Abstract
Objectives
The aim of this study was to evaluate the effect of removing excess of resin-based materials applied to eroded enamel, subjected to erosive challenge.
Methods
Bovine enamel blocks were immersed in HCl 0.01 M, pH 2.3, for 30 s under agitation at 50 rpm in room temperature, in order to form a softened erosion lesion. The blocks were then randomly divided into eight groups ( n = 12) and treated as follows: Cn- and Ce-control without treatment, Hn- and He-fissure resin sealant (Helioseal Clear ® ), An- and Ae-self-etch adhesive (Adhese ® ), In- and Ie-infiltrant (Icon ® ); being n -with excess removal and e-without excess removal of the material. After application of the materials, the blocks were immersed in HCl for 2 min, followed by immersion in artificial saliva for 120 min. This cycle was repeated four times a day for five days. Material thickness and enamel wear were assessed using profilometry. Data were analyzed by two-way ANOVA and Tukey’s test ( P < 0.05).
Results
Groups He, Ae, and Ie resulted in the formation of a layer of material over enamel, being similar effective in inhibiting erosion progression ( P > 0.05). Groups Hn, An, and In (with excess removal) were similar to controls (Cn, Ce) and resulted in near enamel loss after application and after erosive challenge ( P > 0.05).
Conclusions
Resin-based materials are able to protect enamel against erosion only when they are present over enamel, as a physical barrier.
Clinical significance
The resin-based materials demonstrated potential to prevent the progression of erosion lesions when the material remains on the dental surface.
1
Introduction
The high prevalence of dental erosion motivates studies on searching preventive methods and early treatment, aiming at inhibiting the erosion lesion progression.
Traditionally, to prevent enamel erosion some strategies have been proposed for at-risk patients. These strategies are mainly focused on topical application of fluoride and dietary counseling. However, these lesions often progress because of the limited effect of fluoride and improper preventive measures performed by the patients . One alternative treatment reported by the literature is the application of resin-based materials on the erosion lesion surface . This treatment attempts to create a physical barrier to prevent the contact of the erosive agent with the dental surface . The results of studies using resin-based materials to protect dental tissues showed the reduction of erosive demineralization caused by acids . Moreover, the successful preventive effect of adhesives and pit and fissure resin sealants on the occurrence or progression of erosion lesions rely on the presence of the materials on the dental surface .
Resin infiltration was developed for the conservative treatment of white spot lesions . The infiltrant is an special low-viscosity resin that penetrates into the porous lesion body of enamel’s initial carious lesions, blocking the diffusion of acids into the lesion, thereby slowing or arresting the progression of caries . In contrast to conventional sealants that adhere to the enamel surface, resin infiltration penetrates inside the initial carious lesion. In previous study, infiltrant was able to inhibit enamel erosion progression, although the material application did not follow the manufacture instruction regarding the material excess remove over the surface . Thus, the infiltrant protective effect was based on its presence over enamel. When analyzing the penetration of resin-based materials into initial erosion lesion, infiltrant showed the highest penetration depth in enamel (8.24 μm) followed by a pit and fissure resin sealant (6.03 μm) . Even knowing that the penetration is much smaller in depth for erosion lesion when compared to caries lesion, we hypothesized that after the application of resin materials in initial erosive lesions, the presence of the resin material only on the eroded (porous) superficial surface would be enough to inhibit the erosion progression. Furthermore, considering the infiltrant, it would be important to test its action on erosion lesion, following the manufacturer’s recommendations to remove material excess from the enamel surface prior to light-curing.
Accordingly, this in vitro study aimed to evaluate the effectiveness of the application of infiltrant, pit and fissure resin sealant and self-etching adhesive (either with and without excess) on the eroded enamel surface, against the erosive challenge. The null hypothesis is that there is no difference in the inhibition of the erosion progression in vitro among the resin materials applied with and without excess on the enamel.
2
Material and methods
2.1
Study design
This in vitro study evaluated two factors: the surface condition at 2 levels (with/without the use of cotton swab for excess removal on the enamel surface) and the treatment type at 4 levels (control, pit and fissure resin sealant, self-etching adhesive, and infiltrant). The study was conducted with bovine enamel samples previously eroded (immersion in HCl 0.01 M, pH 2.3 for 30 s), randomly distributed among the groups ( n = 12) and treated according to the manufacturer’s instructions. In half of the sample, after the application, the excess of each material was removed prior to light-curing. In control group, a cotton swab was applied over the eroded surface, simulating material excess removal. After treatment, the samples were submitted to erosive cycling: HCl 0.01 M, pH 2.3, for 2 min, followed by the immersion in artificial saliva for 2 h. This process was repeated four times per day during 5 days. The response variable was enamel and/or material loss by profilometry.
2.2
Enamel specimens preparation
The enamel specimens were obtained from bovine teeth (one specimen per tooth). The roots were removed and the crowns were embedded in self-polymerizable resin (JET, Campo Limpo Paulista, SP, Brazil). Next, the specimens were planned and polished by using a metallographic polishing machine (APL 4, Arotec, Cotia) with 300-, 600-, and 1200-grit sandpaper (Extec Corp., Enfield, CT, USA). The enamel finishing was obtained with a felt disc (Extec Corp., Enfield, CT, USA) moistened with 1 μm diamond suspension (Buehler, Ltd., Lake Bluff, IL, USA).
2.3
Evaluation of initial superficial hardness
The initial superficial hardness (Shi) was evaluated to select the specimens, by using microdurometer (HMV-2000/Shimadzu Corporation) coupled to a computer and software for image analysis (Cams-Win-New Age Industries). A Knoop diamond tip with 25-g static load was used for 10 s. Five indentations were made at the central area of each specimen at 100 μm distance. Two hundred specimens showing hardness between 300 and 400 KHN (mean ± SD of 380.56 ± 43.71) were selected and subjected to the artificial erosion lesion formation.
2.4
Initial artificial erosion lesion
The initial erosion lesion was obtained in vitro by immersing the specimens in hydrochloric acid solution (0.01 M, pH 2.3) for 30 s, under agitation at speed of 50 rpm and room temperature (25 °C). In a pilot study, this protocol decreased the surface hardness without detectable wear and the indentations made when enamel was sound were still visualized .
The surface hardness after erosion lesion development (She) was obtained adopting the same parameters of initial superficial hardness measurement (SHe was measured at distances of 100 μm from the SHi). This procedure was performed to confirm the lesion formation, to select and randomize specimens among groups. Ninety-six specimens with hardness between 130 and 230 KHN (mean ± SD of 179.76 ± 24.8) were randomly divided into eight groups: four groups with cotton swab use for excess removal and four groups without cotton swab use.
2.5
Specimens initial profile
The initial profile of the specimens with initial erosion lesion was performed with contact profilometer (Mahr Perthometer, Göttingen, Germany), coupled to a computer. The specimens received marks made with a scalpel blade (Embramac, Itapira, SP, Brazil) delimiting three areas, two lateral reference areas (then protected with nail varnish) and the central area (test area = 2.0 mm 2 ). The marks are necessary to allow accurate repositioning of the stylus on subsequent profile measurements. Each specimen was submitted to five readings, at determined distances of 2.25; 2.0; 1.75; 1.5; and 1.25 μm.
2.6
Treatment of the specimens
Prior to the application of the materials, two thirds of the lateral surface of the specimens were covered by nail varnish, to obtain reference surfaces in the profiles. Next, the materials were applied according to the groups and the manufacturers’ recommendations using microbrushes ( Table 1 ). Half of the groups, including control group, had the use of cotton swab for excess removal on the enamel surface; in the other half, the excess was not removed, as follows:
| Material | Batch number | Aplication steps | Composition a | |
|---|---|---|---|---|
| AdheSE ® : Ivoclar Vivadent, Schan, Liechestein (A) | R82366 | Apply 1 layer of AdheSE-primer for 30 s, air dry 10 s. Apply 1 layer of AdheSE-bond for 5 s, air dry 5 s e and polymerize 10 s | AdheSE-primer: phosphonic acid acrylate bis-acrylamide derivative and additives; | |
| AdheSE-bond: dimethacrylates, HEMA b , SiO 2 , initiators and stabilizers | ||||
| Helioseal Clear ® : Ivoclar Vivadent, Schan, Liechestein (H) |
S04166 | Apply 37% phosphoric acid gel for 30 s, rinse 30 s, air dry 10 s. Apply 1 layer of Helioseal Clear for 15 s e and polymerize 20 s | Bis-GMA c , TEGDMA d and additives. | |
| Icon ® : DMG, Hamburg, Germany (I) |
669742 | Apply Icon-etch for 2 min, rinse 30 s and apply Icon-dry for 30 s, air dry 60 s. Apply the first layer of Icon-infiltrant for 3 min e , polymerize 40 s, apply the second layer for 1 min e and polymerize 40 s | Icon-etch: hydrochloric acid, pyrogenic silicic acid, surface-active substances; Icon-dry: 99% ethanol; Icon-infiltrant: TEGDMA d -based resin, initiators, and additives. | |
a Composition of the materials according to “Safety Data Sheet”, except for Icon ® that is in accordance with the package insert by the manufacturer.
b HEMA, 2-hydroxyethyl methacrylate.
c Bis-GMA, bisphenol A diglycidyl ether dimethacrylate.
d TEGDMA, triethylene glycol dimethacrylate.
e Moment of material removal with the use of a cotton swab, applied without force on the surface, with gentle movements.
Control (C)—without treatment (negative control)/Cn—use of cotton swab to simulate excess removal; Ce—without excess removal;
Pit and fissure resin sealant (H)—Helioseal Clear ® (Ivoclar Vivadent, Schaan/Liechtenstein)/Hn—use of cotton swab to remove material excess; He—without excess removal;
Self-etching adhesive system (A)—Adhese ® (Ivoclar Vivadent, Schaan/Liechtenstein)/An—use of cotton swab to remove material excess; Ae—without excess removal;
Infiltrant (I)—Icon ® (DMG, Hamburg/Germany)/In—use of cotton swab to remove material excess; Ie—without excess removal.
The removal of the material excess was carried out with the aid of a cotton swab applied without force on the surface, with gentle movements. It is worth emphasizing that the excess removal was executed always prior to the light-curing of the material. In control group, the cotton swab was applied likewise over the eroded surface, simulating the mechanical impact of the material excess removal.
2.7
Specimens profile after materials application
After the treatment, the nail varnish was removed from the specimens with the aid of wax spatula. Then, profilometric analysis was performed again at the same sites used for the baseline measurements. The specimens were submitted to profilometry, according to the initial position and using the same software (XCR 20) with the same measurement parameters, with 5 readings, at the pre-determined distances of 2.25; 2.0; 1.75; 1.5; and 1.25 μm.
2.8
Erosive cycling
After recovering the marks and reference areas with nail varnish, the specimens were subjected to erosive cycling for 5 days. Each cycling day was composed by 4 cycles:
- 1.
Demineralization, through immersion in hydrochloric acid solution (pH 2.3; 0.01 M), volume of 17.6 mL per specimen, during 2 min, under environmental temperature inside a plastic flask (approximately 264 mL) on a shaking table . Washing in deionized water (20 s);
- 2.
Immersion in artificial saliva (0.33 g KH 2 PO 4 , 0.34 g Na 2 HPO 4 , 1.27 g KCl, 0.16 g NaSCN, 0.58 g NaCl, 0.17 g CaCl 2 , 0.16 g NH 4 Cl, 0.2 g urea, 0.03 g glucose, 0.002 g ascorbic acid, 2.7 g mucin in 1000 mL of distilled water pH 7) volume of 17.6 mL per specimen, during 2 h ;
- 3.
Washing in deionized water (20 s). At the ending of each cycling day, the specimens were immersed in artificial saliva at 37 °C overnight .
2.9
Specimens profile after the erosive cycling
Elapsed the erosive cycling, the nail varnish was removed from the specimens with the aid of wax spatula to enable another profilometric analysis. The specimens were submitted to profilometry, according to the initial position with the same measurement parameters, with 5 readings, at the pre-determined distances of 2.25; 2.0; 1.75; 1.5; and 1.25 μm.
2.10
Profile analyses
The resin-based material thickness after application and material and/or enamel loss after erosive cycling were quantitatively determined using specific software (MarSurf XCR 20). Since the enamel samples could be precisely repositioned in the wells of the profilometer, it was possible to match the respective baseline and final profiles. The profiles were analyzed by superimposing the initial graphs on the graphs obtained after the material application and those after the erosive cycling. Then, the average thickness of the materials and the depth of the eroded surface relative to the baseline surface profiles were calculated.
2.11
Statistical analysis
The statistical analysis was performed by SigmaPlot software for Windows version 11.0 (Germany). Two-way ANOVA was applied considering the two factors under study (surface condition and treatment type), followed by Tukey’s test, with level of significance of 5%.
2
Material and methods
2.1
Study design
This in vitro study evaluated two factors: the surface condition at 2 levels (with/without the use of cotton swab for excess removal on the enamel surface) and the treatment type at 4 levels (control, pit and fissure resin sealant, self-etching adhesive, and infiltrant). The study was conducted with bovine enamel samples previously eroded (immersion in HCl 0.01 M, pH 2.3 for 30 s), randomly distributed among the groups ( n = 12) and treated according to the manufacturer’s instructions. In half of the sample, after the application, the excess of each material was removed prior to light-curing. In control group, a cotton swab was applied over the eroded surface, simulating material excess removal. After treatment, the samples were submitted to erosive cycling: HCl 0.01 M, pH 2.3, for 2 min, followed by the immersion in artificial saliva for 2 h. This process was repeated four times per day during 5 days. The response variable was enamel and/or material loss by profilometry.
2.2
Enamel specimens preparation
The enamel specimens were obtained from bovine teeth (one specimen per tooth). The roots were removed and the crowns were embedded in self-polymerizable resin (JET, Campo Limpo Paulista, SP, Brazil). Next, the specimens were planned and polished by using a metallographic polishing machine (APL 4, Arotec, Cotia) with 300-, 600-, and 1200-grit sandpaper (Extec Corp., Enfield, CT, USA). The enamel finishing was obtained with a felt disc (Extec Corp., Enfield, CT, USA) moistened with 1 μm diamond suspension (Buehler, Ltd., Lake Bluff, IL, USA).
2.3
Evaluation of initial superficial hardness
The initial superficial hardness (Shi) was evaluated to select the specimens, by using microdurometer (HMV-2000/Shimadzu Corporation) coupled to a computer and software for image analysis (Cams-Win-New Age Industries). A Knoop diamond tip with 25-g static load was used for 10 s. Five indentations were made at the central area of each specimen at 100 μm distance. Two hundred specimens showing hardness between 300 and 400 KHN (mean ± SD of 380.56 ± 43.71) were selected and subjected to the artificial erosion lesion formation.
2.4
Initial artificial erosion lesion
The initial erosion lesion was obtained in vitro by immersing the specimens in hydrochloric acid solution (0.01 M, pH 2.3) for 30 s, under agitation at speed of 50 rpm and room temperature (25 °C). In a pilot study, this protocol decreased the surface hardness without detectable wear and the indentations made when enamel was sound were still visualized .
The surface hardness after erosion lesion development (She) was obtained adopting the same parameters of initial superficial hardness measurement (SHe was measured at distances of 100 μm from the SHi). This procedure was performed to confirm the lesion formation, to select and randomize specimens among groups. Ninety-six specimens with hardness between 130 and 230 KHN (mean ± SD of 179.76 ± 24.8) were randomly divided into eight groups: four groups with cotton swab use for excess removal and four groups without cotton swab use.
2.5
Specimens initial profile
The initial profile of the specimens with initial erosion lesion was performed with contact profilometer (Mahr Perthometer, Göttingen, Germany), coupled to a computer. The specimens received marks made with a scalpel blade (Embramac, Itapira, SP, Brazil) delimiting three areas, two lateral reference areas (then protected with nail varnish) and the central area (test area = 2.0 mm 2 ). The marks are necessary to allow accurate repositioning of the stylus on subsequent profile measurements. Each specimen was submitted to five readings, at determined distances of 2.25; 2.0; 1.75; 1.5; and 1.25 μm.
2.6
Treatment of the specimens
Prior to the application of the materials, two thirds of the lateral surface of the specimens were covered by nail varnish, to obtain reference surfaces in the profiles. Next, the materials were applied according to the groups and the manufacturers’ recommendations using microbrushes ( Table 1 ). Half of the groups, including control group, had the use of cotton swab for excess removal on the enamel surface; in the other half, the excess was not removed, as follows:
