Caries-inhibiting Effect of Microencapsulated Active Components in Pit and Fissure Sealants
The aim of the present in vitro study was to examine the caries-inhibiting effect of a pit and fissure sealant (PFS) containing ion-releasing microcapsules under cariogenic conditions in a biofilm artificial mouth. Forty-eight human third molars were divided into four groups (n=12 per group). Fissures were extended with burs and sealed with experimental PFS. The four groups of specimens were treated as follows: 1) EPFS 1: EPFS (Premier Dental) of increasing viscosity, containing microcapsules loaded with remineralizing agents (calcium, phosphate, and fluoride ions); 2) US: fluoride-releasing PFS (UltraSeal XT plus, UltraDent Products, South Jordan, UT, USA); 3) EPFS 2: experimental PFS of constant viscosity containing microcapsules loaded with calcium, phosphate, and fluoride ions; and 4) FT: glass ionomer cement (GIC) (GC Fuji Triage CAPSULE WHITE glass ionomer cement, GC Europe NV, Leuven, Belgium). FT and US were used as control groups. EPFS 1 and EPFS 2 were the experimental groups. Specimens were stored in distilled water for 14 days at 37°C, subjected to 10,000 thermocycles (5°C and 55°C) and finally exposed to microbiological cycling in a Streptococcus mutans-based artificial mouth for 10 days. Replicas were made for scanning electron microscopic (SEM) evaluation and specimens were cut for fluorescence microscopy. Overall demineralization depths at the margin of Fuji Triage were significantly shallower than in the other groups (p<0.05). Overall demineralization depths adjacent to the experimental pit and fissure sealant EPFS 2 (59±15 μm) were comparable to the values of the resin-based pit and fissure sealant UltraSeal XT plus (58±10 μm, p≥0.05). SEM revealed surface roughness of the GIC-based PFS. The experimental PFS with microcapsules containing active components for remineralization did not show a caries-inhibiting effect compared to a fluoride-releasing resin-based PFS. Lower demineralization depths adjacent to GIC sealants indicate an anticariogenic effect through fluoride ion release.SUMMARY
Objective
Methods and Materials
Results
Conclusions
INTRODUCTION
In recent years, a decline in caries has been observed in many Western industrialized nations.1,2 Epidemiological studies conducted in Germany have supported this trend of decreasing caries prevalence.3,4 The results of the German Oral Health Studies (DMS I–V) showed a decline in the mean decayed, missing, and filled teeth count (DMF[T]) from 4.9 in 1989–1992 (DMS I and II, 13-14-year-olds) to 0.5 (DMS V, 12-year-olds) in 2014.3 Besides the caries decline, a polarization of the caries prevalence with an increased onset of carious lesions among socially deprived children1–3, 5–9 as well as changes in the lesion patterns have been recognized.1,5,6,10–12 In contrast to earlier days, an increasing number of “noncavitated carious lesions” is diagnosed, especially on permanent molars in childhood and adolescence.1
The occlusal surfaces of the permanent molars, and especially the pits and fissures of the first permanent molars, appear to be the tooth surfaces with the highest caries susceptibility in the phase of mixed dentition.1,7,10,13,14 To protect the permanent molars from the onset or the progression of a carious lesion, pit and fissure sealing has proven to be an effective caries-preventive treatment measure.1,11,14 Whereas conventional pit and fissure sealants usually do not contain material-specific caries-preventive ingredients, there are other sealing materials, like fluoride-releasing pit and fissure sealants or GICs, that are supposed to have anti-bacterial and/or remineralizing effects through fluoride-release.15 Children with a high caries risk might especially benefit from the use of these pit and fissure sealants, receiving additional protection against the occurrence of a carious lesion under cariogenic conditions.9
A new approach for remineralization of demineralized dental hard tissue is the incorporation of microcapsules loaded with aqueous solutions of calcium nitrate [Ca(NO3)2], sodium fluoride (NaF) and/or dipotassium phosphate (K2HPO4) in dental materials (rosin varnish, resin glaze, pit and fissure sealant, orthodontic cement). The microcapsules embedded in these materials are supposed to promote remineralization through a sustained, long-term release of bioavailable ions (calcium, phosphate, and fluoride ions).16–19
The caries-preventive properties of newly developed dental materials can be investigated in vitro during preliminary testing, for instance in artificial mouth models simulating the physical circumstances of the human oral cavity in a simplified way.20 Before the launch of new products for use in clinical practice, further in situ and in vivo studies should be conducted to confirm or refute the findings of these laboratory studies.20,21
Against this backdrop, the aim of the present in vitro study was to examine the demineralization-inhibiting properties of a newly developed, resinous pit and fissure sealant containing ion-releasing microcapsules for remineralization (Premier Dental, Plymouth Meeting, PA, USA). The investigation was conducted under cariogenic conditions in a Streptococcus mutans-based artificial mouth model for 10 days.
The null hypothesis tested was that no differences in demineralization-inhibiting effects would be measurable among the four pit and fissure sealants after exposing them to a microbiological load in an artificial mouth model for 10 days. Measurements taken included gap width, demineralization depth, substance loss, and overall demineralization depth (as the sum of the two parameters mentioned above) at the sealing margin and at a 500-μm distance from the margin. Furthermore, it was hypothesized that differences in the surface quality of the pit and fissure sealants would not be discernable by investigation under SEM.
The alternative hypothesis was that the four pit and fissure sealants subjected to 10 days of cariogenic challenge in an artificial mouth model would exhibit different demineralization-inhibiting effects through ion release adjacent to the sealing margin and at a 500-μm distance from the margin. Additionally, it was hypothesized that the microbiological load would lead to qualitative differences in sealant surface quality.
METHODS AND MATERIALS
Preparation of Specimens and Fissure Sealing
Forty-eight caries-free human third molars that had either been extracted or osteotomized for therapeutic reasons were used in this study. After extraction or osteotomy, the teeth were stored in 0.5% chloramine-T solution (Chloramin T Trihydrat, Carl Roth GmbH & Co KG, Karlsruhe, Germany) at 4°C for up to twenty-eight days. The teeth were randomly assigned to four groups (n/group=12). The specimens were then cleaned by air-blasting (PROPHYflex 3, KaVo Dental GmbH, Biberach/Riss, Germany; powder: Clinpro Glycine Prophy Powder, 3M Oral Care, Germany, Neuss, Germany) and rinsed with water spray, and the fissures were prepared using a Fissurotomy-bur (type “original”, Fissurotomy burs, SS White Bur, distributor: atec Dental GmbH, Ebringen, Germany). After air-drying of the occlusal surfaces, the following pit and fissure sealants were used to seal the prepared fissures (Table 1):
- Group 1 (EPFS 1)—
Experimental calcium-, phosphate-, and fluoride-releasing resin-based pit and fissure sealant containing microcapsules loaded with remineralizing agents (Batch No 1, proprietary composition; Premier Dental)
- Group 2 (US)—
UltraSeal XT plus (fluoride-releasing resin-based pit and fissure sealant; Ultradent Products)
- Group 3 (EPFS 2)—
Experimental calcium-, phosphate-, and fluoride-releasing resin-based pit and fissure sealant containing microcapsules loaded with remineralizing agents (Batch No 2, proprietary composition; Premier Dental)
- Group 4 (FT)—
GC Fuji Triage CAPSULE WHITE (glass ionomer cement; GC)
In Group 1 (EPFS 1), Group 2 (US), and Group 3 (EPFS 2), 35 % phosphoric acid in gel form (Ultra-Etch, Ultradent Products) was applied on enamel for 30 seconds and if an exposure of dentin was visible, the dentin was etched for 15 seconds. Then, Ultra-Etch (Ultradent Products) was rinsed off with water spray for minimally 10 seconds, followed by air-drying. The pit and fissure sealants were applied in three layers with a gentle dispersion of each layer to avoid void formation and afterwards polymerized for 30 seconds (Bluephase; Ivoclar Vivadent AG, Schaan, Liechtenstein). The light intensity of the LED curing device was monitored regularly with a radiometer (CURE RITE, Dentsply Caulk, Milford, DE, USA) to ensure a light output of at least 1200 mW/cm2. Finally, the oxygen inhibition layer was removed by wiping the pit and fissure sealant surfaces with a foam pellet (Pele Tim No 2, Voco GmbH, Cuxhaven, Germany).
The application of Fuji Triage (GC Europe NV) in Group 4 was done in one layer, after the product was activated and mixed according to the manufacturer’s specifications. Heliobond (Ivoclar Vivadent) was applied on the pit and fissure sealant surfaces using a Microbrush (Microbrush International, Grafton, WI, USA), and polymerized for 30 seconds (Bluephase; Ivoclar Vivadent) to protect the glass ionomer cement against humidity and dehydration during the setting reaction. Since it took about 24 hours until the setting reaction of the glass ionomer cement was completed, the specimens were first stored in distilled water for 24 hours at 37°C, and then further processing was performed.
Following sealant application, the occlusal surfaces were ground until the cusps were flattened and polished (BUEHLER Beta GRINDER - POLISHER, sandpaper: BUEHLER CarbiMet Grit 600 [P1200 and P4000], ITW Test & Measurement GmbH, Düsseldorf, Germany) in order to attain a smooth surface at the pit and fissure sealant-enamel interface. Thereafter, the specimens were stored in distilled water for 14 days at 37°C (Incubator Type B20, Heraeus Holding GmbH, Hanau, Germany), and subjected to 10,000 thermocycles (5°C and 55°C, dwell time 15 seconds; TCS 30, Syndicad, Munich, Germany). After the apical thirds of the roots were cut off, the teeth were mounted on chewing simulator holders (Festo AG & Co KG, Denkendorf, Germany) with glue wax (Chemical Dental Laboratory, Oppermann-Schwedler, Bonn, Germany), followed by disinfection in 70% ethanol for 120 minutes. The specimens were then exposed to a microbiological load in an automated, S mutans-based artificial mouth model for 10 days with a total of 4 hours of demineralization/day.
Artificial Mouth Model
The computer-controlled artificial mouth model was composed of a reaction chamber (300-4100 Reusable Filter Holder with Receiver, Thermo Scientific Nalgene Labware, Rochester, NY, USA) containing a Teflon holder (Bretthauer GmbH, Dillenburg, Germany) to attach the teeth and a pH-measuring electrode (SI Analytics Electrode N1048 1M – DIN – ID, SI Analytics GmbH, Mainz, Germany; Schott Instruments Lab 870, Schott AG, Mainz, Germany; MultiLab pilot v.4.7.2, WTW GmbH, Weilheim, Germany), an Erlenmeyer flask acting as a bacterial reservoir (Schott AG), two 20 L bottles (Schott AG), one for the nutrient medium and the other for artificial saliva, and a 10 L bottle (Thermo Scientific Nalgene) for liquid waste. Computer-controlled peristaltic pumps (Cyclo II, Carl Roth GmbH & Co KG; LeC, Conrad Electronic SE, Hirschau, Germany) transported the liquids within the experimental setup. To ensure a temperature of 37°C during test-efforts, the artificial mouth model was built inside an incubator (IPS Memmert, Memmert GmbH & Co KG, Schwabach, Germany). All components of the artificial mouth model except the pH measuring electrode could be disassembled and sterilized at 121°C and 2 bars for 15 minutes (autoclave VX-75, Systec GmbH, Linden, Germany). After calibration, the pH measuring electrode was disinfected with 70% ethanol and rinsed with distilled water. To prevent undesirable contamination, the specimens were placed in the Teflon holder inside the reaction chamber under a clean bench (Thermo Fisher Scientific Inc, Waltham, MA, USA). The pH measuring electrode was also attached to the reaction chamber under the clean bench before every episode of testing.
To simulate exposure of specimens to intermittent demineralization and remineralization phases within the artificial mouth model, a nutrient medium, a bacterial strain, and artificial saliva were needed. The nutrient medium utilized for bacterial proliferation within the artificial mouth model was Schaedler Broth (BD, BBL Schaedler Broth, Becton Dickinson and Company, Sparks, MD, USA), which was used according to manufacturer’s specifications (Table 2). To simulate the remineralizing effects of human saliva within the oral cavity, a remineralizing solution as described by Zampatti and others was used as artificial saliva (pH 7; Table 3).22
Freeze-dried S mutans (DSM No: 20523, Leibniz-Institute DSMZ GmbH, Braunschweig, Germany) was stored in glycerin cultures at −80°C. After thawing, bacterial cultures were cultivated on Columbia blood agar (sheep blood, OXOID AGS, Oxoid Limited, Basingstoke, Hampshire, UK) for 48 hours at 37°C under aerobic conditions. Overnight cultures of S mutans dissolved in Schaedler Broth (12 hour incubation, 37°C, aerobic conditions) were then produced and diluted 1:10 (8 hour incubation, 37°C, aerobic conditions). After the optical density of the 1:10 diluted overnight culture, which was targeted to be ~1 at 600 nm, was controlled, the inoculation procedure was conducted by pipetting 1 ml of the bacterial culture into the bacterial reservoir. Following proliferation of S mutans within the bacterial reservoir for 8 hours at 37°C, the microbiological load was begun. While the specimen demineralization was caused by acidogenic S mutans producing organic acids through sugar metabolism during glycolysis, remineralization was simulated by exposure to artificial saliva. During this microbiological stress protocol, the demineralization phases (duration 1 hour, pH Ø 4.2–4.3) and remineralization phases (duration 5 hours, pH Ø 7.0) alternated for 10 days, so that a total of 4 hours of demineralization and 20 hours of remineralization was achieved per day.
To control the microbiological viability of S mutans and the absence of bacterial contamination during the experimental procedures, samples of the bacterial solutions were cultivated on BHI agar plates (BBL, Becton, Dickinson) for 48 hours at 37°C under aerobic conditions.
Following the microbiological loading, specimens were disinfected by storage in 70% ethanol for 3 minutes. In the next step, the specimens were cut bucco-orally in slices of 1-mm thickness by means of a microtome (IsoMet 1000 Precision Saw, Buehler, ITW Test & Measurement GmbH).
Furthermore, two sets of epoxy replicas of each specimen occlusal surface were produced by taking impressions with a double-mix technique using a vinylpolysiloxane (Panasil Putty, Panasil initial contact Light, Kettenbach GmbH & Co KG, Rosbach, Germany) and casting them with AlphaDie MF (Schuetz Dental GmbH, Rosbach, Germany) to optimally visualize the quality of the enamel-pit and fissure sealant interface. The first set of replicas was manufactured after thermocycling, and the second one after microbiological loading.
In addition, fracture specimens of each fissure sealant were prepared by producing beams and fracturing those into halves to assess the surface quality.
Data Evaluation
During fluorescence microscopy evaluation, the demineralization depth, substance loss, and overall demineralization depth as the sum of the previous two parameters were evaluated for the enamel at the fissure sealant margin and at a point 500 μm from it (AZ 100M, Nikon, Tokyo, Japan; FITC-filter: 450–490 nm, spacing: 515–565 nm; NIS Elements for Windows XP, 0.9 μm/px; Figure 1). If a gap was detectable at the fissure sealant-enamel interface, the gap width was assessed. These values were measured adjacent to the fissure sealing on both sides of each tooth slice.
Citation: Operative Dentistry 47, 4; 10.2341/20-048-L
Additionally, the two sets of epoxy replicas were examined with an SEM (Amray Turbo 1610, Amray Inc, Bedford, MA, USA; 10-kV acceleration voltage, 200× magnification) to investigate the quality of the fissure sealant margins. To best investigate, SEM photographs were taken of the fracture preparation surfaces at 500×, 1000×, and 1500× magnifications to examine surface roughness.
One additional specimen of each group was prepared for energy-dispersive X-ray analysis (Quantax spectrometer, X-Flash 5010, Bruker Nano GmbH, Berlin, Germany) with an SEM (JSM-6510, JEOL, Tokyo, Japan) to obtain information about the chemical composition of the pit and fissure sealants. Specimens were attached to aluminum sample trays with a conductive carbon cement (Leit-C, Plano GmbH, Wetzlar, Germany) and sputter-coated with gold (JFC-1200 fine coater, Tokyo, Japan). SEM images of the specimens were captured at 2000× magnification (acceleration voltage: 15 kV) and energy-dispersive X-ray (EDX) analyses were conducted with unchanged settings at count rates of 1 kilocount per second (kcps).
IBM SPSS Statistics 26 (SPSS Inc, Chicago, IL, USA) was used for statistical data evaluation. Normal distribution of the measured values was checked by means of the Kolmogorov-Smirnov test. One-way analysis of variance was performed to verify the existence of statistically significant differences between the fissure sealants used in this study (ANOVA, mod LSD, α=0.05). Homogeneity of variance was found to exist in cases in which ANOVA was calculated (Levene test). The Mann-Whitney test was applied for statistical evaluation in the remaining cases with sensitive disturbances of the normal distribution of data. The significance level was set at α=0.05.
RESULTS
Application of the New Pit and Fissure Sealant
During pit and fissure sealing, EPFS 1 showed viscosity fluctuations, with the sealant being of low viscosity at the beginning of the application and becoming more viscous as the syringe was emptied. These viscosity fluctuations were not observed for the enhanced pit and fissure sealant EPFS 2.
Fluorescence Microscopy
After the 10-day exposure of the specimens to the microbiological load in the artificial mouth model, the overall demineralization depths at the sealant margins for FT (Group 4) were significantly smaller than those of the other groups (ANOVA, mod LSD, p<0.05; Table 4, Figures 2–5). The overall demineralization depths adjacent to the new, experimental pit and fissure sealant EPFS 2 (59±15 μm; Group 3; Figure 4) were comparable to the values of US measured at the sealant margin (58±10 μm; Group 2, Figure 3; ANOVA, mod LSD, p≥0.05).
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At the point 500 μm from the sealant margin, no statistically significant differences in the overall demineralization depths between the groups were detectable (Mann-Whitney test, p≥0.05, Figures 2–5).
Scanning Electron Microscopy
Scanning electron microscopic images taken from representative replicas of all four groups showed the impact of the sealant-biofilm interaction after the 10-day microbiological load. On the one hand, sealant surfaces of the resin-based pit and fissure sealants (EPFS 1, US, EPFS 2) showed smooth surface structures with gap-free margins after biodegradation. On the other hand, images of FT surfaces (group 4) revealed an increased surface roughness with cracks and air voids not seen in the other groups (Figures 6 and 7). Additionally, the surface of the fracture preparations produced with FT appeared to be rougher than those of the other groups (Figure 8).
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Energy-dispersive X-ray Analysis
SEM images and spectra of the EDX analyses are presented in Figure 9. EPFS 1 and EPFS 2 showed granular particles, which were interpreted as microcapsules. In contrast, US exhibited a smooth surface layer. The surface of FT revealed cracks and surface roughness. The comparison of EDX spectra showed that fluorine detection was lowest for US, followed by EPFS 1, EPFS 2, and FT. Calcium elements were detected in EPFS 1 but not in EPFS 2. In contrast, the spectrum of specimen EPFS 2 showed phosphorus elements; these were undetectable in the spectrum of specimen EPFS 1.
Citation: Operative Dentistry 47, 4; 10.2341/20-048-L
DISCUSSION
The present study was designed to investigate potential demineralization-inhibiting effects of a new, experimental pit and fissure sealant after a 10-day exposure to a microbiological load in a completely automated, S mutans-based, artificial mouth model. To assess this effect, the demineralization depths adjacent to the four different pit and fissure sealants were measured during fluorescence microscopy evaluation, and visible changes in the surface quality between two points in time (after thermocycling and after microbiological loading) were checked under SEM.
Recently developed dental materials with microencapsulated remineralizing agents have shown a potential for the sustained, long-term release of calcium, phosphate, and/or fluoride ions16,17 and an ability to remineralize artificial carious lesions in vitro.16 Polyurethane-based microcapsules containing aqueous solutions of Ca(NO3)2, NaF, or K2HPO4 have been found to be capable of releasing ions [Ca2+, F−, or (PO4)3−], whose presence is an important prerequisite for remineralization of demineralized dental hard tissue.16–19 Burbank and others examined the ion release of a pit and fissure sealant containing microencapsulated 5 molar (M) Ca(NO3)2, 0.8 M NaF, and 6.0 M K2HPO4 in aqueous solutions separately or in combination.16 The authors reported a sustained release of Ca2+, F−, and (PO4)3− ions for about 180 days. Moreover, the fluoride uptake into artificially demineralized bovine enamel was significantly higher when specimens were exposed to a pit and fissure sealant containing a mixture of these remineralizing molecules compared to a microcapsule-free sealant. After storage in nanopure water for 90 days, the fluoride content of the bovine enamel had increased from 1.7 ± 0.7 μg F/g to 190 ± 137 μg F/g when the microencapsulated pit and fissure sealant containing 2 w/w% Ca(NO3)2, 2 w/w% NaF, and 1 w/w% K2HPO4 was used.16 To our knowledge, the ability of pit and fissure sealant containing these microencapsulated remineralizing agents to prevent demineralization under cariogenic conditions has not been tested before.
Caries-free human third molars (ICDAS II Code 0) were chosen for this study,23,24 and before sealant application the pits and fissures were prepared with a Fissurotomy-bur, which aimed to achieve the even cavity wall necessary for correct alignment of the specimens during fluorescence microscopic analysis and for a standardized evaluation. In addition, the preparation enabled the definite exclusion of “hidden caries”, dentin caries hidden under an almost intact surface.25 Not least because of the preparation, a higher amount of sealing material could be applied in the pit and fissure system, which was advantageous since a slight reduction of the sealing material had to be expected during grinding of the occlusal surfaces. The occlusal surfaces of the specimens had to be ground and polished to obtain plain and smooth surfaces and guarantee a comparable bacterial adhesion. It has to be mentioned that the cavity preparation was solely performed for experimental purposes in the present study. In daily practice, neither a cavity preparation prior to pit and fissure sealing, nor flattening of cusps should be performed in caries-free teeth.
Resin-based pit and fissure sealants usually contain a small amount of filler (6.5–54.3 wt%), preserving a low viscosity and a low Vickers microhardness of the sealing material.26 For large pit and fissure sealants, the inferior mechanical properties of the material are disadvantageous and flowable composites should be used instead.26 As there was no mechanical stress in terms of chewing or clenching operating on the specimens during the microbiological loading in the artificial mouth model, the pit and fissure sealants were used to seal the prepared pits and fissures in this study. The resin-based pit and fissure sealants were applied in three layers to compensate for polymerization shrinkage and to avoid void formations. It must be remembered that this method was only performed for experimental purposes and does not reflect daily clinical practice, where pit and fissure sealants are applied in one layer,1 and where the use of flowable composites is recommended after fissurotomy because of their more favorable mechanical properties.26
In the present study, one fluoride-releasing resin-based pit and fissure sealant (UltraSeal XT plus) and one GIC (GC Fuji Triage) were chosen as comparators to the experimental pit and fissure sealant. All of these pit and fissure sealants are categorized as ion-releasing, which is a limitation of the present study. The microbiological load in the artificial mouth model simulated highly cariogenic conditions that can be expected in children with a high caries risk. In daily practice, the material of choice in these cases is a fluoride-releasing rather than a conventional pit and fissure sealant as it provides the benefit of a caries-protective effect from fluoride release in addition to its function as a physical barrier within the pit and fissure system.27 It has been shown by Alsaffar and others that the mean mineral loss of enamel adjacent to US was comparable to the values of the control group sealed with a conventional, fluoride-free pit and fissure sealant after 20 days of demineralization with lactic acid gel.27 Delben and others compared the anticariogenic effect of US to four other fluoride- and/or amorphous calcium phosphate-containing pit and fissure sealants. After pH-cycling, specimens of the US group exhibited the lowest surface hardness, the highest mineral loss, and a subsurface lesion formation within adjacent enamel.28 Although US contains fluoride ions, the results of in vitro studies indicate that it behaves like a conventional pit and fissure sealant under simulated cariogenic conditions, which may be attributed to its comparatively low fluoride ion release.29
The glass ionomer cement, GC Fuji Triage, was used as a positive control in the present study. A demineralization-protective effect of this GIC was observed by Alsaffar and others, who demonstrated decreased mineral loss of adjacent enamel after 20 days of demineralization in vitro.27 Additionally, Poggio and others found a significantly higher fluoride release (1.1±0.3 – 8.0±0.6 ppm F−) for FT compared to a conventional and a fluoride-releasing pit and fissure sealant over a period of 49 days.30 Clinical trials support the caries-preventive effect of FT when this GIC of low viscosity and high fluoride release is used as a pit and fissure sealant.31–33
The main difference between the two batches of the experimental pit and fissure sealants was related to the sealants’ flow properties. During the application by syringe of Batch No 1, the material showed an increasing viscosity as the syringe was emptied. The viscosity fluctuations observed in Batch No 1 (EPFS 1) may be attributable to an agglomeration phenomenon of the microcapsules or a separation of the microcapsules from the matrix. Batch No 2 (EPFS 2) showed a uniform viscosity, which enabled good sealant application. The EDX spectra of EPFS 1 and EPFS 2 indicated differences in chemical composition, as calcium elements were only detected in EPFS 1 and phosphorus elements solely in EPFS 2 (Figure 9). Due to the proprietary nature of the compositions, more information about differences between the two batches of the experimental sealant could not be obtained from the manufacturer. In a preliminary microleakage test, the experimental pit and fissure sealant exhibited a sealing ability and a formation of voids that were both comparable to those of Helioseal F (Ivoclar Vivadent AG) and US (ANOVA, mod LSD p≥0.05; unpublished data).
The S mutans-based artificial mouth model used has proven its reproducibility in producing artificial secondary caries-like lesions during earlier studies.34–38 To simulate the daily consumption of four cariogenic meals, demineralization was caused by acidogenic S mutans producing organic acids during glucose digestion (four, 1-hour demineralization phases, pH Ø 4.2–4.3); remineralization was induced by exposing the specimens to artificial saliva (four, 5-hour remineralization phases, pH Ø 7.0). Nevertheless, it has to be mentioned that using an artificial mouth model with one bacterial strain (in this case S mutans, the main pathogenic bacterial species in caries etiology39–45) and causing demineralization through acid production during glycolysis is a simplified experimental setup in comparison to the microbiological diversity and complexity of biofilms in the human oral cavity. This simplification constitutes a weakness of the artificial mouth model, and the results should not be unreservedly transferred to the situation in vivo. Therefore, the results of in vitro studies need to be verified in further in situ and in vivo studies in order to adhere to an experimental hierarchy with an increasing significance for clinical practice.20,21
The fluorescence microscopy evaluation showed that the overall demineralization depths of the FT specimens were significantly smaller at the enamel-pit and fissure sealant interface than those of the other groups (ANOVA, mod LSD, p<0.05; Table 4). While a demineralization-inhibiting effectiveness of GICs is verifiable under simulated cariogenic conditions in vitro,34,37 clinical trials show partially contradictory results.46 It is assumed that an anticariogenic efficacy of fluoride-releasing restoration materials (ie, GICs, compomers) under simulated cariogenic ambient conditions in vitro is caused by disrupting bacterial metabolism due to the release of fluoride ions.46 Fluoride release by the restorative materials depends on material scientific parameters (matrix, fillers, fluoride content, setting reaction) and surrounding factors.46
The new, experimental pit and fissure sealant and US did not show significantly different overall demineralization depths in enamel at the pit and fissure sealant margin (ANOVA, mod LSD, p≥0.05). Therefore, demineralization-inhibiting characteristics of the experimental pit and fissure sealant were not measurable in comparison to US after microbiological loading in the artificial mouth model for 10 days. Under pH-neutral conditions in vitro, pit and fissure sealants containing the ion-releasing microcapsules showed a remineralization of initially demineralized artificial carious lesions after 90 days, measured by the fluoride uptake in enamel.16 The results of the present study showed that under cariogenic conditions in the artificial mouth model, the amount of ion release by the experimental pit and fissure sealant seemed to be insufficient to prevent demineralization.
The surface roughness of glass ionomer cements observed during scanning electron microscopic evaluation has also been described by Yoshihara and others.47 Changes in the surface quality of sealing materials with increasing roughness and crack formation can encourage biofilm formation,47 and might be a reason for unfavorable retention rates of GICs used as pit and fissure sealants. According to a meta-analysis by Kühnisch and others, the five-year pooled retention rate estimate amounted to 1.6% for GIC pit and fissure sealants.48 Because it has an increased surface roughness and shows crack formation, GIC seems best limited to temporary pit and fissure sealing.
Based on the results of the present study, the null hypothesis of no difference in the demineralization-inhibiting effect between the four pit and fissure sealants after microbiological load for 10 days has to be partially rejected and the alternative hypothesis has to be partially accepted, as statistically significant differences among the groups were only measurable at the sealing margins and not at a 500-μm distance from the margin. Additionally, the alternative hypothesis that the four pit and fissure sealants differ in their surface quality has to be partially accepted, as the appearance of the surface structure showed a material dependence as the appearance of the surface structure was dependent on the type of fissure sealant under investigation.
CONCLUSION
Demineralization-inhibiting effects of the experimental pit and fissure sealant containing microcapsules with remineralizing agents could not be measured after a 10-day exposure to a cariogenic load in a S mutans-based artificial mouth model in comparison to a fluoride-releasing resin-based pit and fissure sealant. Within the limitations of this in vitro study, the lower demineralization depths at the glass ionomer cement-enamel interface may be caused by anti-cariogenic effects resulting from a burst of fluoride ions released from the mineral fillers30 during the cariogenic and acidogenic challenge in the artificial mouth model.
Fluorescence microscopic evaluation of gap width, demineralization depth, and substance loss at pit and fissure sealing margin and in 500 μm distance from margin. PFS: Pit and fissure sealant.
Fluorescence microscopic evaluation of demineralization depth at pit and fissure sealant margin and at 500 μm distance from sealing margin (Tooth 6; experimental pit and fissure sealant No 1). PFS: pit and fissure sealant.
Fluorescence microscopic evaluation of demineralization depth at pit and fissure sealant margin and at 500 μm distance from sealant margin (Tooth 19; UltraSeal XT plus). PFS: pit and fissure sealant.
Fluorescence microscopic evaluation of demineralization depth at pit and fissure sealant margin and at 500 μm distance from sealant margin (Tooth 40; experimental pit and fissure sealant No 2). PFS: pit and fissure sealant.
Fluorescence microscopic evaluation of demineralization depth at pit and fissure sealing margin and at 500 μm distance from sealant margin (Tooth 33; Fuji Triage)PFS: pit and fissure sealing.
Example of overlapped SEM pictures taken from the replicas of tooth 26 (experimental pit and fissure sealant No 2) to visualize the quality of the pit and fissure sealant margin after thermocycling (TC) and after microbiological load in the artificial mouth model (AM) Red arrows mark the enamel-pit and fissure sealant interface. PFS: pit and fissure sealing.
Example of overlapped SEM pictures taken from the replicas of tooth 32 (Fuji Triage) to visualize the quality of the pit and fissure sealing margin after thermocycling (TC) and after microbiological load in the artificial mouth model (AM). Red arrows mark the enamel-pit and fissure sealant interface. PFS: pit and fissure sealant.
SEM evaluation of fracture preparations of (A) EPFS 1; (B) US; (C) EPFS 2; (D) FT. The FT surface shows an increased roughness with crack formation. EPFS: experimental pit and fissure sealant; US: UltraSeal XT plus; FT: Fuji Triage.
SEM images and EDX spectra of the pit and fissure sealants: (A): EPFS 1; (B): US; (C): EPFS 2; (D): FT. EPFS: experimental pit and fissure sealant; US: UltraSeal XT plus; FT: Fuji Triage.
Contributor Notes
Clinical Relevance
A bioactive pit and fissure sealant with microcapsules containing remineralizing agents did not show a caries inhibiting effect in an in vitro biofilm model.