d e n t a l m a t e r i a l s 2 4 ( 2 0 0 8 ) 457463

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Crystal growth by uoridated adhesive resins

Masanori Hashimoto , Kouichi Nakamura, Masayuki Kaga, Yasutaka Yawaka
Department of Dentistry for Children and Disabled Persons, Graduate School of Dental Medicine, Hokkaido University, Kita 13, Nishi 7, Kita-ku, Sapporo 060-8586, Hokkaido, Japan

a r t i c l e
Article history:

i n f o

a b s t r a c t
Objective. This investigation was carried out to evaluate the long-term effects of uoridereleasing adhesive resins on structural changes in standardized uid-lled gaps simulating microleakage between the materials and the tooth surface in vitro. Methods. Three commercially available uoride-releasing resin adhesives (One-Up Bond F, OptiBond Solo, and Reactmer Bond) were used in this study. Cured disks of resin adhesive were placed over at human tooth surfaces (enamel and dentin), separated by a standardized 40 m interfacial gap and stored in distilled water for 24 h (control group) or 1000 days

Received 17 February 2006 Received in revised form 2 February 2007 Accepted 4 April 2007

Keywords: Crystal growth Fluoride-releasing resin Long-term Scanning electron microscope Laser-Raman spectroscope

(experimental group). After 1000 days of water storage, the resins were detached from the teeth and the opposing surfaces were examined by scanning electron microscopy (SEM). In addition, chemical structural analysis was performed by laser-Raman spectroscopy. Results. The SEM microphotographs showed numerous crystal types on the enamel, dentin, and resin surfaces after 1000 days of water storage for OptiBond Solo and Reactmer Bond. However, there was no crystal formation in the control specimens and the aged specimens of One-Up Bond F. Raman analysis showed several peaks (463, 618, and 990 cm1 ) from the crystals of OptiBond Solo that were not identied in the enamel, dentin, or cured resin. Signicance. In conclusion, two of the three tested uoride-release resin adhesives (OptiBond Solo and Reactmer Bond) have the ability to induce crystal growth within gaps between the adhesive and teeth in long-term water storage. These results suggest that the two adhesive resins have self-reparative ability with regard to bond leakage. 2007 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

Glass ionomer cements are widely used in dentistry as liners or restorations, but have limited clinical use because of low wear resistance, brittleness, and low bond strength [15]. However, it is well-known that the rates of secondary caries development and marginal discoloration of glass-ionomer cements are lower than those of composite resins. Secondary caries development has been shown to be the major cause of failure of dental restorations [6,7]. Recently, many studies have focused on the effect of uoride on caries prevention. Glassionomer cements are potentially anticariogenic due to their

long-term uoride release. In an effort to develop a more complete understanding of the effects of glass-ionomer cements, a number of studies have provided evidence that uoride effectively prevents caries development by inhibiting demineralization and enhancing remineralization of enamel and dentin in vitro [8,9], and also by having anti-bacterial effects [10]. Recent studies have shown the mineralization of an apatite layer on bone and dentin using glass-ionomer cement containing bioactive glass [1114]. Most recently developed resin adhesives contain uoride with the aim of achieving the same purpose as uoride in glass-ionomer cement. However, studies have shown that the anticariologic and bacterial

Corresponding author. Tel.: +81 11 706 4292; fax: +81 11 706 4307. E-mail address: masanori-h@mue.biglobe.ne.jp (M. Hashimoto). 0109-5641/$ see front matter 2007 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dental.2007.04.014

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Table 1 The three tested adhesives were a total-etch adhesive (OptiBond Solo) and two-bottled self-etching primer adhesives (One-Up Bond F and Reactmer Bond) that contained uoride Material (manufacturer)
Manufactures and chemical formulations of the three adhesives tested Oue-Up Bond F (Tokuyama Co. Ltd., Tokyo, Japan)

Resin adhesive
HEMA, MAC-10, MMA, water, ller, coumarin dye, uoroaluminosilicate glass, photoinitiator HEMA, Bis-GMA, GPDM, ethanol, ller, barium aluminoborosilicate glass, photoinitiator HEMA, 4-AET, 4-AETA, UDMA, F-PRG llers, uoroaluminosilicate glass, water, acetone, photoinitiator

OptiBond Solo (Kerr Corp., Orange, CA, USA)

Reactmer Bond (Shofu Inc., Kyoto, Japan)

AbbreviationsBis-GMA: bisphenol-glycodyl methacrylate; HEMA: 2-hydroyxylethyl methacrylate; MMA: methyl methacrylate; MAC-10: 11 methacryloxy-1,1-undecanedicarboxylic acid; GPDM: glycerol phosphate dimethacrylate; UDMA: urethane dimethacrylate; 4-AET: 4acryloxyethyltrimellitic acid; 4-AETA: 4-acryloxyethltrimellitic anhydride; F-PRG: full-reaction type pre-reacted glass ionomer ller.

effects of uoride-releasing resins are weaker than those of glass-ionomer cement due to poorer uoride-releasing ability [1519]. Hence, more detailed information is required to evaluate the effect of uoride-releasing adhesive resins on caries prevention in long-term clinical use. The objective of this study was to evaluate the effects of uoride-releasing adhesive resins on chemical and structural changes in material and tissue cavity walls between the tooth structure and restorative resins in long-term (1000 days) water storage, using scanning electron microscopy (SEM) and laser-Raman spectroscopy. The null hypothesis here is that there are no chemical or structural changes due to the three adhesive resins tested.

A and B were mixed and then applied to the Teon molds and subsequently light-cured for 10 s. For the OS (total-etch adhesive system), the bonding resin was applied and illuminated by light for 20 s. The adhesive resin side of the cured disk was placed on the human tooth surface but separated by 40 m thick spacers at each end to create standard gaps 40 m wide. The uid-containing/tooth-material disks were stored in distilled water at 37 C for 24 h (control group) or 1000 days (experimental group). Five specimens were prepared for each group. A schematic outline of the experimental design is shown in Fig. 1.

2.
2.1.

Materials and methods

Specimen preparation

Fifteen non-carious human premolars extracted for orthodontic reasons with the patients informed consent were used in this study. The teeth were stored in thymol-saturated isotonic saline at 4 C, and were used within 2 months of extraction. The teeth were sectioned perpendicular to the longitudinal axis using a diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under water, to create a tooth disk from the mid-coronal dentin. The surfaces were ground with 600-grit silicon carbide paper under running water for 30 s. Approximately 1.0 mm thick disks were used in this study. Three commercially available uoride-releasing lightcured resin adhesives (One-Up Bond F/OBF, Tokuyama Co. Ltd., Tokyo, Japan; OptiBond Solo/OS, Kerr Corp., Orange, CA, USA; and Reactmer Bond/RB, Shofu Inc., Kyoto, Japan) were used in this study (Table 1). The adhesive resin was placed in wells in Teon molds to form layers 1.2 cm in diameter and approximately 0.7 mm thick. The bottom surface was covered with a glass cover slip, forming a at surface, and the resin was light-cured for 20 s using a dental curing light (Curing Light XL 3000, 3M, St. Paul, MN, USA) operated at 600 mW/cm2 with the tip 1 mm from the adhesive resin layer. The resin composites (Z100, 3M) were inserted into a Teon mold that was over the thin layer of each adhesive resin (approximately 0.7 mm thick). For OBF and RB (self-etch primer system), adhesives

Fig. 1 Schematic representation of the experimental procedures. Cured disks of the tested compounds (adhesive resin coated Z100 resin composite) were placed on the human tooth surfaces (tooth disk 1 mm thick) with 40 m spacers at each end to create a standard 40 m wide interfacial gap. The uid-containing/tooth-material disks were stored in distilled water at 37 C for 24 h (control group) or 1000 days (experimental group).

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2.2.

Laser-Raman analysis

The disks of teeth and test materials were carefully detached after 24 h or 1000 days of water storage. No pre-treatment (i.e. water rinsing, drying, or xation) was conducted for Raman specimens. To evaluate the chemical structures of crystals, enamel, dentin, adhesive resin, and the crystals on teeth and resin disks, they were analyzed with laser-Raman spectroscopy (NR-1800, Nihon Bunkou, Tokyo, Japan). The focus for the analyzed points was provided by the XYZ stage of an optical microscope (Fig. 2). The specimens were examined with 514.5 nm argon ion laser excitation at the focus of the microscopes objective (40). The output power was 200 mW and the laser beam size was approximately 2 m in diameter. Raman spectra were obtained in the 3001300 cm1 range with 20 repetitions and an integration time of 2.0 s.

2.3.

SEM examination

After Raman analysis, the opposing sides of the teeth and resins were sputter-coated with gold and then observed with an acceleration voltage of 10 kV in a vacuum using a eldemission scanning electron microscope (S-4000, HITACHI Ltd., Tokyo, Japan).

Fig. 2 Optical micrograph showing the area of the point that was analyzed (500). The micrograph is taken from the resin side with the crystals of OptiBond Solo using a light microscope, equipped with a Raman spectroscope. The green dot shows the point at which the crystal was analyzed (argon ion laser; 514.5 nm). (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

3.
3.1.

Results
Laser-Raman analysis
Fig. 3 shows Raman spectra of crystals of OS, enamel, dentin, and cured resin of OS in the 3001300 cm1 region. Symmetric bending vibrations ( 2 mode) of PO4 3 give rise to the peak at 430 cm1 . Series of bands at 581 and 591 cm1 are assigned to symmetric bending vibrations ( 4 mode of PO4 3 )

Fig. 2 shows optical micrographs of crystals and a point analyzed using a Raman spectroscope.

Fig. 3 Raman spectra (3001300 cm1 ), from the top: crystal formed on an OptiBond Solo disk surface, enamel, dentin, and a cured disk of OptiBond Solo. Symmetric bending vibrations ( 2 mode) of PO4 3 are observed at 430 cm1 . A series of bands at 581 and 591 cm1 can be assigned to symmetric bending vibrations ( 4 mode of PO4 3 ). The Raman bands observed at 961 are due to the 1 mode of PO4 3 , and at 1023 and 1069 cm1 to the 3 mode of PO4 3 in the dentin and enamel surface. Peaks at 1113 cm1 due to COC and 1187 cm1 due to gem-dimethyl (CH3 CCH3 ) were observed in the cured resin of the OptiBond Solo. There are relatively strong peaks at 463, 618, and 990 cm1 in the crystals of the resin surface of OptiBond Solo.

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Fig. 4 SEM micrographs of the resin (A and B) and dentin sides (C and D) of Reactmer Bond specimens after 1000 days of water storage. (B and D) Higher magnications of the specimens in (A and C), respectively. There are thin layers of crystals precipitated at both the resin and dentin surfaces.

[2124]. Typical Raman bands were observed at 961 cm1 ( 1 mode of PO4 3 ), 1023 cm1 ( 3 mode of PO4 3 ), and 1069 cm1 ( 3 mode of PO4 3 ) in the dentin and enamel surfaces [2023]. The peaks at 1113 cm1 due to COC, and 1187 cm1 due to

gem-dimethyl (CH3 CCH3 ) [24], were observed in the cured adhesive resin of OS. In the Raman spectra of crystals formed on the OS resin disks, relatively strong peaks could be identied at 463, 618, and 990 cm1 . However, no peaks of the 3

Fig. 5 SEM micrographs of the resin (AE) and dentin sides (F) of OptiBond Solo specimens after 1000 days of water storage. (B) A lower magnication view of crystal formation on the resin disks. The areas marked b and d in (A) are shown in higher magnications in (B and D), respectively. (C and E) The higher magnications of the specimens in (B and D), respectively. Numerous plate-shaped crystals were found at the center part of the resin disks, and large needle-shaped crystals were located around the plate-shaped crystals. (D) The needle-shaped crystals show 100 m on the resin disk. The area marked e in (D) is shown in a higher magnication in (E). The higher magnication view of the crystal surface shows small rod-like crystals growing from the needle-like crystal.

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mode of PO4 3 were identied at 1023 and 1069 cm1 of the spectra of enamel and dentin.

3.2.

SEM examination

Crystal growth was observed inside the gap in the RB (Fig. 4) and OS specimens (Fig. 5) after 1000 days of water storage. However, no crystals were observed in the OBF specimens (Fig. 6). No crystals were found in the control specimens (not shown) that had been stored in water for 24 h. Fig. 4 shows the crystal growth on the surface of an RB resin disk (Fig. 4A and B) and on the dentin side (Fig. 4C and D). The formed layer of crystals was very thin and not continuous. Fig. 5 shows the crystal growth on the surface of an OS resin disk (Fig. 5AE) and dentin (Fig. 5F). There was sporadic crystal growth at the resin side (Fig. 5A and B), and plate-shaped crystalline deposits covered the central portion of the resin disk (Fig. 5AC). There were large needle-shaped crystals on the outer part of the plateshaped crystal deposits. The needle-shaped crystal in Fig. 5D was more than 100 m long and this slender crystal type was typical for this resin (OS) but was not observed with the RB specimens (Fig. 4).

4.

Discussion

An important nding in this study was that crystal formation was a common feature of RB (Fig. 4) and OS (Fig. 5). However, no crystal formation could be observed in the control specimens (not shown) or in the OBF specimens stored for 1000 days in water (Fig. 6). This allowed the rejection of the null hypothesis for RA and OS, but not for OBF. The amount of crystalline deposit that developed on the material opposite the 40 m gap over the dentin was far greater than the amount that developed on the outer surface (not shown). Presumably, crystal formation depends on the ion product being greater than the solubility product constants of the solid phase formed on both resins and teeth under the test conditions.

Fig. 6 SEM micrograph of a One-Up Bond F resin disk specimen after 1000 days of water storage. The surface is smooth and structureless with no crystal growth evident.

Water absorption into cured adhesive resins leads to leaching of ions and swelling that may prolong the rate of release of uoride or other ions. The microenvironment in the 40 m gap may permit higher local ion concentrations than those that develop on external or free surfaces, and exceed the solubility product constants for a number of mineral forms (calcium phosphates, calcium uoride, and others). It may be that this bioactive behavior of uoridated compounds is strongly inuenced by the local environment and, in turn, modies that environment. All the resin adhesives tested in this study contained uoride (i.e. uoroaluminosilicate glass or barium aluminoborosilicate glass) (Table 1). It has been reported that the reaction between the uoride released from glass-ionomer cement and the calcium and phosphate ions enhances remineralization of demineralized tissue close to restorations [25]. Furthermore, uoride ions have a strong effect on apatite formation at very small concentrations during enamel formation in teeth in vitro [26]. Even ppm levels of uoride accelerate the transition of OCP to hydroxyapatite, resulting in the formation of uoride-containing hydroxyapatite [27,28]. The appearance and structure of the crystals resemble enamel apatite crystals [29,30]. However, uoride alone may not be the sole factor responsible for the crystal formation because no crystals were detected in the OBF specimen. Depending on the mechanism involved, both the effect of the uoride release, and the physical properties of the resin adhesives may have affected the results of this study. One reason why crystal formation was found with OS and RB but not with OBF, may be the characteristic hydrophilic nature of the polymer matrix of the cured adhesive resin. It has been shown that the polymer matrix of adhesive resin is hydrophilic and therefore is porous, like a permeable membrane [31,32]. The water sorption and dissolution of adhesive resins probably led to the release of ions into the surrounding aqueous environment. There are water-lled regions and/or hydrophilic polymer domains presented in cured resin adhesives [33], and these channels are thought to be responsible for water movement within the adhesives. In addition, there is micromorphological evidence that the amount of water leakage via this pathway (nanoleakage) increases after more than 1 year of water storage [34,35], because of the combined effects of water sorption and solubility. Water sorption is enhanced by the presence of hydrophilic and ionic resin monomers or solvents, which induce the hydrolysis of adhesive resin that may expedite the degradation of resindentin bonds [36]. Negative effects (the dissolution of resin) caused by hydrolysis of resin with regard to bond strength or nanoleakage formation may act to promote a side effect for crystal growth formation within the resindentin bonds during long-term water storage. Under Raman analysis, the peak positions at 463, 618, and 990 cm1 were not observed in the specimens of enamel, dentin or cured resin disks of OS. For the RB crystal specimens, there were no special peaks observed from 300 to 1300 cm1 (not shown), which may be under the detection limit of Raman spectroscopy. The crystals formed on the OS specimens were sufciently large to enable measurement of the chemical structure by Raman spectroscopy. Careful observation showed peaks approximately 30 cm1 distant from the peaks of dentin and enamel, at 430, 591, and 961 cm1 . How-

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ever, no such peaks derived from the resin adhesive could be identied with the crystals. Based on these results, it may be concluded that the crystals were formed by crystallization due to ions that were mainly derived from the teeth. The detailed chemical structure could not be identied because there are no previous reports of the special peaks at 463, 618, and 990 cm1 . The observations reported here may, however, be helpful in further research. Although many studies have shown the mechanism of bond degradation (i.e. hydrolysis of collagen and bonding resin or resin composite) [34,35,37,38], more detailed investigations that look at the mechanism of crystal formation or chemical compositions of crystals using different restorative materials and test conditions, and their effects on clinically relevant problems such as microleakage or nanoleakage, are required to obtain a more complete understanding and are currently being undertaken. The ability to grow crystals between uoridated restorative compounds and dental hard tissues may protect tooth surfaces in interfacial gaps and contribute to long-term stability. The ability of restorative materials to ll in voids making them self-repairing, is a desirable goal of adhesive dentistry. In summary, it may be concluded that some resin adhesives show a good potential for long-term clinical use with enhanced mineralization within the resindentin interface.

Acknowledgements
The materials (One-Up Bond F, Tokuyama Co. Ltd., Tokyo, Japan; OptiBond Solo, Kerr Corp., Orange, CA, USA; and Reactmer Bond, Shofu Inc., Kyoto, Japan) used in this study were generously supplied by the respective manufacturers. The authors thank Dr. N. Nodasaka of the Center for Electron Microscopy of Hokkaido University for helpful discussion of the SEM results. We gratefully acknowledge Drs. H. Ohno and K. Endo of the Health Sciences University of Hokkaido for technical support with the laser-Raman spectroscopy.

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