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In vitro study of the effects of fluoride-releasing dental materials on remineralization in an enamel erosion model
journal of dentistry 40 (2012) 255–263
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In vitro study of the effects of fluoride-releasing dental materials on remineralization in an enamel erosion model San Ling Zhou a,1, Jun Zhou b,1, Shigeru Watanabe c, Koji Watanabe c, Ling Ying Wen a,*, Kun Xuan a,* a
Department of Pediatric Dentistry, School of Stomatology, Fourth Military Medical University, 145 West Changle Road, Xi’an 710032, China Department of Oral Histology and Pathology, School of Stomatology, The Fourth Military Medical University, 145 West Changle Road, Xi’an, Shaanxi 710032, China c Division of Pediatric Dentistry, Department of Human Development and Fostering, School of Dentistry, Meikai University, 1-1 Keyakita, Sakado, Saitama 3500283, Japan b
article info
abstract
Article history:
Objectives: This study was conducted to compare the remineralization effects of five regi-
Received 21 October 2011
mens on the loss of fluorescence intensity, surface microhardness, roughness and micro-
Received in revised form
structure of bovine enamel after remineralization. We hope that these results can provide
19 December 2011
some basis for the clinical application of these materials.
Accepted 20 December 2011
Methods: One hundred bovine incisors were prepared and divided into the following five groups, which were treated with distinct dental materials: (1) ClinproTM XT varnish (CV), (2) F-varnish (FV), (3) Tooth Mousse (TM), (4) Fuji III LC1 light-cured glass ionomer pit
Keywords:
and fissure sealant (FJ) and (5) Base Cement1 glass polyalkenoate cement (BC). Subse-
Bovine enamel
quently, they were detected using four different methods: quantitative light-induced
Fluoride
fluorescence, microhardness, surface 3D topography and scanning electron microscopy
Remineralization
(SEM). Results: The loss of fluorescence intensity of CV, BC and FJ groups showed significant decreases after remineralization ( p < 0.05). The microhardness values of the BC group were significantly higher than those of the other groups ( p < 0.05) after 6 weeks of remineralization. The CV group’s surface roughness was significantly lower than those of the other groups after 6 weeks of remineralization ( p < 0.05). Regarding microstructure values, the FV group showed many round particles deposited in the bovine enamel after remineralization. However, the other four groups mainly showed needlelike crystals. Conclusions: Glass ionomer cement (GIC)-based dental materials can promote more remineralization of the artificial enamel lesions than can NaF-based dental materials. Resinmodified GIC materials (e.g., CV and FJ) have the potential for more controlled and sustained release of remineralized agents. The effect of TM requires further study. Crown Copyright # 2011 Published by Elsevier Ltd. All rights reserved.
* Corresponding authors. Tel.: +86 29 84776087; fax: +86 29 84776083. E-mail addresses: [email protected] (L.Y. Wen), [email protected] (K. Xuan). 1 These authors contribute equally to this work. 0300-5712/$ – see front matter . Crown Copyright # 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2011.12.016
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1.
journal of dentistry 40 (2012) 255–263
Introduction
The enamel erosion of human teeth can be caused by caries, tooth wear and congenital dental anomalies, all of which result in the irreversible loss of dental hard tissues.1–3 The erosion of tooth surfaces is first seen as white spots, which are particularly vulnerable to progression from non-cavitated to cavitated lesions because of their poor chemical and physical structure and their greater porosity at the enamel surface. The primary mineral in enamel is hydroxyapatite, which is a crystalline calcium phosphate.4 Enamel erosion involves the disintegration of calcium phosphate into calcium, phosphate and hydroxyl ions.5 The most important causes of enamel erosion are the dissolution of acids derived from Streptococcus mutans as well as dietary, occupational and intrinsic sources.6,7 The usefulness of fluoride-releasing materials for enamel remineralization has been demonstrated in various models.8,9 Fluoride catalyses the diffusion of calcium and phosphate into the tooth surface, which in turn remineralizes the crystalline structures in dental cavities.10 The remineralized tooth surfaces contain fluoridated hydroxyapatite and fluorapatite, which both resist acid attack more effectively than the original teeth.11 Enamel remineralization has been studied for approximately 100 years, during which time many successful remineralizing agents have been made available. To date, different types of fluoride-releasing dental materials have appeared on the market, including fluoride rinses, dentifrices, varnishes, sealants, glass ionomer cements and polyacid modified resins (compomers). In the 21st century, new products based on casein phosphopeptide–amorphous calcium phosphate (CPP–ACP) have also appeared on the market. Fluoride-containing dental materials show clear differences in fluoride release and uptake characteristics, and may act as reservoirs to increase fluoride levels in demineralized enamel.12–14 The remineralization efficiency of fluoride-releasing dental materials is related to the fluoride content, fluoride matrices, setting mechanisms and other material components.15,16 Contemporary approaches to the treatment of enamel erosion are based on the idea of ‘‘demineralization and remineralization’’ in a microphase to retain healthy teeth.17,18 Early childhood caries (ECC) may be present in a rampant, acute or progressive manner. The treatment of primary teeth with extensive enamel demineralization is often a difficult procedure and presents a particular challenge to dentists. The desired dental materials should provide efficient, durable, and functional remineralization, which also offers a simple means of use in the management of ECC patients. Although many remineralizing regimens have long been studied, no definitive conclusions have been made. The hypothesis for this in vitro study was that five remineralizing regimens widely used in paediatric dentistry, ClinproTM XT varnish (CV), F-varnish (FV), Tooth Mousse (TM), Fuji III LC1 light-cured glass ionomer pit and fissure sealant (FJ), and Base Cement1 glass polyalkenoate cement (BC), exert different remineralizing effects on bovine enamel erosion. The purpose of this study was to examine and assess the
potential remineralization capacities of five dental materials applied in the artificial enamel erosion model using quantitative light-induced fluorescence, surface microhardness, surface 3D topography and scanning electron microscopy (SEM) methods.
2.
Materials and methods
2.1.
Specimen preparation
One hundred bovine incisors, which were preserved via freezing, were visually examined to confirm the absence of physical damage such as discoloration, surface texture, or cracks. The selected bovine teeth were fixed in 4% formaldehyde solution for 48 h. The samples were then washed and cleaned with normal saline. Cylinder-shaped specimens with 10-mm diameters and 4-mm heights were cut from the crowns of bovine teeth using a slow-speed, water-cooled drill. The specimens were ground and polished using 400-, 600-, and 800-grit silicon carbide abrasive paper lubricated with water to flatten the outer enamel surface. Each specimen was coated with a clear nail varnish, leaving four enamel windows with approximately 2 mm 2 mm exposed in the centre.
2.2.
Preparation of the enamel erosion model
The specimens were placed into a demineralizing solution in an incubator at 37 8C, with 5% CO2 for 48 h. The demineralizing solution contained 0.1 mol/L of lactic acid and 6% carboxy methylcellulose, which was saturated with 50% hydroxyapatite and adjusted to pH 5 using NaOH. After demineralization, the specimens were washed carefully with deionized water and dried at room temperature.
2.3.
Remineralization
One hundred bovine incisors were prepared and divided into five groups, which were treated with the following dental materials: (1) ClinproTM XT varnish (CV, Dental, USA), (2) Fvarnish (FV, Oriental Pharmaceutical, Japan), (3) Tooth Mousse (TM, GC Dental, Japan), (4) Fuji III LC1 light-cured glass ionomer pit and fissure sealant (FJ, GC Dental, Japan) and (5) Base Cement1 glass polyalkenoate cement (BC, Shofu Dental, Japan). For each specimen, a control window was painted with nail varnish after demineralization, whilst the other three windows were covered with the corresponding dental material to remineralize the eroded enamel. The enamel of the three windows was remineralized for 2 weeks, 4 weeks, and 6 weeks in the incubator at 37 8C with 5% CO2. The dental material on one window was removed every 2 weeks, and then the exposed window was painted with nail varnish. Following the 6-week test period, the nail varnish was removed from all of the specimens using acetone. After remineralization, the specimens of each group were divided into four groups and tested by the following four methods: quantitative light-induced fluorescence, microhardness, surface 3D topography and scanning electron microscopy.
journal of dentistry 40 (2012) 255–263
2.4.
Quantitative light-induced fluorescence detection (QLF)
The varnish in each window of the 25 bovine enamels was removed carefully with a surgical blade. Removal was completed with cotton swabs soaked in acetone. The blocks were then washed with deionized water for 1 min. To standardize the QLF measurements, specimens were embedded in a self-cured acrylic resin, and the borders of the resin block were fitted to the windows of the QLF hand-piece to maintain a constant focus. All of the QLF images were captured with the QLFTM Clin System (Inspektor Research Systems, Amsterdam, The Netherlands) using an American Standards Association (ASA) ClassIdark room at room temperature (22 1 8C). The QLF images were then analysed using the same software package. In this study, only the values for the parameter DQ were documented. Annotation: DF (%) is the average fluorescence loss; white spot (WS) area (mm2) is the size of the white spot lesion; DQ = DF WS area (% mm2) is DF integrated by the lesion area in mm2; and reduction rate (%) = (DQ of control windows DQ after remineralization)/DQ of the control windows.
2.5.
Surface microhardness measurements (SMH)
The 25 bovine enamels were treated as described above. The hardness of the 25 specimens was determined using a microhardness tester (15JE Shanghai Optical Instrument Corp., Shanghai, China) with a diamond Vickers indenter. Three Vickers microhardness indentations were performed along the horizontal centre line with a 50-gf load for 10 s. The microhardness values for each specimen were measured in three steps: at baseline, after the induction of carious lesions (demineralization) and after remineralization. In this study, we documented the values of Vickers microhardness (VHN).
2.6.
Surface 3D topography evaluation (3D-ST)
The 25 bovine enamels were treated as described above. The enamel surface was imaged using NANOVEA PS 50, and three height profiles were measured on the surface. The field scanning (in mm) was set at 1.5 1.5 for 3 min. The measurement range of the optical pen was 400 mm, whilst the vertical resolution was 12 nm. The surface image was plane-fit into a 3D image. Microroughness (in mm) was measured on the bovine enamel surface representing surface area roughness (Sa). In this study, we documented the values of Sa.
2.7.
Scanning electron microscopy (SEM)
The 25 bovine enamels were treated as described above. The specimens were mounted and sputter-coated with platinumgold using a Hammer VI cathodic evaporator (Hitachi, E-1045 Ion Sputter, Japan). They were then examined and photographed with a Hitachi S-4800 scanning electron microscope, operating at 15 kV.
2.8.
Statistical analysis
SPSS software (version 11.0) was used to perform all statistical analyses at a standard p value of 0.05. The normality of the
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data from the QLF, SMH and 3D-ST experiments was confirmed using normal probability plots and the SW test. Mean values with standard deviation bars of the data were plotted against the remineralization time (0–6 weeks). The remineralization effect analysis of each treatment group was performed independently, with the remineralization time (in weeks) as the dependent variable and DQ, VHN and 3D-ST values as the independent variables. As a significant interaction was confirmed between the remineralization time and the related values at the corresponding measuring points, multiple comparisons of the treatment groups were conducted to determine differences using a one-way ANOVA test and Tukey’s HSD.
3.
Results
3.1. (QLF)
Quantitative light-induced fluorescence detection
QLF images of the specimens with enamel erosion after remineralization with the dental materials are shown in Fig. 1. As expected, specimens in all of the treatment groups showed increases in enamel fluorescence (DQ values) with remineralization time. The relationships between the mean DQ values with standard deviation bars and remineralization times are shown in Fig. 1. Only in the group receiving TM treatment was a significant difference not observed in the DQ values before or after remineralization. The analysis found that the reduction rate of the mean DQ values in the enamel erosion models treated with FV was significantly higher than those in the enamel erosion models treated with CV and FJ after 2 weeks of remineralization ( p < 0.05). Moreover, the reduction rate in the BC treatment group was significantly higher than those in the other groups after 4 weeks of remineralization ( p < 0.05). After 6 weeks of remineralization, the reduction rate in the FV group was significantly lower than those in the other groups ( p < 0.05). There were no significant differences in the other three groups ( p > 0.05).
3.2.
Surface micro hardness (SMH)
The mean (SD) SMH values of the surfaces at different time points are presented in Table 1. The mean SMH value of the 25 eroded enamel specimens was 146.9. There were significant increases in the SMH values when the eroded enamel specimens were treated with the dental materials in our study ( p < 0.05). The SMH values of all treatment groups demonstrated increases with changes in remineralization time. The SMH values in the FV group were significantly higher than those in the other groups ( p < 0.05), whilst the SMH values in the CV group were significantly lower than those in the other groups ( p < 0.05) after 2 weeks of remineralization. SMH values in the BC group were significantly higher than those in the other groups after 4 weeks of remineralization ( p < 0.05), although there were no significant differences between the FV and FJ groups ( p > 0.05) or between the CV and TM groups ( p > 0.05) after 4 weeks of remineralization. The SMH values in the BC group were still significantly higher than those in the other groups ( p < 0.05) after 6 weeks of
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Fig. 1 – Quantitative light-induced fluorescence detection of the bovine enamel at different times (0 week, 2 weeks, 4 weeks and 6 weeks). The top row images are the bovine enamel discs of the five groups after in vitro experiments measured using QLF before fluorescence excitation, with the squares representing the test areas. The second row shows the same discs after fluorescence excitation, with the rings representing the test areas. The subtraction images were generated with a DF threshold of S5%, colouring only those points with a loss in fluorescence intensity exceeding 5%. The pseudo-colour in the squares and the bright colour areas in the rings represent the significant areas of demineralization. The bar chart below represents the relationships between DQ values and remineralization.
remineralization. However, there were no significant differences between the CV and FJ groups ( p > 0.05) after 6 weeks of remineralization.
3.3.
Surface 3D topography (3D-ST)
The mean (SD) surface area roughness measurements at different time points and pictures of surface 3D topography are shown in Fig. 2. The surface area roughness of the FV, CV and TM groups declined gradually with changes in the time points ( p < 0.05). Interestingly, the surface area roughness of the FJ and BC groups decreased significantly after 2 weeks of remineralization; however, it increased significantly after
both 4 weeks and 6 weeks of remineralization ( p < 0.05). The surface area roughness of the FV group was significantly lower than those in the other groups after both 2 weeks and 4 weeks of remineralization ( p < 0.05). However, the roughness in the CV group was significantly lower than those in the other groups after 6 weeks of remineralization ( p < 0.05). The surface area roughness of both CV and TM groups had no significant differences after 2 weeks of remineralization ( p > 0.05), whilst that of the FJ and BC groups also had no significant differences after 2 weeks of remineralization ( p > 0.05). The bovine enamel surface became less rough after remineralization, as shown in Fig. 2. The CV group displayed more improvement than the other groups.
Table 1 – The mean W SD of the VHN. Time 0 2 4 6
week weeks weeks weeks
Baseline
Group I (CV)
Group II (TM)
Group III (FV)
Group IV (FJ)
Group V (BC)
329.2 1.17
145.04 6.19 185.48 1.44 238.78 2.76 295.52 1.78
145.26 14.82 209.50 1.54 241.58 1.36 268.26 1.55
149.14 3.73 237.22 1.32 254.50 5.41 273.46 2.30
147.70 13.13 212.52 1.51 257.24 3.32 297.48 1.47
147.48 7.19 220.54 1.49 272.54 2.03 303.10 2.96
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Fig. 2 – Surface 3D topography measurements of the bovine enamel at different remineralization times (0 week and 6 weeks). The images at 6 weeks showed a less rough structure than those at 0 week. The CV, TM, FV and BC groups all showed that the distance between the peaks and valleys decreased after demineralization. However, that of the FJ group showed an increased tendency. The table below represents the mean W SD of the Sa values at various experimental stages.
3.4.
Scanning electron microscopy (SEM)
Before demineralization, the enamel surfaces were smooth and devoid of structure. After demineralization, the control windows of the five groups demonstrated different appearances, although they mostly showed a typical fish-scale appearance with the enamel prisms. Moreover, the enamel rods were preferentially etched at the prism cores, as shown in Fig. 3. There were some minerals deposited in the etched enamel rods after 2 weeks of remineralization. The honeycombed-patterned surface, which was visible through the bovine enamel, appeared to have been filled in and partially covered over. Furthermore, the mineral deposition in each group increased gradually with changes in time points. The FV group showed many round particles deposited in the bovine enamel after remineralization. However, mainly needle-like crystals were deposited in the bovine enamel in the other four groups.
4.
Discussion
In the present study, an in vitro model was used to compare the remineralizing efficacy of five regimens on initial caries lesions in bovine enamel. Bovine enamel was used as a substitute for human enamel. Some studies have stated that both the crystallite orientation and weight percentage of the calcium content of bovine enamel match those of human enamel, with the latter showing a similar gradual decrease from the surface to the dentine–enamel junction.19 Several studies have confirmed the preventive effects of topical fluoride application using different methods.20–22 In our study, we compared five dental materials with regard to loss of fluorescence intensity, surface microhardness, roughness and microstructure of the teeth after remineralization. We hope that these results can provide some basis for the clinical application of these materials.
QLF is a visible light system that can quantitatively detect the degree of demineralization and then monitor its progression or regression in a non-destructive way.23 QLF uses the principle of fluorescence to reveal dental erosion. Mineral loss measurement is based on an increase in the fluorescence scattering coefficient due to demineralization. It has been shown that the difference between sound and deficient enamel is significantly greater in fluorescence images than in white light images.24 Thus, the demineralized areas appear as dark spots on the QLF images; furthermore, QLFTM software is used to quantify lesion size, depth and volume from the image of the tooth.25 Previous studies have tested the QLF method in in vitro experiments with transverse microradiography (TMR), using scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDS) and chemical analysis: these studies reported a good correlation when the subsurface loss of mineralization was measured.26,27 Moreover, the assessment conditions of QLF detection should be standardized to eliminate some interference factors such as environmental conditions, operational motion, degree of dehydration and enamel thickness of the specimens.26,28 In the present study, all five dental materials exhibited appropriate remineralizing abilities, which were validated by increases in the DQ values of enamel fluorescence with regard to remineralization time. However, TM demonstrated the lowest values. This finding was likely a result of the key components of TM presenting as amorphous calcium phosphate and casein phosphopeptides. Although TM contains a minute amount of calcium and phosphate, its ability to remineralize cannot compare with that of fluoride-releasing dental materials. SMH provides a relatively simple, non-destructive and rapid method for studies assessing demineralization and remineralization. Therefore, in this study, microhardness values for each specimen were measured in three steps: at baseline, after demineralization and after remineralization.21
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Fig. 3 – Scanning electron microscopy examinations of the bovine enamel at different remineralization times (0 week, 2 weeks, 4 weeks and 6 weeks). The bovine enamel surfaces in all five groups changed progressively from porous flake-like
journal of dentistry 40 (2012) 255–263
The initial mean VHN values for the five groups ranged from 310.1 to 334.7 for enamel. These values fell well within the range of 200–350 that was reported by Wang et al.29 for bovine teeth. After demineralization, the VHN values apparently decreased, and after remineralization, they increased significantly compared to the control windows. However, none of the VHN values reached baseline levels. The most effective regimen was BC. CV was the least effective regimen after 2 weeks of remineralization, although its effect increased gradually with time. Microhardness assessment requires a flat and polished surface to enable accurate measurements; thus, the area subjected to erosion was not an ideal enamel surface. Therefore, we encountered difficulties in accurately detecting the erosion enamel surface hardness, which may have affected our results. 3D Non-Contact Optical Profiles are designed with leading edge white light axial chromatism optical pens to obtain nanometer resolutions for surface inspection as well as highspeed 3D metrology and more precise thickness mapping on a wider range of geometries and materials than any other profilometer. However, only a few studies to date have described the use of optical profilometry, laser scanning microscopy or white light microscopy for investigating enamel erosion.30–32 CV is the most effective regimen for 3D-ST. The effects of FV are better displayed over a short period of time, although they decrease in the long term. We found that the surface roughness of the FJ and BC groups declined after 2 weeks of remineralization but increased after 4 weeks and 6 weeks of remineralization ( p < 0.05). The deposition of many minerals into the porous zones over a short time in the FJ and BC groups likely caused the bovine enamel to flatten, whilst the later deposition of some large components on the flat bovine enamel caused the surface roughness to increase. SEM was one of the first techniques used to measure the in vitro resorption of dental hard tissues, and it is still widely used today. SEM scatters electrons at the sample surface, and the resulting signal that is received provides information about surface topography and composition.33 The results of the SEM observation demonstrated that spherical CaF2 was present only in the FV group, which had apparently precipitated on the enamel surface. The regimens altered the crystal nanostructure from a honeycombed pattern to a needle-like array of fluoridated hydroxyapatite nanocrystals in the other four groups, which was in accordance with Y. Fan’s finding.34 Many studies have demonstrated that calcium fluoride is a major reaction product after topical treatment with high fluoride concentrations.20,35 Spherical CaF2 precipitation can promote fluoride release and remineralization of enamel hydroxyapatite. However, other researchers have found that under controlled conditions, fluoride can be incorporated directly into the fluoridated hydroxyapatite grown on an etched enamel surface without the formation of spherical CaF2 particles.34 Thus, in our study, the results of the SEM observation manifested both phenomena. CV and FV are different fluoride varnish products. Fluoride varnish is composed of a concentrated dose of fluoride as a salt
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or saline preparation in a fast-drying alcohol- and resin-based solution.36 Fluoride varnish has been reported to deposit large amounts of fluoride in the enamel via the formation of CaF2like materials and fluorapatite, which are surrounded by pellicle protein and phosphate that prolong their retention on the enamel surface.37,38 The present study confirmed that both fluoride varnishes caused reductions in the enamel erosion volume when compared to the controls. However, our results showed that CV was more effective than FV in the remineralization of artificial enamel lesions underneath the materials. The efficacy of fluoride varnish depends on the concentration, type of fluoride, and dispensing method.39,40 FV is one type of traditional 5% NaF varnish, whereas CV is durable and has a fluoride-releasing coating, which is based on a liquid/paste glass ionomer technology. Moreover, CV also contains calcium and phosphate, which are necessary and serve as useful supplements in the remineralization process. The main component of FJ and BC is a glass ionomer material as well. BC is a type of conventional glass ionomer cement (GIC) composed of calcium and strontium aluminosilicate glass powder (base) in combination with a water-soluble polymer (acid). FJ is a type of fluoride-releasing pit and fissure sealant comprised of resin-modified glass ionomer cements. Fluoride is released from GIC as ionic F, ionic AlF6 and fluorophosphate compounds.41 Some studies have also verified that glass ionomer-based materials display a great potential for fluoride release in dental caries prevention and remineralization of lesions.42–44 GIC usually has higher fluoride content than does fluoride varnish. Therefore, in our study, the remineralizing ability of FV was significantly lower than that of the GIC materials (e.g., CV, FJ and BC). Furthermore, there were no significant differences amongst the three GIC materials. This finding also supported previous in vitro studies, which had found that the amount of constant fluoride release did not differ much between conventional GIC and resin-modified GIC.45,46 The pattern of fluoride release from conventional GIC is characterized by an initial rapid release, which is followed by a rapid reduction in the rate of release of fluoride after a short period of time.47 Our findings revealed that the three GICbased materials achieved better results than did FV after 6 weeks of remineralization. The reasons for this result were likely as follows: (1) the FV’s efficacious component was in the form of NaF; (2) the lower the content of the GIC-based filler, the more flowable is the fluoride-releasing dental material; and (3) the fluoride-containing dental materials had different release mechanisms. According to the results of our study, FJ and CV exhibited a controlled pattern of fluoride release during remineralization. TM’s component is CPP–ACP. CPP–ACP nanocomplexes are casein-derived peptides in which ACP is stabilized by CPP: these nanocomplexes act as calcium and phosphate reservoirs when incorporated into the dental plaque and on the tooth surface.48 Some studies49,50 have shown that CPP–ACP can reduce demineralization and promote remineralization of
apatite to flat apatite (5000T magnification). The CV, TM, FJ and BC groups showed many tightly bound fluoride apatite deposits on the bovine enamel surface. However, the FV group showed many loosely bound fluoride or CaF2 deposits on the bovine enamel surface (50,000T magnification). Stars indicate the pores from demineralization. The arrow represents the spherical CaF2 crystal. The triangle indicates tightly bound fluorapatite.
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enamel lesions. However, some studies have concluded that the effect of CPP–ACP cannot compare with that of fluoride agents. For example, Lata et al.21 compared the remineralizing potential of fluoride varnish, CPP–ACP, and a combination of fluoride and CPP–ACP on early enamel lesions and concluded that each was effective; however, CPP–ACP had less significant effects than did fluoride varnish in remineralizing early enamel caries at the surface level. In addition, the combination of fluoride varnish and CPP–ACP did not provide any additive remineralization potential when compared to fluoride varnish alone at the surface level. Based on the results of the four detection methods, we found that TM did not have a significant ability to remineralize compared with the fluoridereleasing dental materials. Our findings are in agreement with those of most previous studies.
5.
Conclusion
GIC-based dental materials can promote more remineralization of artificial enamel lesions than can NaF-based dental materials. The resin-modified GIC materials (e.g., CV and FJ) have the potential for more controlled and sustained release of remineralizing agents. The effect of TM requires further studies. We presume that multiple testing methods may better detect the effects of demineralization and remineralization.
Acknowledgment This study was supported by grants from the Nature Science Foundation of China (81072273 and 81171001).
references
1. Ranjitkar S, Rodriguez JM, Kaidonis JA, Richards LC, Townsend GC, Bartlett DW. The effect of casein phosphopeptide–amorphous calcium phosphate on erosive enamel and dentine wear by toothbrush abrasion. Journal of Dentistry 2009;37:250–4. 2. Rees J, Loyn T, Chadwick B. Pronamel and tooth mousse: an initial assessment of erosion prevention in vitro. Journal of Dentistry 2007;35:355–7. 3. Crombie F, Manton D, Kilpatrick N. Aetiology of molarincisor hypomineralization: a critical review. International Journal of Paediatric Dentistry 2009;19:73–83. 4. Palmer LC, Newcomb CJ, Kaltz SR, Spoerke ED, Stupp SI. Biomimetic systems for hydroxyapatite mineralization inspired by bone and enamel. Chemical Reviews 2008;108:4754–83. 5. Hicks J, Garcia-Godoy F, Flaitz C. Biological factors in dental caries enamel structure and the caries process in the dynamic process of demineralization and remineralization. Journal of Clinical Pediatric Dentistry 2004;28:119–24. 6. Cross SE, Kreth J, Wali RP, Sullivan R, Shi W, Gimzewski JK. Evaluation of bacteria-induced enamel demineralization using optical profilometry. Dental Materials 2009;25: 1517–26. 7. Lussi A, Hellwig E, Zero D, Jaeggi T. Erosive tooth wear: diagnosis, risk factors and prevention. American Journal of Dentistry 2006;19:319–25.
8. Burke FM, Ray NJ, McConnell RJ. Fluoride-containing restorative materials. International Dental Journal 2006; 56:33–43. 9. Rodrigues E, Delbem AC, Pedrini D, de Oliveira MS. PHcycling model to verify the efficacy of fluoride-releasing materials in enamel demineralization. Operative Dentistry 2008;33:658–65. 10. Robinson C, Connell S, Kirkham J, Brookes SJ, Shore RC, Smith AM. The effect of fluoride on the developing tooth. Caries Research 2004;38:268–76. 11. Robinson C. Fluoride and the caries lesion: interactions and mechanism of action. European Archives of Paediatric Dentistry 2009;10:136–40. 12. Ikemura K, Tay FR, Endo T, Pashley DH. A review of chemical-approach and ultramorphological studies on the development of fluoride-releasing dental adhesives comprising new pre-reacted glass ionomer (PRG) fillers. Dental Materials Journal 2008;27:315–39. 13. Okuyama K, Murata Y, Pereira PN, Miguez PA, Komatsu H, Sano H. Fluoride release and uptake by various dental materials after fluoride application. American Journal of Dentistry 2006;19:123–7. 14. Wiegand A, Buchalla W, Attin T. Review on fluoridereleasing restorative materials—fluoride release and uptake characteristics, antibacterial activity and influence on caries formation. Dental Materials 2007;23:343–62. 15. Casals E, Boukpessi T, McQueen CM, Eversole SL, Faller RV. Anticaries potential of commercial dentifrices as determined by fluoridation and remineralization efficiency. Journal of Contemporary Dental Practice 2007;8:1–10. 16. ten Cate JM. Current concepts on the theories of the mechanism of action of fluoride. Acta Odontologica Scandinavica 1999;57:325–9. 17. Murdoch-Kinch CA, McLean ME. Minimally invasive dentistry. Journal of the American Dental Association 2003;134:87–95. 18. Malterud MI. Minimally invasive restorative dentistry: a biomimetic approach. Practical Procedures & Aesthetic Dentistry 2006;18:409–14. 19. Turssi CP, Messias DF. Viability of using enamel and dentin from bovine origin as a substitute for human counterparts in an intraoral erosion model. Brazilian Dental Journal 2010;21:332–6. 20. Lee YE, Baek HJ, Choi YH, Jeong SH, Park YD, Song KB. Comparison of remineralization effect of three topic fluoride regimens on enamel initial carious lesions. Journal of Dentistry 2010;38:166–71. 21. Lata S, Varghese NO, Varughese JM. Remineralization potential of fluoride and amorphous calcium phosphate– casein phosphor peptide on enamel lesions: an in vitro comparative evaluation. Journal of Conservative Dentistry 2010;13:42–6. 22. Ahiropoulos V, Helvatjoglu-Antoniades M, Papadogiannis Y. In vitro fluoride uptake by bovine enamel from aesthetic restorative materials. International Journal of Paediatric Dentistry 2008;18:291–9. 23. Angmar-Ma˚nsson B, ten Bosch JJ. Quantitative light-induced fluorescence (QLF): a method for assessment of incipient caries lesions. Dentomaxillofacial Radiology 2001;30:298–307. 24. Feng Y, Yin W, Hu DY, Zhang Y, Pretty IA, Ellwood RP. Detection and prevention of early caries after fluoride dentifrice application using quantitative light-induced fluorescence in vivo. Hua Xi Kou Qiang Yi Xue Za Zhi 2008;26:607–10. 25. Taylor AM, Ellwood RP, Pretty IA, Mohan N. Quantitative stain detection in vivo using fluorescent imaging. Journal of Dentistry 2009;37:397–405. 26. Nakata K, Nikaido T, Ikeda M, Foxton RM, Tagami J. Relationship between fluorescence loss of QLF and depth of
journal of dentistry 40 (2012) 255–263
27. 28.
29.
30.
31. 32.
33.
34.
35.
36.
37. 38.
demineralization in an enamel erosion model. Dental Materials Journal 2009;28:523–9. Elton V, Cooper L, Higham SM, Pender N. Validation of enamel erosion in vitro. Journal of Dentistry 2009;37:336–41. Ando M, Stookey GK, Zero DT. Ability of quantitative lightinduced fluorescence (QLF) to assess the activity of white spot lesions during dehydration. American Journal of Dentistry 2006;19:15–8. Wang R, Hu Y, Ng C. Microstructure and interfacial fracture at the cementum–enamel junctions in equine and bovine enamel. Journal of Materials Research 2006;21:2146–55. Ren Y-F, Zhao Q, Malmstroma H, Barnes V, Xu T. Assessing fluoride treatment and resistance of dental enamel to soft drink erosion in vitro: applications of focus variation 3D scanning microscopy and stylus profilometry. Journal of Dentistry 2009;37:167–76. Mathia TG, Pawlus P, Wieczorowski M. Recent trends in surface metrology. Wear 2011;271:494–508. Belli R, Pelka M, Petschelt A, Lohbauer U. In vitro wear gap formation of self-adhesive resin cements: ACLSM evaluation. Journal of Dentistry 2009;37:984–93. Field J, Waterhouse P, German M. Quantifying and qualifying surface changes on dental hard tissues in vitro. Journal of Dentistry 2010;38:182–90. Fan Y, Sun Z, Moradian-Oldak. J. Effect of fluoride on the morphology of calcium phosphate crystals grown on acidetched human enamel. Caries Research 2009;43:132–6. Gerth HU, Dammaschke T, Scha¨fer E, Zuchner H. A three layer structure model of fluoridated enamel containing CaF2, Ca(OH)2 and FAp. Dental Materials 2007;23:1521–8. Miller EK, Vann Jr WF. The use of fluoride varnish in children: a critical review with treatment recommendations. Journal of Clinical Pediatric Dentistry 2008;32:259–64. Seppa¨ L. Fluoride varnishes in caries prevention. Medical Principles and Practice 2004;13:307–11. Weinstein P, Spiekerman C, Milgrom P. Randomized equivalence trial of intensive and semiannual applications of fluoride varnish in the primary dentition. Caries Research 2009;43:484–90.
263
39. Seppa¨ L, Po¨lla¨nen L, Hausen H. Caries-preventive effect of fluoride varnish with different fluoride concentrations. Caries Research 1994;28:64–7. 40. Marinho VC. Evidence-based effectiveness of topical fluorides. Advances in Dental Research 2008;20:3–7. 41. Billington RW, Williams JA, Pearson GJ. Ion processes in glass ionomer cements. Journal of Dentistry 2006;34: 544–55. 42. Francisconi LF, Scaffa PM, de Barros VR, Coutinho M, Francisconi PA. Glass ionomer cements and their role in the restoration of non-carious cervical lesions. Journal of Applied Oral Science 2009;17:364–9. 43. Thanjal NK, Billington RW, Shahid S, Luo J, Hill RG, Pearson GJ. Kinetics of fluoride ion release from dental restorative glass ionomer cements: the influence of ultrasound, radiant heat and glass composition. Journal of Materials Science Materials in Medicine 2010;21:589–95. 44. Luo J, Billington RW, Pearson GJ. Kinetics of fluoride release from glass components of glass ionomers. Journal of Dentistry 2009;37:495–501. 45. Rafeek RN. The effects of heat treatment on selected properties of a conventional and a resin-modified glass ionomer cement. Journal of Materials Science Materials in Medicine 2008;19:1913–20. 46. Nicholson JW, Czarnecka B. The biocompatibility of resinmodified glass-ionomer cements for dentistry. Dental Materials 2008;24:1702–8. 47. Cildir SK, Sandalli N. Fluoride release/uptake of glassionomer cements and polyacid-modified composite resins. Dental Materials Journal 2005;24:92–7. 48. Srinivasan N, Kavitha M, Loganathan SC. Comparison of the remineralization potential of CPP–ACP and CPP–ACP with 900 ppm fluoride on eroded human enamel: an in situ study. Archives of Oral Biology 2010;55:541–4. 49. Zero DT. RecaldentTM—evidence for clinical activity. Advances in Dental Research 2009;21:30–4. 50. Reynolds EC. Casein phosphopeptide–amorphous calcium phosphate: the scientific evidence. Advances in Dental Research 2009;21:25–9.
Available online at www.sciencedirect.com
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In vitro study of the effects of fluoride-releasing dental materials on remineralization in an enamel erosion model San Ling Zhou a,1, Jun Zhou b,1, Shigeru Watanabe c, Koji Watanabe c, Ling Ying Wen a,*, Kun Xuan a,* a
Department of Pediatric Dentistry, School of Stomatology, Fourth Military Medical University, 145 West Changle Road, Xi’an 710032, China Department of Oral Histology and Pathology, School of Stomatology, The Fourth Military Medical University, 145 West Changle Road, Xi’an, Shaanxi 710032, China c Division of Pediatric Dentistry, Department of Human Development and Fostering, School of Dentistry, Meikai University, 1-1 Keyakita, Sakado, Saitama 3500283, Japan b
article info
abstract
Article history:
Objectives: This study was conducted to compare the remineralization effects of five regi-
Received 21 October 2011
mens on the loss of fluorescence intensity, surface microhardness, roughness and micro-
Received in revised form
structure of bovine enamel after remineralization. We hope that these results can provide
19 December 2011
some basis for the clinical application of these materials.
Accepted 20 December 2011
Methods: One hundred bovine incisors were prepared and divided into the following five groups, which were treated with distinct dental materials: (1) ClinproTM XT varnish (CV), (2) F-varnish (FV), (3) Tooth Mousse (TM), (4) Fuji III LC1 light-cured glass ionomer pit
Keywords:
and fissure sealant (FJ) and (5) Base Cement1 glass polyalkenoate cement (BC). Subse-
Bovine enamel
quently, they were detected using four different methods: quantitative light-induced
Fluoride
fluorescence, microhardness, surface 3D topography and scanning electron microscopy
Remineralization
(SEM). Results: The loss of fluorescence intensity of CV, BC and FJ groups showed significant decreases after remineralization ( p < 0.05). The microhardness values of the BC group were significantly higher than those of the other groups ( p < 0.05) after 6 weeks of remineralization. The CV group’s surface roughness was significantly lower than those of the other groups after 6 weeks of remineralization ( p < 0.05). Regarding microstructure values, the FV group showed many round particles deposited in the bovine enamel after remineralization. However, the other four groups mainly showed needlelike crystals. Conclusions: Glass ionomer cement (GIC)-based dental materials can promote more remineralization of the artificial enamel lesions than can NaF-based dental materials. Resinmodified GIC materials (e.g., CV and FJ) have the potential for more controlled and sustained release of remineralized agents. The effect of TM requires further study. Crown Copyright # 2011 Published by Elsevier Ltd. All rights reserved.
* Corresponding authors. Tel.: +86 29 84776087; fax: +86 29 84776083. E-mail addresses: [email protected] (L.Y. Wen), [email protected] (K. Xuan). 1 These authors contribute equally to this work. 0300-5712/$ – see front matter . Crown Copyright # 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2011.12.016
256
1.
journal of dentistry 40 (2012) 255–263
Introduction
The enamel erosion of human teeth can be caused by caries, tooth wear and congenital dental anomalies, all of which result in the irreversible loss of dental hard tissues.1–3 The erosion of tooth surfaces is first seen as white spots, which are particularly vulnerable to progression from non-cavitated to cavitated lesions because of their poor chemical and physical structure and their greater porosity at the enamel surface. The primary mineral in enamel is hydroxyapatite, which is a crystalline calcium phosphate.4 Enamel erosion involves the disintegration of calcium phosphate into calcium, phosphate and hydroxyl ions.5 The most important causes of enamel erosion are the dissolution of acids derived from Streptococcus mutans as well as dietary, occupational and intrinsic sources.6,7 The usefulness of fluoride-releasing materials for enamel remineralization has been demonstrated in various models.8,9 Fluoride catalyses the diffusion of calcium and phosphate into the tooth surface, which in turn remineralizes the crystalline structures in dental cavities.10 The remineralized tooth surfaces contain fluoridated hydroxyapatite and fluorapatite, which both resist acid attack more effectively than the original teeth.11 Enamel remineralization has been studied for approximately 100 years, during which time many successful remineralizing agents have been made available. To date, different types of fluoride-releasing dental materials have appeared on the market, including fluoride rinses, dentifrices, varnishes, sealants, glass ionomer cements and polyacid modified resins (compomers). In the 21st century, new products based on casein phosphopeptide–amorphous calcium phosphate (CPP–ACP) have also appeared on the market. Fluoride-containing dental materials show clear differences in fluoride release and uptake characteristics, and may act as reservoirs to increase fluoride levels in demineralized enamel.12–14 The remineralization efficiency of fluoride-releasing dental materials is related to the fluoride content, fluoride matrices, setting mechanisms and other material components.15,16 Contemporary approaches to the treatment of enamel erosion are based on the idea of ‘‘demineralization and remineralization’’ in a microphase to retain healthy teeth.17,18 Early childhood caries (ECC) may be present in a rampant, acute or progressive manner. The treatment of primary teeth with extensive enamel demineralization is often a difficult procedure and presents a particular challenge to dentists. The desired dental materials should provide efficient, durable, and functional remineralization, which also offers a simple means of use in the management of ECC patients. Although many remineralizing regimens have long been studied, no definitive conclusions have been made. The hypothesis for this in vitro study was that five remineralizing regimens widely used in paediatric dentistry, ClinproTM XT varnish (CV), F-varnish (FV), Tooth Mousse (TM), Fuji III LC1 light-cured glass ionomer pit and fissure sealant (FJ), and Base Cement1 glass polyalkenoate cement (BC), exert different remineralizing effects on bovine enamel erosion. The purpose of this study was to examine and assess the
potential remineralization capacities of five dental materials applied in the artificial enamel erosion model using quantitative light-induced fluorescence, surface microhardness, surface 3D topography and scanning electron microscopy (SEM) methods.
2.
Materials and methods
2.1.
Specimen preparation
One hundred bovine incisors, which were preserved via freezing, were visually examined to confirm the absence of physical damage such as discoloration, surface texture, or cracks. The selected bovine teeth were fixed in 4% formaldehyde solution for 48 h. The samples were then washed and cleaned with normal saline. Cylinder-shaped specimens with 10-mm diameters and 4-mm heights were cut from the crowns of bovine teeth using a slow-speed, water-cooled drill. The specimens were ground and polished using 400-, 600-, and 800-grit silicon carbide abrasive paper lubricated with water to flatten the outer enamel surface. Each specimen was coated with a clear nail varnish, leaving four enamel windows with approximately 2 mm 2 mm exposed in the centre.
2.2.
Preparation of the enamel erosion model
The specimens were placed into a demineralizing solution in an incubator at 37 8C, with 5% CO2 for 48 h. The demineralizing solution contained 0.1 mol/L of lactic acid and 6% carboxy methylcellulose, which was saturated with 50% hydroxyapatite and adjusted to pH 5 using NaOH. After demineralization, the specimens were washed carefully with deionized water and dried at room temperature.
2.3.
Remineralization
One hundred bovine incisors were prepared and divided into five groups, which were treated with the following dental materials: (1) ClinproTM XT varnish (CV, Dental, USA), (2) Fvarnish (FV, Oriental Pharmaceutical, Japan), (3) Tooth Mousse (TM, GC Dental, Japan), (4) Fuji III LC1 light-cured glass ionomer pit and fissure sealant (FJ, GC Dental, Japan) and (5) Base Cement1 glass polyalkenoate cement (BC, Shofu Dental, Japan). For each specimen, a control window was painted with nail varnish after demineralization, whilst the other three windows were covered with the corresponding dental material to remineralize the eroded enamel. The enamel of the three windows was remineralized for 2 weeks, 4 weeks, and 6 weeks in the incubator at 37 8C with 5% CO2. The dental material on one window was removed every 2 weeks, and then the exposed window was painted with nail varnish. Following the 6-week test period, the nail varnish was removed from all of the specimens using acetone. After remineralization, the specimens of each group were divided into four groups and tested by the following four methods: quantitative light-induced fluorescence, microhardness, surface 3D topography and scanning electron microscopy.
journal of dentistry 40 (2012) 255–263
2.4.
Quantitative light-induced fluorescence detection (QLF)
The varnish in each window of the 25 bovine enamels was removed carefully with a surgical blade. Removal was completed with cotton swabs soaked in acetone. The blocks were then washed with deionized water for 1 min. To standardize the QLF measurements, specimens were embedded in a self-cured acrylic resin, and the borders of the resin block were fitted to the windows of the QLF hand-piece to maintain a constant focus. All of the QLF images were captured with the QLFTM Clin System (Inspektor Research Systems, Amsterdam, The Netherlands) using an American Standards Association (ASA) ClassIdark room at room temperature (22 1 8C). The QLF images were then analysed using the same software package. In this study, only the values for the parameter DQ were documented. Annotation: DF (%) is the average fluorescence loss; white spot (WS) area (mm2) is the size of the white spot lesion; DQ = DF WS area (% mm2) is DF integrated by the lesion area in mm2; and reduction rate (%) = (DQ of control windows DQ after remineralization)/DQ of the control windows.
2.5.
Surface microhardness measurements (SMH)
The 25 bovine enamels were treated as described above. The hardness of the 25 specimens was determined using a microhardness tester (15JE Shanghai Optical Instrument Corp., Shanghai, China) with a diamond Vickers indenter. Three Vickers microhardness indentations were performed along the horizontal centre line with a 50-gf load for 10 s. The microhardness values for each specimen were measured in three steps: at baseline, after the induction of carious lesions (demineralization) and after remineralization. In this study, we documented the values of Vickers microhardness (VHN).
2.6.
Surface 3D topography evaluation (3D-ST)
The 25 bovine enamels were treated as described above. The enamel surface was imaged using NANOVEA PS 50, and three height profiles were measured on the surface. The field scanning (in mm) was set at 1.5 1.5 for 3 min. The measurement range of the optical pen was 400 mm, whilst the vertical resolution was 12 nm. The surface image was plane-fit into a 3D image. Microroughness (in mm) was measured on the bovine enamel surface representing surface area roughness (Sa). In this study, we documented the values of Sa.
2.7.
Scanning electron microscopy (SEM)
The 25 bovine enamels were treated as described above. The specimens were mounted and sputter-coated with platinumgold using a Hammer VI cathodic evaporator (Hitachi, E-1045 Ion Sputter, Japan). They were then examined and photographed with a Hitachi S-4800 scanning electron microscope, operating at 15 kV.
2.8.
Statistical analysis
SPSS software (version 11.0) was used to perform all statistical analyses at a standard p value of 0.05. The normality of the
257
data from the QLF, SMH and 3D-ST experiments was confirmed using normal probability plots and the SW test. Mean values with standard deviation bars of the data were plotted against the remineralization time (0–6 weeks). The remineralization effect analysis of each treatment group was performed independently, with the remineralization time (in weeks) as the dependent variable and DQ, VHN and 3D-ST values as the independent variables. As a significant interaction was confirmed between the remineralization time and the related values at the corresponding measuring points, multiple comparisons of the treatment groups were conducted to determine differences using a one-way ANOVA test and Tukey’s HSD.
3.
Results
3.1. (QLF)
Quantitative light-induced fluorescence detection
QLF images of the specimens with enamel erosion after remineralization with the dental materials are shown in Fig. 1. As expected, specimens in all of the treatment groups showed increases in enamel fluorescence (DQ values) with remineralization time. The relationships between the mean DQ values with standard deviation bars and remineralization times are shown in Fig. 1. Only in the group receiving TM treatment was a significant difference not observed in the DQ values before or after remineralization. The analysis found that the reduction rate of the mean DQ values in the enamel erosion models treated with FV was significantly higher than those in the enamel erosion models treated with CV and FJ after 2 weeks of remineralization ( p < 0.05). Moreover, the reduction rate in the BC treatment group was significantly higher than those in the other groups after 4 weeks of remineralization ( p < 0.05). After 6 weeks of remineralization, the reduction rate in the FV group was significantly lower than those in the other groups ( p < 0.05). There were no significant differences in the other three groups ( p > 0.05).
3.2.
Surface micro hardness (SMH)
The mean (SD) SMH values of the surfaces at different time points are presented in Table 1. The mean SMH value of the 25 eroded enamel specimens was 146.9. There were significant increases in the SMH values when the eroded enamel specimens were treated with the dental materials in our study ( p < 0.05). The SMH values of all treatment groups demonstrated increases with changes in remineralization time. The SMH values in the FV group were significantly higher than those in the other groups ( p < 0.05), whilst the SMH values in the CV group were significantly lower than those in the other groups ( p < 0.05) after 2 weeks of remineralization. SMH values in the BC group were significantly higher than those in the other groups after 4 weeks of remineralization ( p < 0.05), although there were no significant differences between the FV and FJ groups ( p > 0.05) or between the CV and TM groups ( p > 0.05) after 4 weeks of remineralization. The SMH values in the BC group were still significantly higher than those in the other groups ( p < 0.05) after 6 weeks of
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journal of dentistry 40 (2012) 255–263
Fig. 1 – Quantitative light-induced fluorescence detection of the bovine enamel at different times (0 week, 2 weeks, 4 weeks and 6 weeks). The top row images are the bovine enamel discs of the five groups after in vitro experiments measured using QLF before fluorescence excitation, with the squares representing the test areas. The second row shows the same discs after fluorescence excitation, with the rings representing the test areas. The subtraction images were generated with a DF threshold of S5%, colouring only those points with a loss in fluorescence intensity exceeding 5%. The pseudo-colour in the squares and the bright colour areas in the rings represent the significant areas of demineralization. The bar chart below represents the relationships between DQ values and remineralization.
remineralization. However, there were no significant differences between the CV and FJ groups ( p > 0.05) after 6 weeks of remineralization.
3.3.
Surface 3D topography (3D-ST)
The mean (SD) surface area roughness measurements at different time points and pictures of surface 3D topography are shown in Fig. 2. The surface area roughness of the FV, CV and TM groups declined gradually with changes in the time points ( p < 0.05). Interestingly, the surface area roughness of the FJ and BC groups decreased significantly after 2 weeks of remineralization; however, it increased significantly after
both 4 weeks and 6 weeks of remineralization ( p < 0.05). The surface area roughness of the FV group was significantly lower than those in the other groups after both 2 weeks and 4 weeks of remineralization ( p < 0.05). However, the roughness in the CV group was significantly lower than those in the other groups after 6 weeks of remineralization ( p < 0.05). The surface area roughness of both CV and TM groups had no significant differences after 2 weeks of remineralization ( p > 0.05), whilst that of the FJ and BC groups also had no significant differences after 2 weeks of remineralization ( p > 0.05). The bovine enamel surface became less rough after remineralization, as shown in Fig. 2. The CV group displayed more improvement than the other groups.
Table 1 – The mean W SD of the VHN. Time 0 2 4 6
week weeks weeks weeks
Baseline
Group I (CV)
Group II (TM)
Group III (FV)
Group IV (FJ)
Group V (BC)
329.2 1.17
145.04 6.19 185.48 1.44 238.78 2.76 295.52 1.78
145.26 14.82 209.50 1.54 241.58 1.36 268.26 1.55
149.14 3.73 237.22 1.32 254.50 5.41 273.46 2.30
147.70 13.13 212.52 1.51 257.24 3.32 297.48 1.47
147.48 7.19 220.54 1.49 272.54 2.03 303.10 2.96
journal of dentistry 40 (2012) 255–263
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Fig. 2 – Surface 3D topography measurements of the bovine enamel at different remineralization times (0 week and 6 weeks). The images at 6 weeks showed a less rough structure than those at 0 week. The CV, TM, FV and BC groups all showed that the distance between the peaks and valleys decreased after demineralization. However, that of the FJ group showed an increased tendency. The table below represents the mean W SD of the Sa values at various experimental stages.
3.4.
Scanning electron microscopy (SEM)
Before demineralization, the enamel surfaces were smooth and devoid of structure. After demineralization, the control windows of the five groups demonstrated different appearances, although they mostly showed a typical fish-scale appearance with the enamel prisms. Moreover, the enamel rods were preferentially etched at the prism cores, as shown in Fig. 3. There were some minerals deposited in the etched enamel rods after 2 weeks of remineralization. The honeycombed-patterned surface, which was visible through the bovine enamel, appeared to have been filled in and partially covered over. Furthermore, the mineral deposition in each group increased gradually with changes in time points. The FV group showed many round particles deposited in the bovine enamel after remineralization. However, mainly needle-like crystals were deposited in the bovine enamel in the other four groups.
4.
Discussion
In the present study, an in vitro model was used to compare the remineralizing efficacy of five regimens on initial caries lesions in bovine enamel. Bovine enamel was used as a substitute for human enamel. Some studies have stated that both the crystallite orientation and weight percentage of the calcium content of bovine enamel match those of human enamel, with the latter showing a similar gradual decrease from the surface to the dentine–enamel junction.19 Several studies have confirmed the preventive effects of topical fluoride application using different methods.20–22 In our study, we compared five dental materials with regard to loss of fluorescence intensity, surface microhardness, roughness and microstructure of the teeth after remineralization. We hope that these results can provide some basis for the clinical application of these materials.
QLF is a visible light system that can quantitatively detect the degree of demineralization and then monitor its progression or regression in a non-destructive way.23 QLF uses the principle of fluorescence to reveal dental erosion. Mineral loss measurement is based on an increase in the fluorescence scattering coefficient due to demineralization. It has been shown that the difference between sound and deficient enamel is significantly greater in fluorescence images than in white light images.24 Thus, the demineralized areas appear as dark spots on the QLF images; furthermore, QLFTM software is used to quantify lesion size, depth and volume from the image of the tooth.25 Previous studies have tested the QLF method in in vitro experiments with transverse microradiography (TMR), using scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDS) and chemical analysis: these studies reported a good correlation when the subsurface loss of mineralization was measured.26,27 Moreover, the assessment conditions of QLF detection should be standardized to eliminate some interference factors such as environmental conditions, operational motion, degree of dehydration and enamel thickness of the specimens.26,28 In the present study, all five dental materials exhibited appropriate remineralizing abilities, which were validated by increases in the DQ values of enamel fluorescence with regard to remineralization time. However, TM demonstrated the lowest values. This finding was likely a result of the key components of TM presenting as amorphous calcium phosphate and casein phosphopeptides. Although TM contains a minute amount of calcium and phosphate, its ability to remineralize cannot compare with that of fluoride-releasing dental materials. SMH provides a relatively simple, non-destructive and rapid method for studies assessing demineralization and remineralization. Therefore, in this study, microhardness values for each specimen were measured in three steps: at baseline, after demineralization and after remineralization.21
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journal of dentistry 40 (2012) 255–263
Fig. 3 – Scanning electron microscopy examinations of the bovine enamel at different remineralization times (0 week, 2 weeks, 4 weeks and 6 weeks). The bovine enamel surfaces in all five groups changed progressively from porous flake-like
journal of dentistry 40 (2012) 255–263
The initial mean VHN values for the five groups ranged from 310.1 to 334.7 for enamel. These values fell well within the range of 200–350 that was reported by Wang et al.29 for bovine teeth. After demineralization, the VHN values apparently decreased, and after remineralization, they increased significantly compared to the control windows. However, none of the VHN values reached baseline levels. The most effective regimen was BC. CV was the least effective regimen after 2 weeks of remineralization, although its effect increased gradually with time. Microhardness assessment requires a flat and polished surface to enable accurate measurements; thus, the area subjected to erosion was not an ideal enamel surface. Therefore, we encountered difficulties in accurately detecting the erosion enamel surface hardness, which may have affected our results. 3D Non-Contact Optical Profiles are designed with leading edge white light axial chromatism optical pens to obtain nanometer resolutions for surface inspection as well as highspeed 3D metrology and more precise thickness mapping on a wider range of geometries and materials than any other profilometer. However, only a few studies to date have described the use of optical profilometry, laser scanning microscopy or white light microscopy for investigating enamel erosion.30–32 CV is the most effective regimen for 3D-ST. The effects of FV are better displayed over a short period of time, although they decrease in the long term. We found that the surface roughness of the FJ and BC groups declined after 2 weeks of remineralization but increased after 4 weeks and 6 weeks of remineralization ( p < 0.05). The deposition of many minerals into the porous zones over a short time in the FJ and BC groups likely caused the bovine enamel to flatten, whilst the later deposition of some large components on the flat bovine enamel caused the surface roughness to increase. SEM was one of the first techniques used to measure the in vitro resorption of dental hard tissues, and it is still widely used today. SEM scatters electrons at the sample surface, and the resulting signal that is received provides information about surface topography and composition.33 The results of the SEM observation demonstrated that spherical CaF2 was present only in the FV group, which had apparently precipitated on the enamel surface. The regimens altered the crystal nanostructure from a honeycombed pattern to a needle-like array of fluoridated hydroxyapatite nanocrystals in the other four groups, which was in accordance with Y. Fan’s finding.34 Many studies have demonstrated that calcium fluoride is a major reaction product after topical treatment with high fluoride concentrations.20,35 Spherical CaF2 precipitation can promote fluoride release and remineralization of enamel hydroxyapatite. However, other researchers have found that under controlled conditions, fluoride can be incorporated directly into the fluoridated hydroxyapatite grown on an etched enamel surface without the formation of spherical CaF2 particles.34 Thus, in our study, the results of the SEM observation manifested both phenomena. CV and FV are different fluoride varnish products. Fluoride varnish is composed of a concentrated dose of fluoride as a salt
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or saline preparation in a fast-drying alcohol- and resin-based solution.36 Fluoride varnish has been reported to deposit large amounts of fluoride in the enamel via the formation of CaF2like materials and fluorapatite, which are surrounded by pellicle protein and phosphate that prolong their retention on the enamel surface.37,38 The present study confirmed that both fluoride varnishes caused reductions in the enamel erosion volume when compared to the controls. However, our results showed that CV was more effective than FV in the remineralization of artificial enamel lesions underneath the materials. The efficacy of fluoride varnish depends on the concentration, type of fluoride, and dispensing method.39,40 FV is one type of traditional 5% NaF varnish, whereas CV is durable and has a fluoride-releasing coating, which is based on a liquid/paste glass ionomer technology. Moreover, CV also contains calcium and phosphate, which are necessary and serve as useful supplements in the remineralization process. The main component of FJ and BC is a glass ionomer material as well. BC is a type of conventional glass ionomer cement (GIC) composed of calcium and strontium aluminosilicate glass powder (base) in combination with a water-soluble polymer (acid). FJ is a type of fluoride-releasing pit and fissure sealant comprised of resin-modified glass ionomer cements. Fluoride is released from GIC as ionic F, ionic AlF6 and fluorophosphate compounds.41 Some studies have also verified that glass ionomer-based materials display a great potential for fluoride release in dental caries prevention and remineralization of lesions.42–44 GIC usually has higher fluoride content than does fluoride varnish. Therefore, in our study, the remineralizing ability of FV was significantly lower than that of the GIC materials (e.g., CV, FJ and BC). Furthermore, there were no significant differences amongst the three GIC materials. This finding also supported previous in vitro studies, which had found that the amount of constant fluoride release did not differ much between conventional GIC and resin-modified GIC.45,46 The pattern of fluoride release from conventional GIC is characterized by an initial rapid release, which is followed by a rapid reduction in the rate of release of fluoride after a short period of time.47 Our findings revealed that the three GICbased materials achieved better results than did FV after 6 weeks of remineralization. The reasons for this result were likely as follows: (1) the FV’s efficacious component was in the form of NaF; (2) the lower the content of the GIC-based filler, the more flowable is the fluoride-releasing dental material; and (3) the fluoride-containing dental materials had different release mechanisms. According to the results of our study, FJ and CV exhibited a controlled pattern of fluoride release during remineralization. TM’s component is CPP–ACP. CPP–ACP nanocomplexes are casein-derived peptides in which ACP is stabilized by CPP: these nanocomplexes act as calcium and phosphate reservoirs when incorporated into the dental plaque and on the tooth surface.48 Some studies49,50 have shown that CPP–ACP can reduce demineralization and promote remineralization of
apatite to flat apatite (5000T magnification). The CV, TM, FJ and BC groups showed many tightly bound fluoride apatite deposits on the bovine enamel surface. However, the FV group showed many loosely bound fluoride or CaF2 deposits on the bovine enamel surface (50,000T magnification). Stars indicate the pores from demineralization. The arrow represents the spherical CaF2 crystal. The triangle indicates tightly bound fluorapatite.
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enamel lesions. However, some studies have concluded that the effect of CPP–ACP cannot compare with that of fluoride agents. For example, Lata et al.21 compared the remineralizing potential of fluoride varnish, CPP–ACP, and a combination of fluoride and CPP–ACP on early enamel lesions and concluded that each was effective; however, CPP–ACP had less significant effects than did fluoride varnish in remineralizing early enamel caries at the surface level. In addition, the combination of fluoride varnish and CPP–ACP did not provide any additive remineralization potential when compared to fluoride varnish alone at the surface level. Based on the results of the four detection methods, we found that TM did not have a significant ability to remineralize compared with the fluoridereleasing dental materials. Our findings are in agreement with those of most previous studies.
5.
Conclusion
GIC-based dental materials can promote more remineralization of artificial enamel lesions than can NaF-based dental materials. The resin-modified GIC materials (e.g., CV and FJ) have the potential for more controlled and sustained release of remineralizing agents. The effect of TM requires further studies. We presume that multiple testing methods may better detect the effects of demineralization and remineralization.
Acknowledgment This study was supported by grants from the Nature Science Foundation of China (81072273 and 81171001).
references
1. Ranjitkar S, Rodriguez JM, Kaidonis JA, Richards LC, Townsend GC, Bartlett DW. The effect of casein phosphopeptide–amorphous calcium phosphate on erosive enamel and dentine wear by toothbrush abrasion. Journal of Dentistry 2009;37:250–4. 2. Rees J, Loyn T, Chadwick B. Pronamel and tooth mousse: an initial assessment of erosion prevention in vitro. Journal of Dentistry 2007;35:355–7. 3. Crombie F, Manton D, Kilpatrick N. Aetiology of molarincisor hypomineralization: a critical review. International Journal of Paediatric Dentistry 2009;19:73–83. 4. Palmer LC, Newcomb CJ, Kaltz SR, Spoerke ED, Stupp SI. Biomimetic systems for hydroxyapatite mineralization inspired by bone and enamel. Chemical Reviews 2008;108:4754–83. 5. Hicks J, Garcia-Godoy F, Flaitz C. Biological factors in dental caries enamel structure and the caries process in the dynamic process of demineralization and remineralization. Journal of Clinical Pediatric Dentistry 2004;28:119–24. 6. Cross SE, Kreth J, Wali RP, Sullivan R, Shi W, Gimzewski JK. Evaluation of bacteria-induced enamel demineralization using optical profilometry. Dental Materials 2009;25: 1517–26. 7. Lussi A, Hellwig E, Zero D, Jaeggi T. Erosive tooth wear: diagnosis, risk factors and prevention. American Journal of Dentistry 2006;19:319–25.
8. Burke FM, Ray NJ, McConnell RJ. Fluoride-containing restorative materials. International Dental Journal 2006; 56:33–43. 9. Rodrigues E, Delbem AC, Pedrini D, de Oliveira MS. PHcycling model to verify the efficacy of fluoride-releasing materials in enamel demineralization. Operative Dentistry 2008;33:658–65. 10. Robinson C, Connell S, Kirkham J, Brookes SJ, Shore RC, Smith AM. The effect of fluoride on the developing tooth. Caries Research 2004;38:268–76. 11. Robinson C. Fluoride and the caries lesion: interactions and mechanism of action. European Archives of Paediatric Dentistry 2009;10:136–40. 12. Ikemura K, Tay FR, Endo T, Pashley DH. A review of chemical-approach and ultramorphological studies on the development of fluoride-releasing dental adhesives comprising new pre-reacted glass ionomer (PRG) fillers. Dental Materials Journal 2008;27:315–39. 13. Okuyama K, Murata Y, Pereira PN, Miguez PA, Komatsu H, Sano H. Fluoride release and uptake by various dental materials after fluoride application. American Journal of Dentistry 2006;19:123–7. 14. Wiegand A, Buchalla W, Attin T. Review on fluoridereleasing restorative materials—fluoride release and uptake characteristics, antibacterial activity and influence on caries formation. Dental Materials 2007;23:343–62. 15. Casals E, Boukpessi T, McQueen CM, Eversole SL, Faller RV. Anticaries potential of commercial dentifrices as determined by fluoridation and remineralization efficiency. Journal of Contemporary Dental Practice 2007;8:1–10. 16. ten Cate JM. Current concepts on the theories of the mechanism of action of fluoride. Acta Odontologica Scandinavica 1999;57:325–9. 17. Murdoch-Kinch CA, McLean ME. Minimally invasive dentistry. Journal of the American Dental Association 2003;134:87–95. 18. Malterud MI. Minimally invasive restorative dentistry: a biomimetic approach. Practical Procedures & Aesthetic Dentistry 2006;18:409–14. 19. Turssi CP, Messias DF. Viability of using enamel and dentin from bovine origin as a substitute for human counterparts in an intraoral erosion model. Brazilian Dental Journal 2010;21:332–6. 20. Lee YE, Baek HJ, Choi YH, Jeong SH, Park YD, Song KB. Comparison of remineralization effect of three topic fluoride regimens on enamel initial carious lesions. Journal of Dentistry 2010;38:166–71. 21. Lata S, Varghese NO, Varughese JM. Remineralization potential of fluoride and amorphous calcium phosphate– casein phosphor peptide on enamel lesions: an in vitro comparative evaluation. Journal of Conservative Dentistry 2010;13:42–6. 22. Ahiropoulos V, Helvatjoglu-Antoniades M, Papadogiannis Y. In vitro fluoride uptake by bovine enamel from aesthetic restorative materials. International Journal of Paediatric Dentistry 2008;18:291–9. 23. Angmar-Ma˚nsson B, ten Bosch JJ. Quantitative light-induced fluorescence (QLF): a method for assessment of incipient caries lesions. Dentomaxillofacial Radiology 2001;30:298–307. 24. Feng Y, Yin W, Hu DY, Zhang Y, Pretty IA, Ellwood RP. Detection and prevention of early caries after fluoride dentifrice application using quantitative light-induced fluorescence in vivo. Hua Xi Kou Qiang Yi Xue Za Zhi 2008;26:607–10. 25. Taylor AM, Ellwood RP, Pretty IA, Mohan N. Quantitative stain detection in vivo using fluorescent imaging. Journal of Dentistry 2009;37:397–405. 26. Nakata K, Nikaido T, Ikeda M, Foxton RM, Tagami J. Relationship between fluorescence loss of QLF and depth of
journal of dentistry 40 (2012) 255–263
27. 28.
29.
30.
31. 32.
33.
34.
35.
36.
37. 38.
demineralization in an enamel erosion model. Dental Materials Journal 2009;28:523–9. Elton V, Cooper L, Higham SM, Pender N. Validation of enamel erosion in vitro. Journal of Dentistry 2009;37:336–41. Ando M, Stookey GK, Zero DT. Ability of quantitative lightinduced fluorescence (QLF) to assess the activity of white spot lesions during dehydration. American Journal of Dentistry 2006;19:15–8. Wang R, Hu Y, Ng C. Microstructure and interfacial fracture at the cementum–enamel junctions in equine and bovine enamel. Journal of Materials Research 2006;21:2146–55. Ren Y-F, Zhao Q, Malmstroma H, Barnes V, Xu T. Assessing fluoride treatment and resistance of dental enamel to soft drink erosion in vitro: applications of focus variation 3D scanning microscopy and stylus profilometry. Journal of Dentistry 2009;37:167–76. Mathia TG, Pawlus P, Wieczorowski M. Recent trends in surface metrology. Wear 2011;271:494–508. Belli R, Pelka M, Petschelt A, Lohbauer U. In vitro wear gap formation of self-adhesive resin cements: ACLSM evaluation. Journal of Dentistry 2009;37:984–93. Field J, Waterhouse P, German M. Quantifying and qualifying surface changes on dental hard tissues in vitro. Journal of Dentistry 2010;38:182–90. Fan Y, Sun Z, Moradian-Oldak. J. Effect of fluoride on the morphology of calcium phosphate crystals grown on acidetched human enamel. Caries Research 2009;43:132–6. Gerth HU, Dammaschke T, Scha¨fer E, Zuchner H. A three layer structure model of fluoridated enamel containing CaF2, Ca(OH)2 and FAp. Dental Materials 2007;23:1521–8. Miller EK, Vann Jr WF. The use of fluoride varnish in children: a critical review with treatment recommendations. Journal of Clinical Pediatric Dentistry 2008;32:259–64. Seppa¨ L. Fluoride varnishes in caries prevention. Medical Principles and Practice 2004;13:307–11. Weinstein P, Spiekerman C, Milgrom P. Randomized equivalence trial of intensive and semiannual applications of fluoride varnish in the primary dentition. Caries Research 2009;43:484–90.
263
39. Seppa¨ L, Po¨lla¨nen L, Hausen H. Caries-preventive effect of fluoride varnish with different fluoride concentrations. Caries Research 1994;28:64–7. 40. Marinho VC. Evidence-based effectiveness of topical fluorides. Advances in Dental Research 2008;20:3–7. 41. Billington RW, Williams JA, Pearson GJ. Ion processes in glass ionomer cements. Journal of Dentistry 2006;34: 544–55. 42. Francisconi LF, Scaffa PM, de Barros VR, Coutinho M, Francisconi PA. Glass ionomer cements and their role in the restoration of non-carious cervical lesions. Journal of Applied Oral Science 2009;17:364–9. 43. Thanjal NK, Billington RW, Shahid S, Luo J, Hill RG, Pearson GJ. Kinetics of fluoride ion release from dental restorative glass ionomer cements: the influence of ultrasound, radiant heat and glass composition. Journal of Materials Science Materials in Medicine 2010;21:589–95. 44. Luo J, Billington RW, Pearson GJ. Kinetics of fluoride release from glass components of glass ionomers. Journal of Dentistry 2009;37:495–501. 45. Rafeek RN. The effects of heat treatment on selected properties of a conventional and a resin-modified glass ionomer cement. Journal of Materials Science Materials in Medicine 2008;19:1913–20. 46. Nicholson JW, Czarnecka B. The biocompatibility of resinmodified glass-ionomer cements for dentistry. Dental Materials 2008;24:1702–8. 47. Cildir SK, Sandalli N. Fluoride release/uptake of glassionomer cements and polyacid-modified composite resins. Dental Materials Journal 2005;24:92–7. 48. Srinivasan N, Kavitha M, Loganathan SC. Comparison of the remineralization potential of CPP–ACP and CPP–ACP with 900 ppm fluoride on eroded human enamel: an in situ study. Archives of Oral Biology 2010;55:541–4. 49. Zero DT. RecaldentTM—evidence for clinical activity. Advances in Dental Research 2009;21:30–4. 50. Reynolds EC. Casein phosphopeptide–amorphous calcium phosphate: the scientific evidence. Advances in Dental Research 2009;21:25–9.