- Email: [email protected]
Effects of ammonium hexafluorosilicate concentration on dentin tubule occlusion and composition of the precipitate
d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 29–34
available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/dema
Effects of ammonium hexafluorosilicate concentration on dentin tubule occlusion and composition of the precipitate Toshiyuki Suge a,∗ , Akiko Kawasaki a , Kunio Ishikawa b , Takashi Matsuo a , Shigeyuki Ebisu c a
Department of Conservative Dentistry, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima 770-8504, Japan b Department of Biomaterials, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan c Department of Restorative Dentistry and Endodontology, Division of Oral Infections and Disease Control, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan
a r t i c l e
i n f o
a b s t r a c t
Article history:
Ammonium hexafluorosilicate [SiF: (NH4 )2 SiF6 ] was prepared in order to overcome the tooth
Received 6 August 2008
discoloration caused by diamine silver fluoride [AgF: (NH3 )2 AgF] application. We employed
Accepted 23 August 2009
a single concentration of SiF solution in our previous study; therefore, it is still unclear how the concentration of SiF solution affects the occlusion of dentin tubules and composition of the precipitate.
Keywords:
Objective. The aim of this study was to evaluate the effects of changing the concentration of
Ammonium hexafluorosilicate
SiF on its clinical use as a dentin hypersensitivity treatment.
Dentin tubules
Methods. To simulate dentin tubules subject to dentin hypersensitivity, dentin disks were
Occlusion
treated with EDTA for 2 min. Then, the disks were treated with several concentrations of SiF
Dentin sensitivity
solution (from 100 to 19,400 ppm) for 3 min. The occlusion of dentin tubules was evaluated
Calcium phosphate
using scanning electron microscopy (SEM), and the composition of the precipitate formed in
Silica
the tubules after SiF treatment was assessed using energy dispersive X-ray analysis (EDXA). Results. SEM photographs demonstrated that dentin tubules after treatment with SiF were occluded homogeneously and fully regardless of the concentration of SiF solution. The Ca/P molar ratio of the precipitate formed in dentin tubules after SiF treatment was increased with the concentration of SiF solution. Significance. It was concluded that the capacity to occlude dentin tubules was the same regardless of the concentration of SiF solution. However, the composition of the precipitate formed in the tubules was dependent on the concentration of SiF solution. © 2009 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Fluoride is effective for the prevention of dental caries, and sometimes it has also been used for the treatment of dentin hypersensitivity. The representative fluoride solution appli-
∗
cable for both treatments is diamine silver fluoride [AgF: (NH3 )2 AgF] (Saforide® , Beebland Medico Dental Inc., Osaka, Japan), which is widely used in dental clinics in Japan [1–4]. However, AgF causes tooth discoloration, especially problematic with regard to permanent teeth. Ammonium hexafluorosilicate [SiF: (NH4 )2 SiF6 ] was subsequently prepared in
Corresponding author. Tel.: +81 88 633 7340; fax: +81 88 633 7340. E-mail address: [email protected] (T. Suge). 0109-5641/$ – see front matter © 2009 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dental.2009.08.011
30
d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 29–34
order to overcome tooth discoloration caused by treatment with AgF [5]. Acid resistance was significantly increased in both enamel and dentin after SiF treatment, similar to that following AgF treatment, being much more effective than NaF or acidulated phosphate fluoride (APF) [6]. Also, open dentin tubules within simulated hypersensitive teeth were completely occluded with a silica–calcium phosphate precipitate to the depth of approximately 20 m from the dentin surface immediately after SiF treatment, and induced apatitic precipitation on the dentin surface in the simulated oral environment when the dentin disk was immersed in synthetic saliva. Therefore, SiF solution shows a strong potential for use in dentin hypersensitivity treatment [7,8]. The same concentration of SiF solution (9000 ppm) has been used throughout our study to compare its fluoridation effect with that of acidulated phosphate fluoride. Therefore, the effects of SiF concentration on the occlusion of dentin tubules and composition of the precipitate forming within tubules have not been studied up to now. The aim of this study was, therefore, to evaluate the effects of changing the SiF concentration and to identify an optimal concentration prior to its clinical use as a treatment for dentin hypersensitivity.
2.
Materials and methods
2.1.
Preparation of SiF solution
Several concentrations of SiF solution (from 100 to 19,400 ppm) were prepared from reagent grade chemicals (Kanto Chemical Co., Inc., Tokyo, Japan) following a previous study protocol [5]. Then, the pH of each SiF solution was measured at room temperature with a pH meter (DKK·TOA Co., Tokyo, Japan).
2.2.
Dentin disk treatment
Dentin disks were obtained from healthy adults with informed consent, through caries-free human molars extracted for periodontal reasons at the Tokushima University Hospital. Each tooth was sectioned horizontally below the cementoenamel junction. To prepare the disks from the middle of the coronal dentin, enamel was removed with a high-speed, water-cooled handpiece, followed by sectioning with a low-speed watercooled diamond saw (Buehler Ltd., Evanston, IL, USA) to a thickness of approximately 1.5 and 0.5 mm for SEM observation and dentin permeability measurement, respectively [7,8]. To remove the smear layer and prepare open dentin tubules to simulate dentin hypersensitivity, the disks were immersed in 0.5 mol/L EDTA (pH = 7.4) for 2 min [8]. The disks were then washed with distilled water for 1 min and dried using a three-way syringe. These specimens served as a control. Several SiF solutions at different concentrations were applied to each pretreated dentin disk with a cotton swab for 3 min. After SiF treatment, the tooth was washed with distilled water for 1 min.
2.3.
SEM observation
The occlusion of dentin tubules due to SiF treatment was evaluated through scanning electron microscope observation (Hitachi Co., Tokyo, Japan). The specimens obtained following
the above procedures were dried with a critical point dryer (Hitachi Co., Tokyo, Japan). After the specimens were coated with gold, the surface and fractured surface were observed using SEM. Occlusion was evaluated in five specimens for each treatment concentration.
2.4.
Dentin permeability
The ability of SiF to occlude dentin tubules was also evaluated from the dentin permeability measurements, made using an apparatus described by Pashley and Galloway [9]. The rate of fluid flow through the dentin disk was measured with this apparatus. Fluid flow was measured by following the progress of an air bubble in a micropipette at a pressure of 98 kPa. Dentin permeability was measured after EDTA treatment (Control), just after SiF treatment. Dentin permeability was presented as a percentage value against the control (before SiF treatment) specimen. This permeability was taken as an averaged value from 10 specimens.
2.5.
EDXA analysis
EDXA apparatus attached to a transmission electron microscope (H-500; Hitachi Co., Tokyo, Japan) was used to analyze the precipitate. The specimens after being treated with several concentrations of SiF solution were mounted on carbon holders and then carbon-coated. The carbon-coated specimens were analyzed with an accelerating voltage of 10 kV, a spot size of 100 nm, and a counting time of 100 s. The Ca/P molar ratio obtained with EDXA was the average value of ten specimens.
2.6.
Statistical analysis
For statistical analysis, one-way factorial ANOVA and Fisher’s PLSD method, used as a post hoc test, were performed using the program “Stat View 4.02” (Abacus Concepts Inc., Berkeley, CA, USA). p-Values < 0.05 were considered to indicate significant differences.
3.
Results
The pH of several SiF solutions is shown in Fig. 1. All solutions were acidic, with pH values ranging from 2.2 to 3.4. No correlation was observed between the pH value and concentration of SiF solution. A typical SEM image immediately after EDTA treatment is shown in Fig. 2. The dentin tubules had opened, similar to hypersensitive dentin. Following the application of several concentrations of SiF solution for 3 min, the open dentin tubules were completely occluded by the precipitate irrespective of the SiF concentration (Fig. 3). The surface morphology of the precipitate which occluded the dentin tubules was smooth and homogeneous regardless of the concentration of SiF solution. No morphological differences were observed among SiF treatments. SEM images of a longitudinally sectioned surface are shown in Fig. 4. Both conventional (9000 ppm) and highly concentrated (19,400 ppm) SiF treatment caused the homogeneous occlusion of dentin tubules from the dentin surface to a depth of approximately 10 m. In addition, many globular crystals were observed in a deep part of the tubules. The depth of occlusion after
d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 29–34
Fig. 1 – The pH of several concentrations of SiF solution.
100 ppm SiF treatment was shallower than that after 9000 and 19,400 ppm SiF treatment, although dentin tubules were filled densely with the precipitate. In conjunction, dentin permeability was reduced by approximately 90% after conventional SiF treatment (9000 ppm) (Table 1). Also, both weakly (100 ppm) and strongly (19,400 ppm) concentrated SiF solution significantly reduced dentin permeability to the same level as that with conventional SiF treatment. No significant difference was
31
Fig. 2 – Typical scanning electron micrograph of the surface of a dentin specimen after treatment with EDTA for 2 min. Bar represents 30 m.
observed among the three different concentrations of SiF solution. The Ca/P molar ratios of the precipitate formed in the dentin tubules after SiF treatments are shown in Fig. 5. The Ca/P molar ratio was gradually increased from 1.5 to 2.1 with an increase in the concentration of SiF solution.
Fig. 3 – Typical scanning electron micrograph of the surface of dentin after treatment with several concentrations of SiF: (a) 100 ppm; (b) 1000 ppm; (c) 3000 ppm; (d) 5000 ppm; (e) 9000 ppm; (f) 11,000 ppm; (g) 13,000 ppm; (h) 15,000 ppm; and (i) 19,400 ppm. Bar represents 30 m.
32
d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 29–34
Fig. 4 – Typical scanning electron micrograph of the longitudinally divided surface of specimens treated with several concentrations of SiF: (a) 100 ppm; (b) 1000 ppm; (c) 9000 ppm; (d) 19,400 ppm. (a and b) Bar represents 10 m. (c and d) Bar represents 20 m.
Table 1 – Measurement of dentin permeability before and after SiF treatment. Treatment Before (EDTA 2 min) 100 ppm SiF 9000 ppm SiF 19,400 ppm SiF
Dentin permeability (%) 100 14.1 (4.2) 11.3 (2.5) 9.9 (4.9)
Mean (SD), N = 10. No significant difference was observed among the three SiF treatments (p < 0.05).
4.
Discussion
Open dentin tubules are closely related to the development of hypersensitive teeth [10,11], and most of the therapy blocks the open tubules to reduce hydrodynamic stimuli with the application of a drug [12]. The application of an MS coat (Pain Free® in Europe and the United States) is a typical treatment method for hypersensitive teeth, its action mechanism is to block dentin tubules with MS emulsion; however, it has the ability to inhibit the mineralization of the dentin surface since substances formed in dentin tubules or on dentin surfaces
Fig. 5 – Relationship between concentration of SiF solution and Ca/P molar ratio of the precipitate.
after MS coat treatment are separated from the tooth component. It is preferable for open dentin tubules to be blocked with calcium phosphate, which is the main component of teeth. This does not inhibit the spontaneous remineralization of the tooth surface. SiF treatment occludes the open dentin tubules with silica–calcium phosphate complexes. Its ability to occlude dentin tubules and promote the acid resistance of teeth has been evaluated using a fixed concentration of SiF solution (9000 ppm) up to now in order to compare its fluoridation effects with other fluoride solutions such as an acidulated phosphate fluoride [6–8]. No attempt has been made to determine the optimal concentration of SiF solution; therefore, the effect of varying concentrations of SiF solutions on the occlusion of dentin tubules and composition of precipitate formed in the tubules is still uncertain. In the present study, the open dentin tubules were completely occluded by the low and high concentrations of SiF solution as well as the conventional SiF solution (9000 ppm). As a mechanism of dentin tubule occlusion using SiF solution, SiF is an acidic solution, and so it is likely that etched dentin, ionized calcium, and phosphate were deposited in dentin tubules. Therefore, it might be expected that the amount of precipitate would be increased when the low pH solution is applied, due to an increase in the volume of dissolved calcium phosphate from dentin. The pH values in the weakly concentrated (from 100 to 3000 ppm) SiF solutions were slightly high, exceeding 3.0. The mid-range SiF solutions (from 5000 to 13,000 ppm) showed a lower pH value, below 2.5. However, the pH values were increased in high concentrated SiF solutions (over 17,000 ppm). There was no correlation between the pH and concentration of SiF solution. In weakly concentrated SiF solution, the amount of tooth demineralization would be decreased; in consequence, the amount of crystal deposition in dentin tubules would be decreased due to the relatively high pH as well as the low concentrations of silica and fluoride. However, there was no correlation between the ability to occlude dentin tubules and concentration of SiF solution. In consequence, a consistent occlusion of dentin tubules was achieved irrespective of concentration of SiF solution. From the results of SEM observations, crystalline sedimentation was almost absent at sites of intertubular dentin; therefore, SiF solution seems to selectively react with peritubular dentin. The degree of mineralization of peritubular dentin is significantly higher than that of intertubular dentin,
d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 29–34
whereas peritubular dentin is more easily demineralized by acid than intertubular dentin. Therefore, SiF solution seems to readily fill up the dentin tubules. In light of this selective chemical reaction between SiF solution and peritubular dentin, the level of the crystal deposition in dentin tubules does not significantly decrease even with the application of low concentrations of SiF solution. Although the surface SEM images and dentin permeability after treatment with several SiF solutions did not reveal significant differences, the depth of occlusion after treatment with low concentrations of SiF solution showed a tendency to be shallower than that with conventional and high concentrations. With an increasing SiF concentration, globules were observed below the dense precipitate in the dentin tubules. In general, the application of a highly concentrated fluoride solution leads to the formation of calcium fluoride or calcium fluoride-like minerals on the tooth surface easily. Calcium fluoride or calcium fluoride-like minerals reportedly formed globules in a microscopic study, with a particle size ranging from 50 to 200 nm [13]. The globules attached to dentin tubule wall in the present study showed slightly larger particle sizes (approximately 100–800 nm). EDXA analysis showed that the composition of the precipitate formed in dentin tubules after treatment with several SiF solutions gradually changed based on the shifting Ca/P molar ratio. The Ca/P molar ratio was 1.5 after the application of the 100 ppm SiF solution, and the value gradually increased with the rising concentration of the SiF solution, reaching 2.1 with 19,400 ppm. As a result of analyzing the crystal composition after treatment using 9000 ppm SiF solution in our previous study, silica, calcium, and phosphate were detected in the precipitate, seeming to comprise a mixture of fluoridated apatite and calcium fluoride or calcium fluoride-like minerals. Based on the EDXA results with an increasing Ca/P molar ratio, the amount of calcium fluoride or calcium fluoride-like minerals in the crystal would rise on increasing the concentration of SiF solution. The application of a low concentration SiF solution led to the formation of a precipitate with a low Ca/P molar ratio, and it seems that the content of calcium fluoride in the precipitate was relatively low compared to the amount of fluoridated apatite. Calcium fluoride or calcium fluoride-like minerals are known to act as a fluoride reservoir in the oral environment; however, these would wash away and/or be dissolved easily in the oral environment compared to fluoridated apatite [14,15]. The duration of dentin tubule occlusion due to SiF treatment seems to decrease with an increasing amount of calcium fluoride in the precipitate. On the other hand, fluoridated apatite is stable and not dissolved in saliva, since it is supersaturated with respect to saliva; therefore, continuous dentin tubule occlusion would be achieved being similar to our previous report [8]. In addition, it was reported that some silica compounds induce apatite formation from simulated body fluids and synthetic saliva [16–18]; if the silica content of the precipitate formed in dentin tubules is increased, we may expect further mineralization of the dentin surface and, consequently, a longer duration of tubule occlusion. Prior to its clinical application, it is mandatory for the safety and stability of SiF treatment to be unequivocally established. The results of the present study showed that the optimal concentration of SiF solution for clinical use seems to be from 1000 to 9000 ppm based on clinical safety and efficacy. A low concentration flu-
33
oride solution can be used everyday, e.g., for mouth rinsing; therefore, SiF solution can be used for not only the treatment of hypersensitive dentin but also for the prevention of dental caries. Although a more detailed evaluation is indispensable prior to its clinical use, the results of this study further support the clinical application of SiF solution. Based on the results of this study, further in vivo investigations are warranted.
Acknowledgment This investigation was supported, in part, by a Grant-in-Aid for Scientific Research (19592203) from the Ministry of Education, Science, Sports and Culture, Japan.
references
[1] Nishino M, Yoshida S, Sobue S, Kato J, Nishida M. Effect of topically applied ammonical silver fluoride on dental caries in children. J Osaka Univ Dent Sch 1969;9:149–55. [2] Nishino M, Ono S, Kita Y, Tsuchitani Y. Caries prevention in pits and fissures with diamine silver fluoride solution and fissure sealant—sealing properties of pits and fissures and adhesive characteristics to enamel. J Osaka Univ Dent Sch 1974;14:1–8. [3] Suzuki T, Nishida M, Sobue S, Moriwaki Y. Effects of diamine silver fluoride on tooth enamel. J Osaka Univ Dent Sch 1974;14:61–72. [4] Yamaga R, Nishino M, Yoshida S, Yokomizo I. Diamine silver fluoride and its clinical application. J Osaka Univ Dent Sch 1972;12:1–20. [5] Murata H, Ishikawa K, Tenshin S, Horiuchi S, Nakanishi M, Asaoka K, et al. Fluoridation of hydroxyapatite powder by ammonium hexafluorosilicate. Caries Res 1996;30:465–70. [6] Kawasaki A, Suge T, Ishikawa K, Ozaki K, Matsuo T, Ebisu S. Ammonium hexafluorosilicate increased acid resistance of bovine enamel and dentin. J Mater Sci: Mater Med 2005;16:461–6. [7] Suge T, Kawasaki A, Ishikawa K, Matsuo T, Ebisu S. Effect of ammonium hexafluorosilicate on dentin tubule occlusion for the treatment of dentin hypersensitivity. Am J Dent 2006;19:248–52. [8] Suge T, Kawasaki A, Ishikawa K, Matsuo T, Ebisu S. Ammonium hexafluorosilicate elicits calcium phosphate precipitation and shows continuous dentin tubule occlusion. Dent Mater 2008;24:192–8. [9] Pashley DH, Galloway SE. The effects of oxalate treatment on the smear layer of ground surfaces of human dentine. Arch Oral Biol 1985;30:731–7. [10] Yoshiyama M, Noiri Y, Ozaki K, Uchida A, Ishikawa Y, Ishida H. Transmission electron microscopic characterization of hypersensitive human radicular dentin. J Dent Res 1990;69:1293–7. [11] Yoshiyama M, Masada J, Uchida A, Ishida H. Scanning electron microscopic characterization of sensitive vs. insensitive human radicular dentin. J Dent Res 1989;68:1498–502. [12] Olusile AO, Bamise CT, Oginni AO, Dosumu OO. Short-term clinical evaluation of four desensitizing agents. J Contemp Dent Pract 2008;9:22–9. [13] Arends J, Reintsema H, Dijkman TG. ‘Calcium fluoride-like’ material formed in partially demineralized human enamel in vivo owing to the action of fluoridated toothpastes. Acta Odontol Scand 1988;46:347–53. [14] Sieck B, Takagi S, Chow LC. Assessment of loosely-bound and firmly-bound fluoride uptake by tooth enamel from
34
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topically applied fluoride treatments. J Dent Res 1990;69:1261–5. [15] Takagi S, Chow LC, Sieck BA. Deposition of loosely bound and firmly bound fluoride on tooth enamel by an acidic gel containing fluorosilicate and monocalcium phosphate monohydrate. Caries Res 1992;26:321–7. [16] Li P, Nakanishi K, Kokubo T, de Groot K. Induction and morphology of hydroxyapatite, precipitated from metastable
simulated body fluids on sol–gel prepared silica. Biomaterials 1993;14:963–8. [17] Damen JJM, Ten Cate JM. The effect of silicic acid on calcium phosphate precipitation. J Dent Res 1989;68:1355–9. [18] Damen JJM, Ten Cate JM. Silica-induced precipitation of calcium phosphate in the presence of inhibitors of hydroxyapatite formation. J Dent Res 1992;71:453–7.
available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/dema
Effects of ammonium hexafluorosilicate concentration on dentin tubule occlusion and composition of the precipitate Toshiyuki Suge a,∗ , Akiko Kawasaki a , Kunio Ishikawa b , Takashi Matsuo a , Shigeyuki Ebisu c a
Department of Conservative Dentistry, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima 770-8504, Japan b Department of Biomaterials, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan c Department of Restorative Dentistry and Endodontology, Division of Oral Infections and Disease Control, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan
a r t i c l e
i n f o
a b s t r a c t
Article history:
Ammonium hexafluorosilicate [SiF: (NH4 )2 SiF6 ] was prepared in order to overcome the tooth
Received 6 August 2008
discoloration caused by diamine silver fluoride [AgF: (NH3 )2 AgF] application. We employed
Accepted 23 August 2009
a single concentration of SiF solution in our previous study; therefore, it is still unclear how the concentration of SiF solution affects the occlusion of dentin tubules and composition of the precipitate.
Keywords:
Objective. The aim of this study was to evaluate the effects of changing the concentration of
Ammonium hexafluorosilicate
SiF on its clinical use as a dentin hypersensitivity treatment.
Dentin tubules
Methods. To simulate dentin tubules subject to dentin hypersensitivity, dentin disks were
Occlusion
treated with EDTA for 2 min. Then, the disks were treated with several concentrations of SiF
Dentin sensitivity
solution (from 100 to 19,400 ppm) for 3 min. The occlusion of dentin tubules was evaluated
Calcium phosphate
using scanning electron microscopy (SEM), and the composition of the precipitate formed in
Silica
the tubules after SiF treatment was assessed using energy dispersive X-ray analysis (EDXA). Results. SEM photographs demonstrated that dentin tubules after treatment with SiF were occluded homogeneously and fully regardless of the concentration of SiF solution. The Ca/P molar ratio of the precipitate formed in dentin tubules after SiF treatment was increased with the concentration of SiF solution. Significance. It was concluded that the capacity to occlude dentin tubules was the same regardless of the concentration of SiF solution. However, the composition of the precipitate formed in the tubules was dependent on the concentration of SiF solution. © 2009 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Fluoride is effective for the prevention of dental caries, and sometimes it has also been used for the treatment of dentin hypersensitivity. The representative fluoride solution appli-
∗
cable for both treatments is diamine silver fluoride [AgF: (NH3 )2 AgF] (Saforide® , Beebland Medico Dental Inc., Osaka, Japan), which is widely used in dental clinics in Japan [1–4]. However, AgF causes tooth discoloration, especially problematic with regard to permanent teeth. Ammonium hexafluorosilicate [SiF: (NH4 )2 SiF6 ] was subsequently prepared in
Corresponding author. Tel.: +81 88 633 7340; fax: +81 88 633 7340. E-mail address: [email protected] (T. Suge). 0109-5641/$ – see front matter © 2009 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dental.2009.08.011
30
d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 29–34
order to overcome tooth discoloration caused by treatment with AgF [5]. Acid resistance was significantly increased in both enamel and dentin after SiF treatment, similar to that following AgF treatment, being much more effective than NaF or acidulated phosphate fluoride (APF) [6]. Also, open dentin tubules within simulated hypersensitive teeth were completely occluded with a silica–calcium phosphate precipitate to the depth of approximately 20 m from the dentin surface immediately after SiF treatment, and induced apatitic precipitation on the dentin surface in the simulated oral environment when the dentin disk was immersed in synthetic saliva. Therefore, SiF solution shows a strong potential for use in dentin hypersensitivity treatment [7,8]. The same concentration of SiF solution (9000 ppm) has been used throughout our study to compare its fluoridation effect with that of acidulated phosphate fluoride. Therefore, the effects of SiF concentration on the occlusion of dentin tubules and composition of the precipitate forming within tubules have not been studied up to now. The aim of this study was, therefore, to evaluate the effects of changing the SiF concentration and to identify an optimal concentration prior to its clinical use as a treatment for dentin hypersensitivity.
2.
Materials and methods
2.1.
Preparation of SiF solution
Several concentrations of SiF solution (from 100 to 19,400 ppm) were prepared from reagent grade chemicals (Kanto Chemical Co., Inc., Tokyo, Japan) following a previous study protocol [5]. Then, the pH of each SiF solution was measured at room temperature with a pH meter (DKK·TOA Co., Tokyo, Japan).
2.2.
Dentin disk treatment
Dentin disks were obtained from healthy adults with informed consent, through caries-free human molars extracted for periodontal reasons at the Tokushima University Hospital. Each tooth was sectioned horizontally below the cementoenamel junction. To prepare the disks from the middle of the coronal dentin, enamel was removed with a high-speed, water-cooled handpiece, followed by sectioning with a low-speed watercooled diamond saw (Buehler Ltd., Evanston, IL, USA) to a thickness of approximately 1.5 and 0.5 mm for SEM observation and dentin permeability measurement, respectively [7,8]. To remove the smear layer and prepare open dentin tubules to simulate dentin hypersensitivity, the disks were immersed in 0.5 mol/L EDTA (pH = 7.4) for 2 min [8]. The disks were then washed with distilled water for 1 min and dried using a three-way syringe. These specimens served as a control. Several SiF solutions at different concentrations were applied to each pretreated dentin disk with a cotton swab for 3 min. After SiF treatment, the tooth was washed with distilled water for 1 min.
2.3.
SEM observation
The occlusion of dentin tubules due to SiF treatment was evaluated through scanning electron microscope observation (Hitachi Co., Tokyo, Japan). The specimens obtained following
the above procedures were dried with a critical point dryer (Hitachi Co., Tokyo, Japan). After the specimens were coated with gold, the surface and fractured surface were observed using SEM. Occlusion was evaluated in five specimens for each treatment concentration.
2.4.
Dentin permeability
The ability of SiF to occlude dentin tubules was also evaluated from the dentin permeability measurements, made using an apparatus described by Pashley and Galloway [9]. The rate of fluid flow through the dentin disk was measured with this apparatus. Fluid flow was measured by following the progress of an air bubble in a micropipette at a pressure of 98 kPa. Dentin permeability was measured after EDTA treatment (Control), just after SiF treatment. Dentin permeability was presented as a percentage value against the control (before SiF treatment) specimen. This permeability was taken as an averaged value from 10 specimens.
2.5.
EDXA analysis
EDXA apparatus attached to a transmission electron microscope (H-500; Hitachi Co., Tokyo, Japan) was used to analyze the precipitate. The specimens after being treated with several concentrations of SiF solution were mounted on carbon holders and then carbon-coated. The carbon-coated specimens were analyzed with an accelerating voltage of 10 kV, a spot size of 100 nm, and a counting time of 100 s. The Ca/P molar ratio obtained with EDXA was the average value of ten specimens.
2.6.
Statistical analysis
For statistical analysis, one-way factorial ANOVA and Fisher’s PLSD method, used as a post hoc test, were performed using the program “Stat View 4.02” (Abacus Concepts Inc., Berkeley, CA, USA). p-Values < 0.05 were considered to indicate significant differences.
3.
Results
The pH of several SiF solutions is shown in Fig. 1. All solutions were acidic, with pH values ranging from 2.2 to 3.4. No correlation was observed between the pH value and concentration of SiF solution. A typical SEM image immediately after EDTA treatment is shown in Fig. 2. The dentin tubules had opened, similar to hypersensitive dentin. Following the application of several concentrations of SiF solution for 3 min, the open dentin tubules were completely occluded by the precipitate irrespective of the SiF concentration (Fig. 3). The surface morphology of the precipitate which occluded the dentin tubules was smooth and homogeneous regardless of the concentration of SiF solution. No morphological differences were observed among SiF treatments. SEM images of a longitudinally sectioned surface are shown in Fig. 4. Both conventional (9000 ppm) and highly concentrated (19,400 ppm) SiF treatment caused the homogeneous occlusion of dentin tubules from the dentin surface to a depth of approximately 10 m. In addition, many globular crystals were observed in a deep part of the tubules. The depth of occlusion after
d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 29–34
Fig. 1 – The pH of several concentrations of SiF solution.
100 ppm SiF treatment was shallower than that after 9000 and 19,400 ppm SiF treatment, although dentin tubules were filled densely with the precipitate. In conjunction, dentin permeability was reduced by approximately 90% after conventional SiF treatment (9000 ppm) (Table 1). Also, both weakly (100 ppm) and strongly (19,400 ppm) concentrated SiF solution significantly reduced dentin permeability to the same level as that with conventional SiF treatment. No significant difference was
31
Fig. 2 – Typical scanning electron micrograph of the surface of a dentin specimen after treatment with EDTA for 2 min. Bar represents 30 m.
observed among the three different concentrations of SiF solution. The Ca/P molar ratios of the precipitate formed in the dentin tubules after SiF treatments are shown in Fig. 5. The Ca/P molar ratio was gradually increased from 1.5 to 2.1 with an increase in the concentration of SiF solution.
Fig. 3 – Typical scanning electron micrograph of the surface of dentin after treatment with several concentrations of SiF: (a) 100 ppm; (b) 1000 ppm; (c) 3000 ppm; (d) 5000 ppm; (e) 9000 ppm; (f) 11,000 ppm; (g) 13,000 ppm; (h) 15,000 ppm; and (i) 19,400 ppm. Bar represents 30 m.
32
d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 29–34
Fig. 4 – Typical scanning electron micrograph of the longitudinally divided surface of specimens treated with several concentrations of SiF: (a) 100 ppm; (b) 1000 ppm; (c) 9000 ppm; (d) 19,400 ppm. (a and b) Bar represents 10 m. (c and d) Bar represents 20 m.
Table 1 – Measurement of dentin permeability before and after SiF treatment. Treatment Before (EDTA 2 min) 100 ppm SiF 9000 ppm SiF 19,400 ppm SiF
Dentin permeability (%) 100 14.1 (4.2) 11.3 (2.5) 9.9 (4.9)
Mean (SD), N = 10. No significant difference was observed among the three SiF treatments (p < 0.05).
4.
Discussion
Open dentin tubules are closely related to the development of hypersensitive teeth [10,11], and most of the therapy blocks the open tubules to reduce hydrodynamic stimuli with the application of a drug [12]. The application of an MS coat (Pain Free® in Europe and the United States) is a typical treatment method for hypersensitive teeth, its action mechanism is to block dentin tubules with MS emulsion; however, it has the ability to inhibit the mineralization of the dentin surface since substances formed in dentin tubules or on dentin surfaces
Fig. 5 – Relationship between concentration of SiF solution and Ca/P molar ratio of the precipitate.
after MS coat treatment are separated from the tooth component. It is preferable for open dentin tubules to be blocked with calcium phosphate, which is the main component of teeth. This does not inhibit the spontaneous remineralization of the tooth surface. SiF treatment occludes the open dentin tubules with silica–calcium phosphate complexes. Its ability to occlude dentin tubules and promote the acid resistance of teeth has been evaluated using a fixed concentration of SiF solution (9000 ppm) up to now in order to compare its fluoridation effects with other fluoride solutions such as an acidulated phosphate fluoride [6–8]. No attempt has been made to determine the optimal concentration of SiF solution; therefore, the effect of varying concentrations of SiF solutions on the occlusion of dentin tubules and composition of precipitate formed in the tubules is still uncertain. In the present study, the open dentin tubules were completely occluded by the low and high concentrations of SiF solution as well as the conventional SiF solution (9000 ppm). As a mechanism of dentin tubule occlusion using SiF solution, SiF is an acidic solution, and so it is likely that etched dentin, ionized calcium, and phosphate were deposited in dentin tubules. Therefore, it might be expected that the amount of precipitate would be increased when the low pH solution is applied, due to an increase in the volume of dissolved calcium phosphate from dentin. The pH values in the weakly concentrated (from 100 to 3000 ppm) SiF solutions were slightly high, exceeding 3.0. The mid-range SiF solutions (from 5000 to 13,000 ppm) showed a lower pH value, below 2.5. However, the pH values were increased in high concentrated SiF solutions (over 17,000 ppm). There was no correlation between the pH and concentration of SiF solution. In weakly concentrated SiF solution, the amount of tooth demineralization would be decreased; in consequence, the amount of crystal deposition in dentin tubules would be decreased due to the relatively high pH as well as the low concentrations of silica and fluoride. However, there was no correlation between the ability to occlude dentin tubules and concentration of SiF solution. In consequence, a consistent occlusion of dentin tubules was achieved irrespective of concentration of SiF solution. From the results of SEM observations, crystalline sedimentation was almost absent at sites of intertubular dentin; therefore, SiF solution seems to selectively react with peritubular dentin. The degree of mineralization of peritubular dentin is significantly higher than that of intertubular dentin,
d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 29–34
whereas peritubular dentin is more easily demineralized by acid than intertubular dentin. Therefore, SiF solution seems to readily fill up the dentin tubules. In light of this selective chemical reaction between SiF solution and peritubular dentin, the level of the crystal deposition in dentin tubules does not significantly decrease even with the application of low concentrations of SiF solution. Although the surface SEM images and dentin permeability after treatment with several SiF solutions did not reveal significant differences, the depth of occlusion after treatment with low concentrations of SiF solution showed a tendency to be shallower than that with conventional and high concentrations. With an increasing SiF concentration, globules were observed below the dense precipitate in the dentin tubules. In general, the application of a highly concentrated fluoride solution leads to the formation of calcium fluoride or calcium fluoride-like minerals on the tooth surface easily. Calcium fluoride or calcium fluoride-like minerals reportedly formed globules in a microscopic study, with a particle size ranging from 50 to 200 nm [13]. The globules attached to dentin tubule wall in the present study showed slightly larger particle sizes (approximately 100–800 nm). EDXA analysis showed that the composition of the precipitate formed in dentin tubules after treatment with several SiF solutions gradually changed based on the shifting Ca/P molar ratio. The Ca/P molar ratio was 1.5 after the application of the 100 ppm SiF solution, and the value gradually increased with the rising concentration of the SiF solution, reaching 2.1 with 19,400 ppm. As a result of analyzing the crystal composition after treatment using 9000 ppm SiF solution in our previous study, silica, calcium, and phosphate were detected in the precipitate, seeming to comprise a mixture of fluoridated apatite and calcium fluoride or calcium fluoride-like minerals. Based on the EDXA results with an increasing Ca/P molar ratio, the amount of calcium fluoride or calcium fluoride-like minerals in the crystal would rise on increasing the concentration of SiF solution. The application of a low concentration SiF solution led to the formation of a precipitate with a low Ca/P molar ratio, and it seems that the content of calcium fluoride in the precipitate was relatively low compared to the amount of fluoridated apatite. Calcium fluoride or calcium fluoride-like minerals are known to act as a fluoride reservoir in the oral environment; however, these would wash away and/or be dissolved easily in the oral environment compared to fluoridated apatite [14,15]. The duration of dentin tubule occlusion due to SiF treatment seems to decrease with an increasing amount of calcium fluoride in the precipitate. On the other hand, fluoridated apatite is stable and not dissolved in saliva, since it is supersaturated with respect to saliva; therefore, continuous dentin tubule occlusion would be achieved being similar to our previous report [8]. In addition, it was reported that some silica compounds induce apatite formation from simulated body fluids and synthetic saliva [16–18]; if the silica content of the precipitate formed in dentin tubules is increased, we may expect further mineralization of the dentin surface and, consequently, a longer duration of tubule occlusion. Prior to its clinical application, it is mandatory for the safety and stability of SiF treatment to be unequivocally established. The results of the present study showed that the optimal concentration of SiF solution for clinical use seems to be from 1000 to 9000 ppm based on clinical safety and efficacy. A low concentration flu-
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oride solution can be used everyday, e.g., for mouth rinsing; therefore, SiF solution can be used for not only the treatment of hypersensitive dentin but also for the prevention of dental caries. Although a more detailed evaluation is indispensable prior to its clinical use, the results of this study further support the clinical application of SiF solution. Based on the results of this study, further in vivo investigations are warranted.
Acknowledgment This investigation was supported, in part, by a Grant-in-Aid for Scientific Research (19592203) from the Ministry of Education, Science, Sports and Culture, Japan.
references
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