2.1

Specimens preparation

Twenty extracted, sound bovine incisors obtained from a local slaughter house (Yokohama, Japan) and checked to be free from any evidence of enamel cracks were collected and used according to a protocol approved by the Institutional Review Board of Tokyo Medical and Dental University for animal studies. The teeth were cleaned with deionized water to remove any surface debris and stored at −25 °C until needed. Enamel blocks 6 mm × 3 mm × 3 mm (length × width × depth) were cut from the bovine incisors using a low speed diamond saw (Isomet; Buehler, Lake Bluff, IL, USA) under running water, and embedded in epoxy resin (Epoxycure resin; Buehler). The outer surface was slightly polished with 800-grit silicon carbide (SiC) paper (Sankyo, Saitama, Japan) to remove the superficial layer and expose enamel.

2.2

Coating materials

According to the study design, the enamel blocks were divided into 4 groups ( n = 5/group) corresponding to the materials used and control. In the control group, specimens received no treatment, three different resin-based materials were used to coat enamel in other groups; giomer coating PRG Barrier Coat (PBC; Shofu, Kyoto, Japan), resin-modified glass-ionomer Clinpro XT Varnish (CXT; 3M ESPE, St. Paul, MN, USA); and two-step self-etch adhesive resin Clearfil SE Protect (SEP; Kuraray Noritake Dental, Tokyo, Japan). Two areas, namely coated (C), and uncoated (UC), were assigned on the polished enamel surface of each block. Half surface of each block was carefully treated with the materials in accordance with the instructions supplied by the manufacturers as listed in Table 1 for all groups that served as the C area while the other half stayed intact as UC area. The specimens were then stored in water for 24 h at 37 °C.

2.3

Thermo cycling procedure

All specimens were placed in wire-mesh basket and subjected to 5000 thermal cycles between 5 °C and 55 °C (Yamato Scientific, Tokyo, Japan), with 30 s dwell time at each temperature with an exchange time of 5 s between baths. This was aimed to allow the materials complete polymerization and water sorption under thermal aging.

2.4

Demineralization procedure

All samples were subjected to demineralization solution (CaCl ₂ 1.5 mM, KH ₂ PO ₄ 0.9 mM, CH ₃ COOH 50.0 mM, NaN ₃ 3.08 mM) at pH 4.5 at 37 °C for one week. The pH of the solution was checked every day . The volume of demineralization solution was 140 ml per each five samples. The solution was not changed to clarify the ion releasing and buffering effect of each material ( Fig. 1 ).

Schematic drawing for the sample preparation and NI test. Bovine enamel blocks were embedded in epoxy resin; one of the coating materials (PBC, CXT and SEP) was applied on half of the enamel surface and then specimens were secondly embedded after the challenges, cross-sectioned and polished for SEM observation and NI test. Hardness was obtained after 5000 thermal cycles plus one-week demineralization for all groups. C: coated; UC: uncoated; NI: nanoindentation.

2.5

Nanoindentation test

The epoxy resin embedded enamel specimens were secondly embedded in polyester resin (Rigolac, Oken, Tokyo, Japan) to prevent breaking the surface or chipping of the specimen edges, and cross-sectioned along the longer edge of specimen using the low speed diamond saw into two halves to perform NI evaluation. To produce a smooth surface, the cross-section was sequentially polished by SiC papers #600, #800, #1000, #1200, #1500, and #2000, followed by diamond slurries with particle sizes of 6 μm, 3 μm, 1 μm, 0.5 μm, and 0.25 μm, with a lapping machine (Maruto, Tokyo, Japan). Every specimen was observed by confocal laser scanning microscope (CLSM; 1LM21H/W, Lasertec, Yokohama, Japan) after polishing to ensure there was no scratch on the surface.

On one cross-section obtained from each enamel block, 3 areas of interest, each 100 μm (lateral dimension) by 200 μm (axial dimension or depth) were defined to map hardness and draw hardness versus depth profiles at the center of C and UC area as well as the intermediate (INT) areas at the edge of coating (INT-C and INT-UC) using a NI device (ENT-1100a; Elionix, Tokyo, Japan). The maximum load was 2 mN at a loading rate of 0.2 mN/s and 1 s holding segment with a Berkovich diamond tip at chamber temperature of 27.5 °C. For this purpose, indentations were performed along 22 rows in each area, where each row included 10 points, with an axial spacing of 2.5 μm between each 2 neighboring points for the first 50 μm depth, then 50 μm and 100 μm spacing for the last two lines up to 200 μm from the surface. The lateral spacing between points on each row was 10 μm, and the rows were programmed to avoid overlapping of neighboring points. This NI mapping strategy is schematically presented in Fig. 2 . To calculate Martens hardness (HM) in MPa, the force was divided over the projected area under maximum load, which would include both elastic and plastic deformations, as described previously .

(a–e) Schematic representations of the indentation points on cross-sectioned enamel. (a) Two matrices programmed on 20 lines with axial pitch distances of 2.5 μm (first 50 μm), 50 and 100 μm among rows for enamel (C, INT-C, INT-UC and UC). The total distance of the last raw from the surface was 200 μm. (b) The magnified area represents indentations programmed for hardness measurement. (c–e) A real optical microscopic image of polished of enamel surface after 50-μm depth NI of C, INT and UC enamel respectively. C: coated; UC: uncoated; INT: intermediate; NI: nanoindentation.

2.6

Statistical analysis

Two variables were defined and subjected to statistical analysis in this study; surface zone hardness (SZH) that considered the average enamel hardness at the first 10 μm, and integrated hardness (INH), which was defined to compare the overall hardness values by calculating the area under the hardness profile curve at the first 50 μm, corresponding to the demineralized lesion body. Statistical analyses were performed using 2-way ANOVAs with experimental groups (PBC, CXT, SEP and control) and different areas of enamel (C, INT-C, INT-UC and UC) as factors, followed by multiple comparisons between each two pairs with Bonferroni correction. All the statistical procedures were performed at a significance level of α = 0.05 with the statistical package for social science (SPSS for windows, Version 16.0, SPSS, IL, USA).

2.7

Scanning electron microscope (SEM) examination

The observation was performed on the cross-sectioned surfaces either after polishing or after NI test. The specimens were cleaned ultrasonically in distilled water for 3 min, dehydrated at room temperature for 24 h, gold sputter-coated (300 Å), and finally observed in SEM (JSM-5310LV, JEOL, Tokyo, Japan).

2.8

Fluoride ion release

After one week of demineralization challenge, 10 ml of the demineralization solution in each container (PBC, CXT, SEP and control) was mixed with 1 ml of buffer solution (sodium acetate trihydrate 0.1 M, pH 5.5). The test solution was placed in a beaker over a non-heating magnetic stirrer, and readings were taken after a 5-min immersion period using F ion selective electrode (8010-10C, 2060A-10T, HORIBA, Kyoto, Japan) connected to an ion analyzer (F-53, HORIBA). Standards were also prepared from sodium fluoride solution with concentrations of 0.05, 0.1, 0.5, 1, 10 and 100 ppm to which a buffer solution was added in order to obtain a constant background ionic strength. These standard solutions were used to plot the calibration graphs. The temperature of the solution was maintained at 25 °C. F ion concentration in each solution was measured 3 times, and the mean value was reported in ppm.

2.1

Specimens preparation

Twenty extracted, sound bovine incisors obtained from a local slaughter house (Yokohama, Japan) and checked to be free from any evidence of enamel cracks were collected and used according to a protocol approved by the Institutional Review Board of Tokyo Medical and Dental University for animal studies. The teeth were cleaned with deionized water to remove any surface debris and stored at −25 °C until needed. Enamel blocks 6 mm × 3 mm × 3 mm (length × width × depth) were cut from the bovine incisors using a low speed diamond saw (Isomet; Buehler, Lake Bluff, IL, USA) under running water, and embedded in epoxy resin (Epoxycure resin; Buehler). The outer surface was slightly polished with 800-grit silicon carbide (SiC) paper (Sankyo, Saitama, Japan) to remove the superficial layer and expose enamel.

2.2

Coating materials

According to the study design, the enamel blocks were divided into 4 groups ( n = 5/group) corresponding to the materials used and control. In the control group, specimens received no treatment, three different resin-based materials were used to coat enamel in other groups; giomer coating PRG Barrier Coat (PBC; Shofu, Kyoto, Japan), resin-modified glass-ionomer Clinpro XT Varnish (CXT; 3M ESPE, St. Paul, MN, USA); and two-step self-etch adhesive resin Clearfil SE Protect (SEP; Kuraray Noritake Dental, Tokyo, Japan). Two areas, namely coated (C), and uncoated (UC), were assigned on the polished enamel surface of each block. Half surface of each block was carefully treated with the materials in accordance with the instructions supplied by the manufacturers as listed in Table 1 for all groups that served as the C area while the other half stayed intact as UC area. The specimens were then stored in water for 24 h at 37 °C.

Table 1

Materials used in this study.

Material	Brand	Code (Lot#)	Composition	Application instruction	Hardness (MPa)
Giomer-based material	PRG Barrier Coat (Shofu, Kyoto, Japan)	PBC 051101	Base: glass powder, purified water, methacrylate monomer, S-PRG filler, phosphonic acid monomer Activator: methacrylate acid monomer, bis-MPEPP, carboxylic acid, TEGDMA, catalyzer.	Put one drop of activator into base and mix together. Apply thin layer of the mixture, Light-cure for 10 s.	157.7 ± 35.4
Resin-modified glass-ionomer	Clinpro XT Varnish (3M ESPE, St. Paul, MN, USA)	CXT N352376	Liquid: HEMA, water, camphorquinone, calcium glycerophosphate and polyalkenoic acid Paste: HEMA, Bis-GMA, water, initiators and fluoroaluminosilicate glass.	Apply acid etchant for 15 s with 35% phosphoric acid. Rinse with water. Apply air for 5 s. Mix paste/liquid components together rapidly for 15 s (2.5 min working time). Apply thin layer to tooth surface. Light cure for 20 s. Wipe the coating by a moist cotton applicator.	442.1 ± 46.5
Two-step, self-etch adhesive	Clearfil SE Protect (Kuraray Noritake Dental, Tokyo, Japan)	SEP Primer:00103A Bond: 00165B	Primer: MDP, MDPB, HEMA, hydrophilic dimethacrylate, water Bond: MDP, Bis-GMA, HEMA, dimethacrylate hydrophobic, di-camphorquinone, N,N-diethanol-p-toluidine, silanated colloidal silica, surface treated sodium fluoride.	Apply primer and leave for 20 s. Dry with gentle air flow. Apply bond. Air flow gently. Light-cure for 10 s.	223.2 ± 31.2

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