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Origin of Interfacial Droplets with One-step Adhesives

¹ Leuven BIOMAT Research Cluster, Department of Conservative Dentistry, School of Dentistry, Oral Pathology and Maxillo-Facial Surgery, Catholic University of Leuven, Kapucijnenvoer 7, B-3000 Leuven, Belgium;
² Department of Chemistry, Catholic University of Leuven, Celestijnenlaan 200G, B-3001 Heverlee, Belgium; and
³ Department of Biomaterials, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8525, Japan

Correspondence: ^* corresponding author, bart.vanmeerbeek{at}med.kuleuven.be

	ABSTRACT

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Contemporary one-step self-etch adhesives are often documented with interfacial droplets. The objective of this study was to research the origin of these droplets. Two HEMA-rich and one HEMA-free adhesive were applied to enamel and dentin, with the lining composite either immediately cured or cured only after 20 min. All one-step adhesives exhibited droplets at the interface; however, the droplets had two different origins. With the HEMA-free adhesives, droplets were located throughout the adhesive layer and were stable in number over time. With the HEMA-rich adhesives, the droplets were observed exclusively at the adhesive resin/composite interface, and their number increased significantly when the composite was delay-cured. Only the latter droplets caused a significant drop in bond strength after delayed curing. While the droplets in the HEMA-free one-step adhesives should be ascribed to phase separation, those observed with HEMA-rich adhesives resulted from water absorption from dentin through osmosis.

Key Words: adhesion • one-step self-etch adhesive • droplets • HEMA • osmosis • phase separation • diffusion • origin of droplets • water

	INTRODUCTION

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The application procedure of one-step self-etch adhesives (1-SEAs), in particular those that consist of one component, may take considerably less time compared with their multi-step counterparts. Especially in busy dental practices, this may be an important advantage. Nevertheless, ever since the commercialization of these 1-SEAs, many observations against their use have been reported in the literature.

They have repeatedly been reported to exhibit high permeability, resulting in water flow through the adhesive. This has been attributed to their rather hydrophilic nature and the lack of a cured hydrophobic layer lining the adhesive (King et al., 2005). Ultra-morphologically, the presence of so-called "water-trees" is assumed to be a manifestation of this permeability (Tay et al., 2004a). Also, scanning-electron-microscopic observations of blisters on top of 1-SEAs bonded to dentin (Chersoni et al., 2004), and underneath the adhesive resin on enamel after immersion in water (Tay et al., 2004b), have been considered to be the result of their high permeability. Apart from these SEM observations, other sorts of droplets and blisters have been observed. Tay et al. (2001, 2002a) found droplets near the interface between the adhesive layer and the lining composite in 1-SEAs when light-curing of the lining composite was delayed for 20 min. Droplets could also be found when chemical-curing composites were used in conjunction with 1-SEAs (Sanares et al., 2001). In addition, recent research revealed that droplets originating from a phase-separation reaction could remain trapped in the adhesive layer after curing (Van Landuyt et al., 2005). This reaction has been shown to occur as soon as the solvent starts to evaporate, and is clearly visible to the naked eye as the adhesive loses its translucency and becomes opaque.

The objective of this study was to research the occurrence and the origin of interfacial droplets with 1-SEAs. The hypothesis was that all interfacial droplets are the result of phase separation.

	MATERIALS & METHODS

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One HEMA-free (G-bond, GC, Tokyo, Japan) and one HEMA-rich commercial 1-SEA (Clearfil S³Bond, Kuraray, Tokyo, Japan) were selected. Both are one-component self-etch solutions with "mild" acidity (pH > 2). An experimental HEMA-rich version of G-Bond (G-Bond/HEMA) was also tested, the composition of which differed from that of the commercial G-Bond only by the addition of 36% of HEMA (Appendix Table 1).

The three-step etch-and-rinse adhesive (3-E&R) Optibond FL (Kerr, Danbury, CT, USA) was used as control.

Light Microscopy
After being dispensed onto a glass plate, all 1-SEA solutions were examined uncured for a phase-separation reaction and the presence of droplets by light microscopy (LM), as previously described (Van Landuyt et al., 2005).

Bond-strength Testing
Non-carious human third molars (gathered after informed consent was obtained from donors, according to a protocol approved by the Commission for Medical Ethics, KU Leuven) were stored in 0.5% chloramine/water at 4°C and used within 1 mo after extraction. To prepare dentin samples, we removed the occlusal crown third with a diamond saw (Isomet 1000, Buehler, Lake Bluff, IL, USA), thereby exposing a flat mid-coronal dentin surface. We produced a standardized bur-cut smear layer by removing a thin layer of the surface using a Micro-Specimen Former (University of Iowa, Iowa City, USA), equipped with a high-speed regular-grit (100 µm) diamond bur (842, KOMET, Schaumburg, IL, USA). For enamel, a flat surface was ground with the same bur at the buccal and lingual surfaces of the tooth. The adhesive was applied according to the instructions of the manufacturer. After the adhesive was light-cured, the teeth were assigned to a "delay" or a "non-delay" group. In the non-delay group, a layer of 1–2 mm of lining composite (Clearfil AP-X, Kuraray) was immediately applied on top of the adhesive and light-cured (Demetron Optilux500, 550 mW/cm² for 40 sec), and composite build-ups were made in 3 or 4 layers to a height of 5–6 mm. In the delay group, the first layer of composite was applied but not light-cured, and was protected from light. After 20 min, this first layer was cured, and the composite build-up was made. After 24-hour storage in distilled water (37°C), micro-specimens with an hourglass constriction at the interface were prepared so that we could determine the µTBS according to a protocol described previously (Van Landuyt et al., 2006). Kruskal-Wallis statistical analysis ( = 0.05) was performed. The mode of failure was determined by stereo-microscopy at 50x magnification. Representative fractured enamel/dentin and composite surfaces, exhibiting the most frequently observed failure mode and a µTBS close to the mean, were processed for field-emission-gun scanning electron microscopy (Feg-SEM; Philips XL30), by common specimen processing, as described previously (Perdigão et al., 1995).

To evaluate the effect of intrinsic water in dentin, we prepared additional dehydrated dentin surfaces. After removing the occlusal crown third and smear-layer preparation, we removed the root to obtain smaller specimens. These were subsequently dehydrated in ascending concentrations of ethanol (20, 50, 75, 90, and 100%), and air-dried in a dessicator. Clearfil S³Bond and G-Bond were applied, followed by application and light-curing of the composite with or without delay. To obtain fractured surfaces, we prepared micro-specimens after 24 hrs and tested them according to the µTBS protocol. The µTBS results of these specimens were not taken into consideration, because of the unreliable effect of dehydrated dentin. They were processed for SEM as described above.

TEM/SEM Interface Characterization
Both delay and non-delay specimens of all tested adhesives, on enamel and dentin, were prepared for TEM. Whereas flowable composite (Protect Liner F, Kuraray) was used for the specimens in the non-delay group, to ease specimen preparation, Clearfil AP-X composite was used for the delay group. As such, all TEM specimens were prepared similarly to the µTBS specimens in the delay group. Further specimen processing was according to the procedure described by Van Meerbeek et al.(1998). Non-demineralized ultra-thin sections were cut (Ultracut UCT, Leica, Heidelberg, Germany) and examined by TEM (Philips CM10). Additional adhesive-enamel/dentin interfaces, stained with 50 wt% ammoniacal silver-nitrate solution, were prepared as described by Tay et al.(2002b). Because samples of the delay group of Clearfil S³Bond and G-Bond/HEMA often fractured during TEM sectioning, the epoxy-resin-embedded tooth-resin interfaces that remained after diamond-knife ultramicrotomy were also examined by SEM.

	RESULTS

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We confirmed, by light microscopy, that all adhesive solutions were free of phase separation except for the commercial HEMA-free G-Bond.

When adhesives were bonded to enamel, no difference in bond strength could be observed between the delay and non-delay groups for all tested adhesives (Fig. 1). As to dentin, the bond strength of the HEMA-rich 1-SEAs Clearfil S³Bond and G-Bond/HEMA decreased significantly when light-curing of the composite was delayed for 20 min (Fig. 1). There was no difference in bond strength for the HEMA-free G-Bond and the 3-E&R Optibond FL (control). The drop in bond strength was partly due to an increased number of pre-testing failures (14 for Clearfil S³Bond and 9 for G-Bond/HEMA).

View larger version (31K): [in this window] [in a new window]   Figure 1. Box-whisker plot (min-[lower quartile-median-upper quartile]-max) of the µTBS to enamel (top) and dentin (bottom) (mean ± standard deviation; n = total number of specimens; ptf = pre-testing failure). The diamonds represent the mean µTBS. NS = not statistically significant, p < 0.05 indicates statistical significance. Superscript letters indicate significant differences (Kruskal-Wallis non-parametric statistical analysis).

View larger version (105K): [in this window] [in a new window]   Figure 2. Fracture analysis by LM and SEM (Appendix Table 2). SEM analysis confirmed the initial LM observations. Most frequently, the tested adhesives exhibited a mixed failure pattern. A striking shift was seen in the delay group of Clearfil S3Bond and G-Bond/HEMA, where failure occurred predominantly in the adhesive layer. Further SEM investigations showed that a large number of droplets within the adhesive layer of these HEMA-rich adhesives caused these fractures. SEM images of Clearfil S3Bond, G-Bond, and G-Bond/HEMA after µTBS are shown. a1 and a2 show a representative sample of the non-delay group of Clearfil S3Bond on dentin, with a1 the dentin side and a2 the composite side. Typical accumulations of small droplets (from 300 nm to 1.5 µm) can be seen in some areas. Notice the linear pattern parallel to the scratches of the smear layer (arrows). b1 and b2 show Clearfil S3Bond after delayed curing of the composite. In contrast to the non-delay group, almost the entire adhesive layer was affected by droplets, which had sometimes coalesced to larger droplets. No linear pattern was seen. c1 and c2, respectively, show G-Bond on dentin in the non-delay group and the delay group. c1 is a detail of a dentin side, showing the presence of droplets at different levels throughout the adhesive layer. In contrast to Clearfil S3Bond and G-Bond/HEMA, a mixture of both small and large droplets (from 0.5 to 10 µm) was observed. In the delay group (c2) (composite side of G-Bond on dentin), quantities of droplets comparable with those in the non-delay group were observed. d1 and d2 are images of G-Bond/HEMA on dentin in the non-delay and delay groups, respectively. d1 SEM revealed similar droplets in G-Bond/HEMA without delayed curing. After delayed curing (d2), similarly to Clearfil S3Bond, the adhesive layer was completely affected by droplets, which coalesced and had a disruptive effect. Abbreviations: Ar, Adhesive resin; Hy, Hybrid layer.

View larger version (115K): [in this window] [in a new window]   Figure 3. TEM and SEM interface analysis. TEM was the most appropriate tool to determine the exact location of the droplets at the interface with reference to the adhesive layer. However, because a great many samples failed during diamond-knife ultra-microtomy for TEM, the TEM blocks themselves were also prepared for SEM, after a very smooth surface was obtained by ultra-microtomy. (a) An SEM image of Clearfil S3Bond on dentin with a flowable composite (Protect Liner, Kuraray) applied without delay. Yet, along the adhesive resin-composite interface, a distinct line of droplets can be seen (arrows). (b,c) TEM images of Clearfil S3Bond applied on dentin, showing samples of the delay group. Both samples failed before being embedded in epoxy resin, since epoxy resin is observed between the composite (AP-X) and the adhesive resin. (b) Detail of the adhesive resin on dentin. Notice how the dentin surface is irregular, due to the scratches of the smear layer. The adhesive resin, however, does not follow this pattern of "valleys" and "peaks" and has an even upper surface. As a consequence, at the "peaks" of the smear layer, the thickness of the adhesive layer was greatly reduced. (c) Detail of the composite, which was detached from the dentin side. In the delay group, most often a small zone of droplets (in contrast to a line of droplets in the non-delay group) was found. (d) Overview of the adhesive layer of G-Bond in the delay group. This sample was dyed with silver-nitrate. Notice the presence of a large droplet in the adhesive layer. Some small droplets are visible near the top of the hybrid layer, and were stained with silver (arrows). However, it must be noted that silver-staining was most inconsistent, with some droplets and areas heavily stained and other droplets and locations remaining unstained. Notice how this section was made perpendicular to the smear-layer pattern, showing that the thickness of the adhesive layer was variable. Also notable was the absence of an oxygen inhibition layer (also in samples of the delay group of Clearfil S3Bond and G-Bond/HEMA), which was easily detectable in non-delay samples (not shown). This may be due to the use, in the delay group, of a viscous composite (Clearfil AP-X) that was applied with pressure, in contrast to the flowable composite that was applied without pressure in the non-delay group. (e) SEM of G-Bond/HEMA on dentin after delayed curing of the composite (AP-X). A zone of droplets similar to that found with Clearfil S3Bond delay can be seen. (f) SEM of Clearfil S3Bond applied to dehydrated dentin (non-delay group). No droplets could be found. Abbreviations: Ar, Adhesive resin; C, composite; E, Epoxy resin; Hy, Hybrid layer; Ud, Unaffected dentin.

View larger version (30K): [in this window] [in a new window]   Figure 4. Theoretical scheme illustrating the osmosis process in the non-delay and delay groups. A perpendicular section of dentin is shown. Due to the parallel scratches made by the diamond bur, the dentin surface is irregular and consists of "valleys" and "peaks". The upper part of the adhesive layer, however, does not follow this pattern and has an even upper surface. The uppermost part of the adhesive resin (1–3 µm) is not cured, due to oxygen inhibition. As a consequence, this "oxygen-inhibition layer" consisted mainly of uncured monomers. As such, this layer represents a zone with a "hypertonic" solution (high concentration of molecules and low concentration of water). The dentin and its intrinsic water (in the dentinal tubules and in smear crevices) represent a "hypotonic" area. Diffusion of water from the dentin through the cured adhesive layer will occur (osmosis), and the cured adhesive resin functions as a semi-permeable membrane. In HEMA-rich adhesives, uncured small HEMA-molecules must be important components of the oxygen-inhibition layer. The strong hydrophilic character and the small dimensions of this monomer explain why osmosis is fast and easy in HEMA-rich adhesives. In the non-delay group, water is able to reach the oxygen inhibition layer only in areas where the adhesive resin is minimal in thickness, which also explains the linear pattern of droplets according to the scratches of the smear layer. When the composite is cured after 20 min, water is able to diffuse, even in areas where the adhesive resin is maximal in thickness. The large accumulations of droplets have a severely weakening effect on the bond between the adhesive layer and the lining composite, resulting in low bond strengths. In HEMA-free adhesives, a similar osmotic process cannot be excluded. However, even if it occurs, it must be to a limited extent, since the bond strength is not reduced after delayed curing of the composite. HEMA-free adhesives do exhibit droplets, but, in contrast to HEMA-rich adhesives, these droplets are due to phase separation. Unlike the droplets in HEMA-rich adhesives, these droplets do not increase with time, but are usually larger in size and are located throughout the adhesive resin.

The exact location of the droplets with reference to the adhesive layer could be determined by TEM. The droplets with Clearfil S³Bond and G-Bond/HEMA were situated exclusively at the interface between the adhesive resin and the lining composite, whereas the droplets with the commercial HEMA-free G-Bond could be found throughout the adhesive layer. Whereas in the non-delay group the droplets in the HEMA-rich 1-SEAs often appeared in a single row at the interface, a small zone of droplets (2.5 µm) was observed after delay (Figs. 3c, 3e). Often, samples failed near this zone during processing.

In the delay samples, no evident oxygen-inhibition layer could be observed in the 1-SEAs, in contrast to the non-delay samples.

	DISCUSSION

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The bond strengths of the HEMA-rich adhesives Clearfil S³Bond and G-Bond/HEMA dropped significantly for dentin in the delay group. These µTBSs can be clearly correlated to the increased number of droplets that appeared when light-curing of the composite was delayed. These droplets were situated exclusively at the adhesive resin-composite interface. This finding is consistent with results from a previous study (Tay et al., 2002a). However, in that study, all tested 1-SEAs underwent a decrease in bond strength when light-curing was delayed, and no droplets were seen in the non-delay group.

Even though the commercial HEMA-free G-Bond also exhibited droplets, there was no drop in bond strength after delay. The fact that there was no increase in droplets after the 20-minute delay clarifies these stable µTBS results. In addition, in contrast to the HEMA-rich 1-SEAs, droplets could be observed on both enamel and dentin, and were located throughout the adhesive layer.

This indicates that the droplets with G-Bond have a different origin and should be attributed to a phase-separation reaction, when, upon evaporation of the solvent, water separates from the other adhesive ingredients (Van Landuyt et al., 2005). Since these droplets disappear only after several min, they can be trapped in the adhesive resin when cured. The fact that they could also be found on enamel confirms that they originate from phase separation.

Since neither Clearfil S³Bond nor G-Bond/HEMA exhibited droplets on a glass plate (LM), it is unlikely that the interfacial droplets with these 1-SEAs were caused by phase separation. In addition, the increase in number of droplets in the delay group indicates that their occurrence is time-dependent. To reveal the origin of these droplets, we included G-Bond/HEMA in the study. This adhesive did not exhibit phase separation, since the high concentration of HEMA kept the ingredients in solution (Van Landuyt et al., 2005). When one considers that this experimental adhesive differed only from the commercial G-Bond for HEMA, this low-molecular-weight monomer must have an important influence. The presence of water also plays a paramount role, since, on dehydrated dentin, no droplets were observed with both HEMA-rich adhesives, even after a 20-minute delay in curing. This indicates that the droplets with the HEMA-rich 1-SEAs contain water that was absorbed from the underlying dentin. This also explains why no droplets were found on enamel, which contains only minute amounts of water. These findings indeed indicate that an osmotic process caused these droplets, as suggested previously (Tay et al., 2002a). The adhesive layer would then function as a semi-permeable membrane, which allows for the transmission of small molecules, such as water (Chersoni et al., 2004). Water is transmitted from dentin following a diffusion process, characterized by initially rapid progression and slowing with time (exponential progress). This explains the time-dependent accumulation of the droplets, and also their presence in the no-delay group. The location of the droplets near the adhesive resin-composite interface indicates that a "hypertonic" (high concentration of molecules and low concentration of water) solution is present there. This area corresponds to the oxygen-inhibition layer, which exists of uncured monomers (Rueggeberg and Margeson, 1990). The low molecular weight and the strong hydrophilic character of HEMA explain why such osmosis occurs in HEMA-containing adhesives. Since the monomers in the oxygen-inhibition layer polymerize when the composite is light-cured, further water absorption should be arrested. A similar osmotic reaction in the HEMA-free G-Bond, however, cannot be excluded. This may be the reason why there seemed to be fewer droplets on dehydrated dentin. Nevertheless, the stable µTBS results show that osmosis is negligible with G-Bond.

In spite of some small droplets in the non-delay group, Clearfil S³Bond obtained a significantly higher bond strength than the other 1-SEAs. Hence, more research is warranted to assess the relevance of these droplets.

Droplets were also observed when 1-SEAs were combined with self-curing composites (Sanares et al., 2001). The resemblance of these droplets to those in Clearfil S³Bond and G-Bond/HEMA is striking, especially since they were also situated on top of the adhesive resin. An acid-base reaction between tertiary amines in the composite and acidic monomers in the adhesive has been suggested as a possible cause. However, the droplets may as well have been caused by osmosis, since the chemically cured composite was also cured after a delay period.

To conclude, this study showed that there are two distinct kinds of droplets with 1-SEAs, characterized by differences in appearance, location, and behavior. The hypothesis—that all droplets in 1-SEAs originate from phase separation—must therefore be rejected. The droplets in G-Bond occurred on both enamel and dentin, were situated throughout the adhesive layer, and did not increase in quantity after delayed curing. These droplets must be attributed to phase separation. Conversely, the droplets in the two HEMA-rich adhesives could be found only on dentin, were located near the adhesive resin-composite interface, and were generally smaller. When light-curing of the composite was delayed, their number increased significantly. This resulted in a fragile zone and, subsequently, in easy interfacial fracture. HEMA and the presence of water in the bonding substrate were found to be important influencing factors for these droplets. These droplets must be attributed to osmosis.

	ACKNOWLEDGMENTS

 
K.L. Van Landuyt is appointed as Aspirant of the Fund for Scientific Research of Flanders. We thank Kuraray and GC for providing the adhesives and composite material.

	FOOTNOTES

 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received for publication September 11, 2006. Revision received February 20, 2007. Accepted for publication March 29, 2007.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Dental Research, Vol. 86, No. 8, 739-744 (2007)
DOI: 10.1177/154405910708600810


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This Article Abstract Figures Only Full Text (PDF) Data Supplement Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Van Landuyt, K.L. Articles by Van Meerbeek, B. Search for Related Content PubMed PubMed Citation Articles by Van Landuyt, K.L. Articles by Van Meerbeek, B. Social Bookmarking                         What's this?