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Determination of residual double bonds in resin–dentin interface by Raman spectroscopy
dental materials Dental Materials 19 (2003) 245±251
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Determination of residual double bonds in resin±dentin interface by Raman spectroscopy M. Miyazaki a,*, H. Onose a, N. Iida b, H. Kazama b a
Department of Operative Dentistry, Nihon University School of Dentistry, 1-8-13, Kanda Surugadai, Chiyoda-Ku, Tokyo 101-8310, Japan b Research Laboratories, Tokuyama Corp., 40, Wadai, Tsukuba-city, Ibaraki 300-4247, Japan Received 30 January 2001; revised 6 September 2001; accepted 4 December 2001
Abstract Objectives: The quality of the hybrid layer is believed to be more important than the thickness of this layer. The purpose of this study was to investigate a method to analyze the percentage of adhesive resin residual double bonds in the dentin±resin interface using laser Raman spectroscopy. Methods: Bovine dentin was treated with dentin adhesives and resin composite was bonded according to the manufacturers' instructions. The specimens were sectioned parallel to dentinal tubules and the surfaces were then polished to 1 mm diamond pastes. Raman spectra were recorded along a line perpendicular to the dentin±resin interface in steps of 0.2 mm. The measurement of residual CyC bond was made on a relative basis by comparing the CyC unpolymerized methacrylate stretching vibration (1638 cm 21) against the CyO stretching mode of the ester group (1719 cm 21). The percentage of residual double bonds including pendant and monomeric double bonds was calculated by comparing the obtained ratio with that of uncured adhesive resin. Results: The amount of residual double bonds in the hybrid layer varied from 10 to 25% compared to the uncured adhesives, a relatively higher percentage was detected for Fluoro Bond (12.3±23.6%) and Single Bond (9.5±21.8%), and lower for Mac Bond II (10.6±18.0%) and Mega Bond (10.7±16.3%). No relationship was seen between the percentage of remaining double bonds and the location within the resin± dentin interface. Signi®cance: Laser Raman microscopy used was a useful tool for measuring the residual double bonds in the dentin±resin interface. q 2003 Academy of Dental Materials. Published by Elsevier Science Ltd. All rights reserved. Keywords: Laser Raman microscopy; Residual double bond; Dentin bonding; Hybrid layer
1. Introduction A strong and durable bond between resin restoration and tooth is needed to prevent microleakage and formation of marginal gap. Excellent adaptation of resin to dentin with monomer penetration has been reported with scanning and transmission electron microscopic studies [1±4]. The hybrid layer is believed to be one of the most effective mechanisms in dentin bonding [5]. Morphological examination of the hybrid layer has revealed that the bond strength and sealing ability of bonding systems are not related to the thickness of this layer but seem to correlate with the quality of the dentin substrate [6±8]. Although many advances have been made in understanding the hybridization process, several important questions remain with respect to physical and mechanical properties of this layer. * Corresponding author. Tel.: 181-3-3219-8141; fax: 181-3-3219-8347. E-mail address: [email protected] (M. Miyazaki).
If the super®cial dentin is completely demineralized and the resin monomer in®ltration is complete, the hybrid layer consists of approximately 70 vol% of resin and 30 vol% of collagen ®bers [9]. Mechanical properties of the adhesive resin in the hybrid layer should play an important role in creation of a durable bond [10]. Due to the solubility parameter theory that controls resin in®ltration [11,12], and the presence of collagen ®bers, there is a probability that incomplete resin penetration may occur [13,14]. The existence of a decalci®ed collagen layer not impregnated by resin at the base of the hybrid layer might weaken the dentin±resin bond [15]. Even if the full penetration into the decalci®ed dentin occurs, the degree of double bond conversion of adhesive resin in the hybrid layer is not known. The presence of intrinsic water in dentin and solvents such as ethanol and acetone in bonding agent may affect polymerization of resinous components in the hybrid layer [16]. Inadequate polymerization of the adhesive monomers or small oligomers might attract
0109-5641/03/$30.00 + 0.00 q 2003 Academy of Dental Materials. Published by Elsevier Science Ltd. All rights reserved. PII: S 0109-564 1(02)00039-8
0ABJ Filtek Z250 6AB
J225 Pal®que Estelite 004
00687 A Clear®l AP-X 0056
050113 Lite-Fil II A 0797
FB Bond (4-AET, HEMA, UDMA, Filler, CQ) Bond (MDP, HEMA, BisGMA, ®ller, CQ) Bonding Agent (MAC-10, Bis-GMA CQ) Single Bond (Bis-GMA, HEMA, polyalkenoic copolymer water, ethanol, CQ)
Resin Lot no. Adhesive (main components)
Lot. no.
M. Miyazaki et al. / Dental Materials 19 (2003) 245±251
water, leading to their slow extraction and diminished bonding properties. However, due to thin thickness of the hybrid layer, insuf®cient information with respect to the quality of the resin±dentin interface is available. The presence of oxygen is known to inhibit the polymerization of resin composite. The bonding resin, which is placed in a very thin layer, might be more susceptible to oxygen inhibition of the polymerization. It has been reported that air thinning the bonding resin prior to light irradiation signi®cantly reduced dentin bond strength [17]. The thicker layer is necessary to make the inner region of the bonding resin to be less in¯uenced by oxygen inhibition. The effect of oxygen was responsible for the formation of an inhibited zone on the surface of the resin in contact with environmental air. The thickness of the oxygen inhibited layer is related to monomer composition, mode of activation, and viscosities of the resin [18]. It was the purpose of this study to investigate the degree of remaining uncured methacrylate carbon±carbon double bonds (RDB) in the resin±dentin interface including the hybrid layer by use of layer Raman microscopy. The hypothesis tested here was that the RDB in the resin±dentin interface are different among the resin±dentin interface.
7EC
A: 005 B: 003
A: 038 B:048
Imperva Fluoro Bond
Clear®l Mega Bond
Mac Bond II
Single Bond
FB
LB
MB
SB
FB Primer (4-AET, HEMA, water, ethanol) Primer (5-MNSA, HEMA, water, ethanol) Primer (MAC-10, HEMA, water, ethanol) Etchant (35% phosphoric acid)
A: 079726 B:079732
2.1. Specimen preparation
System
Conditioner (main components)
Lot no.
2. Materials and methods
Code
Table 1 Bonding systems used in this study (4-AET: 4-acryloxyethyltrimellitic acid, HEMA: 2-hydroxyethyl methacrylate, UDMA: urethane di-methacrylate, 5-MNSA: N-methacryloyl 5-aminosalicylic acid, Phenyl-P: 2-methacryloyloxyethyl phenyl hydrogen phosphate, MDP: 10-methacryloyloxydecyl dihydrogen phosphate; Bis-GMA: bisphenol-glycidyl methacrylate, MAC-10: 11-methacryloxy-11-undecarboxylic acid, CQ: camphorquinone)
246
The combination of the bonding systems/resin composites used were three self-etching primer systems Mac Bond II/Pal®que Estelite (Tokuyama Co., Tokyo, Japan), Clear®l Mega Bond/Clear®l AP-X (Kuraray Co., Osaka, Japan), and Imperva Fluoro Bond/Lite-Fil II A (Shofu Inc., Kyoto, Japan), and a total-etch adhesive system Single Bond/Z250 (3M Dental Products Division, St Paul, MN, USA) as listed in Table 1. The same adhesive resins without photoinitiators were provided by each manufacturer as controls to calculate the percentage of RDB. A dental photo-curing unit (Optilux 500, Demetron/Kerr, Danbury, CT, USA) was connected to a variable transformer in order to adjust the light intensity to 600 mW/cm 2 as measured with a dental radiometer (Model 100, Demetron/Kerr). Mandibular incisors extracted from 2 to 3 year old cattle and stored frozen for up to 2 weeks were used as a substitute for human teeth in this study [19±21]. It has been reported that the adhesion to the super®cial layer of dentin showed no signi®cant differences between human and bovine dentin, and the dentin bond strength decreased with the depth of dentin because of the lower density of dentinal tubules [21]. Because the differences in tubule diameters and the number of lateral branches may have some effect on dentin bond strength [22], bovine super®cial dentin was used as a substitute for human dentin. After removing the roots with a low-speed saw (Isomet, Buehler Ltd, Lake Bluff, IL, USA), the pulps were removed,
M. Miyazaki et al. / Dental Materials 19 (2003) 245±251
247
Fig. 1. Typical Raman spectrum obtained from Si plate. Because the semiconductor such as Si is very simple consisting of only one Raman peak, this peak was used for calibration the equipment before measurement.
and the labial surfaces were ground on wet 240-grit silicon carbide paper to expose dentin. Final ®nish was accomplished with 600-grit silicon carbide paper followed by ultrasonic cleaning with distilled water for 1 min. The dentin surfaces were treated with primer/etchant and adhesives were applied according to manufacturers' directions. Resin composite as listed in Table 1 was condensed into a Te¯on mold (4 mm diameter, 2 mm high) and exposed for 40 s. After 24 h in distilled water at 37 8C, the specimens were embedded in self-curing epoxy resin (Epon 812, Nisshin EM, Tokyo, Japan) and stored at 37 8C for 12 h. After setting, the epoxy-embedded specimens were sectioned parallel to dentinal tubules. The sectioned surfaces of the cut halves were polished to high gloss (Ecomet 4/ Automet 2, Buehler Ltd) using silicon carbide papers of 600-, 1200- and 4000-grit, successively. Then, the surface was mirror polished on a special soft cloth (Technofron, Heraus Kulzer GmbH, Wehrheim, Germany) with diamond paste (Hyprez Diamond Compounds, Engis Corp., Morton Grove, IL, USA) to a particle size of 1 mm. Three specimens originating from different bovine teeth were prepared for each adhesive system. 2.2. Laser Raman microscopy Raman spectra of the unpolymerized adhesive resins and polymerized resins in the hybrid layer were obtained with a computer-controlled laser Raman microscope (System 2000, Renishaw, UK), equipped to analyze the dentin± resin interface. The sample was excited at a wavelength of 632.8 nm with the output level of 75 mW by a He±Ne laser (GLG-5900, NEC Co., Tokyo, Japan) through an optical microscope. The focus of the laser beam in conjunction
with the backthinned CCD camera provided a spatial resolution of 0.6±0.8 mm determined as follows. The spectral resolution of the laser Raman microscopy was determined by use of a silicon solid plate, ion-coated with thin Au. The plate was placed on a precision X±Y stage and moved across from Au-coated to Au-uncoated areas using 0.2 mm steps. Spectra of Si (520 cm 21) were obtained at positions corresponding to each measuring point and the relative intensities of the spectra were calculated (Fig. 1). Using curve ®tting data of this spectrum, the spatial resolution was 1.6±1.9 mm, hence the spectral resolution was determined to be spatial resolution of 0.6± 0.8 mm. Variations of Raman intensities of partially the Au-coated silicon plate as a function of measuring points are shown in Fig. 2. From the acquired data, a differential rate was calculated, and the spatial resolution of the test instrumentation was determined as 0.6±0.8 mm. This device was also designed as a confocal microscope and sampling depth of the Raman scattering from the 1 £ 1 mm 2 area was calculated as 2 mm. 2.3. Residual double bonds calculation The specimen was placed on the X±Y stage and the laser beam was focused on the specimen surface through a 100 £ microscope objective. The specimen was moved relative to the layer spot position by steps of 0.2 mm from the dentin towards the adhesive resin, and the spectra were obtained at positions corresponding to this interval across the dentin± adhesive interface. When the measurement was performed on dentin, the laser beam was focused on intertubular dentin, avoiding dentinal tubules ®lled with adhesive resin. Raman spectra of the uncured adhesive resins were also recorded as the control. Measurements were repeated
248
M. Miyazaki et al. / Dental Materials 19 (2003) 245±251
Fig. 3. The Raman spectrum of cured MB in the region of 1560± 1800 cm 21. A few intense peaks were observed in this area. Fig. 2. Obtained Raman intensities of Si as a function of each measuring point from Au-coated to Au-uncoated areas by 0.2 mm steps. Differential rates were calculated from the acquired data, and the spatial resolution of the test instrumentation was determined as 0.6±0.8 mm.
three times with different specimens for each bonding system. The acquired spectra in the region of interest (1500± 1800 cm 21) were analyzed using a curve ®tting program with the Raman microscope software (Renishaw Raman Software ver. 3.3.1., Renishaw, UK). Before curve ®tting, a strong peak at 960 cm 21 (P±O stretching mode of hydroxyapatite) was eliminated from the obtained spectrum to reduce the background effect, and then the spectrum of pure bovine collagen dentin was subtracted. The measurement of RDB was made on a relative basis by comparing the CyC unpolymerized methacrylate stretching vibration (1638 cm 21) to that of the CyO stretching mode of the ester group (1719 cm 21). A band whose intensity does not change during polymerization should be selected as a reference band. In this study, the CyO stretching mode of the ester group at 1719 cm 21 was used as an internal standard, because some of the adhesive resins do not use aromatic ring containing monomers as base resins. It has been reported that the CyC stretching mode at 1640 cm 21 can be in¯uenced by overlapping with the CyO stretching mode at 1719 cm 21, whose intensity has tended to broaden during polymerization [23]. The frequency of the CyO is affected by intra- and intermolecular hydrogen bonding, so that the CyO vibration sometimes is observed as a broadened doublet [24]. To eliminate this effect, the area ratio after curve ®tting was used to calculate the intensity of the bands [25]. The intensities for the individual bands (1638 and 1719 cm 21) were then obtained from the peak areas by the curve ®tting software, which was designed to calculate a best-®t curve. In this way, the ratios of the two band areas were calculated at each measuring point, from dentin side to resin side of the dentin±resin interface. The percentage of RDB in the hybrid layer was calculated using the following
equations: RDB % Rcured =Runcured where Rcured
CyC absorbance area of hybrid layer CyO absorbance area of hybrid layer
Runcured
CyC absorbance area of uncured resin adhesive CyO absorbance area of uncured resin adhesive
Care should be taken that the obtained percentages were based on the assumption that the linearity of the ratios between uncured and cured states existed. The area of dentin, hybrid layer, and adhesive resin were determined by observing changes in Raman intensities at 960 cm 21 (P±O stretching mode of hydroxyapatite) and 1450 cm 21 (CH scissoring vibration of alkyl group). The intensities of the peaks were plotted relatively from dentin to resin [26±32]. The gradual increase in intensity of the Raman peak at 960 cm 21 (hydroxyapatite) from resin to dentin side of the resin±dentin interface was observed. On the other hand, the intensity of the Raman peak at 1450 cm 21 (organic substrate) across the interface gradually decreased, indicating gradual impregnation of bonding resin into the demineralized dentin. From the gradual changes of these Raman peaks, the width of each area was determined. One-way analysis of variance and Duncan multiple range test was used to statistically evaluate the RDB among the bonding systems used a 0:05: 3. Results The Raman spectrum of cured adhesive resin of Mac Bond II adhesive in the spectral region of 1560± 1800 cm 21 is shown in Fig. 3. Several intense peaks in the Raman spectrum were observed. The band area at
M. Miyazaki et al. / Dental Materials 19 (2003) 245±251
Fig. 4. Representative intensity curves of the selected Raman bands scanned across the resin±dentin interface. Since the C±H alkyl groups also appeared in the collagen spectrum in dentinal substrate, the changes in relative intensities of this peak were considered from the dentin to the resin side.
1638 cm 21, which is assigned to CyC stretching vibration of methacrylate was compared to the area at 1719 cm 21, which is assigned to CyO stretching vibration of ester, used as an internal standard. Intensity curves of the selected Raman bands scanned across the resin±dentin interface obtained with the P±O peak at 960 cm 21 and the C±H peak at 1450 cm 21 (Fig. 4) allowed the estimated depth of resin impregnation into dentin. Since the C±H band obtained at 1450 cm 21 also appeared in the collagen spectrum, the changes in relative intensities of this peak were considered from the dentin to the resin. These depths were 2.2±2.4 mm for Mac Bond II, 2.0±2.2 mm for Mega Bond and Fluoro Bond, 6.4±6.8 mm for Single Bond. Fig. 5 shows the changes in percentages of RDB in the hybrid layer of each bonding system. The percent of residual double bonds detected in the hybrid layer ranged from 9.5 to 23.6%, and was different among the bonding systems used in this study. A relatively higher percentage of RDB was detected for Fluoro Bond (12.3±23.6%) and Single bond (9.5±21.8%), and lower for Mac Bond II (10.6±18.0%) and Mega Bond (10.7±16.3%). No relationship was seen between the percentage of remaining double bonds and the location within the resin±dentin interface. 4. Discussion The laser Raman spectroscopy is a useful analytical technique for study of the bonding structure of samples and determining their composition. Using this technique, the problems associated with morphological analysis of the dentin±resin interface with infrared spectroscopy (IR) can be avoided [33]. The specimens can be observed under normal atmospheric conditions without ultra-thin sectioning
249
Fig. 5. Percentage of residual double bonds in the hybrid layer of the bonding systems used in this study.
or other preparatory procedures that might damage the interface. In contrast to conventional IR microscopy, water is a weak Raman scatterer so that Raman spectra can be acquired from moist dentin specimens. Despite these advantages, the background from ¯uorescence dominates the weaker Raman signal. The ¯uorescence is due to electronic excitation of the organic component when irradiated by laser radiation. To help overcome the problem of high background noise, a He±Ne laser was used as an excitation source in this study. In addition, the light generated from a He±Ne laser (632.8 nm) does not contribute to initiating the polymerization reaction of visible-light cured resins that use campohroquinone as a photoinitiator [34]. Several approaches are employed for measurement of conversion in resin composites; the majority of analytical studies have been done with the use of Fourier-Transmit IR (FTIR) [35±38]. As a vibrational technique like FTIR, Raman microscopy has been used to investigate conversion pro®les of resin composite [23,24,39±41]. Since Raman microscopy involves scattering rather than absorption, and laser emission can be focused on the specimen surface, specimens of any geometry and thickness can be analyzed without destructive procedures. The molecular vibration frequencies observed by FTIR and Raman microscopy are nearly the same, and Raman scattering due to a symmetrical vibration (such as CyC) is more sensitive than FTIR scattering [33]. The micromechanical entrapment of resin in the demineralized dentin substrate is of importance to create a hybrid layer. The dentin±resin interface has been investigated by use of micromorphological techniques and the importance of resin penetration through the entire depth of decalci®ed dentin has been emphasized. After in®ltration of the resin monomers into the decalci®ed dentin, subsequent polymerization of monomers is required to create a stable bond. If the polymerization of these monomers is not complete, hydrophilic monomers or small oligomers might be extracted or hydrolyzed by the presence of nanoleakage
250
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[13]. The quality of the hybrid layer and uniformity of resin impregnation are important factors to understand contributions of the hybrid layer for dentin bonding mechanism. The presence of water inside the dentin substrate might interfere with the polymerization of resin adhesives [16]. Residual resin monomers may act as a plasticizer to alter the mechanical properties of adhesive resin as well as hybrid layer, leading to lower dentin bond strength. From the results of this study, the amount of RDB of the adhesive resin in the hybrid layer varied from 10 to 25%, which is lower than reported for the restorative composite resins at 25±55% [42]. The amount of RDB in methacrylate groups depends on the extent of polymerization reaction. For a light-cured resin, the degree of conversion is affected by light intensity, which provides the energy to excite the photoinitiator. It is well known that the distance from the light tip and the resin surface, and the thickness of the material are the factors that would affect the conversion [43]. Since the adhesive resin and the hybrid layer have micron-sized thickness, light emission from the activator light should be intense enough to polymerize the monomers in this layer. Another reason for relatively lower amounts of residual double bonds in the hybrid layer would be an elution of monomers into water. The rate of elution of components from composite resins has been reported to be rapid during the initial period of immersion in solvent, leading to substantial solution within hours [44]. Uncured monomers in the hybrid layer may leach during the specimen preparation or storage in water thus reducing the amount of uncured RDB found. Relatively higher amounts of RDB in the hybrid layer were observed with Fluoro Bond and Single Bond. The degree of conversion appears to depend upon the monomer composition of the resin and the concentration of initiator/ inhibitor. The conversion of a bis-GMA/TEGDMA mixture is about 20% higher than that of UDMA/TGDMA mixture [45]. The Fluoro Bond adhesive system utilizes UDMA as a base resin. For Single bond, phosphoric acid was used as a demineralizing treatment to remove the smear layer and to expose collagen ®bers. The adhesive resin penetrates into the demineralized dentin by displacing water inside the collagen network. After in®ltration into the dentin, the resin monomers should polymerize to create a strong bond. It is well known that the presence of oxygen from ambient air during placement of resin composite inhibits the resin polymerization to some extent [46]. There might be a possibility that oxygen present in the water around collagen ®bers acts as an inhibitor of bonding resin polymerization for the bonding system, which utilizes a wet bonding technique. Due to the micron-size scale and compositional complexity of the hybrid layer, it has been dif®cult earlier to analyze the nature of the hybrid layer. With the use of the laser Raman spectroscopy, the remaining double bonds and the relative extent of conversion of commercial dentin bonding agents in hybrid layer has been assessed. Further studies are
needed to determine the relationship between the amount of remaining double bonds and bond strength durability.
Acknowledgements The authors are indebted to Mr Kimura (Tokuyama Co., Tokyo, Japan) for his technical assistance. This work was supported, in part, by grant in aid (C)(2) 12671861 from the Ministry of Education, Science, Sports and Culture of Japan.
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Determination of residual double bonds in resin±dentin interface by Raman spectroscopy M. Miyazaki a,*, H. Onose a, N. Iida b, H. Kazama b a
Department of Operative Dentistry, Nihon University School of Dentistry, 1-8-13, Kanda Surugadai, Chiyoda-Ku, Tokyo 101-8310, Japan b Research Laboratories, Tokuyama Corp., 40, Wadai, Tsukuba-city, Ibaraki 300-4247, Japan Received 30 January 2001; revised 6 September 2001; accepted 4 December 2001
Abstract Objectives: The quality of the hybrid layer is believed to be more important than the thickness of this layer. The purpose of this study was to investigate a method to analyze the percentage of adhesive resin residual double bonds in the dentin±resin interface using laser Raman spectroscopy. Methods: Bovine dentin was treated with dentin adhesives and resin composite was bonded according to the manufacturers' instructions. The specimens were sectioned parallel to dentinal tubules and the surfaces were then polished to 1 mm diamond pastes. Raman spectra were recorded along a line perpendicular to the dentin±resin interface in steps of 0.2 mm. The measurement of residual CyC bond was made on a relative basis by comparing the CyC unpolymerized methacrylate stretching vibration (1638 cm 21) against the CyO stretching mode of the ester group (1719 cm 21). The percentage of residual double bonds including pendant and monomeric double bonds was calculated by comparing the obtained ratio with that of uncured adhesive resin. Results: The amount of residual double bonds in the hybrid layer varied from 10 to 25% compared to the uncured adhesives, a relatively higher percentage was detected for Fluoro Bond (12.3±23.6%) and Single Bond (9.5±21.8%), and lower for Mac Bond II (10.6±18.0%) and Mega Bond (10.7±16.3%). No relationship was seen between the percentage of remaining double bonds and the location within the resin± dentin interface. Signi®cance: Laser Raman microscopy used was a useful tool for measuring the residual double bonds in the dentin±resin interface. q 2003 Academy of Dental Materials. Published by Elsevier Science Ltd. All rights reserved. Keywords: Laser Raman microscopy; Residual double bond; Dentin bonding; Hybrid layer
1. Introduction A strong and durable bond between resin restoration and tooth is needed to prevent microleakage and formation of marginal gap. Excellent adaptation of resin to dentin with monomer penetration has been reported with scanning and transmission electron microscopic studies [1±4]. The hybrid layer is believed to be one of the most effective mechanisms in dentin bonding [5]. Morphological examination of the hybrid layer has revealed that the bond strength and sealing ability of bonding systems are not related to the thickness of this layer but seem to correlate with the quality of the dentin substrate [6±8]. Although many advances have been made in understanding the hybridization process, several important questions remain with respect to physical and mechanical properties of this layer. * Corresponding author. Tel.: 181-3-3219-8141; fax: 181-3-3219-8347. E-mail address: [email protected] (M. Miyazaki).
If the super®cial dentin is completely demineralized and the resin monomer in®ltration is complete, the hybrid layer consists of approximately 70 vol% of resin and 30 vol% of collagen ®bers [9]. Mechanical properties of the adhesive resin in the hybrid layer should play an important role in creation of a durable bond [10]. Due to the solubility parameter theory that controls resin in®ltration [11,12], and the presence of collagen ®bers, there is a probability that incomplete resin penetration may occur [13,14]. The existence of a decalci®ed collagen layer not impregnated by resin at the base of the hybrid layer might weaken the dentin±resin bond [15]. Even if the full penetration into the decalci®ed dentin occurs, the degree of double bond conversion of adhesive resin in the hybrid layer is not known. The presence of intrinsic water in dentin and solvents such as ethanol and acetone in bonding agent may affect polymerization of resinous components in the hybrid layer [16]. Inadequate polymerization of the adhesive monomers or small oligomers might attract
0109-5641/03/$30.00 + 0.00 q 2003 Academy of Dental Materials. Published by Elsevier Science Ltd. All rights reserved. PII: S 0109-564 1(02)00039-8
0ABJ Filtek Z250 6AB
J225 Pal®que Estelite 004
00687 A Clear®l AP-X 0056
050113 Lite-Fil II A 0797
FB Bond (4-AET, HEMA, UDMA, Filler, CQ) Bond (MDP, HEMA, BisGMA, ®ller, CQ) Bonding Agent (MAC-10, Bis-GMA CQ) Single Bond (Bis-GMA, HEMA, polyalkenoic copolymer water, ethanol, CQ)
Resin Lot no. Adhesive (main components)
Lot. no.
M. Miyazaki et al. / Dental Materials 19 (2003) 245±251
water, leading to their slow extraction and diminished bonding properties. However, due to thin thickness of the hybrid layer, insuf®cient information with respect to the quality of the resin±dentin interface is available. The presence of oxygen is known to inhibit the polymerization of resin composite. The bonding resin, which is placed in a very thin layer, might be more susceptible to oxygen inhibition of the polymerization. It has been reported that air thinning the bonding resin prior to light irradiation signi®cantly reduced dentin bond strength [17]. The thicker layer is necessary to make the inner region of the bonding resin to be less in¯uenced by oxygen inhibition. The effect of oxygen was responsible for the formation of an inhibited zone on the surface of the resin in contact with environmental air. The thickness of the oxygen inhibited layer is related to monomer composition, mode of activation, and viscosities of the resin [18]. It was the purpose of this study to investigate the degree of remaining uncured methacrylate carbon±carbon double bonds (RDB) in the resin±dentin interface including the hybrid layer by use of layer Raman microscopy. The hypothesis tested here was that the RDB in the resin±dentin interface are different among the resin±dentin interface.
7EC
A: 005 B: 003
A: 038 B:048
Imperva Fluoro Bond
Clear®l Mega Bond
Mac Bond II
Single Bond
FB
LB
MB
SB
FB Primer (4-AET, HEMA, water, ethanol) Primer (5-MNSA, HEMA, water, ethanol) Primer (MAC-10, HEMA, water, ethanol) Etchant (35% phosphoric acid)
A: 079726 B:079732
2.1. Specimen preparation
System
Conditioner (main components)
Lot no.
2. Materials and methods
Code
Table 1 Bonding systems used in this study (4-AET: 4-acryloxyethyltrimellitic acid, HEMA: 2-hydroxyethyl methacrylate, UDMA: urethane di-methacrylate, 5-MNSA: N-methacryloyl 5-aminosalicylic acid, Phenyl-P: 2-methacryloyloxyethyl phenyl hydrogen phosphate, MDP: 10-methacryloyloxydecyl dihydrogen phosphate; Bis-GMA: bisphenol-glycidyl methacrylate, MAC-10: 11-methacryloxy-11-undecarboxylic acid, CQ: camphorquinone)
246
The combination of the bonding systems/resin composites used were three self-etching primer systems Mac Bond II/Pal®que Estelite (Tokuyama Co., Tokyo, Japan), Clear®l Mega Bond/Clear®l AP-X (Kuraray Co., Osaka, Japan), and Imperva Fluoro Bond/Lite-Fil II A (Shofu Inc., Kyoto, Japan), and a total-etch adhesive system Single Bond/Z250 (3M Dental Products Division, St Paul, MN, USA) as listed in Table 1. The same adhesive resins without photoinitiators were provided by each manufacturer as controls to calculate the percentage of RDB. A dental photo-curing unit (Optilux 500, Demetron/Kerr, Danbury, CT, USA) was connected to a variable transformer in order to adjust the light intensity to 600 mW/cm 2 as measured with a dental radiometer (Model 100, Demetron/Kerr). Mandibular incisors extracted from 2 to 3 year old cattle and stored frozen for up to 2 weeks were used as a substitute for human teeth in this study [19±21]. It has been reported that the adhesion to the super®cial layer of dentin showed no signi®cant differences between human and bovine dentin, and the dentin bond strength decreased with the depth of dentin because of the lower density of dentinal tubules [21]. Because the differences in tubule diameters and the number of lateral branches may have some effect on dentin bond strength [22], bovine super®cial dentin was used as a substitute for human dentin. After removing the roots with a low-speed saw (Isomet, Buehler Ltd, Lake Bluff, IL, USA), the pulps were removed,
M. Miyazaki et al. / Dental Materials 19 (2003) 245±251
247
Fig. 1. Typical Raman spectrum obtained from Si plate. Because the semiconductor such as Si is very simple consisting of only one Raman peak, this peak was used for calibration the equipment before measurement.
and the labial surfaces were ground on wet 240-grit silicon carbide paper to expose dentin. Final ®nish was accomplished with 600-grit silicon carbide paper followed by ultrasonic cleaning with distilled water for 1 min. The dentin surfaces were treated with primer/etchant and adhesives were applied according to manufacturers' directions. Resin composite as listed in Table 1 was condensed into a Te¯on mold (4 mm diameter, 2 mm high) and exposed for 40 s. After 24 h in distilled water at 37 8C, the specimens were embedded in self-curing epoxy resin (Epon 812, Nisshin EM, Tokyo, Japan) and stored at 37 8C for 12 h. After setting, the epoxy-embedded specimens were sectioned parallel to dentinal tubules. The sectioned surfaces of the cut halves were polished to high gloss (Ecomet 4/ Automet 2, Buehler Ltd) using silicon carbide papers of 600-, 1200- and 4000-grit, successively. Then, the surface was mirror polished on a special soft cloth (Technofron, Heraus Kulzer GmbH, Wehrheim, Germany) with diamond paste (Hyprez Diamond Compounds, Engis Corp., Morton Grove, IL, USA) to a particle size of 1 mm. Three specimens originating from different bovine teeth were prepared for each adhesive system. 2.2. Laser Raman microscopy Raman spectra of the unpolymerized adhesive resins and polymerized resins in the hybrid layer were obtained with a computer-controlled laser Raman microscope (System 2000, Renishaw, UK), equipped to analyze the dentin± resin interface. The sample was excited at a wavelength of 632.8 nm with the output level of 75 mW by a He±Ne laser (GLG-5900, NEC Co., Tokyo, Japan) through an optical microscope. The focus of the laser beam in conjunction
with the backthinned CCD camera provided a spatial resolution of 0.6±0.8 mm determined as follows. The spectral resolution of the laser Raman microscopy was determined by use of a silicon solid plate, ion-coated with thin Au. The plate was placed on a precision X±Y stage and moved across from Au-coated to Au-uncoated areas using 0.2 mm steps. Spectra of Si (520 cm 21) were obtained at positions corresponding to each measuring point and the relative intensities of the spectra were calculated (Fig. 1). Using curve ®tting data of this spectrum, the spatial resolution was 1.6±1.9 mm, hence the spectral resolution was determined to be spatial resolution of 0.6± 0.8 mm. Variations of Raman intensities of partially the Au-coated silicon plate as a function of measuring points are shown in Fig. 2. From the acquired data, a differential rate was calculated, and the spatial resolution of the test instrumentation was determined as 0.6±0.8 mm. This device was also designed as a confocal microscope and sampling depth of the Raman scattering from the 1 £ 1 mm 2 area was calculated as 2 mm. 2.3. Residual double bonds calculation The specimen was placed on the X±Y stage and the laser beam was focused on the specimen surface through a 100 £ microscope objective. The specimen was moved relative to the layer spot position by steps of 0.2 mm from the dentin towards the adhesive resin, and the spectra were obtained at positions corresponding to this interval across the dentin± adhesive interface. When the measurement was performed on dentin, the laser beam was focused on intertubular dentin, avoiding dentinal tubules ®lled with adhesive resin. Raman spectra of the uncured adhesive resins were also recorded as the control. Measurements were repeated
248
M. Miyazaki et al. / Dental Materials 19 (2003) 245±251
Fig. 3. The Raman spectrum of cured MB in the region of 1560± 1800 cm 21. A few intense peaks were observed in this area. Fig. 2. Obtained Raman intensities of Si as a function of each measuring point from Au-coated to Au-uncoated areas by 0.2 mm steps. Differential rates were calculated from the acquired data, and the spatial resolution of the test instrumentation was determined as 0.6±0.8 mm.
three times with different specimens for each bonding system. The acquired spectra in the region of interest (1500± 1800 cm 21) were analyzed using a curve ®tting program with the Raman microscope software (Renishaw Raman Software ver. 3.3.1., Renishaw, UK). Before curve ®tting, a strong peak at 960 cm 21 (P±O stretching mode of hydroxyapatite) was eliminated from the obtained spectrum to reduce the background effect, and then the spectrum of pure bovine collagen dentin was subtracted. The measurement of RDB was made on a relative basis by comparing the CyC unpolymerized methacrylate stretching vibration (1638 cm 21) to that of the CyO stretching mode of the ester group (1719 cm 21). A band whose intensity does not change during polymerization should be selected as a reference band. In this study, the CyO stretching mode of the ester group at 1719 cm 21 was used as an internal standard, because some of the adhesive resins do not use aromatic ring containing monomers as base resins. It has been reported that the CyC stretching mode at 1640 cm 21 can be in¯uenced by overlapping with the CyO stretching mode at 1719 cm 21, whose intensity has tended to broaden during polymerization [23]. The frequency of the CyO is affected by intra- and intermolecular hydrogen bonding, so that the CyO vibration sometimes is observed as a broadened doublet [24]. To eliminate this effect, the area ratio after curve ®tting was used to calculate the intensity of the bands [25]. The intensities for the individual bands (1638 and 1719 cm 21) were then obtained from the peak areas by the curve ®tting software, which was designed to calculate a best-®t curve. In this way, the ratios of the two band areas were calculated at each measuring point, from dentin side to resin side of the dentin±resin interface. The percentage of RDB in the hybrid layer was calculated using the following
equations: RDB % Rcured =Runcured where Rcured
CyC absorbance area of hybrid layer CyO absorbance area of hybrid layer
Runcured
CyC absorbance area of uncured resin adhesive CyO absorbance area of uncured resin adhesive
Care should be taken that the obtained percentages were based on the assumption that the linearity of the ratios between uncured and cured states existed. The area of dentin, hybrid layer, and adhesive resin were determined by observing changes in Raman intensities at 960 cm 21 (P±O stretching mode of hydroxyapatite) and 1450 cm 21 (CH scissoring vibration of alkyl group). The intensities of the peaks were plotted relatively from dentin to resin [26±32]. The gradual increase in intensity of the Raman peak at 960 cm 21 (hydroxyapatite) from resin to dentin side of the resin±dentin interface was observed. On the other hand, the intensity of the Raman peak at 1450 cm 21 (organic substrate) across the interface gradually decreased, indicating gradual impregnation of bonding resin into the demineralized dentin. From the gradual changes of these Raman peaks, the width of each area was determined. One-way analysis of variance and Duncan multiple range test was used to statistically evaluate the RDB among the bonding systems used a 0:05: 3. Results The Raman spectrum of cured adhesive resin of Mac Bond II adhesive in the spectral region of 1560± 1800 cm 21 is shown in Fig. 3. Several intense peaks in the Raman spectrum were observed. The band area at
M. Miyazaki et al. / Dental Materials 19 (2003) 245±251
Fig. 4. Representative intensity curves of the selected Raman bands scanned across the resin±dentin interface. Since the C±H alkyl groups also appeared in the collagen spectrum in dentinal substrate, the changes in relative intensities of this peak were considered from the dentin to the resin side.
1638 cm 21, which is assigned to CyC stretching vibration of methacrylate was compared to the area at 1719 cm 21, which is assigned to CyO stretching vibration of ester, used as an internal standard. Intensity curves of the selected Raman bands scanned across the resin±dentin interface obtained with the P±O peak at 960 cm 21 and the C±H peak at 1450 cm 21 (Fig. 4) allowed the estimated depth of resin impregnation into dentin. Since the C±H band obtained at 1450 cm 21 also appeared in the collagen spectrum, the changes in relative intensities of this peak were considered from the dentin to the resin. These depths were 2.2±2.4 mm for Mac Bond II, 2.0±2.2 mm for Mega Bond and Fluoro Bond, 6.4±6.8 mm for Single Bond. Fig. 5 shows the changes in percentages of RDB in the hybrid layer of each bonding system. The percent of residual double bonds detected in the hybrid layer ranged from 9.5 to 23.6%, and was different among the bonding systems used in this study. A relatively higher percentage of RDB was detected for Fluoro Bond (12.3±23.6%) and Single bond (9.5±21.8%), and lower for Mac Bond II (10.6±18.0%) and Mega Bond (10.7±16.3%). No relationship was seen between the percentage of remaining double bonds and the location within the resin±dentin interface. 4. Discussion The laser Raman spectroscopy is a useful analytical technique for study of the bonding structure of samples and determining their composition. Using this technique, the problems associated with morphological analysis of the dentin±resin interface with infrared spectroscopy (IR) can be avoided [33]. The specimens can be observed under normal atmospheric conditions without ultra-thin sectioning
249
Fig. 5. Percentage of residual double bonds in the hybrid layer of the bonding systems used in this study.
or other preparatory procedures that might damage the interface. In contrast to conventional IR microscopy, water is a weak Raman scatterer so that Raman spectra can be acquired from moist dentin specimens. Despite these advantages, the background from ¯uorescence dominates the weaker Raman signal. The ¯uorescence is due to electronic excitation of the organic component when irradiated by laser radiation. To help overcome the problem of high background noise, a He±Ne laser was used as an excitation source in this study. In addition, the light generated from a He±Ne laser (632.8 nm) does not contribute to initiating the polymerization reaction of visible-light cured resins that use campohroquinone as a photoinitiator [34]. Several approaches are employed for measurement of conversion in resin composites; the majority of analytical studies have been done with the use of Fourier-Transmit IR (FTIR) [35±38]. As a vibrational technique like FTIR, Raman microscopy has been used to investigate conversion pro®les of resin composite [23,24,39±41]. Since Raman microscopy involves scattering rather than absorption, and laser emission can be focused on the specimen surface, specimens of any geometry and thickness can be analyzed without destructive procedures. The molecular vibration frequencies observed by FTIR and Raman microscopy are nearly the same, and Raman scattering due to a symmetrical vibration (such as CyC) is more sensitive than FTIR scattering [33]. The micromechanical entrapment of resin in the demineralized dentin substrate is of importance to create a hybrid layer. The dentin±resin interface has been investigated by use of micromorphological techniques and the importance of resin penetration through the entire depth of decalci®ed dentin has been emphasized. After in®ltration of the resin monomers into the decalci®ed dentin, subsequent polymerization of monomers is required to create a stable bond. If the polymerization of these monomers is not complete, hydrophilic monomers or small oligomers might be extracted or hydrolyzed by the presence of nanoleakage
250
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[13]. The quality of the hybrid layer and uniformity of resin impregnation are important factors to understand contributions of the hybrid layer for dentin bonding mechanism. The presence of water inside the dentin substrate might interfere with the polymerization of resin adhesives [16]. Residual resin monomers may act as a plasticizer to alter the mechanical properties of adhesive resin as well as hybrid layer, leading to lower dentin bond strength. From the results of this study, the amount of RDB of the adhesive resin in the hybrid layer varied from 10 to 25%, which is lower than reported for the restorative composite resins at 25±55% [42]. The amount of RDB in methacrylate groups depends on the extent of polymerization reaction. For a light-cured resin, the degree of conversion is affected by light intensity, which provides the energy to excite the photoinitiator. It is well known that the distance from the light tip and the resin surface, and the thickness of the material are the factors that would affect the conversion [43]. Since the adhesive resin and the hybrid layer have micron-sized thickness, light emission from the activator light should be intense enough to polymerize the monomers in this layer. Another reason for relatively lower amounts of residual double bonds in the hybrid layer would be an elution of monomers into water. The rate of elution of components from composite resins has been reported to be rapid during the initial period of immersion in solvent, leading to substantial solution within hours [44]. Uncured monomers in the hybrid layer may leach during the specimen preparation or storage in water thus reducing the amount of uncured RDB found. Relatively higher amounts of RDB in the hybrid layer were observed with Fluoro Bond and Single Bond. The degree of conversion appears to depend upon the monomer composition of the resin and the concentration of initiator/ inhibitor. The conversion of a bis-GMA/TEGDMA mixture is about 20% higher than that of UDMA/TGDMA mixture [45]. The Fluoro Bond adhesive system utilizes UDMA as a base resin. For Single bond, phosphoric acid was used as a demineralizing treatment to remove the smear layer and to expose collagen ®bers. The adhesive resin penetrates into the demineralized dentin by displacing water inside the collagen network. After in®ltration into the dentin, the resin monomers should polymerize to create a strong bond. It is well known that the presence of oxygen from ambient air during placement of resin composite inhibits the resin polymerization to some extent [46]. There might be a possibility that oxygen present in the water around collagen ®bers acts as an inhibitor of bonding resin polymerization for the bonding system, which utilizes a wet bonding technique. Due to the micron-size scale and compositional complexity of the hybrid layer, it has been dif®cult earlier to analyze the nature of the hybrid layer. With the use of the laser Raman spectroscopy, the remaining double bonds and the relative extent of conversion of commercial dentin bonding agents in hybrid layer has been assessed. Further studies are
needed to determine the relationship between the amount of remaining double bonds and bond strength durability.
Acknowledgements The authors are indebted to Mr Kimura (Tokuyama Co., Tokyo, Japan) for his technical assistance. This work was supported, in part, by grant in aid (C)(2) 12671861 from the Ministry of Education, Science, Sports and Culture of Japan.
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