Relationship between microtensile bond strength and submicron hiatus at the composite–dentin interface using CLSM visualization technique

Abstract

Objectives

“Submicron hiatus” represents a potential space between the base of the collagenous network and the mineralized dentin when it is acid etched for bonding. This study evaluated the relationship between microtensile bond strength (μTBS) and occurrence of submicron hiatus formations at the resin–dentin interface using the same specimens.

Methods

Resin–dentin bonded micro-specimens (sticks with a size of 300 μm × 300 μm × 8 mm) were prepared using one of two material combinations (group I: Syntac Classic/Tetric Ceram Cavifil: n = 51 group II: Prime & Bond NT/Tetric Ceram Cavifil: n = 56). After labeling the primer component with a tiny amount of rhodamine-B-isothiocyanate, submicron hiatus formations were imaged nondestructively using a confocal laser scanning microscope (CLSM). Subsequently specimens were subjected to a μTBS test.

Results

For the influence of submicron hiatus formations on μTBS with the Syntac Classic group, the nonparametric Spearman’s correlation was −0.329 at p = 0.02. For the Prime & Bond NT group, the nonparametric Spearman’s correlation was −0.356 at p = 0.007. Analyzing the effect of submicron hiatus on without discriminating by group resulted in a Spearman’s correlation coefficient of −0.341 at p = 0.001; μTBS and quality of hybrid layer showed a correlation coefficient of 0.849 at p = 0.001, and μTBS and quality of tag formation showed a correlation coefficient of 0.474 at p = 0.001.

Significance

The degree of submicron hiatus formations had an influence on microtensile bond strength for both the Syntac Classic and the Prime & Bond NT group.

Introduction

Attaching composite to tooth substances, especially dentin, is well established in dentistry . New materials are able to produce a durable connection between composite and tooth substances. Tensile bond strength of adhesive systems regarding adhesion to dentin depends most on the so-called hybrid layer . Thickness of hybrid layer formation is 1–8 μm and it consists of resin penetrating into the organic structure of the dentin (collagenous network), which is exposed using acid-etching techniques (e.g. phosphoric acid, 37.5%). The tensile bond strength of an adhesive system is mostly influenced by the hybrid layer, followed by resin tags in the dentinal tubuli and finally by chemical bonding .

In previous studies, formations of a so-called submicron hiatus have been described . This structure ( Fig. 3 ) represents a potential space between the bottom of the collagenous networks and mineralized dentin when performing acid-etching techniques for bonding purposes and has to be divided from complete breaks of specimens. In the first case, tag formations are still connected between dentin and adhesive layer, while in the last case, these formations are broken, too.

It is reported, that collagenous fibres are destroyed, while some others do remain embedded in resin connected to un-demineralised dentin . Until now, these spaces were only observed in studies applying scanning electron microscopy (SEM). In contrast, this phenomenon has never been identified in studies using transmission electron microscopy (TEM) for evaluating the dentin hybrid layers. TEM requires an embedding of the specimens while SEM can only show the surface details brought into relief by an etching process. Preparing dentin-etched specimens for SEM, a drying procedure (e.g. critical-point-drying) has to be performed. Hence, submicron hiatus formations have been discussed to be an artifact of desiccation while preparing specimens for SEM observation .

So far, not very much is known about submicron hiatus formation. Furthermore it is not known if it is linked with reduced bonding ability. Therefore, the objective of the present study was to image and analyze the adhesive–dentin junction nondestructively without any kind of drying procedure before fracturing the very same specimens performing a modified microtensile bond strength (μTBS) test, reducing the cross-section size of the specimens to less than 1/10 of a square millimeter.

Materials and methods

Preliminary testing

Prior to the experiments, 5 randomly selected teeth were proceeded as described above, but only sectioned perpendicular once per tooth and not cut into beams. These hemi-sections were subjected to CLSM observation. This was performed to pick up information regarding occurrence of submicron hiatus formations or any other kind of leakage. Furthermore, this procedure was undertaken to guarantee observation of faultless adhesive interfaces.

Preparation of the specimens

Human third molars without carious lesions were kept in 50% ethanol for a maximum of one week to avoid microbial contamination. This storage medium was chosen because it has only minor effects on the dentin . Prior to the experiments, teeth were cleaned using scalers and put into water for 24 h at 20 °C. Subsequently, the teeth were glued (Renfert Cyanoacrylate, Hilzingen, Germany) into a microtome saw (Leica 1600, Leica Microsystems, Wetzlar, Germany) upside down ( Fig. 1 A ). The first cut was near the highest pulp horn ( Fig. 1 B) to remove the roots, leaving the coronal crown segment. To obtain a crown segment free of pulp tissue, parallel horizontal slicing was continued until no pulp cave remained in the dentin. Then the segment was removed from the acrylic ( Fig. 1 C). Finally, the residual crown segment was divided into two dentin discs using the microtome saw ( Fig. 1 D and E). To achieve two identical specimens from one tooth the upper dentin disk was inverted to expose the mid-coronal dentin for processing ( Fig. 1 F).

Fig. 1
Sequence of specimen preparation.

Both dentin discs were acid etched with 36% phosphoric acid for 15 s (Conditioner 36, Dentsply, DeTrey, Konstanz, Germany, LOT: 0209001571), rinsed with water and bonded while the etched surfaces were moist with water . Both adhesives were processed according to the manufacturer’s instructions and light cured (650 mW/cm 2 ) for 20 s, with the addition of a very tiny amount of rhodamine-B-isothiocyanate into the primer component of the adhesive systems before its application . The following material combinations were used to attach some increments of composite (each of about 1 mm) to the mid-coronal dentin surface of the discs: (i) for disc #1 Syntac Classic (Ivoclar Vivadent, Ellwangen, Germany; primer LOT: E030561; adhesive LOT: E 30794; Heliobond LOT: E30212) and the composite Tetric Ceram Cavifil (Ivoclar Vivadent, Ellwangen, Germany, LOT E54847), (ii) for disc #2 Prime & Bond NT (Dentsply DeTrey, Konstanz, Germany, LOT: 0005216) and the composite Tetric Ceram Cavifil (Ivoclar Vivadent, Ellwangen, Germany, LOT E54847). Each increment was light cured for 40 s. The flattened top of the last increment of resin composite was glued to a perpendicular cube of transparent acrylic (Paladur transparent, Kulzer, Wehrheim, Germany) to facilitate the attachment of the assembly to the microtome saw ( Fig. 1 G and H). The resin–dentin end of the assembly was cut into multiple parallel slabs with a thickness of 300 μm ( Fig. 1 I). After rotating the assembly about 90°, another series of parallel cuts were made in intervals of 300 μm ( Fig. 1 J). To remove the resin–dentin sticks from the acrylic cube a final cut was performed within the composite buildup ( Fig. 1 K) resulting in resin–dentin sticks with a diameter of 300 μm × 300 μm and a length of 6–8 mm ( Fig. 1 L and Fig. 2 ). The specimens were kept humid at all time.

Fig. 2
Four specimens (SEM-picture; 17.7× magnification) dark parts = dentin, light parts = composite. Differences in specimen width could be detected in the further analyzing progress by CLSM.

Evaluation of submicron hiatus, tag formation and hybrid layer

To visualize submicron hiatus and bonding morphology, rhodamine-B-isothiocyanate (Merck, Darmstadt, Germany) was mixed with the primer in a concentration of approximately 0.1% prior to application procedure. The fabricated sticks were mounted onto microscope slides (Histobond, Marienfeld, Lauda-Königshofen, Germany) and documented using a confocal laser scanning microscope (CLSM; Leica Diaplan, Bensheim, Germany). Occurrence of submicron hiatus was scored on an arbitrary scale by one experienced examiner (TP). The shape of the hybrid layer and tag formation was evaluated as secondary outcome variables by the same examiner again using an arbitrary scale-model (3 categories). The criteria applied for evaluation are listed in Table 1 . Two opposite sides per stick were examined independently and each specimen was characterized by the mean of both values for submicron hiatus, hybrid layer, and tag formation, respectively ( Fig. 3 ).

Table 1
Criteria applied for examination of the specimens.
Tags Category 1 Low; only few, inhomogeneous tags, narrow basis
Category 2 Medium; average amount, quite homogeneous tags, average basis
Category 3 Good; many homogeneous tags, broad basis
Hybrid layer Category 1 Low; inhomogeneous shape
Category 2 Medium; average shape and homogeneity
Category 3 Good; homogenous and regular shape
Submicron hiatus Category 0 None
Category 1 Locally isolated
Category 2 <50% of the specimen a
Category 3 >50% of the specimen a

a Percentage of the hybrid layer.

Fig. 3
Submicron hiatus formation (CLSM picture; fluorescence mode; arrows). The adhesive layer shows a strong fluorescent signal due to added dye before application. The tags are intact. Some penetrated zone at the bottom of the collagenous network remains faultless and can be detected as continuous reddish line.

Microtensile bond strength (μTBS) test

By employing CLSM for evaluation specimens could be examined nondestructively. Thus, the identical specimens could be used for the following microtensile bond strength (μTBS) test. Specimens displaying air bubbles or artifacts near the hybrid layer during CLSM observation were rejected from the μTBS test. The test was performed by an examiner who was blinded to the results of CLSM examination (PFD) and each specimen was randomly given a number. Because of the very small diameter of the sticks a modified μTBS test was applied to fracture the specimens. Small acrylic slices were used as jigs to fix the bonded specimens (cyanoacrylate) to the tensile testing machine (Zwicki 1120, Zwick, Ulm, Germany). Tensile testing was performed with a crosshead speed of 1 mm/min. Finally, the μTBS values were decoded to permit correlation to submicron hiatus morphology.

To guarantee reliable results, another fracturing test was performed to examine the influence of the fluorescent dye on μTBS. Two groups of specimens were formed ( n = 25 each). Both groups were prepared and tested as described above, but one group was not exposed to the fluorescent dye rhodamine-B-isothiocyanate.

Statistical analysis

Statistical analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). Values for μTBS, submicron hiatus, tag formation and quality of hybrid layer were analyzed descriptively. The data for μTBS showed a normal distribution; t -test for independent groups was used to compare the mean of both material combination groups. To test the differences in ranks for tag formation, submicron hiatus, and hybrid layer and to assess the influence of the fluorescent dye on μTBS nonparametric Mann–Whitney U -test was applied. Scatter plots together with Spearman’s correlation coefficient were used to assess the relationship between submicron hiatus and μTBS as primary outcome variable and of tags and hybrid layer on μTBS as secondary outcome of this study.

Materials and methods

Preliminary testing

Prior to the experiments, 5 randomly selected teeth were proceeded as described above, but only sectioned perpendicular once per tooth and not cut into beams. These hemi-sections were subjected to CLSM observation. This was performed to pick up information regarding occurrence of submicron hiatus formations or any other kind of leakage. Furthermore, this procedure was undertaken to guarantee observation of faultless adhesive interfaces.

Preparation of the specimens

Human third molars without carious lesions were kept in 50% ethanol for a maximum of one week to avoid microbial contamination. This storage medium was chosen because it has only minor effects on the dentin . Prior to the experiments, teeth were cleaned using scalers and put into water for 24 h at 20 °C. Subsequently, the teeth were glued (Renfert Cyanoacrylate, Hilzingen, Germany) into a microtome saw (Leica 1600, Leica Microsystems, Wetzlar, Germany) upside down ( Fig. 1 A ). The first cut was near the highest pulp horn ( Fig. 1 B) to remove the roots, leaving the coronal crown segment. To obtain a crown segment free of pulp tissue, parallel horizontal slicing was continued until no pulp cave remained in the dentin. Then the segment was removed from the acrylic ( Fig. 1 C). Finally, the residual crown segment was divided into two dentin discs using the microtome saw ( Fig. 1 D and E). To achieve two identical specimens from one tooth the upper dentin disk was inverted to expose the mid-coronal dentin for processing ( Fig. 1 F).

Nov 30, 2017 | Posted by in Dental Materials | Comments Off on Relationship between microtensile bond strength and submicron hiatus at the composite–dentin interface using CLSM visualization technique
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