Line analysis of interface layer on dentin by means of electron-probe microanalysis Tadao Fukushima* and Takashi Horibe Department of Dental Materials and Devices, Fukuoka Dental College, 700 Ta, Sawara-ku, Fukuoka 814-01, Japan Three types of brominated methacrylates, SBPPM, BPylM, and BNEM, were synthesized. The distribution of Br, Ca, and Fe at the interface between dentin treated with the 10-3 solution and resin containing these monomers was analyzed using an electron-probe microanalyzer (EPMA) in order to predict the composition and thickness of the interface layer on dentin for the corresponding unbrominated methacrylates. There was no significant difference between the brominated and unbrominated methacrylates in either the
bond strength to the treated dentin or in the thickness of the interface layer on dentin observed with a scanning electron microscope (SEM). The thickness determined with EPMA was equivalent to that observed with SEM. The concentration of each brominated methacrylate in the interface layer was higher than the original concentration in the resin monomers, and BNEM showed higher concentration than the others. The presence of Fe in the layer was confirmed by EPMA.
INTRODUCTION
One problem in dentistry has been the poor bond strength of dental restorative materials to Many dentin treating agents4-’ and adhesive monomer^"^-^' have been developed to improve the bond strength of restorative materials. Recently, dentin treating agents containing ferric salts have been developed in an attempt to further improve the bond strength of restorative material^^,^,'^ and tests of some of these agents have shown that they greatly increased the bond strengthP” A 10% citric acid and 3% ferric chloride solution called 10-3 solution, which was developed by Nakabayashi et al.,’3 greatly enhanced the bond strength of the MMA-TBB resin system to dentin. They clarify14that the increase in bond strength was the result of the formation of an interface layer in the treated dentin called resin-reinforced dentin. In a previous study,” the interface between the resin (MMA-TBB resin system) containing 2-bromoethyl methacrylate and the dentin treated with the 10-3 solution was observed with a scanning electron microscope (SEM). The distribution of Br, Ca, and Fe at the interface between the resin and dentin *To whom correspondence should be addressed. Journal of Biomedical Materials Research, Vol. 25, 129-140 (1991) CCC 0021-9304/91/010129-12$04.00 0 1991 John Wiley & Sons, Inc.
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was analyzed with an electron-probe microanalyzer (EPMA) in order to clearly confirm the presence of, and measure the thickness of, the interface layer. A layer approximately 3 pm in thickness consisting of resin and dentin was identified by SEM and EPMA studies. Therefore, it was presumed that if methacrylate monomers to be used as bonding agents for composite resins or other resin systems were labeled with halogens such as Br and C1, and the distribution of halogen at the interface between resin and dentin was analyzed with EPMA, the permeability of each original monomer into dentin treated with various etchants could be predicted. In the present study, three types of aromatic methacrylates and the corresponding brominated methacrylates were synthesized. The bond strength to dentin treated with the 10-3 solution and thickness of the layer at the interface between the resin and dentin observed with SEM were compared to each other in order to investigate the effect of bromine on the bonding and the formation of the interface layer. Additionally, the distribution of Br, Ca, and Fe at the interface between resin containing the brominated methacrylates and dentin was analyzed with EPMA in order to determine the composition and thickness of the layer. MATERIALS AND METHODS
Preparation of monomers Preparation of 2-succinoxy-3-phenoxypropylmethacrylate (SPPM) and 2-succinoxy-3-(p-bromophenoxy)propylmethacrylate (SBPPM): SPPM and SBPPM were prepared by the reaction of 2-hydroxy-3-phenoxypropylmethacrylate or 2-hydroxy-3-(p-bromophenoxy)propy1methacrylate, which were synthesized by the method of Masuhara," with succinoxy anhydride according to the previously described method." Preparation of 2-phenylethyl methacrylate (PylEM), 2-(4-bromophenyl) ethyl methacrylate (BPylEM), 2-(l-naphthyl)ethyl methacrylate (NEM) and 2- (4-br omo-l-naphthy1)ethyl methacrylate (BNEM): PylEM, BPylEM, N EM, and BNEM were prepared by the reaction of P-phenethyl alcohol, 4bromophenethyl alcohol, 2-(l-naphthyl)ethanol, and 2-(4-bromo-l-naphthyl)ethanol, which was obtained by the action of bromine on 2-(l-naphthyl)ethan01,'~with methacryloyl chloride according to the previously described method.l8 The structural formulae of synthesized monomers are shown in Figure 1. Preparation of specimens Mixtures of synthesized monomers (2 mol%)with MMA were used as resin monomers. Monomer liquid/PMMA powder (Sun Medical Co., Kyoto, Japan) mixture was cured by partially oxidized tri-n-butyl borane (TBB-0: Sun Medical Co., Kyoto, Japan).
LINE ANALYSIS OF INTERFACE LAYER ON DENTIN
I
COCH,CHCH,O
II 0
I 0
0 x
131
X:H Br
CCH,CH,COOH I1
0 H:SPPM Br: SB P P M
H : Pyl E M 6r:BPvlEM
1 COCH,CH,
II 0
8 0X
H:NEM 6r:BN EM
Figure 1. Structural formulae of synthesized monomers.
Measurement of tensile bond strength
Preparation of tooth surface Freshly extracted bovine anterior teeth substituted for human teeth were used as adherents.” All teeth with exposed dentin surfaces were embedded in acrylic tubes with acrylic resin. The dentin surface was finely polished with 600-grit Sic paper under running water, and etched with the 10-3 solution for 30 s. All the etched dentin surfaces were washed with water for 10 s after etching. They were then dried with oil-free compressed air for 20 s.
Preparation of specimens Split-silicone with a 5mm-diameter hole was placed on the dried dentin surface. Resin monomer (1.2 g) and TBB-0 (5 wt%)were stirred well and then mixed with PMMA powder (1.0 g) for 5 s at 20°C. With a brush, liquid/PMMA powder mixture was applied to the dentin. Next, Orthodontic resin (L.D. Caulk Co., Milford, DE) was placed over the cured resin and an acrylic rod with a retaining hole was pressed onto Orthodontic resin. Upon curing and
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removing the silicone, the specimens shown in Figure 2 were immersed in 37°C water for 1 day and 30 days. Tensile bond tests were performed on a universal testing machine (IS-5000, Shimazu Co., Kyoto, Japan) at the cross-head speed of 1 mm/min using the apparatus shown in the previous paper.” The mean bond strength was calculated using the test results of 10 specimens for each bonding resin. The results were analyzed by analysis of variance and a Scheffe’s multiple comparison test at a significance level of 0.05. SEM observation To prepare the etched dentin surface for SEM observation in cross section, a 2-mm-thick section of etched dentin was split in a labiolingual direction. The specimen was fixed in a 5% glutaraldehyde-0.25 M cacodylate buffer at room temperature for 24 h and dehydrated through a graded series of ethanol. The ethanol was replaced with isoamyl acetate and the specimen was dried with liquid carbon dioxide using a critical point dryer. To prepare the interface layer on dentin, for SEM observation, the specimens were prepared in the same way as the specimens used for bond tests, and were then labiolingually cut using a diamond saw (ISOMET, Buehler Ltd., IL). The cut surface was finely polished with 600-grit Sic paper under running water, and was brushed with MMA liquid to remove the surface layer of the resin. Subsequently, the surface was etched with a 6 N HCl solution for 30 s. After being etched, and washed with water, the specimens were dried in a desiccator under vacuum for 24 h. The surface was coated with a thin layer of gold-palladium and was then observed with SEM (JSM-T330,JEOL Ltd., Tokyo, Japan). Line analyses of the distribution of Br, Ca, and Fe at the resin-dentin interface The specimens, which were cut in the same way as the specimens used for SEM observation, were finely polished with diamond paste, cleaned in water
Figure 2. Diagrams of specimen (A) PMMA rods, (B) orthodontic resin, (C) bonding resin, (D) split-silicone, (E) tooth, and (F) acrylic tube.
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with ultrasonic agitation for 1 min, and dried in a desiccator for 24 h under vacuum. After drying, they were coated with evaporated carbon. The distribution of Br, Ca, and Fe at 20 points in the resin-dentin interface for each specimen was analyzed with EPMA (EMX-SM, Shimazu Co., Kyoto, Japan). Line scan analyses of Br, Ca, and Fe with an accelerating voltage of 15 kV and specimen current of 0.01 pA were carried out on the line which ran perpendicular to the interface from the dentin side. The thickness of the layer was determined by the distance between the point at which Ca cps approaches zero and the point at which Br cps increases in line scan analysis profiles. The concentration of Br in the layer was estimated from the maximum intensity of the Br x-rays. Calibration of bromine x-ray intensity Mixtures of 2, 5, and 10 mol% brominated monomers with MMA were polymerized by 0.5 wt% benzoyl peroxide(BP0) in a glass tube, which was sealed under vacuum, at 110°C for 24 h. The polymer rods were cut with a diamond saw. The specimens were prepared in the same way as the specimens used for the line analyses. The bromine x-ray intensity for each specimen was calibrated with EPMA. RESULTS
Bond strength to etched dentin There was no significant difference in the bond strength between the brominated and unbrominated methacrylates under all conditions, regardless of structure (Table I). The bond strength did not decrease statistically in 30 days in water. SEM observation
The treatment of the 10-3 solution for 30 s resulted in a porous layer 1-2 pm thick in the upper dentin surface, where collagen was clearly visible as shown in Figure 3. A layer 2-3 pm thick which withstood the MMA liquid and the acid was visible at the resin-dentin interface for each specimen as shown in Figure 4. The thickness of the layer for each brominated methacrylate was the same as the original methacrylate. EPMA analyses
The typical line analyses of Br, Ca, and Fe and the calibration curves are shown in Figures 5 and 6, and in Figure 7, respectively. It was found that the
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TABLE I Bond Strength to Dentin Treated with the 10-3 Solution (MPa) 1 Day
30 Days
SPPM SBPPM
17.7 (2.3) 17.2 (2.3)
15.3 (4.1) 15.2 (3.3)
PylEM BPylEM
15.9 (1.5) 13.5 (4.0)
15.8 (3.9) 15.2 (3.4)
NEM BNEM
14.9 (3.1) 13.5 (4.7)
13.8 (2.9) 13.5 (4.5)
Note. Mean (standard deviation).
Figure 3. SEM micrograph of fractured surface of dentin, the top of which has been treated with the 10-3 solution.
Figure 4. SEM micrographs of typical interface layer on dentin for bonding resin with (left) and without (right) brominated methacrylate.
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Figure 5. SEM micrographs and line analyses of interface between dentin and resin containing the brominated methacrylates (bar = 3 pm).
thickness of the layer for all specimens was approximately 3 pm, keeping in mind that the beam size is about 1 pm in diameter. Iron was present in the layer. The concentration of SBPPM, BPylM, and BNEM in the layer ranged from 2.2 to 5.5 mol%,from 2.6 to 5.5 mol%, and from 2.6 to 9.5 mol%, respectively, and was higher than the original concentration in the resin monomers. The mean concentration of SBPPM, BPylM, and BNEM in the layer was 3.6, 4.6,
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6
5
4 3 2 1
-
0
N
zx
a
% m
v)
n
Resin
0
Dent in
6
5
Ca
4 3
I
2 1
0
Figure 6. Typical distribution patterns of Br, Ca, and Fe at interface between dentin and resin. (Top) High Br x-ray intensity and (bottom) low Br x-ray intensity.
and 6.1 mol%, respectively (Table 11). The concentration of BNEM was higher than that of SBPPM and BPylM. DISCUSSION
All the bonding resins showed a bond strength greater than 13 MPa and formed an interface layer with about 3 pm thickness, which was slightly thicker than that of the porous layer in the etched upper dentin surface, at the resin-dentin interface. These results, which concur with those obtained
LINE ANALYSIS OF INTERFACE LAYER ON DENTIN
4
137
-1
0
I
,
I
1
2
5
1‘0
C 0 NC ENT R AT I0 N (mo1%) Figure 7. Relationship between the concentration of the brominated monomers and cps of Br Ka ( 0 )SBPPM, (A)BPylEM, and (a)BNEM. Regression coefficients were 0.999 for all monomers.
in a previous study,” strongly indicate that the bond strength is the result of the formation of the interface layer on dentin. Therefore, it should be possible to significantly increase the bond strength of any bonding resin system in which the interface layer is easily formed. However, the mechanism of the layer formation is presently unknown, and confirmation of and analysis of the layer are critical if understanding of the mechanism is to be achieved. In this study, the upper thin layer of the resin and dentin surfaces at the resin-dentin interface was removed with the acid solution and the MMA liquid so that the clear confirmation of the interface layer with SEM became possible. However, it is difficult to confirm the presence of the interface layer with SEM for a composite resin system because its base monomers are TABLE I1 The Mean Concentration of Brominated Methacrylates in the Interface Layer Concentration (mol %) SBPPM BPylEM BNEM
4.6 3.6 (0.8) 6.1 (2.2)
1
Note. Mean (standard deviation). Bonding resins connected by bar showed no significant difference.
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dimethacrylates such as Bis-GMA and Tri-EDMA, whose cross-linked polymers are stable to organic solvents. EPMA or an energy-dispersive x-ray spectrometer (EDS), which are widely used for line analyses of elements at porcelain-metal interfaces,212’ are able to provide more information on composition and thickness of the interface layer than SEM. However, it is very difficult to prove that the composition and thickness of the interface layer were clearly determined by the line analyses for carbon and oxygen because most dental polymers and dentin contain these elements. Therefore, three types of methacrylates were labeled with bromine, because the 10-3 solution contains chlorine and, therefore, the composition and thickness of the interface layer for each original methacrylate could be predicted by means of the combination of the brominated methacrylate and EPMA. There was no significant difference in bond strength or thickness of the interface layer between the brominated methacrylate and the corresponding unbrominated methacrylate, regardless of structure. Additionally, the thickness of the layer determined with EPMA was equivalent to that observed with SEM. EPMA provided the expected information on the concentration of the brominated methacrylates as well as composition of the layer. The layer had higher concentration of brominated methacrylates than the resin monomers, and BNEM showed a higher concentration in the layer than the others. The variation in concentration of monomers may be caused by a difference in the diffusion of monomers into PMMA powder. It was also found that Fe remained in the layer. However, the major role of Fe is unknown although some hypotheses have been ~ u g g e s t e d . ~ ~ - ~ ~ The results obtained from this study suggest that bromine in the monomers does not adversely affect the bonding and the formation of the interface layer, and combining a halogenated monomer and EPMA provides a useful technique to investigate the mechanism of bonding for any bonding systems.
CONCLUSIONS
The method of combining the brominated methacrylates and EPMA was used to attempt to predict the composition and thickness of the interface layer between resin containing the corresponding unbrominated methacrylates and dentin treated with the 10-3 solution. (1)The bond strength and the thickness of the layer were comparable for the brominated and unbrominated methacrylates. (2) The thickness of the layer determined with EPMA was very similar to that observed with SEM. (3) The EPMA analyses showed that the concentration of the brominated methacrylates in the layer was higher than the original concentration in the resin monomers. BNEM had higher concentration in the layer than the others. (4)The presence of Fe in the layer was clearly confirmed with EPMA.
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References 1. D. R. Beech, "Adhesion in the oral environment: biophysical and biochemical considerations," Int. Dent. J., 28, 338-347 (1978). L.
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B. E. Causton, D.Y. D. Samara-Wickrama, and N.W. Johnson, "Effect of calcifying fluid on bonding of cements and composites to dentin in vitro," Brit. Dent. I., 140, 339-342 (1976). B. E. Causton, "Improved bonding of composite restorative to dentin," Brit. Dent. I., 156, 93-95 (1984). R. L. Bowen, "Adhesive bonding of various materials to hard tooth tissues. XXII. The effects of a cleanser, mordant, and polySAC on adhesion between a composite resin and dentin," J. Dent. Res., 59, 809-814 (1980). H. S. Shalabi, E. Asmussen, and K. D. Jorgensen, "Increased bonding of a glass-ionomer cement to dentin by means of FeC13," Scund. J. Dent. Res., 89, 348-353 (1981). M. Peddey, "The bond strength of polycarboxylic acid cements to dentin: Effect of surface modification and time after extraction," Aust. Dent. J., 26, 178-180 (1981). R. L. Bowen, E. N. Cobb, and J. E. Rapson, 'Adhesive bonding of various materials to hard tooth tissues: Improvement in bond strength to dentin," J. Dent Res., 61, 1070-1076 (1982). E.C. Munksgaard and E. Asmussen, "Bond strength between dentin and restorative resins mediated by mixtures of HEMA and glutaraldehyde," J. Dent. Res., 63, 1087-1089 (1984). G.C. Eliades, A. A. Caputo, and G. J. Vougiouklakis, "Composition, wetting properties and bond strength with dentin of 6 new dentin adhesives," Dent. Muter., 1, 170-176 (1985). T. Fukushima, M. Kawaguchi, Y. Inoue, K. Miyazaki, and T. Horibe, "Application of functional monomers for dental use (Part-9) Syntheses of succinoxy methacrylates and their adhesion to polished and etched tooth surfaces," Dent. Mater. J., 4, 33-39 (1985). T. Fukushima, Y. Inoue, and T. Horibe, "Synthesis of 2-methacryloxyethyl hydrogen maleate and its bonding to tooth surfaces," J. Jpn.Dent. Mater., 7, 675-679 (1988). Y. Inoue, T. Fukushima, K. Miyazaki, and T. Horibe, "Bond strength of organic cements to tooth structure-Effect of various mordants on bonding to dentin," Dent. Muter. J., 5, 276-282 (1986). N. Nakabayashi, M. Takeyama, K. Kojima, and E. Masuhara, "Studies on dental self-curing resins (19)-Adhesion of 4-META/MMA-TBB resin to pretreated dentine," 1.Jpn. SOC.Dent. Appar. Muter., 23, 29-33 (1982). N. Nakabayashi, M. Takeyama, K. Kojima, and E. Masuhara, "Studies on dental self-curing resins (20)-Adhesion mechanism of 4-META/ MMA-TBB resin to dentin," J. Jpn. SOC. Dent. Appar. Muter., 23, 34-39 (1982). T. Fukushima, Y. Inoue, and T. Horibe, "Bonding of various methacrylates to dentin treated with 10% citric acid-3% ferric chloride solution," J. Jpn. Dent. Muter., 7, 592-598 (1988). E. Masuhara, N. Nakabayashi, and K. Furuta, "Synthesis of 2-hydroxypropylmethacrylate," J. Syn. Org. Chem. Jpn., 33, 52-55 (1975). H.T. Clarke and M. R. Brethen, "a-Bromonaphthalene," in Organic Syntheses, collective vol. 1, 2nd Ed., A. H. Blatt (ed.), Wiley, New York, 1941, pp. 121-123. M. Kawaguchi, T. Fukushima, K. Miyazaki, and T. Horibe, "Syntheses and physical properties of polyfunctional methacrylates (6) Physical properties of spiro dimethacrylate copolymer," Dent. Muter. J., 4, 201207 (1985).
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19. I. Nakamichi, M. Iwaku, and T. Fusayama, “Bovine teeth as possible substitutes in the adhesion test,” J. Dent. Res., 62, 1076-1081 (1983). 20. T. Fukushima, M. Kawaguchi, Y. Inoue, K. Miyazaki, and T. Horibe, ”Application of functional monomers for dental use (part-8) Adhesion of alkylene glycol monomethacrylates to etched enamel and dentin surfaces,” Dent. Muter. I., 3, 49-55 (1984). 21. H. Ohno, 0. Miyakawa, K. Watanabe, and N. Shiokawa, ”The structure of oxide formed by high-temperature oxidation of commercial J. Dent. Res., 61, 1255-1261 gold alloys for p o r c e l a i ~ - m ~boAding,” al (19821. 22. K. J. Anusavice, J. A. Horner, and C.W. Fairhurst, ‘Adherence controlling elements in ceramic-metal systems. 1. Precious alloys,” J. Dent. Res., 56, 1045-1052 (1977). 23. J.R. Jedrychowski, A.A. Caputo, and J. Prola, ”Influence of a ferric chloride mordant solution on resin-dentin retention,” J. Dent. Res., 60, 134-138 (1981). 24. Y. Kadoma, K. Kojima, and E. Masuhara, ”Studies on dental self-curing resins (25) Effect of ferric chloride or cupric chloride on the polymerization of methyl methacrylate,” J. Jpn. Dent. Muter., 2, 495-502 (1983). 25. T. Mizunuma and N. Nakabayashi, “Relationship between bond strength and structure of dentin collagen-Adhesion to dentin with 4-METAIMMA-TBB resin,” J. Jpn. Dent. Muter., 5, 471-474 (1986).
Received December 1988 Accepted August 21,1990
bond strength to the treated dentin or in the thickness of the interface layer on dentin observed with a scanning electron microscope (SEM). The thickness determined with EPMA was equivalent to that observed with SEM. The concentration of each brominated methacrylate in the interface layer was higher than the original concentration in the resin monomers, and BNEM showed higher concentration than the others. The presence of Fe in the layer was confirmed by EPMA.
INTRODUCTION
One problem in dentistry has been the poor bond strength of dental restorative materials to Many dentin treating agents4-’ and adhesive monomer^"^-^' have been developed to improve the bond strength of restorative materials. Recently, dentin treating agents containing ferric salts have been developed in an attempt to further improve the bond strength of restorative material^^,^,'^ and tests of some of these agents have shown that they greatly increased the bond strengthP” A 10% citric acid and 3% ferric chloride solution called 10-3 solution, which was developed by Nakabayashi et al.,’3 greatly enhanced the bond strength of the MMA-TBB resin system to dentin. They clarify14that the increase in bond strength was the result of the formation of an interface layer in the treated dentin called resin-reinforced dentin. In a previous study,” the interface between the resin (MMA-TBB resin system) containing 2-bromoethyl methacrylate and the dentin treated with the 10-3 solution was observed with a scanning electron microscope (SEM). The distribution of Br, Ca, and Fe at the interface between the resin and dentin *To whom correspondence should be addressed. Journal of Biomedical Materials Research, Vol. 25, 129-140 (1991) CCC 0021-9304/91/010129-12$04.00 0 1991 John Wiley & Sons, Inc.
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was analyzed with an electron-probe microanalyzer (EPMA) in order to clearly confirm the presence of, and measure the thickness of, the interface layer. A layer approximately 3 pm in thickness consisting of resin and dentin was identified by SEM and EPMA studies. Therefore, it was presumed that if methacrylate monomers to be used as bonding agents for composite resins or other resin systems were labeled with halogens such as Br and C1, and the distribution of halogen at the interface between resin and dentin was analyzed with EPMA, the permeability of each original monomer into dentin treated with various etchants could be predicted. In the present study, three types of aromatic methacrylates and the corresponding brominated methacrylates were synthesized. The bond strength to dentin treated with the 10-3 solution and thickness of the layer at the interface between the resin and dentin observed with SEM were compared to each other in order to investigate the effect of bromine on the bonding and the formation of the interface layer. Additionally, the distribution of Br, Ca, and Fe at the interface between resin containing the brominated methacrylates and dentin was analyzed with EPMA in order to determine the composition and thickness of the layer. MATERIALS AND METHODS
Preparation of monomers Preparation of 2-succinoxy-3-phenoxypropylmethacrylate (SPPM) and 2-succinoxy-3-(p-bromophenoxy)propylmethacrylate (SBPPM): SPPM and SBPPM were prepared by the reaction of 2-hydroxy-3-phenoxypropylmethacrylate or 2-hydroxy-3-(p-bromophenoxy)propy1methacrylate, which were synthesized by the method of Masuhara," with succinoxy anhydride according to the previously described method." Preparation of 2-phenylethyl methacrylate (PylEM), 2-(4-bromophenyl) ethyl methacrylate (BPylEM), 2-(l-naphthyl)ethyl methacrylate (NEM) and 2- (4-br omo-l-naphthy1)ethyl methacrylate (BNEM): PylEM, BPylEM, N EM, and BNEM were prepared by the reaction of P-phenethyl alcohol, 4bromophenethyl alcohol, 2-(l-naphthyl)ethanol, and 2-(4-bromo-l-naphthyl)ethanol, which was obtained by the action of bromine on 2-(l-naphthyl)ethan01,'~with methacryloyl chloride according to the previously described method.l8 The structural formulae of synthesized monomers are shown in Figure 1. Preparation of specimens Mixtures of synthesized monomers (2 mol%)with MMA were used as resin monomers. Monomer liquid/PMMA powder (Sun Medical Co., Kyoto, Japan) mixture was cured by partially oxidized tri-n-butyl borane (TBB-0: Sun Medical Co., Kyoto, Japan).
LINE ANALYSIS OF INTERFACE LAYER ON DENTIN
I
COCH,CHCH,O
II 0
I 0
0 x
131
X:H Br
CCH,CH,COOH I1
0 H:SPPM Br: SB P P M
H : Pyl E M 6r:BPvlEM
1 COCH,CH,
II 0
8 0X
H:NEM 6r:BN EM
Figure 1. Structural formulae of synthesized monomers.
Measurement of tensile bond strength
Preparation of tooth surface Freshly extracted bovine anterior teeth substituted for human teeth were used as adherents.” All teeth with exposed dentin surfaces were embedded in acrylic tubes with acrylic resin. The dentin surface was finely polished with 600-grit Sic paper under running water, and etched with the 10-3 solution for 30 s. All the etched dentin surfaces were washed with water for 10 s after etching. They were then dried with oil-free compressed air for 20 s.
Preparation of specimens Split-silicone with a 5mm-diameter hole was placed on the dried dentin surface. Resin monomer (1.2 g) and TBB-0 (5 wt%)were stirred well and then mixed with PMMA powder (1.0 g) for 5 s at 20°C. With a brush, liquid/PMMA powder mixture was applied to the dentin. Next, Orthodontic resin (L.D. Caulk Co., Milford, DE) was placed over the cured resin and an acrylic rod with a retaining hole was pressed onto Orthodontic resin. Upon curing and
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removing the silicone, the specimens shown in Figure 2 were immersed in 37°C water for 1 day and 30 days. Tensile bond tests were performed on a universal testing machine (IS-5000, Shimazu Co., Kyoto, Japan) at the cross-head speed of 1 mm/min using the apparatus shown in the previous paper.” The mean bond strength was calculated using the test results of 10 specimens for each bonding resin. The results were analyzed by analysis of variance and a Scheffe’s multiple comparison test at a significance level of 0.05. SEM observation To prepare the etched dentin surface for SEM observation in cross section, a 2-mm-thick section of etched dentin was split in a labiolingual direction. The specimen was fixed in a 5% glutaraldehyde-0.25 M cacodylate buffer at room temperature for 24 h and dehydrated through a graded series of ethanol. The ethanol was replaced with isoamyl acetate and the specimen was dried with liquid carbon dioxide using a critical point dryer. To prepare the interface layer on dentin, for SEM observation, the specimens were prepared in the same way as the specimens used for bond tests, and were then labiolingually cut using a diamond saw (ISOMET, Buehler Ltd., IL). The cut surface was finely polished with 600-grit Sic paper under running water, and was brushed with MMA liquid to remove the surface layer of the resin. Subsequently, the surface was etched with a 6 N HCl solution for 30 s. After being etched, and washed with water, the specimens were dried in a desiccator under vacuum for 24 h. The surface was coated with a thin layer of gold-palladium and was then observed with SEM (JSM-T330,JEOL Ltd., Tokyo, Japan). Line analyses of the distribution of Br, Ca, and Fe at the resin-dentin interface The specimens, which were cut in the same way as the specimens used for SEM observation, were finely polished with diamond paste, cleaned in water
Figure 2. Diagrams of specimen (A) PMMA rods, (B) orthodontic resin, (C) bonding resin, (D) split-silicone, (E) tooth, and (F) acrylic tube.
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133
with ultrasonic agitation for 1 min, and dried in a desiccator for 24 h under vacuum. After drying, they were coated with evaporated carbon. The distribution of Br, Ca, and Fe at 20 points in the resin-dentin interface for each specimen was analyzed with EPMA (EMX-SM, Shimazu Co., Kyoto, Japan). Line scan analyses of Br, Ca, and Fe with an accelerating voltage of 15 kV and specimen current of 0.01 pA were carried out on the line which ran perpendicular to the interface from the dentin side. The thickness of the layer was determined by the distance between the point at which Ca cps approaches zero and the point at which Br cps increases in line scan analysis profiles. The concentration of Br in the layer was estimated from the maximum intensity of the Br x-rays. Calibration of bromine x-ray intensity Mixtures of 2, 5, and 10 mol% brominated monomers with MMA were polymerized by 0.5 wt% benzoyl peroxide(BP0) in a glass tube, which was sealed under vacuum, at 110°C for 24 h. The polymer rods were cut with a diamond saw. The specimens were prepared in the same way as the specimens used for the line analyses. The bromine x-ray intensity for each specimen was calibrated with EPMA. RESULTS
Bond strength to etched dentin There was no significant difference in the bond strength between the brominated and unbrominated methacrylates under all conditions, regardless of structure (Table I). The bond strength did not decrease statistically in 30 days in water. SEM observation
The treatment of the 10-3 solution for 30 s resulted in a porous layer 1-2 pm thick in the upper dentin surface, where collagen was clearly visible as shown in Figure 3. A layer 2-3 pm thick which withstood the MMA liquid and the acid was visible at the resin-dentin interface for each specimen as shown in Figure 4. The thickness of the layer for each brominated methacrylate was the same as the original methacrylate. EPMA analyses
The typical line analyses of Br, Ca, and Fe and the calibration curves are shown in Figures 5 and 6, and in Figure 7, respectively. It was found that the
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TABLE I Bond Strength to Dentin Treated with the 10-3 Solution (MPa) 1 Day
30 Days
SPPM SBPPM
17.7 (2.3) 17.2 (2.3)
15.3 (4.1) 15.2 (3.3)
PylEM BPylEM
15.9 (1.5) 13.5 (4.0)
15.8 (3.9) 15.2 (3.4)
NEM BNEM
14.9 (3.1) 13.5 (4.7)
13.8 (2.9) 13.5 (4.5)
Note. Mean (standard deviation).
Figure 3. SEM micrograph of fractured surface of dentin, the top of which has been treated with the 10-3 solution.
Figure 4. SEM micrographs of typical interface layer on dentin for bonding resin with (left) and without (right) brominated methacrylate.
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Figure 5. SEM micrographs and line analyses of interface between dentin and resin containing the brominated methacrylates (bar = 3 pm).
thickness of the layer for all specimens was approximately 3 pm, keeping in mind that the beam size is about 1 pm in diameter. Iron was present in the layer. The concentration of SBPPM, BPylM, and BNEM in the layer ranged from 2.2 to 5.5 mol%,from 2.6 to 5.5 mol%, and from 2.6 to 9.5 mol%, respectively, and was higher than the original concentration in the resin monomers. The mean concentration of SBPPM, BPylM, and BNEM in the layer was 3.6, 4.6,
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6
5
4 3 2 1
-
0
N
zx
a
% m
v)
n
Resin
0
Dent in
6
5
Ca
4 3
I
2 1
0
Figure 6. Typical distribution patterns of Br, Ca, and Fe at interface between dentin and resin. (Top) High Br x-ray intensity and (bottom) low Br x-ray intensity.
and 6.1 mol%, respectively (Table 11). The concentration of BNEM was higher than that of SBPPM and BPylM. DISCUSSION
All the bonding resins showed a bond strength greater than 13 MPa and formed an interface layer with about 3 pm thickness, which was slightly thicker than that of the porous layer in the etched upper dentin surface, at the resin-dentin interface. These results, which concur with those obtained
LINE ANALYSIS OF INTERFACE LAYER ON DENTIN
4
137
-1
0
I
,
I
1
2
5
1‘0
C 0 NC ENT R AT I0 N (mo1%) Figure 7. Relationship between the concentration of the brominated monomers and cps of Br Ka ( 0 )SBPPM, (A)BPylEM, and (a)BNEM. Regression coefficients were 0.999 for all monomers.
in a previous study,” strongly indicate that the bond strength is the result of the formation of the interface layer on dentin. Therefore, it should be possible to significantly increase the bond strength of any bonding resin system in which the interface layer is easily formed. However, the mechanism of the layer formation is presently unknown, and confirmation of and analysis of the layer are critical if understanding of the mechanism is to be achieved. In this study, the upper thin layer of the resin and dentin surfaces at the resin-dentin interface was removed with the acid solution and the MMA liquid so that the clear confirmation of the interface layer with SEM became possible. However, it is difficult to confirm the presence of the interface layer with SEM for a composite resin system because its base monomers are TABLE I1 The Mean Concentration of Brominated Methacrylates in the Interface Layer Concentration (mol %) SBPPM BPylEM BNEM
4.6 3.6 (0.8) 6.1 (2.2)
1
Note. Mean (standard deviation). Bonding resins connected by bar showed no significant difference.
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dimethacrylates such as Bis-GMA and Tri-EDMA, whose cross-linked polymers are stable to organic solvents. EPMA or an energy-dispersive x-ray spectrometer (EDS), which are widely used for line analyses of elements at porcelain-metal interfaces,212’ are able to provide more information on composition and thickness of the interface layer than SEM. However, it is very difficult to prove that the composition and thickness of the interface layer were clearly determined by the line analyses for carbon and oxygen because most dental polymers and dentin contain these elements. Therefore, three types of methacrylates were labeled with bromine, because the 10-3 solution contains chlorine and, therefore, the composition and thickness of the interface layer for each original methacrylate could be predicted by means of the combination of the brominated methacrylate and EPMA. There was no significant difference in bond strength or thickness of the interface layer between the brominated methacrylate and the corresponding unbrominated methacrylate, regardless of structure. Additionally, the thickness of the layer determined with EPMA was equivalent to that observed with SEM. EPMA provided the expected information on the concentration of the brominated methacrylates as well as composition of the layer. The layer had higher concentration of brominated methacrylates than the resin monomers, and BNEM showed a higher concentration in the layer than the others. The variation in concentration of monomers may be caused by a difference in the diffusion of monomers into PMMA powder. It was also found that Fe remained in the layer. However, the major role of Fe is unknown although some hypotheses have been ~ u g g e s t e d . ~ ~ - ~ ~ The results obtained from this study suggest that bromine in the monomers does not adversely affect the bonding and the formation of the interface layer, and combining a halogenated monomer and EPMA provides a useful technique to investigate the mechanism of bonding for any bonding systems.
CONCLUSIONS
The method of combining the brominated methacrylates and EPMA was used to attempt to predict the composition and thickness of the interface layer between resin containing the corresponding unbrominated methacrylates and dentin treated with the 10-3 solution. (1)The bond strength and the thickness of the layer were comparable for the brominated and unbrominated methacrylates. (2) The thickness of the layer determined with EPMA was very similar to that observed with SEM. (3) The EPMA analyses showed that the concentration of the brominated methacrylates in the layer was higher than the original concentration in the resin monomers. BNEM had higher concentration in the layer than the others. (4)The presence of Fe in the layer was clearly confirmed with EPMA.
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Received December 1988 Accepted August 21,1990
