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Fluid Movement across the Resin-Dentin Interface during and after Bonding

¹ Division of Pediatric Dentistry, Hokkaido University, Graduate School of Dental Medicine, Kita 13, Nishi 7, Kita-ku, Sapporo 060-8586, Hokkaido, Japan;
² Department of Conservative Dentistry, Health Sciences University of Hokkaido, School of Dentistry, Ishikari-Tobetsu, Hokkaido, Japan;
³ Department of Conservative Dentistry, Faculty of Dentistry, University of Hong Kong, Hong Kong SAR, China;
⁴ Department of Operative Dentistry, University of Sacred Heart, Bauru, SP, Brazil;
⁵ Division of Cariology and Endodontology, Hokkaido University, Graduate School of Dental Medicine, Sapporo, Hokkaido, Japan; and
⁶ Department of Oral Biology and Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, GA, USA;

^* corresponding author, masanori-h{at}mue.biglobe.ne.jp

	ABSTRACT

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This study evaluated the extent of water penetration through resin-dentin interfaces before and after being sealed with adhesives. Four adhesive resin systems (2 total-etch adhesives and 2 self-etching primer adhesives) were used in this study. Dentin disks were placed in a split-chamber device, and in situ fluid movement across dentin was measured, with and without physiological pressure, during bonding procedures or 24 hrs after bonding. The fluid movement across dentin occurs via dentin tubules after acid-etching. Large outward or inward fluid shifts across dentin were observed during air-drying and light-curing for resin application. The amount of fluid movement across resin-bonded dentin when total-etch adhesives were used was significantly greater than that with self-etching adhesives. The milder acid-etching effects of self-etching primers may retain hybridized smear plugs within the tubules that reduce outward fluid flow, resulting in superior dentin sealing.

KEY WORDS: leakage • total-etch adhesive • self-etching primer • hydraulic conductance • smear plug

	INTRODUCTION

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Recent dental adhesive systems can be classified into two types: total-etch and self-etching adhesives. Total-etch adhesives seem to leave exposed collagen fibrils beneath an incompletely infiltrated dentin matrix (Spencer and Swafford, 1999; Yoshida et al., 1999), resulting in hydrolysis of collagen over long periods (Burrow et al., 1996; Hashimoto et al., 2000, 2003a, Hashimoto et al., b,c; De Munck et al., 2003). Moreover, the elution of unconverted monomers within the hybrid and bonding resin layers and their replacement by water cause deleterious effects to bonds for both total-etch and self-etching adhesives (Sano et al., 1999; Okuda et al., 2002; Hashimoto et al., 2002, 2003b, Hashimoto et al., c).

Nanoleakage studies with silver nitrate have shown the presence of water-filled regions and/or hydrophilic polymer domains within the hybrid and adhesive resin layers. These channels are thought to be responsible for water movement from the underlying hydrated dentin via leaky resin tags, through the adhesive layer to the adhesive-composite interface (Tay et al., 2002a,b, 2003; Tay and Pashley, 2003). Moreover, the extent of nanoleakage increased over 12 mos of water storage, due to water sorption (Tay et al, 2003). However, little research has attempted to correlate water penetration through the adhesive via nanoleakage channels. In addition, there has been no research evaluating fluid movement within the resin-dentin bonds during bonding procedures.

Therefore, the purpose of this study was to evaluate in situ fluid movement across the resin-dentin interface during and after bonding. The null hypotheses that were tested were: (1) that there is no difference in the permeability of dentin sealed with total-etch adhesive vs. self-etching adhesive systems, and (2) that there is no water movement across resin-dentin bonds during bonding procedures.

	MATERIALS & METHODS

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Dentin Disk Formation
Eighty unerupted human third molars were collected after the patients’ informed consent had been obtained under a protocol reviewed and approved by the Human Assurance Committee of the Medical College of Georgia. The teeth were stored in thymol-saturated isotonic saline at 4°C, and were used within 2 mos following extraction. Each tooth was sectioned perpendicular to its longitudinal axis by means of an Isomet saw (Buehler Ltd., Lake Bluff, IL, USA) under water, to create a dentin disk from mid-coronal crown. Each surface was ground with 600-grit SiC paper under running water for 30 sec. Disks were 0.3 mm thick, as measured to the nearest 0.01 mm by means of a digital micrometer.

Test Apparatus
The pulp sides of dentin disks were acid-conditioned by 35% H₃PO₄ for 15 sec for removal of the smear layer, leaving the smear layer on the upper surface intact. After being rinsed with water, the disk was placed in the split-chamber device (Pashley et al., 1984). The test area of each dentin disk was limited by identical silicone ‘O’-rings, giving a surface area of 0.28 cm² (Fig. 1). Fluid flow was measured by means of an automated apparatus (Flodec, DeMarco Engineering, Geneva, Switzerland) incorporating a glass capillary tube (0.7 mm, inside diameter) (Ciucchi et al, 1995). An infrared beam was passed through one side of the tube, and a photosensitive diode, positioned on the opposite side of the tube, detected movement of any air bubbles. Fluid movement was calculated as: fluid movement = Jv/At, where: Jv = fluid flow in µL, A = resin surface area in cm^–2, t = time in min, and fluid movement units = µL cm^–2 min^–1.

View larger version (30K): [in this window] [in a new window]   Figure 1. Schematic diagram of split-chamber device and water pathway. The water penetrates from the pulp side of the chamber device via dentinal tubules and then nanoleakage pathways within the hybrid layer or bonding resin. Finally, the water penetrates the gap between the silicone ring and bonding resin or resin composite layer. The gap is formed by polymerization shrinkage of resin, due to lack of adhesion between the resin and the silicone ring.

Bonding Procedures
For SB and PBNT, the dentin surfaces were etched with phosphoric acid (34% for SB and 37% for PBNT) for 15 sec and thoroughly washed in water. Excess water was blot-dried with an absorbent pellet, leaving the dentin surface visibly moist (wet-bonding). Two coats of adhesive were applied, and the liquid was then gently air-dried for 3 sec at a distance of 5 cm, which allowed the solvent to evaporate and form a slightly shiny adhesive film. The adhesive layer was then irradiated for 30 sec with a light-curing unit (Curing Light XL 3000, 3M/ESPE) with a light output not less than 600 mW/cm².

For CPB, the self-etching primer was applied and left undisturbed for 20 sec. The primer was air-dried for 3 sec to remove volatile solvents. The bonding adhesive was applied to the primed dentin surface for 10 sec and light-cured for 30 sec.

For APL, the self-etching adhesive was applied to the dentin twice, and left undisturbed for 30 sec. The surface was then gently air-dried for 3 sec at a distance of 5 cm, and the surface was then light-illuminated for 30 sec.

After bonding occurred, a short air blast was applied to the polymerized resin-bonded dentin surface, and about 10 mm³ of flowable resin composite (Protect Liner F, Kuraray) was placed on all of the resin-covered dentin surfaces and then light-illuminated for 30 sec.

Measurements of Fluid Movement
The experimental design involved measurement of convective fluid flow under a physiological hydrostatic pressure gradient (Ciucchi et al., 1995) of 20 cm H₂O for 10 min for measurement of the fluid conductance of smear-layer-covered dentin under a film of water. Subsequently, phosphoric acid was applied for 15 sec without a simulated pulpal pressure (0 cm H₂O) and rinsed, and the fluid conductance was then re-measured for 10 min under a pulpal pressure of 20 cm H₂O, with water covering the top of the dentin to prevent any evaporative fluid movement. After 10-minute measurements, the water covering the dentin was removed, and a total-etch adhesive was applied as previously described, with and without pressure, for 4 min. The water permeability of the resin-dentin interface was measured for another 10 min at 20 cm H₂O. For the self-etching systems, the fluid conductance of smear-layer-covered dentin was measured, but the acid-etching step was omitted. The dentin was bonded with the self-etching adhesives as previously described at 0 cm H₂O.

After being light-cured, the resin-sealed dentin specimens in the split-chamber devices were stored without pressure (0 cm H₂O) for 1 hr. The pulpal pressure was then raised to 20 cm H₂O while the specimens were stored in a 37°C water bath. After 24 hrs of water storage, the fluid conductance was re-measured for 10 min at 20 cm H₂O.

Statistics
We used two-way ANOVA and Tukey’s multiple-comparison tests to compare fluid movement for each group (p < 0.05, n = 10 for each group). We used regression analysis and Pearson’s correlation coefficient to examine the correlation between fluid flow across acid-etched dentin and that of resin-bonded specimens of total-etch adhesives (n = 10 for each group).

	RESULTS

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In situ Fluid Movement
The in situ water movement across dentin during bonding procedures in a single specimen is shown in Fig. 2. There was a slight outward movement of water across the smear-layer-covered dentin (Fig. 2). When the smear layer was removed by acid (acid-etched in Figs. 2A, 2B), there was a large (ca. 10-fold) increase in outward water flow. Then, when the filtration pressure was reduced to zero, the fluid movement fell to zero. During bonding procedures, the air-blasts induced a large outward fluid flow (labeled Air in Fig. 2). In contrast, subsequent light-illumination of the adhesive produced a large inward fluid shift that was repeated again following application of the resin composite (labeled Light in Fig. 2). After polymerization of PBNT, the outward fluid flow was similar to that seen after acid-etching just prior to bonding. In SB-bonded dentin, the post-bonding fluid flow was only 40% of the pre-bonded value (Fig. 2B). After the APL bonding resin was light-cured, the outward fluid flow rate was twice as high as that seen in smear-layer-covered dentin (Fig. 2C), although the value was lower than the post-bonded value for PBNT or SB. The post-bonded permeability of CPB was near zero (Fig. 2D). Similar fluid movement in response to the air-blasts or light-illumination was recorded in the specimens bonded under 20 cm H₂O pressure.

View larger version (41K): [in this window] [in a new window]   Figure 2. Outward (–) and inward (+) fluid movement across dentin during bonding procedures. The bonding procedures conducted in the absence of pulpal pressure (0 cm H2O) included air-blast, application of adhesive, and light-curing. Negative values indicate outward fluid movements that were recorded when air-blasts (Air) were applied at the surface of smear-layer- or adhesive-resin-covered dentin. Positive values indicated the inward fluid movement that was observed during light-curing (Light) for polymerization of the adhesive resins or resin composites. Smear = smear-layer-covered dentin under 20 cm H2O pulpal pressure; Acid-etched = acid-etched dentin under 20 cm H2O pulpal pressure; Resin = storage of resin-bonded specimens under 20 cm H2O pulpal pressure; Air = air-blast; Light = light illumination.

View larger version (24K): [in this window] [in a new window]   Figure 3. Summary of changes in the fluid movement of smear-layer-covered dentin before and after being bonded with total-etch vs. self-etch adhesives, with and without pulpal pressure. The fluid movement of smear-layer-covered dentin was not significantly different (p > 0.05) among all groups. After dentin was acid-etched with phosphoric acid, the fluid movement increased approximately 20-fold, due to the removal of the smear layer in the PBNT and SB groups. The water movement across self-etching adhesive bonds (APL or CPB) made under 0 cm H2O pulpal pressure was significantly lower (p < 0.05) than that of total-etch adhesives (PBNT or SB) 24 hrs after being bonded. Smear = smear-layer-covered dentin; Acid-etched = acid-etched dentin; Bonded 0 min = resin-/dentin-bonded specimens measured immediately after being bonded; Bonded 24 hrs = resin-/dentin-bonded specimens 24 hrs after being bonded. Values are means ± standard deviation. Groups that were significantly different are indicated by different lower-case letters (two-way ANOVA and Tukey’s multiple-comparison tests, p < 0.05, n = 10 for each group).

For APL (Fig. 3C), the fluid movement of the specimens measured after 0 min and 24 hrs following bonding without pressure was significantly lower (p < 0.05) than that of the specimens bonded under pulp pressure. The fluid flow of APL-bonded specimens 24 hrs after being bonded was significantly lower (p < 0.05) than smear layer values (Fig. 3). The permeability immediately after bonding occurred was significantly lower (p < 0.05) than that of the acid-conditioned dentin surfaces of total-etch adhesives. For specimens bonded with CPB (Fig. 3D), the permeability of resin-dentin-bonded specimens was significantly lower (p < 0.05) than that of smear-layer-covered dentin bonded under no pressure. However, when bonding occurred under 20 cm H₂O, the fluid flow across CPB was similar to that of smear-layer-covered dentin. The fluid movement of the bonds produced by both self-etch adhesive systems created without pulpal pressure was significantly lower (p < 0.05) than that of total-etch adhesives.

Regression Analysis
The results of regression analysis between the permeability of acid-conditioned dentin and that of resin-dentin bonds measured immediately and after 24 hrs are shown in the Table. The permeability of resin-bonded specimens correlated positively with that of acid-etched values (p < 0.05) in all groups.

	DISCUSSION

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Although the adhesive resins apparently bonded to dentin, fluid flow was recorded through or around the bonded interfaces after bonding occurred for all tested adhesives (Figs. 2, 3), although it was much greater for total-etch adhesives. Moreover, this study clearly showed inward fluid movement across dentin induced by light-illumination of adhesive or resin composite, and outward fluid movement produced by air-blasts during solvent evaporation (Fig. 2). The former effect is presumably due to thermal expansion of dentinal fluid, while the latter is due to evaporation of water from both the adhesive and the underlying dentin (Matthews et al., 1993). Although these outward and inward fluid movements were transient events, these fluid shifts may be large enough to create water trees that are thought to contribute to degradation of resin-dentin bonds (Tay et al., 2003). In addition, such water trees may have facilitated subsequent water sorption during the next 24 hrs. Large fluid shifts during bonding, especially seen in acid-etched dentin, may permit water from dentin to mix with the hydrophilic comonomers during evaporation of solvent and during light-curing, creating nanoleakage pathways within the adhesives (Fig. 2).

The etching-effects of self-etching primers depend on the concentration and pH of their acidic monomers (Tay and Pashley, 2001; Chan et al., 2003). The mild etching effect of the self-etching primer (i.e., CPB) leaves residual mineral crystals within the hybrid layer and retains smear plugs in the orifices of dentinal tubules (Tay et al., 2002b). Published reports have shown that APL completely dissolved the smear layer and smear plugs and formed thick hybrid layers similar to those formed in phosphoric-acid-conditioned dentin (Frankenberger et al., 2001; Pashley et al., 2002). However, the fluid movement across APL-bonded specimens 24 hrs after being bonded without pressure was lower than that of the 2 total-etch adhesives (Fig. 3), even though its depth of etching and hybrid layer thickness were similar to those of the total-etch adhesive systems. Because self-etching systems such as APL and CPB do not utilize a rinsing step, the interfibrillar spaces in the walls of etched tubules are not filled with water, as when total-etch adhesives are used. The presence of water in interfibrillar spaces probably dilutes and interferes with resin infiltration in total-etch systems. This would not occur in self-etch adhesives that do not use a water-rinsing step. The extremely low rate of fluid flow through CPB-bonded specimens (Fig. 3) is thought to be due to the use of a separate, solvent-free, relatively hydrophobic adhesive layer placed over the hydrophilic primer as a sealer (Tay and Pashley, 2003a).

It is well-known that there is often a discrepancy between the depth of acid-etching and resin infiltration into exposed collagen webs (Spencer and Swafford, 1999; Yoshida et al., 1999). It may be difficult fully to penetrate water-filled interfibrillar spaces in the collagen network and fill them with adhesive resin. However, fluid movement from dentin across bonds after 24 hrs was lower than the zero-hour values (Fig. 3), indicating that water sorption may swell the resin tags and the overlying resin. This would decrease convective fluid flow across bonded interfaces, even though it might increase nanoleakage as long as those voids were not continuous.

Immediately after bonding occurred, or 24 hrs later, specimen fluid flow prior to bonding correlated with that after bonding for total-etch adhesives (Table). If tubules were perfectly sealed by resin tags, there would be no such significant correlation. This provides additional evidence that the water penetrated the adhesive interface from unsealed dentinal tubules in specimens bonded with total-etch adhesives. Incomplete hybridization of resin tags to tubule walls may permit fluid movement from dentinal tubules to adhesive interfaces in total-etch adhesives (Elgalaid et al., 2004). However, self-etching primers (e.g., CPB) that retain smear plugs within the tubules create superior dentin sealing. There was no correlation between pre- and post-bonding fluid flow rates with self-etching systems (data not shown). Based on these results, we must reject both null hypotheses.

In summary, this study revealed that fluid flow across dentin bonded with self-etching adhesive systems was lower than that of total-etch adhesives. The inward and outward water movement created by air-blasts or light-illumination during bonding procedures may contribute to nanoleakage or water tree formation.

	ACKNOWLEDGMENTS

 
This work was supported by USPHS grants DE 014911 and DE 015306 (to DHP) from the National Institute of Dental and Craniofacial Research. The materials (Single Bond and Adper Prompt L-Pop, 3M/ESPE; Prime & Bond NT, Dentsply/DeTrey; and Clearfil Protect Bond, Kuraray Co., Ltd.) used in this study were generously supplied by their manufacturers. None of the investigators received any remuneration in the form of consultation fees or any other arrangements.

	FOOTNOTES

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

Received January 14, 2004; Last revision July 2, 2004; Accepted August 30, 2004

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This Article Abstract Figures Only Full Text (PDF) Appendix Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (37) Citing Articles via Google Scholar Google Scholar Articles by Hashimoto, M. Articles by Pashley, D.H. Search for Related Content PubMed PubMed Citation Articles by Hashimoto, M. Articles by Pashley, D.H.