours for the pH to rise above pH 6 with BS and IIILC. IIILC is a resin-modified glass-ionomer, while BS includes S-PRG fillers which have surface-layers of prereacted glass-ionomer around the core fillers.13,14 Glass-ionomer has been reported to neutralize acidic solution11 by releasing multiple ions,15,16 but it has not been assessed with tooth substrate. Previous studies only extrapolated a potential capacity of the material to neutralize the environment without using teeth in the vicinity. The results of the present study which included tooth substrate showed that both BS and IIILC could neutralize solution more rapidly than teeth. The calcium-ion concentration of solutions with BS and IIILC were significantly lower than soaking teeth alone (LA). During the neutralization with BS or IIILC, multiple ions were released into solution. Hence, glass-ionomer was a factor in contributing to the neutralization of acidic conditions, and the phenomenon of multiple ion release would have been the same as that with the materials encountering acid. Because the dissolving action of glass-ionomer is its neutralizing effect, the neutralization effect would sustain as far as the structure remain. In this study, we could measure Ca in case of PFS with tooth (Figure3.1), although PFS did not include Ca (Figure3.2). Therefore, it was thought that specimens of teeth were demineralized by acidic solution of pH4.0. SEM observations revealed that BS and IIILC preserved enamel debris and its form on a nonaffected enamel surface. In the current study, the pH curve of solutions with TM was lower in acidity than others due to the acid supplied. Although the teeth were demineralized and countered
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with released H+, dissolution of tooth substrates did not keep up the proton release from metacryloyl fluoride with methyl methacrylate (MF-MMA); MF-MMA polymer in the paste of TM releases both ionic fluoride and H+ by hydrolysis in aqueous solution.17,18 As a result, calcium solute with TM showed a significantly higher concentration than with the others, and significant demineralization was observed by SEM. A former report showed that TM used in permanent teeth clinically sealed not only sound fissures but also cavitated fissures.19 The authors reported that incomplete sealant penetration was observed.19 If water exists in the enclosed area which they called a “black area” in their images, it is possible that cured TM continues to decalcify. Ionic fluoride has been the main means of preventing caries for a long time. Previously, “alkaline-earth” factors in the soil have been a prime focus in caries prevention.1 Subsequently, multiple ions have been targeted for research on caries prevention. Although dental caries research is often divided into hard tissue and microbiological evaluation, comprehensive caries prevention protocols should encompass fluoride and other agents affecting the de-/re-mineralization balance as well as antimicrobial strategies.5 Fluoride has been the focus of mainstream caries prevention procedures for a long time. However, the concentration of fluoride and pH of solution in relation to fluoride has recently been of interest in caries prevention.11 In clinical research reports, the effectiveness of treatment using glass-ionomer was superior to that of resin.6,8,20 These studies, however, assumed that this was due to the effect of fluoride only. Fluoride has a possibility to prevent dental caries. Ionized fluoride does not counter acid directly. Salts affect the acidic balance due to their dissolution. Hence, glass-ionomer controlled acidity from a harmful level to a neutral one.21 The acidity control for local areas which have a predilection for dental caries is a different approach from fluoridation for caries prevention. The function of materials is both neutralization due to dissolution as well as bioactivity towards microbes and teeth by the ions released. Neutralization is related to the process of ionization, but the function of neutralization is different from the function of ions. Tooth activity by neutralization is neither an action of anti-microbial nor a morphological modification of tooth construction. This study suggested that neutralization could occur prior to the effect of the ions. Neutralization is one factor that helps conserve tooth substrate. Neutralization by glass-ionomer maintained the homeostasis of tooth structure in this study. In the oral environment, saliva and its contents act in a buffering capacity against environmental acidity responsible for tooth erosion. The physical function of saliva is neutralization from acid to a neutral situation in the oral cavity. The function of neutralization of glass-ionomer is in an artificial buffering capacity. It is proposed that glass-ionomer cements have two caries preventive properties. One is in terms of an artificial buffering capacity which alters to a given buffering capacity of saliva. The other is a function of the ions released, such as fluoride. These two properties are not independent of each other but correlate with each other as the ions released are due to the dissolution of the glassionomer. The curing system of BS is more resin-like; however, BS includes glass-ionomer
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ingredients in its formulation. Therefore, it is not surprising that the acidity change with BS was similar to IIILC. In conclusion, the behaviour of various PFSs in acidic solutions differs. Hence, the null hypothesis was rejected. An artificial buffering capacity due to neutralization by a cured paste of PFS may have the potential to conserve tooth substrate. On the other hands, although this study showed that a resin modified glass-ionomer cement (IIILC) and a resin composing glass-ionomer filler material (BS) had a potential of neutralization, this study did not explain that the specific component or process contributed to neutralization. Future research is expected to solve that unexplained mechanism.
Acknowledgements This work was supported by JSPS KAKENHI Grant Number 26861587. The authors would like to thank the generous donations of materials by the relevant manufacturers, as well as to Yusuke Ida, Hirokazu Toshima, Masanori Hashimoto, Kazuhiko Endo and Masayuki Kaga who worked with us in an early stage of this study.
Reference 1. Scherp HW. Dental caries: prospects for prevention. Science 1971;173:1199-205. 2. Martins C, Castro GF, Siqueira MF, Xiao Y, Yamaguti PM, Siqueira WL. Effect of Dialyzed Saliva on Human Enamel Demineralization. Caries Research 2012;47:56-62. 3. Clarke NG, Fanning EA. Plaque pH and calcium sucrose phosphate: a telemetric study. Australian Dental Journal 1971;16:13-6. 4. Takahashi N, Washio J. Metabolomic effects of xylitol and fluoride on plaque biofilm in vivo. Journal of Dental Research 2011;90:1463-8. 5. Ten Cate JM. Novel anticaries and remineralizing agents: prospects for the future. Journal of Dental Research 2012;91:813-5. 6. Antonson SA, Antonson DE, Brener S, Crutchfield J, Larumbe J, Michaud C, Yazici AR, Hardigan PC, Alempour S, Evans D, Ocanto R. Twenty-four month clinical evaluation of fissure sealants on partially erupted permanent first molars: glass-ionomer versus resin-based sealant. Journal of the American Dental Association 2012;143:115-22. 7. Beauchamp J , Caufield PW, Crall JJ, Donly KJ, Feigal R, Gooch B, Ismail A, Kohn W, Siegal M, Simonsen R. Evidence-based clinical recommendations for the use of pit-and-fissure sealants: a report of the American Dental Association Council on Scientific Affairs. Journal of the American Dental Association 2009;139:257-68. 8. Trairatvorakul C, Kladkaew S, Songsiripradabboon S. Active management of incipient caries and choice of materials. Journal of Dental Research 2008;87:228-32. 9. Frencken JE, Wolke J. Clinical and SEM assessment of ART high-viscosity glass-ionomer sealants after 8-13 years in 4 teeth. Journal of Dentistry 2010;38:59-64. 10. Nicholson JW, Aggarwal A, Czarnecka B, Limanowska-Shaw H. The rate of change of pH of lactic acid exposed to glass-ionomer dental cements. Biomaterials 2000;21:1989-93. 11. Nakajo K, Imazato S, Takahashi Y, Kiba W, Ebisu S, Takahashi N. Fluoride released from glass-ionomer cement is responsible to inhibit the acid production of caries-related oral streptococci. Dental Materials 2009;25:703-8. 12. Stephan RM. Intra-oral hydrogen-ion concentrations associated with dental caries activity. Journal of Dental Research 1944;23:257-66. 13. Fujimoto Y, Iwasa M, Murayama R, Miyazaki M, Nagafuji A, Nakatsuka T. Detection of ions released from S-PRG fillers and their modulation effect. Dental Materials Journal 2010;29:392-7. 14. Ito S, Iijima M, Hashimoto M, Tsukamoto N, Mizoguchi I, Saito T. Effects of surface pre7
reacted glass-ionomer fillers on mineral induction by phosphoprotein. Journal of Dentistry 2011;39:72-9. 15. Czarnecka B, Limanowska-Shaw H, Nicholson JW. Buffering and ion-release by a glassionomer cement under near-neutral and acidic conditions. Biomaterials 2002;23:2783-8. 16. Czarnecka B, Nicholson JW. Ion release by resin-modified glass-ionomer cements into water and lactic acid solutions. Journal of Dentistry 2006;34:539-43. 17. Kadoma Y, Kojima K, Masuhara E. Studies on dental fluoride-releasing polymers. IV: Fluoridation of human enamel by fluoride-containing sealant. Biomaterials 1983;4:89-93. 18. Tanaka M, Ono H, Kadoma Y, Imai Y. Journal of Dental Research 1987;66:1591-3. 19. Hevinga MA, Opdam NJ, Frencken JE, Bronkhorst EM, Truin GJ. Can caries fissures be sealed as adequately as sound fissures? Journal of Dental Research 2008;87:495-8. 20. Miller BH, Komatsu H, Nakajima H, Okabe T. Effect of glass-ionomer manipulation on early fluoride release. American Journal of Dentistry 1995;8:182-6. 21. Imazato S. Bio-active restorative materials with antibacterial effects: new dimension of innovation in restorative dentistry. Dental Materials Journal 2009;28:11-9.
Figure legends Table 1 Pit and fissure sealant materials used Figure 1 Schematic description for method of present study: Teeth fragments were cut and polished. Sealant pastes were put into a frame, were cured with dental curing-light and were polished. Distilled water was conditioned for certain acidity with lactic acid at room temperature. After that, 5ml of conditioned aqueous was into a tube. And, fragments of both teeth and sealant were put into the tube at the same time. Figure 2 Daily changes of acidity: Abbreviations; BS: acidity of solution with BeautiSealant and fragment of human enamel, IIILC: acidity of solution with Fuji IIILC and fragment of human enamel, LA: acidity of exclusively lactic acid solution without any sealant fragments, TM: acidity of solution with TEETHMATE F-12.0 and fragment of human enamel Figure 3 Measurement of respective released ions in aqueous after 7days storage: Figure 3.1 showed concentrations of various ions (Na, B, Al, Si, P, Ca and Sr) in solution after 7 days as determined with ICP-AES. The tested solution included a tooth fragment. Figure 3.2 showed concentrations of the ions in the solutions without tooth fragments as determined with ICP-AES. Figure 3.3 showed the concentration of fluoride (F) in the solution with tooth fragments, as determined by the Ion-Electrode. Figure 3.4 showed the concentration of fluoride (F) in the solutions without tooth fragments, as determined by the Ion-Electrode. Abbreviations; BS: BeautiSealant; IIILC: Fuji III LC; LA: lactic acid without any sealant fragments; TM: TEETHMATE F-12.0 Figure 4 Field emission scanning electron microscopic images enamel surfaces: All of them were observed at 10,000x magnification. “a” was enamel surface just after preparation. “b”, “c”, “d” and “e” were enamel surfaces of “after 7-days’ soaking” with/without PFS cured-disk 8
(b; with BeautiSealant, c; with Fuji III LC, d; exclusively lactic acid, e; with TEETHMATE F-12.0)
Fig. 1
9
Fig. 2
Fig. 3
10
Fig. 4
Material / Manufacturer
Main composition
FUJI III LC / GC CORPORATION, Japan
Powder: Alumino-fluoro-silicate glass (amorphous)
Liquid: Methacrylic acid ester、Polyacrylic acid、Distilled water
BeautiSealant (Paste) / SHOFU INC., Japan
S-PRG filler particles,UDMA,TEGDMA
TEETHMATE F-12.0 / Kuraray Noritake Dental Inc., Japan
TEGDMA, HEMA, 10-MDP, MF-MMA
Abbreviation: S-PRG; Surface pre-reacted glass, UDMA; Urethane dimethacrylate, TEGDMA; Triethylene glycol dimethacrylate, HEMA; 2-hydroxyethyl methacrylate, 10-MDP; 10-Methacryloyloxydecyl dihydrogen phosphate, MF-MMA; Methacryloylfluoride-methyl methacrylate copolymer
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