Original Paper Received: May 2, 2015 Accepted after revision: November 27, 2015 Published online: February 3, 2016
Caries Res 2016;50:32–39 DOI: 10.1159/000442896
Synergistic Inhibition of Enamel Demineralization by Peptide 8DSS and Fluoride Yang Yang a, b Xueping Lv a Wenyuan Shi c Xuedong Zhou a Jiyao Li a Linglin Zhang a
a
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, and Department of General Dentistry and Emergency, School of Stomatology, The Fourth Military Medical University, Xi’an, PR China; c Dental Research Service Center, UCLA School of Dentistry, Los Angeles, Calif., USA
b
Abstract The biomimetic peptide 8DSS has shown beneficial effects in promoting remineralization of demineralized enamel in vitro. Here we examined the ability of 8DSS alone and in combination with fluoride to inhibit enamel demineralization during pH-cycling mimicking intraoral conditions. Enamel blocks were subjected to 9 days of pH-cycling in the presence of 1,000 ppm NaF (positive control), distilled-deionized water (DDW; negative control), 25 μM 8DSS alone, 25 μM 8DSS with 500 ppm NaF (8DSS-FL) or 25 μM 8DSS with 1,000 ppm NaF (8DSS-FH) twice daily for 1 min each time. The blocks were analyzed in terms of surface microhardness (SMH), fluoride uptake and mineral content. The 8DSS-treated blocks showed significantly lower mineral loss, shallower lesions and higher SMH than the DDW-treated blocks. No significant differences were observed between the blocks treated with 8DSS alone or fluoride alone. The blocks treated with 8DSS alone or DDW showed similar amounts of fluoride uptake,
© 2016 S. Karger AG, Basel 0008–6568/16/0501–0032$39.50/0 E-Mail [email protected] www.karger.com/cre
which was the lowest of all the treatment groups. The blocks treated with 8DSS-FL or 8DSS-FH did not differ significantly, and both groups showed significantly greater SMH and fluoride uptake as well as significantly lower mineral loss and shallower lesions than the NaF-treated blocks. Mineral content was significantly higher in the 8DSS-treated blocks than in the DDW-treated blocks from the surface layer (10 μm) to the lesion depth (110 μm), and it was significantly higher in the blocks treated with 8DSS-FL or 8DSS-FH than in the NaFtreated blocks from 10 to 90 μm. These findings illustrate the potential of 8DSS for inhibiting enamel demineralization and for enhancing the anticaries effect of NaF. © 2016 S. Karger AG, Basel
Dental caries remains one of the most common diseases in the world. Numerous efforts to address this problem have focused on inhibiting enamel demineralization and promoting the remineralization of early enamel caries. Biomimetic mineralization has shown strong potential for regenerating the hierarchical enamel microstructure using such agents as calcium phosphate paste containing hydrogen peroxide [Yamagishi et al., 2005], Linglin Zhang State Key Laboratory of Oral Diseases, West China Hospital of Stomatology Sichuan University, No. 14, 3rd Section, Renmin Nan Lu Chengdu 610041 (PR China) E-Mail zhll_sc @ 163.com
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Key Words Aspartate-serine-serine · Caries · Combined effect · Dentin phosphoprotein · Enamel demineralization · Fluoride
amelogenin [Fan et al., 2007], leucine-rich amelogenin peptide [Le Norcy et al., 2011], polydopamine [Liu et al., 2014] and a synthetic self-assembling oligopeptide amphiphile [Li et al., 2014]. Of the various biomimetic approaches to remineralization, perhaps the most promising are methods based on dentin phosphoprotein (DPP). Dentin cells synthesize DPP and release it into the dentin mineralization front, where it serves as a major noncollagenous component of extracellular matrix. The loss or mutation of the DPP gene in humans and mice is associated with mineralization defects in dentin and bone [Prasad et al., 2010], leading to suggestions that the protein plays a significant role in biomineralization. This led to the proposal that incorporating DPP into engineered tissue may help induce biomimetic mineral formation [Zurick et al., 2013]. Indeed, human DPP has been shown to induce dentin mineralization when tightly combined with certain supports [Luo et al., 2003]. DPP may also help prevent demineralization in vivo, since small amounts of protein have been detected inside tubules far from carious lesions in human enamel in vitro [Martini et al., 2013]. While full-length DPP may be clinically useful for promoting mineralization, short functional peptides based on DPP may be superior because: (1) peptides can be designed to adopt desired structures, (2) peptides can be efficiently produced in large amounts and high purity and (3) using synthetic peptides avoids the risks associated with animal-derived proteins, including allergies, immunogenicity and potential disease [Hsu et al., 2011a]. DPP contains numerous repeats of the sequence aspartate-serine-serine (DSS), and these repeats are believed to promote the formation of hydroxyapatite. Short peptides containing several DSS repeats have been shown to perform comparably well to full-length DPP for remineralizing bovine enamel in vitro. Such peptides bind to calcium phosphate compounds with higher affinity than do other DPP-derived peptides (e.g. DAA, ASS, NAA), and conjugating them to polystyrene beads recruits calcium phosphate to the bead surface [Yarbrough et al., 2010]. Of the various DPP-derived peptides described so far, which contain 2–8 DSS repeats, peptides containing 8 repeats (8DSS) appear to be the most active in promoting biomineralization [Yarbrough et al., 2010]. 8DSS promotes mineral deposition onto human enamel and improves the surface properties of demineralized enamel [Hsu et al., 2011a]. Applying the peptides to demineralized enamel promotes the uniform deposition of small
Preparation of Materials Bovine permanent incisors without any lesions, cracks or fluoric mottle were obtained. On each incisor, a flat and uncontaminated surface was created using a diamond-coated band saw with continuous water cooling (Struers Minitom; Struers, Copenhagen, Denmark) and water-cooled carborundum discs of waterproof silicon carbide paper (800, 1,000, 1,200, 2,400 and 4,000 grit; Struers), which led to removal of about 150 μm of the outer enamel layer. In this way flat surfaces were obtained without surface contamination. The blocks were embedded in polymethylmethacrylate and painted with two layers of acid-resistant nail varnish, leaving a 4 × 4 mm window exposed on the labial enamel surface. Then the initial surface microhardness (SMH1) of the prepared enamel blocks was measured using a microhardness tester (Duramin-1/-2, Struers) and a Knoop indenter at a load of 50 g for 15 s. A total of 75 enamel blocks with an SMH1 of 350.6 ± 21.3 Knoop hardness numbers (KHN) were selected for further study.
Synergistic Inhibitory Effect of 8DSS and Fluoride
Caries Res 2016;50:32–39 DOI: 10.1159/000442896
apatite crystals, protecting them from deformation, thereby reducing average surface roughness and increasing average nanohardness and elastic modulus [Chung et al., 2012]. Furthermore, our previous research has shown that 8DSS promotes remineralization of demineralized enamel in an in vitro pH-cycling system that simulates the oral environment [Yang et al., 2014]. Together, these studies provide strong evidence that 8DSS efficiently promotes remineralization of early caries lesions. However, whether the peptide can prevent the demineralization of sound enamel is unknown. This is an intriguing question given that several other agents have been shown not only to do just that, but also to boost the fluoride-mediated inhibition of demineralization. These other agents include casein phosphopeptide-amorphous calcium phosphate [Hamba et al., 2011], Ca3SiO5 [Wang et al., 2012], zinc [Lippert, 2012], strontium and aluminum [KoletsiKounari et al., 2012], tin [Rakhmatullina et al., 2013], milk [Arnold et al., 2014] and sodium trimetaphosphate [Manarelli et al., 2014]. This led us to investigate the ability of 8DSS to inhibit enamel demineralization in sound bovine enamel. We examined the effects of the peptide alone and in combination with fluoride to examine whether the peptide could potentiate the well-established antidemineralizing effects of NaF in vitro pH-cycling system. It was hypothesized that 8DSS alone could inhibit the demineralization of sound enamel, and the significant synergistic inhibition of enamel demineralization by peptide 8DSS and fluoride should be observed. The results of the present study should bring us closer to a biomimetic mineralization approach to prevent caries onset.
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Materials and Methods
pH-Cycling Regime All 75 enamel samples were treated to a standard regime of controlled pH-cycling [Featherstone et al., 1988] involving a demineralization solution at pH 4.4 [2.0 mM Ca(NO3)2 · 4H2O, 2.0 mM KH2PO4, 75.0 mM CH3COOH] and a remineralization solution at pH 7.0 [1.5 mM Ca(NO3)2 · 4H2O, 0.9 mM KH2PO4, 130.0 mM KCl, 20.0 mM NaC2H6AsO2 · 3H2O]. During each 24-hour period, the blocks were immersed for 16 h in the remineralization solution (20 ml per block) and 6 h in the demineralization solution (40 ml per block). The blocks were rinsed in DDW between solution changes. During pH-cycling, sections were randomly divided into five treatment groups (15 specimens/group) and treated with the following agents: the positive control group: 1,000 ppm NaF (aqueous); the negative control group: DDW; the 8DSS group: 25 μM 8DSS alone; the 8DSS-FL group: 25 μM 8DSS and 500 ppm NaF, and the 8DSS-FH group: 25 μM 8DSS and 1,000 ppm NaF. These treatments were carried out twice daily, before demineralization at 8: 00 a.m. and after demineralization at 3: 00 p.m. During each treatment, each block was immersed for 1 min in 8 ml of solution. The samples were rinsed before and after each treatment with DDW. Each treatment, together with the associated washes before and after, lasted approximately 1 h. The pH-cycling included 9 cycles and was performed for a total of 9 days in sealed containers maintained at 37 ° C with continuous, low-speed magnetic stirring (100 rpm). All solutions were freshly prepared daily.
b
NaF
c
c
DDW
8DSS
a
a
8DSS-FL
8DSS-FH
Fig. 1. Means and 25th and 75th percentiles with maximum and minimum of fluoride uptake in enamel blocks after pH-cycling in the presence of NaF, DDW, 8DSS, 8DSS-FL and 8DSS-FH. Different letters (a–c) indicate significant differences (p < 0.05).
Table 1. SMH of bovine enamel blocks before and after pH-cycling
in the presence of NaF, DDW, 8DSS, 8DSS-FL or 8DSS-FH
Postcycling SMH SMH of samples was measured again after pH-cycling (SMH2). Five indentations spaced 100 μm apart were made. Fluoride Uptake Assay After pH-cycling, fluoride uptake into enamel blocks was assayed using the microdrill biopsy technique [White, 1987]. This procedure was performed prior to analysis by transverse microradiography (TMR; see below). The samples were fixed on the platform and a cylindrical hole was drilled in the outer third of the sample; the hole had a diameter of 50 ± 5 μm and a depth of 50 μm. The enamel powder obtained by drilling was then dissolved in 150 μl of 0.5 M HClO4. TISAB buffer (150 μl) and 1 N NaOH (100 μl) were added to this solution, and the pH was adjusted to approximately 5.2. The levels of fluoride in the enamel solution were measured using a fluoride electrode (Orion 9609BN, Thermo Scientific, Mass., USA). Measurements were taken in the region from the enamel surface to a lesion depth of 50 μm and were reported as micrograms of fluoride/unit area. Transverse Microradiography After pH-cycling, slices approximately 500 μm thick were cut from the inner two-thirds of enamel blocks using a hard-tissue microtome (PCF310; Precise Corporation, USA). All slices were made vertical to the window on the enamel surface and ground to a uniform thickness of 80–100 μm using a diamond-coated band saw (Struers). All slices were cleaned by ultrasound (Jiahui, Suzhou, PR China) to remove any dust particles. The thickness of ground sections was verified in a microscope with a 50× lens and
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130 120 110 100 90 80 70 60 50 40 30 20 10 0
Caries Res 2016;50:32–39 DOI: 10.1159/000442896
Treatments
Before pH-cycling
After pH-cycling
NaF DDW 8DSS 8DSS-FL 8DSS-FH
351 ± 25a 351 ± 18a 351 ± 22a 350 ± 19a 351 ± 21a
265 ± 17b 223 ± 15c 262 ± 26b 284 ± 13d 287 ± 16d
Values shown are means ± SD (n = 15). Different superscript letters denote significant differences between groups at the same time point (p < 0.05).
a 10× measuring eyepiece; the cut surface of the section was positioned at right angles to the optical axis. Each slice was fixed on a Plexiglas slide in a TMR sample holder (Inspektor Research Systems, Amsterdam, The Netherlands). Slices were microradiographed alongside an aluminum calibration step wedge with 14 steps using a monochromatic CuK X-ray source (Philips, Eindhoven, The Netherlands) operated at 20 kV and 20 mA and an exposure time of 25 s. Lesion depth and mineral loss were analyzed using imaging software (Transversal Microradiography Software 2006; Inspektor Research Systems). Five TMR traces were measured on each slice and five slices were analyzed from each enamel block. The software calculated the mineral loss in the lesion (volume percent, vol% × micrometers) relative to sound tissue. The lesion depth was determined as the distance from the enamel surface to the point at which the mineral content reached 87% that of sound enamel.
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Fluoride uptake (μg/cm2)
The nonphosphorylated peptide 8DSS was synthesized by GL Biochem (Shanghai, PR China). 8DSS was dissolved in distilleddeionized water (DDW; pH 7.0) to a final concentration of 25 μM.
b
c
d
Color version available online
a
e
Fig. 2. TMR images of all treatment groups after pH-cycling. a NaF. b DDW. c 8DSS. d 8DSS-FL. e 8DSS-FH.
b
Mineral loss (vol% × μm)
2,500
Fig. 3. Mineral loss and lesion depth in enamel blocks after pH-cycling in the presence of NaF, DDW, 8DSS, 8DSS-FL or 8DSS-FH. Different letters (a–c) indicate significant differences (p < 0.05).
Mineral loss
c b
b
120
Lesion depth
b
100
2,000
80
1,500
a
a
a
a
60
1,000
40
500
20
0
NaF
DDW
8DSS
8DSS-FL
8DSS-FH
0
Table 1 shows SMH in all five groups before (SMH1) and after (SMH2) pH-cycling. Before pH-cycling, the samples showed no significant differences. After pH-cycling, the samples in the 8DSS and NaF groups had similar SMH2, which was greater than that of samples in the DDW group. SMH2 was similar in the 8DSS-FL and
8DSS-FH groups and higher in those groups than in any of the others. Following pH-cycling, the enamel blocks in the 8DSSFL and 8DSS-FH groups showed similar amounts of fluoride uptake, which were greater than the uptake into the blocks treated with NaF alone (fig. 1). As expected, the enamel blocks treated with 8DSS alone or DDW showed similar amounts of fluoride uptake, which were the lowest of all treatment groups. After pH-cycling, TMR was used to compare the thickness of the radiopaque tissue (surface layer) and the depth of radiolucent tissue (lesion body) across the five groups of enamel blocks (fig. 2). The radiopaque belt in the lesion region was thinnest in the DDW-treated blocks and thickest in the blocks of the 8DSS-FL and 8DSS-FH groups. The radiopaque belt was of intermediate thickness in the blocks treated with NaF or 8DSS alone. Consistent with these results, the radiolucent region was widest in the DDW-treated blocks among all treatment
Synergistic Inhibitory Effect of 8DSS and Fluoride
Caries Res 2016;50:32–39 DOI: 10.1159/000442896
Statistical Analysis Data were analyzed using SPSS 17.0 (IBM, Chicago, Ill., USA) and charts were plotted using Origin 8.0 (OriginLab, Northampton, Mass., USA). The normal distribution of the data was verified using the Shapiro-Wilk test (p > 0.05). The average mineral loss, lesion depth and mineral content of different depths after pH-cycling were statistically analyzed by one-way analysis of variance (ANOVA), followed by the Student-Newman-Keuls test. Post hoc power analysis was performed using PASS 11.0 (NCSS, USA). The threshold of statistical significance was set at 0.05.
Results
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c
Lesion depth (μm)
3,000
ly powered and that the results show a low likelihood of type II error. Thus, the results indicate that 8DSS alone or in combination with fluoride may inhibit the demineralization of sound enamel.
100 90
70 60
Discussion
50 40 30 NaF DDW 8DSS 8DSS-FL 8DSS-FH
20 10 0 0
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 Lesion depth (μm)
Fig. 4. Mineral content (vol%) vs. depth (micrometers) for lesions
subjected to 9 days of pH-cycling involving daily treatment with NaF, DDW, 8DSS, 8DSS-FL or 8DSS-FH. Data shown are means ± SD (n = 15 per group, 5 scans per sample).
groups, it was of intermediate width in blocks treated with NaF or 8DSS alone, and it was narrowest in blocks from the 8DSS-FL and 8DSS-FH groups. No significant differences were observed between the blocks treated with NaF or 8DSS alone, or between the blocks treated with 8DSS-FL or 8DSS-FH. Consistent with these results, mineral loss and lesion depth after pH-cycling (fig. 3) were greatest in the DDWtreated blocks, intermediate in the blocks treated with NaF or 8DSS alone and smallest in the blocks from the 8DSS-FL and 8DSS-FH groups. Figure 4 shows the mineral content at different depths after pH-cycling for all treatment groups. As expected, the DDW group showed the lowest mineral content after cycling. The 8DSS and NaF samples showed a significantly greater mineral content than the DDW samples at depths of 10–110 μm, corresponding to the lesion. The 8DSS-FL and 8DSS-FH samples showed a similar mineral content from 10 to 90 μm, which was significantly greater than that of the NaF samples. At depths of 110–130 μm, the blocks treated with NaF, DDW or 8DSS showed no significant differences. The mineral content in all groups was similar at depths beyond 130 μm. In these analyses, post hoc power was higher than 80% for all pairwise comparisons, except for comparisons between NaF and 8DSS and comparisons between 8DSS-FL and 8DSS-FH. This suggests that the study was adequate36
Caries Res 2016;50:32–39 DOI: 10.1159/000442896
The present study provides in vitro evidence that the biomimetic 8DSS peptide on its own inhibits enamel demineralization, and that it potentiates the ability of fluoride to do the same. Studies of SMH, fluoride uptake and TMR indicate that the best results were obtained when 8DSS was used together with NaF. These findings illustrate the potential of 8DSS for inhibiting enamel demineralization and for enhancing the anticaries effect of NaF. In addition, these results also corroborate our hypothesis strongly. The progression of enamel demineralization should be considered the result of two partly independent processes. One is penetration, which is when prism junction material dissolves and which involves negligible mineral loss and greatly increases the size of prism junction pores. The second process is mineral loss, which occurs as the intraprismatic mineral dissolves; the rate of mineral loss depends on the degree of undersaturation with respect to the intraprismatic mineral and is limited by slow diffusion between crystals [Shellis, 1996]. In our study, 8DSS may exert its effects after permeating the inner enamel by passing through the larger pores, consistent with previous studies showing that it can promote the remineralization of demineralized enamel [Chang et al., 2006]. The peptide is predicted to have an overall negative charge at pH 7, since serine and aspartic acid are negatively charged under those conditions. As a result, 8DSS may combine with positively charged calcium ions on the surface of acid-attacked enamel, creating a diffusion barrier that reduces calcium dissolution into the surrounding medium, thereby inhibiting demineralization [Gregory et al., 1991]. At the same time, 8DSS can promote the capture of cations like Ca2+ by the enamel surface and initiate mineral deposition on human tooth surfaces, as shown in an experiment in which 8DSS was used together with a commercial remineralization product to treat partially demineralized dentin [Hsu et al., 2011b]. Fluoride, the classic anticaries agent, has been shown to inhibit the demineralization of sound enamel through several mechanisms. In one mechanism, fluoride ions embedded in the bacterial biofilm decrease the critical Yang/Lv/Shi/Zhou/Li/Zhang
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Mineral content (vol%)
80
The present study relied on Featherstone’s pH-cycling method to mimic intraoral conditions. In this model, two 1-min treatments per day mimic the general frequency and duration of toothbrushing, while daily demineralization lasting altogether 6 h simulates the time to form a cariogenic environment caused when the patient ingests cariogenic products and does not remove the biofilm. In spite of this, the model only simulates the pH changes of the oral cavity and does not take into account saliva and salivary proteins, which can profoundly affect enamel demineralization. Saliva and biofilm are the main factors causing the demineralization of tooth enamel. Under physiological conditions saliva and dental biofilms have a pH near neutrality (pH 7). The oral fluids contain ionic calcium and phosphates and their levels together with the pH, which can be impacted by sugars metabolized in oral biofilms, determine whether a mineral will precipitate or dissolve [ten Cate, 2013]. Furthermore, there are a lot of soluble enzymes in saliva. Therefore, it is not clear whether 8DSS will be easily hydrolyzed by some enzymes in the oral cavity. In addition, in our study, we used an aqueous solution as the treatment agent, which could take the most effect of 8DSS and fluorine. However, in clinical practice, whether this application procedure is feasible also remains unclear. In the future, therefore, more application procedures should be found, such as the addition of 8DSS to gels, fluorocoatings or fluoride varnishes and applied by the dentist under controlled conditions. It may also be possible to add 8DSS to fluoride toothpaste that individuals can use daily. We tested both 500 and 1000 ppm fluoride in our study because although higher levels of fluoride are widely used, it is unclear whether levels above 500 ppm show clinical benefit in adults. The sustained use of high-concentration fluoride is currently recommended [Schlueter et al., 2009], and most commercial toothpastes contain 1,000–1,100 ppm [Nagpal and Damle, 2007]. Several studies suggest that although increasing the fluoride concentration from 250 to approximately 550–600 ppm may increase its therapeutic effects [Featherstone et al., 1988], higher concentrations do not bring further benefit [Abdullah et al., 2008]. This is consistent with our results, which showed similar effects on enamel in groups treated with 500 or 1,000 ppm fluoride in the presence of 8DSS. We speculate that rapid mineral precipitation onto the surface pores of the lesion block access to the lesion interior; thus, 500 ppm fluoride may have ‘saturated’ the lesion surface under our experimental conditions, such that increasing the concentration to
Synergistic Inhibitory Effect of 8DSS and Fluoride
Caries Res 2016;50:32–39 DOI: 10.1159/000442896
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pH for the dissolution of Ca2+ and PO43–; in another, fluoride adsorbs to apatite crystal surfaces, where it replaces Ca2+ to form acid-resistant mineral fluorapatite with low solubility [ten Cate, 2013]. While the present study provides strong in vitro evidence that 8DSS and fluoride interact to reduce the demineralization of sound enamel, how this interaction occurs is unclear. One possibility is that 8DSS, which by itself already binds tightly to calcium phosphate compounds, can capture even more calcium and phosphate ions by binding to fluoride. The 8DSSmediated deposition of fluoride on the enamel surface, most likely in a form similar to CaF2, increases the formation of fluorapatite. This fluoride on the enamel surface may be released from the surface during acid attack to reduce demineralization and enhance remineralization [Liu et al., 2013]. These effects occur because Ca2+ and PO43− react with the kinks, steps and defects on the partially dissolved apatite surface, leading to the growth of existing crystallite remnants rather than the de novo formation of new crystallites, thereby ensuring that new growth conserves the original orientation of the enamel [Siddiqui et al., 2014]. 8DSS may enhance these effects by increasing the deposition of CaF2 on the enamel surface. After lesion initiation, 8DSS may also increase the availability of fluoride in the liquid contacting enamel hydroxyapatite at the surface, prim sheaths and intraprismatic enamel. Future studies should analyze whether one or more of these mechanisms explain the apparent synergistic inhibition of caries progression by 8DSS and fluoride. The present study used bovine enamel as a substitute for human enamel; the two types of enamel show similar chemical composition and physical features [Falla-Sotelo et al., 2005]. Bovine enamel is easier to obtain than human enamel and it presents a larger surface area with more uniform enamel thickness [Lippert and Lynch, 2014]. At the same time, bovine enamel contains a larger amount of interprismatic substances than human enamel; these substances in bovine enamel have ‘fibril-like’ structures. This makes bovine enamel more porous and translates into approximately 40% faster demineralization than with human enamel [Lippert and Lynch, 2014]. We speculate that when 8DSS arrives at the surface of bovine enamel, it diffuses via these pores into the inner enamel, where it creates a barrier that reduces the dissolution of calcium and phosphate ions from the enamel. Future studies should verify whether 8DSS can penetrate the pores of normal human enamel and exert anticaries effects at the peripheries of prisms that are not in contact with the enamel surface.
1,000 ppm did not cause additional inhibition. Whatever the explanation for this lack of additional effect, our results suggest that higher fluoride concentrations in the presence of 8DSS do not necessarily reduce demineralization further. This possibility may be particularly relevant for the dental care of children and adolescents, in whom fluoride may significantly prevent caries only at concentrations of 1,000 ppm and higher, with preventive effects increasing with concentration [Walsh et al, 2010]. Using such high concentrations increases the risk of fluorosis in young individuals. It is possible that adding 8DSS to a low-fluoride toothpaste may lead to a similar anticaries efficacy as a high-fluoride conventional toothpaste containing 1,000– 1,100 ppm fluoride. This may help to reduce the risk of dental fluorosis in young people. In conclusion, the present study presents strong evidence for two findings relevant for clinical practice: 8DSS alone can inhibit the demineralization of sound enamel, and its combined use with NaF renders fluoride even more effective at inhibiting demineralization. As a result of this synergistic interaction, it may be helpful to use lower fluoride concentrations for caries prevention.
Acknowledgments The authors are grateful for the support received from the National Natural Science Foundation of China (No. 81470734), the New Century Excellent Talents University Support Program and the Key Technology Program of Sichuan Province (No. 2014SZ0024). The authors also thank the Crest Research Laboratory of Procter & Gamble Technology (Beijing) Co. Ltd. for help with TMR. The funders had no role in the study design, data collection or analysis, preparation of the manuscript or the decision to publish it.
Author Contributions Linglin Zhang, Wenyuan Shi, Xuedong Zhou and Jiyao Li conceived and designed the experiments. Yang Yang and Xueping Lv contributed to the acquisition, analysis and interpretation of data. The paper was written by Yang Yang. Final approval was given by Linglin Zhang, Wenyuan Shi, Xuedong Zhou, Jiyao Li, Yang Yang and Xueping Lv.
Disclosure Statement The authors report no conflict of interest in the study design, data collection, analysis or interpretation, manuscript writing or the decision to submit the manuscript for publication.
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Schlueter N, Klimek J, Ganss C: Effect of stannous and fluoride concentration in a mouth rinse on erosive tissue loss in enamel in vitro. Arch Oral Biol 2009;54:432–436. Shellis RP: A scanning electron-microscopic study of solubility variations in human enamel and dentine. Arch Oral Biol 1996; 41: 473– 484. Siddiqui S, Anderson P, Al-Jawad M: Recovery of crystallographic texture in remineralized dental enamel. PLoS One 2014;9:e108879. ten Cate JM: Contemporary perspective on the use of fluoride products in caries prevention. Br Dent J 2013;214:161–167. Walsh T, Worthington HV, Glenny AM, Appelbe P, Marinho VC, Shi X: Fluoride toothpastes of different concentrations for preventing dental caries in children and adolescents. Cochrane Database Syst Rev 2010;20:CD007868. Wang Y, Li X, Chang J, Wu C, Deng Y: Effect of tricalcium silicate (Ca3SiO5) bioactive material on reducing enamel demineralization: an in vitro pH-cycling study. J Dent 2012; 40: 1119–1126.
Caries Res 2016;50:32–39 DOI: 10.1159/000442896
White DJ: Reactivity of fluoride dentifrices with artificial caries. I. Effects on early lesions: F uptake, surface hardening and remineralization. Caries Res 1987;21:126–140. Yamagishi K, Onuma K, Suzuki T, Okada F, Tagami J, Otsuki M, Senawangse P: Materials chemistry: a synthetic enamel for rapid tooth repair. Nature 2005;433:819. Yang Y, Lv XP, Shi W, Li JY, Li DX, Zhou XD, Zhang LL: 8DSS-promoted remineralization of initial enamel caries in vitro. J Dent Res 2014;93:520–524. Yarbrough DK, Hagerman E, Eckert R, He J, Choi H, Cao N: Specific binding and mineralization of calcified surfaces by small peptides. Calcif Tissue Int 2010;86:58–66. Zurick KM, Qin C, Matthew T, Bernards MT: Mineralization induction effects of osteopontin, bone sialoprotein, and dentin phosphoprotein on a biomimetic collagen substrate. J Biomed Mater Res A 2013;101:1571–1581.
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Manarelli MM, Delbem AC, Lima TM, Castilho FC, Pessan JP: In vitro remineralizing effect of fluoride varnishes containing sodium trimetaphosphate. Caries Res 2014; 48: 299– 305. Martini D, Trirè A, Breschi L, Mazzoni A, Teti G, Falconi M, Ruggeri A Jr: Dentin matrix protein 1 and dentin sialophosphoprotein in human sound and carious teeth: an immunohistochemical and colorimetric assay. Eur J Histochem 2013;57:216–223. Nagpal DI, Damle SG: Comparison of salivary fluoride levels following use of dentifrices containing different concentrations of fluoride. J Indian Soc Pedod Prev Dent 2007; 25: 20–22. Prasad M, Butler WT, Qin C: Dentin sialophosphoprotein in biomineralization. Connect Tissue Res 2010;51:404–417. Rakhmatullina E, Beyeler B, Lussi A: Inhibition of enamel erosion by stannous and fluoride containing rinsing solutions. Schweiz Monatsschr Zahnmed 2013;23:192–198.
Caries Res 2016;50:32–39 DOI: 10.1159/000442896
Synergistic Inhibition of Enamel Demineralization by Peptide 8DSS and Fluoride Yang Yang a, b Xueping Lv a Wenyuan Shi c Xuedong Zhou a Jiyao Li a Linglin Zhang a
a
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, and Department of General Dentistry and Emergency, School of Stomatology, The Fourth Military Medical University, Xi’an, PR China; c Dental Research Service Center, UCLA School of Dentistry, Los Angeles, Calif., USA
b
Abstract The biomimetic peptide 8DSS has shown beneficial effects in promoting remineralization of demineralized enamel in vitro. Here we examined the ability of 8DSS alone and in combination with fluoride to inhibit enamel demineralization during pH-cycling mimicking intraoral conditions. Enamel blocks were subjected to 9 days of pH-cycling in the presence of 1,000 ppm NaF (positive control), distilled-deionized water (DDW; negative control), 25 μM 8DSS alone, 25 μM 8DSS with 500 ppm NaF (8DSS-FL) or 25 μM 8DSS with 1,000 ppm NaF (8DSS-FH) twice daily for 1 min each time. The blocks were analyzed in terms of surface microhardness (SMH), fluoride uptake and mineral content. The 8DSS-treated blocks showed significantly lower mineral loss, shallower lesions and higher SMH than the DDW-treated blocks. No significant differences were observed between the blocks treated with 8DSS alone or fluoride alone. The blocks treated with 8DSS alone or DDW showed similar amounts of fluoride uptake,
© 2016 S. Karger AG, Basel 0008–6568/16/0501–0032$39.50/0 E-Mail [email protected] www.karger.com/cre
which was the lowest of all the treatment groups. The blocks treated with 8DSS-FL or 8DSS-FH did not differ significantly, and both groups showed significantly greater SMH and fluoride uptake as well as significantly lower mineral loss and shallower lesions than the NaF-treated blocks. Mineral content was significantly higher in the 8DSS-treated blocks than in the DDW-treated blocks from the surface layer (10 μm) to the lesion depth (110 μm), and it was significantly higher in the blocks treated with 8DSS-FL or 8DSS-FH than in the NaFtreated blocks from 10 to 90 μm. These findings illustrate the potential of 8DSS for inhibiting enamel demineralization and for enhancing the anticaries effect of NaF. © 2016 S. Karger AG, Basel
Dental caries remains one of the most common diseases in the world. Numerous efforts to address this problem have focused on inhibiting enamel demineralization and promoting the remineralization of early enamel caries. Biomimetic mineralization has shown strong potential for regenerating the hierarchical enamel microstructure using such agents as calcium phosphate paste containing hydrogen peroxide [Yamagishi et al., 2005], Linglin Zhang State Key Laboratory of Oral Diseases, West China Hospital of Stomatology Sichuan University, No. 14, 3rd Section, Renmin Nan Lu Chengdu 610041 (PR China) E-Mail zhll_sc @ 163.com
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Key Words Aspartate-serine-serine · Caries · Combined effect · Dentin phosphoprotein · Enamel demineralization · Fluoride
amelogenin [Fan et al., 2007], leucine-rich amelogenin peptide [Le Norcy et al., 2011], polydopamine [Liu et al., 2014] and a synthetic self-assembling oligopeptide amphiphile [Li et al., 2014]. Of the various biomimetic approaches to remineralization, perhaps the most promising are methods based on dentin phosphoprotein (DPP). Dentin cells synthesize DPP and release it into the dentin mineralization front, where it serves as a major noncollagenous component of extracellular matrix. The loss or mutation of the DPP gene in humans and mice is associated with mineralization defects in dentin and bone [Prasad et al., 2010], leading to suggestions that the protein plays a significant role in biomineralization. This led to the proposal that incorporating DPP into engineered tissue may help induce biomimetic mineral formation [Zurick et al., 2013]. Indeed, human DPP has been shown to induce dentin mineralization when tightly combined with certain supports [Luo et al., 2003]. DPP may also help prevent demineralization in vivo, since small amounts of protein have been detected inside tubules far from carious lesions in human enamel in vitro [Martini et al., 2013]. While full-length DPP may be clinically useful for promoting mineralization, short functional peptides based on DPP may be superior because: (1) peptides can be designed to adopt desired structures, (2) peptides can be efficiently produced in large amounts and high purity and (3) using synthetic peptides avoids the risks associated with animal-derived proteins, including allergies, immunogenicity and potential disease [Hsu et al., 2011a]. DPP contains numerous repeats of the sequence aspartate-serine-serine (DSS), and these repeats are believed to promote the formation of hydroxyapatite. Short peptides containing several DSS repeats have been shown to perform comparably well to full-length DPP for remineralizing bovine enamel in vitro. Such peptides bind to calcium phosphate compounds with higher affinity than do other DPP-derived peptides (e.g. DAA, ASS, NAA), and conjugating them to polystyrene beads recruits calcium phosphate to the bead surface [Yarbrough et al., 2010]. Of the various DPP-derived peptides described so far, which contain 2–8 DSS repeats, peptides containing 8 repeats (8DSS) appear to be the most active in promoting biomineralization [Yarbrough et al., 2010]. 8DSS promotes mineral deposition onto human enamel and improves the surface properties of demineralized enamel [Hsu et al., 2011a]. Applying the peptides to demineralized enamel promotes the uniform deposition of small
Preparation of Materials Bovine permanent incisors without any lesions, cracks or fluoric mottle were obtained. On each incisor, a flat and uncontaminated surface was created using a diamond-coated band saw with continuous water cooling (Struers Minitom; Struers, Copenhagen, Denmark) and water-cooled carborundum discs of waterproof silicon carbide paper (800, 1,000, 1,200, 2,400 and 4,000 grit; Struers), which led to removal of about 150 μm of the outer enamel layer. In this way flat surfaces were obtained without surface contamination. The blocks were embedded in polymethylmethacrylate and painted with two layers of acid-resistant nail varnish, leaving a 4 × 4 mm window exposed on the labial enamel surface. Then the initial surface microhardness (SMH1) of the prepared enamel blocks was measured using a microhardness tester (Duramin-1/-2, Struers) and a Knoop indenter at a load of 50 g for 15 s. A total of 75 enamel blocks with an SMH1 of 350.6 ± 21.3 Knoop hardness numbers (KHN) were selected for further study.
Synergistic Inhibitory Effect of 8DSS and Fluoride
Caries Res 2016;50:32–39 DOI: 10.1159/000442896
apatite crystals, protecting them from deformation, thereby reducing average surface roughness and increasing average nanohardness and elastic modulus [Chung et al., 2012]. Furthermore, our previous research has shown that 8DSS promotes remineralization of demineralized enamel in an in vitro pH-cycling system that simulates the oral environment [Yang et al., 2014]. Together, these studies provide strong evidence that 8DSS efficiently promotes remineralization of early caries lesions. However, whether the peptide can prevent the demineralization of sound enamel is unknown. This is an intriguing question given that several other agents have been shown not only to do just that, but also to boost the fluoride-mediated inhibition of demineralization. These other agents include casein phosphopeptide-amorphous calcium phosphate [Hamba et al., 2011], Ca3SiO5 [Wang et al., 2012], zinc [Lippert, 2012], strontium and aluminum [KoletsiKounari et al., 2012], tin [Rakhmatullina et al., 2013], milk [Arnold et al., 2014] and sodium trimetaphosphate [Manarelli et al., 2014]. This led us to investigate the ability of 8DSS to inhibit enamel demineralization in sound bovine enamel. We examined the effects of the peptide alone and in combination with fluoride to examine whether the peptide could potentiate the well-established antidemineralizing effects of NaF in vitro pH-cycling system. It was hypothesized that 8DSS alone could inhibit the demineralization of sound enamel, and the significant synergistic inhibition of enamel demineralization by peptide 8DSS and fluoride should be observed. The results of the present study should bring us closer to a biomimetic mineralization approach to prevent caries onset.
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Materials and Methods
pH-Cycling Regime All 75 enamel samples were treated to a standard regime of controlled pH-cycling [Featherstone et al., 1988] involving a demineralization solution at pH 4.4 [2.0 mM Ca(NO3)2 · 4H2O, 2.0 mM KH2PO4, 75.0 mM CH3COOH] and a remineralization solution at pH 7.0 [1.5 mM Ca(NO3)2 · 4H2O, 0.9 mM KH2PO4, 130.0 mM KCl, 20.0 mM NaC2H6AsO2 · 3H2O]. During each 24-hour period, the blocks were immersed for 16 h in the remineralization solution (20 ml per block) and 6 h in the demineralization solution (40 ml per block). The blocks were rinsed in DDW between solution changes. During pH-cycling, sections were randomly divided into five treatment groups (15 specimens/group) and treated with the following agents: the positive control group: 1,000 ppm NaF (aqueous); the negative control group: DDW; the 8DSS group: 25 μM 8DSS alone; the 8DSS-FL group: 25 μM 8DSS and 500 ppm NaF, and the 8DSS-FH group: 25 μM 8DSS and 1,000 ppm NaF. These treatments were carried out twice daily, before demineralization at 8: 00 a.m. and after demineralization at 3: 00 p.m. During each treatment, each block was immersed for 1 min in 8 ml of solution. The samples were rinsed before and after each treatment with DDW. Each treatment, together with the associated washes before and after, lasted approximately 1 h. The pH-cycling included 9 cycles and was performed for a total of 9 days in sealed containers maintained at 37 ° C with continuous, low-speed magnetic stirring (100 rpm). All solutions were freshly prepared daily.
b
NaF
c
c
DDW
8DSS
a
a
8DSS-FL
8DSS-FH
Fig. 1. Means and 25th and 75th percentiles with maximum and minimum of fluoride uptake in enamel blocks after pH-cycling in the presence of NaF, DDW, 8DSS, 8DSS-FL and 8DSS-FH. Different letters (a–c) indicate significant differences (p < 0.05).
Table 1. SMH of bovine enamel blocks before and after pH-cycling
in the presence of NaF, DDW, 8DSS, 8DSS-FL or 8DSS-FH
Postcycling SMH SMH of samples was measured again after pH-cycling (SMH2). Five indentations spaced 100 μm apart were made. Fluoride Uptake Assay After pH-cycling, fluoride uptake into enamel blocks was assayed using the microdrill biopsy technique [White, 1987]. This procedure was performed prior to analysis by transverse microradiography (TMR; see below). The samples were fixed on the platform and a cylindrical hole was drilled in the outer third of the sample; the hole had a diameter of 50 ± 5 μm and a depth of 50 μm. The enamel powder obtained by drilling was then dissolved in 150 μl of 0.5 M HClO4. TISAB buffer (150 μl) and 1 N NaOH (100 μl) were added to this solution, and the pH was adjusted to approximately 5.2. The levels of fluoride in the enamel solution were measured using a fluoride electrode (Orion 9609BN, Thermo Scientific, Mass., USA). Measurements were taken in the region from the enamel surface to a lesion depth of 50 μm and were reported as micrograms of fluoride/unit area. Transverse Microradiography After pH-cycling, slices approximately 500 μm thick were cut from the inner two-thirds of enamel blocks using a hard-tissue microtome (PCF310; Precise Corporation, USA). All slices were made vertical to the window on the enamel surface and ground to a uniform thickness of 80–100 μm using a diamond-coated band saw (Struers). All slices were cleaned by ultrasound (Jiahui, Suzhou, PR China) to remove any dust particles. The thickness of ground sections was verified in a microscope with a 50× lens and
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130 120 110 100 90 80 70 60 50 40 30 20 10 0
Caries Res 2016;50:32–39 DOI: 10.1159/000442896
Treatments
Before pH-cycling
After pH-cycling
NaF DDW 8DSS 8DSS-FL 8DSS-FH
351 ± 25a 351 ± 18a 351 ± 22a 350 ± 19a 351 ± 21a
265 ± 17b 223 ± 15c 262 ± 26b 284 ± 13d 287 ± 16d
Values shown are means ± SD (n = 15). Different superscript letters denote significant differences between groups at the same time point (p < 0.05).
a 10× measuring eyepiece; the cut surface of the section was positioned at right angles to the optical axis. Each slice was fixed on a Plexiglas slide in a TMR sample holder (Inspektor Research Systems, Amsterdam, The Netherlands). Slices were microradiographed alongside an aluminum calibration step wedge with 14 steps using a monochromatic CuK X-ray source (Philips, Eindhoven, The Netherlands) operated at 20 kV and 20 mA and an exposure time of 25 s. Lesion depth and mineral loss were analyzed using imaging software (Transversal Microradiography Software 2006; Inspektor Research Systems). Five TMR traces were measured on each slice and five slices were analyzed from each enamel block. The software calculated the mineral loss in the lesion (volume percent, vol% × micrometers) relative to sound tissue. The lesion depth was determined as the distance from the enamel surface to the point at which the mineral content reached 87% that of sound enamel.
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Fluoride uptake (μg/cm2)
The nonphosphorylated peptide 8DSS was synthesized by GL Biochem (Shanghai, PR China). 8DSS was dissolved in distilleddeionized water (DDW; pH 7.0) to a final concentration of 25 μM.
b
c
d
Color version available online
a
e
Fig. 2. TMR images of all treatment groups after pH-cycling. a NaF. b DDW. c 8DSS. d 8DSS-FL. e 8DSS-FH.
b
Mineral loss (vol% × μm)
2,500
Fig. 3. Mineral loss and lesion depth in enamel blocks after pH-cycling in the presence of NaF, DDW, 8DSS, 8DSS-FL or 8DSS-FH. Different letters (a–c) indicate significant differences (p < 0.05).
Mineral loss
c b
b
120
Lesion depth
b
100
2,000
80
1,500
a
a
a
a
60
1,000
40
500
20
0
NaF
DDW
8DSS
8DSS-FL
8DSS-FH
0
Table 1 shows SMH in all five groups before (SMH1) and after (SMH2) pH-cycling. Before pH-cycling, the samples showed no significant differences. After pH-cycling, the samples in the 8DSS and NaF groups had similar SMH2, which was greater than that of samples in the DDW group. SMH2 was similar in the 8DSS-FL and
8DSS-FH groups and higher in those groups than in any of the others. Following pH-cycling, the enamel blocks in the 8DSSFL and 8DSS-FH groups showed similar amounts of fluoride uptake, which were greater than the uptake into the blocks treated with NaF alone (fig. 1). As expected, the enamel blocks treated with 8DSS alone or DDW showed similar amounts of fluoride uptake, which were the lowest of all treatment groups. After pH-cycling, TMR was used to compare the thickness of the radiopaque tissue (surface layer) and the depth of radiolucent tissue (lesion body) across the five groups of enamel blocks (fig. 2). The radiopaque belt in the lesion region was thinnest in the DDW-treated blocks and thickest in the blocks of the 8DSS-FL and 8DSS-FH groups. The radiopaque belt was of intermediate thickness in the blocks treated with NaF or 8DSS alone. Consistent with these results, the radiolucent region was widest in the DDW-treated blocks among all treatment
Synergistic Inhibitory Effect of 8DSS and Fluoride
Caries Res 2016;50:32–39 DOI: 10.1159/000442896
Statistical Analysis Data were analyzed using SPSS 17.0 (IBM, Chicago, Ill., USA) and charts were plotted using Origin 8.0 (OriginLab, Northampton, Mass., USA). The normal distribution of the data was verified using the Shapiro-Wilk test (p > 0.05). The average mineral loss, lesion depth and mineral content of different depths after pH-cycling were statistically analyzed by one-way analysis of variance (ANOVA), followed by the Student-Newman-Keuls test. Post hoc power analysis was performed using PASS 11.0 (NCSS, USA). The threshold of statistical significance was set at 0.05.
Results
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c
Lesion depth (μm)
3,000
ly powered and that the results show a low likelihood of type II error. Thus, the results indicate that 8DSS alone or in combination with fluoride may inhibit the demineralization of sound enamel.
100 90
70 60
Discussion
50 40 30 NaF DDW 8DSS 8DSS-FL 8DSS-FH
20 10 0 0
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 Lesion depth (μm)
Fig. 4. Mineral content (vol%) vs. depth (micrometers) for lesions
subjected to 9 days of pH-cycling involving daily treatment with NaF, DDW, 8DSS, 8DSS-FL or 8DSS-FH. Data shown are means ± SD (n = 15 per group, 5 scans per sample).
groups, it was of intermediate width in blocks treated with NaF or 8DSS alone, and it was narrowest in blocks from the 8DSS-FL and 8DSS-FH groups. No significant differences were observed between the blocks treated with NaF or 8DSS alone, or between the blocks treated with 8DSS-FL or 8DSS-FH. Consistent with these results, mineral loss and lesion depth after pH-cycling (fig. 3) were greatest in the DDWtreated blocks, intermediate in the blocks treated with NaF or 8DSS alone and smallest in the blocks from the 8DSS-FL and 8DSS-FH groups. Figure 4 shows the mineral content at different depths after pH-cycling for all treatment groups. As expected, the DDW group showed the lowest mineral content after cycling. The 8DSS and NaF samples showed a significantly greater mineral content than the DDW samples at depths of 10–110 μm, corresponding to the lesion. The 8DSS-FL and 8DSS-FH samples showed a similar mineral content from 10 to 90 μm, which was significantly greater than that of the NaF samples. At depths of 110–130 μm, the blocks treated with NaF, DDW or 8DSS showed no significant differences. The mineral content in all groups was similar at depths beyond 130 μm. In these analyses, post hoc power was higher than 80% for all pairwise comparisons, except for comparisons between NaF and 8DSS and comparisons between 8DSS-FL and 8DSS-FH. This suggests that the study was adequate36
Caries Res 2016;50:32–39 DOI: 10.1159/000442896
The present study provides in vitro evidence that the biomimetic 8DSS peptide on its own inhibits enamel demineralization, and that it potentiates the ability of fluoride to do the same. Studies of SMH, fluoride uptake and TMR indicate that the best results were obtained when 8DSS was used together with NaF. These findings illustrate the potential of 8DSS for inhibiting enamel demineralization and for enhancing the anticaries effect of NaF. In addition, these results also corroborate our hypothesis strongly. The progression of enamel demineralization should be considered the result of two partly independent processes. One is penetration, which is when prism junction material dissolves and which involves negligible mineral loss and greatly increases the size of prism junction pores. The second process is mineral loss, which occurs as the intraprismatic mineral dissolves; the rate of mineral loss depends on the degree of undersaturation with respect to the intraprismatic mineral and is limited by slow diffusion between crystals [Shellis, 1996]. In our study, 8DSS may exert its effects after permeating the inner enamel by passing through the larger pores, consistent with previous studies showing that it can promote the remineralization of demineralized enamel [Chang et al., 2006]. The peptide is predicted to have an overall negative charge at pH 7, since serine and aspartic acid are negatively charged under those conditions. As a result, 8DSS may combine with positively charged calcium ions on the surface of acid-attacked enamel, creating a diffusion barrier that reduces calcium dissolution into the surrounding medium, thereby inhibiting demineralization [Gregory et al., 1991]. At the same time, 8DSS can promote the capture of cations like Ca2+ by the enamel surface and initiate mineral deposition on human tooth surfaces, as shown in an experiment in which 8DSS was used together with a commercial remineralization product to treat partially demineralized dentin [Hsu et al., 2011b]. Fluoride, the classic anticaries agent, has been shown to inhibit the demineralization of sound enamel through several mechanisms. In one mechanism, fluoride ions embedded in the bacterial biofilm decrease the critical Yang/Lv/Shi/Zhou/Li/Zhang
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Mineral content (vol%)
80
The present study relied on Featherstone’s pH-cycling method to mimic intraoral conditions. In this model, two 1-min treatments per day mimic the general frequency and duration of toothbrushing, while daily demineralization lasting altogether 6 h simulates the time to form a cariogenic environment caused when the patient ingests cariogenic products and does not remove the biofilm. In spite of this, the model only simulates the pH changes of the oral cavity and does not take into account saliva and salivary proteins, which can profoundly affect enamel demineralization. Saliva and biofilm are the main factors causing the demineralization of tooth enamel. Under physiological conditions saliva and dental biofilms have a pH near neutrality (pH 7). The oral fluids contain ionic calcium and phosphates and their levels together with the pH, which can be impacted by sugars metabolized in oral biofilms, determine whether a mineral will precipitate or dissolve [ten Cate, 2013]. Furthermore, there are a lot of soluble enzymes in saliva. Therefore, it is not clear whether 8DSS will be easily hydrolyzed by some enzymes in the oral cavity. In addition, in our study, we used an aqueous solution as the treatment agent, which could take the most effect of 8DSS and fluorine. However, in clinical practice, whether this application procedure is feasible also remains unclear. In the future, therefore, more application procedures should be found, such as the addition of 8DSS to gels, fluorocoatings or fluoride varnishes and applied by the dentist under controlled conditions. It may also be possible to add 8DSS to fluoride toothpaste that individuals can use daily. We tested both 500 and 1000 ppm fluoride in our study because although higher levels of fluoride are widely used, it is unclear whether levels above 500 ppm show clinical benefit in adults. The sustained use of high-concentration fluoride is currently recommended [Schlueter et al., 2009], and most commercial toothpastes contain 1,000–1,100 ppm [Nagpal and Damle, 2007]. Several studies suggest that although increasing the fluoride concentration from 250 to approximately 550–600 ppm may increase its therapeutic effects [Featherstone et al., 1988], higher concentrations do not bring further benefit [Abdullah et al., 2008]. This is consistent with our results, which showed similar effects on enamel in groups treated with 500 or 1,000 ppm fluoride in the presence of 8DSS. We speculate that rapid mineral precipitation onto the surface pores of the lesion block access to the lesion interior; thus, 500 ppm fluoride may have ‘saturated’ the lesion surface under our experimental conditions, such that increasing the concentration to
Synergistic Inhibitory Effect of 8DSS and Fluoride
Caries Res 2016;50:32–39 DOI: 10.1159/000442896
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pH for the dissolution of Ca2+ and PO43–; in another, fluoride adsorbs to apatite crystal surfaces, where it replaces Ca2+ to form acid-resistant mineral fluorapatite with low solubility [ten Cate, 2013]. While the present study provides strong in vitro evidence that 8DSS and fluoride interact to reduce the demineralization of sound enamel, how this interaction occurs is unclear. One possibility is that 8DSS, which by itself already binds tightly to calcium phosphate compounds, can capture even more calcium and phosphate ions by binding to fluoride. The 8DSSmediated deposition of fluoride on the enamel surface, most likely in a form similar to CaF2, increases the formation of fluorapatite. This fluoride on the enamel surface may be released from the surface during acid attack to reduce demineralization and enhance remineralization [Liu et al., 2013]. These effects occur because Ca2+ and PO43− react with the kinks, steps and defects on the partially dissolved apatite surface, leading to the growth of existing crystallite remnants rather than the de novo formation of new crystallites, thereby ensuring that new growth conserves the original orientation of the enamel [Siddiqui et al., 2014]. 8DSS may enhance these effects by increasing the deposition of CaF2 on the enamel surface. After lesion initiation, 8DSS may also increase the availability of fluoride in the liquid contacting enamel hydroxyapatite at the surface, prim sheaths and intraprismatic enamel. Future studies should analyze whether one or more of these mechanisms explain the apparent synergistic inhibition of caries progression by 8DSS and fluoride. The present study used bovine enamel as a substitute for human enamel; the two types of enamel show similar chemical composition and physical features [Falla-Sotelo et al., 2005]. Bovine enamel is easier to obtain than human enamel and it presents a larger surface area with more uniform enamel thickness [Lippert and Lynch, 2014]. At the same time, bovine enamel contains a larger amount of interprismatic substances than human enamel; these substances in bovine enamel have ‘fibril-like’ structures. This makes bovine enamel more porous and translates into approximately 40% faster demineralization than with human enamel [Lippert and Lynch, 2014]. We speculate that when 8DSS arrives at the surface of bovine enamel, it diffuses via these pores into the inner enamel, where it creates a barrier that reduces the dissolution of calcium and phosphate ions from the enamel. Future studies should verify whether 8DSS can penetrate the pores of normal human enamel and exert anticaries effects at the peripheries of prisms that are not in contact with the enamel surface.
1,000 ppm did not cause additional inhibition. Whatever the explanation for this lack of additional effect, our results suggest that higher fluoride concentrations in the presence of 8DSS do not necessarily reduce demineralization further. This possibility may be particularly relevant for the dental care of children and adolescents, in whom fluoride may significantly prevent caries only at concentrations of 1,000 ppm and higher, with preventive effects increasing with concentration [Walsh et al, 2010]. Using such high concentrations increases the risk of fluorosis in young individuals. It is possible that adding 8DSS to a low-fluoride toothpaste may lead to a similar anticaries efficacy as a high-fluoride conventional toothpaste containing 1,000– 1,100 ppm fluoride. This may help to reduce the risk of dental fluorosis in young people. In conclusion, the present study presents strong evidence for two findings relevant for clinical practice: 8DSS alone can inhibit the demineralization of sound enamel, and its combined use with NaF renders fluoride even more effective at inhibiting demineralization. As a result of this synergistic interaction, it may be helpful to use lower fluoride concentrations for caries prevention.
Acknowledgments The authors are grateful for the support received from the National Natural Science Foundation of China (No. 81470734), the New Century Excellent Talents University Support Program and the Key Technology Program of Sichuan Province (No. 2014SZ0024). The authors also thank the Crest Research Laboratory of Procter & Gamble Technology (Beijing) Co. Ltd. for help with TMR. The funders had no role in the study design, data collection or analysis, preparation of the manuscript or the decision to publish it.
Author Contributions Linglin Zhang, Wenyuan Shi, Xuedong Zhou and Jiyao Li conceived and designed the experiments. Yang Yang and Xueping Lv contributed to the acquisition, analysis and interpretation of data. The paper was written by Yang Yang. Final approval was given by Linglin Zhang, Wenyuan Shi, Xuedong Zhou, Jiyao Li, Yang Yang and Xueping Lv.
Disclosure Statement The authors report no conflict of interest in the study design, data collection, analysis or interpretation, manuscript writing or the decision to submit the manuscript for publication.
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