Effects of coating materials on nanoindentation hardness of enamel and adjacent areas

Abstract

Objectives

Materials that can be applied as thin coatings and actively release fluoride or other bioavailable ions for reinforcing dental hard tissue deserve further investigation. In this study we assessed the potential of resin coating materials in protection of underlying and adjacent enamel against demineralization challenge using nanoindentation.

Methods

Enamel was coated using Giomer (PRG Barrier Coat, PBC), resin-modified glass-ionomer (Clinpro XT Varnish, CXT), two-step self-etch adhesive (Clearfil SE Protect, SEP) or no coating (control). After 5000 thermal cycles and one-week demineralization challenge, Martens hardness of enamel beneath the coating, uncoated area and intermediate areas was measured using a Berkovich tip under 2 mN load up to 200 μm depth. Integrated hardness and 10-μm surface zone hardness were compared among groups.

Results

Nanoindentation and scanning electron microscopy suggested that all materials effectively prevented demineralization in coated area. Uncoated areas presented different hardness trends; PBC showed a remarkable peak at the surface zone before reaching as low as the control, while CXT showed relatively high hardness values at all depths.

Significance

Ion-release from coating materials affects different layers of enamel. Coatings with fluoride-releasing glass fillers contributed to reinforcement of adjacent enamel. Surface prereacted glass filler-containing PBC superficially protected neighboring enamel against demineralization, while resin-modified glass-ionomer with calcium (CXT) improved in-depth protection. Cross-sectional hardness mapping of enamel on a wide range of locations revealed minute differences in its structure.

Introduction

Dental enamel is the hardest tissue of the human body but susceptible to dissolution of the mineral phase by acid. In the caries process, acid produced from bacterial metabolism diffuses into enamel and then dentin, and dissolves the mineral. White spot lesions are the early stage of caries development characterized by an enamel surface zone with subsurface demineralization . Once this surface is broken due to continued effect of caries development, the lesions are cavitated and generally need to be restored. On the other hand, dental erosion is different from caries; it is defined as the chemical dissolution of dental enamel without bacterial involvement due to effects of acidic foods and drinks and considered as an increasingly common problem among different ages . Subjects with gastric disorder may also suffer from sever loss of tooth enamel caused by acid reflux .

Emerging concepts in management of oral health involve preventive strategies as well as new approaches to protect the teeth against demineralization, and to provide non-surgical management options for early lesions . Tooth surface coverage appears to be an immediate, simple and effective way to protect at-risk enamel against acid. Historically, pit and fissure sealants in newly erupted teeth have been well known to decrease caries prevalence . Areas with frequently extensive plaque accumulations adjacent to bonded orthodontic brackets have also been suggested to benefit from extending proper coating materials .

In addition to physical protection effects, newly developed dental resins may act as a reservoir of bioactive ingredients such as fluoride (F) and calcium (Ca) ions or other elements . F-releasing sealants have been suggested to provide additional caries inhibition effect, since fluoride inhibits demineralization and favors the remineralization processes . However, the potential advantages of newly developed thin coating materials that release F or other ions are unknown and deserve further investigation .

Enamel consists of 95 wt.% minerals, mainly an impure calcium hydroxyapatite (Ap) crystals (100–1000 nm in length, 25–90 nm in thickness) make up larger formations as prisms (3–5 μm in diameter) . The microstructure of enamel has been adapted to withstand mechanical and abrasive stresses. Loss of mineral content makes enamel vulnerable to deformation under mechanical load . Moreover, enamel may be physically reinforced through remineralization and improvements in crystalline structure depending on the composition of the surrounding environment, such as degree of saturation with regard to the minerals . In the dental literature, transverse microradiography (TMR) method has been widely employed to estimate the mineral content of dental hard tissue based on their radio opacity . The cross-sectional microhardness measurement has been traditionally accepted as an alternative method to evaluate enamel in de/remineralization studies . Nanoindentation (NI) technique has enabled investigations of local mechanical properties of materials under various loading regimes based on load displacement data of indentations on submicron scale . Measurement of hardness by this technique has been suggested as advantageous over the conventional microhardness test methods for its high resolution of force and accurate indent positioning. This technique has been employed in assessing enamel erosion and demineralization/remineralization , and has shown a good sensitivity to hardness changes at different depths of enamel .

Hardness mapping of enamel beneath and adjacent to the bioactive materials can reveal information on potential benefits of the ions and compounds released from these materials. Thus, the aim of current laboratory study was to evaluate the effect of resin coating materials on nanoindentation hardness of coated enamel and adjacent area after demineralization challenge. The null hypotheses proposed were that covering enamel by resin material does not influence hardness of the enamel and adjacent area, and that there were no differences among the materials investigated.

Materials and methods

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.

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
Abbreviations : HEMA, 2-hydroxyethyl methacrylate; Bis-GMA, bisphenol-A-diglycidyl methacrylate; S-PRG, surface pre-reacted glass inomer fillers; Bis-MPEPP, 2,2-Bis[4-(2-methacryloyloxyethoxy)phenyl] propane; TEGDMA, triethyleneglycoldimethacrylate; MDP, 10-methacryloyloxydecyl dihydrogen phosphate; MDPB, 12-methacryloyloxydodecyl pyridinium bromide.

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.

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 ).

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.

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 .

Fig. 2
(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.

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).

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).

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.

Materials and methods

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.

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
Only gold members can continue reading. Log In or Register to continue

Nov 23, 2017 | Posted by in Dental Materials | Comments Off on Effects of coating materials on nanoindentation hardness of enamel and adjacent areas
Premium Wordpress Themes by UFO Themes