monomers in self-etching primers

Journal of Dentistry (2005) xx, 1 11 www.intl.elsevierhealth.com/journals/jden 13 C NMR analysis of the etching efficacy of acidic monomers in self-etching primers Kou Fujita a,c, *, Norihiro Nishiyama b,c a Department of Dental Caries Control and Aesthetic Dentistry, Nihon University School of Dentistry at Matsudo, 870-1 Sakaecho, Nishi 2, Matsudo, Chiba 271-8587, Japan b Department of Dental Biomaterials, Nihon University School of Dentistry at Matsudo, 870-1 Sakaecho, Nishi 2, Matsudo, Chiba 271-8587, Japan c Research Institute of Oral Science, Nihon University School of Dentistry at Matsudo, 870-1 Sakaecho, Nishi 2, Matsudo, Chiba 271-8587, Japan Received 24 May 2004; accepted 27 April 2005 KEYWORDS Demineralisation aspect; Self-etching primer; Mega bond primer; MDP; UniFil bond primer; 4-MET; 13 C NMR Summary It is well understood that the application of a self-etching primer enhances the bonding of the resin to the tooth. In this study, the demineralisation aspects by the Mega Bond Primer (MB) or the UniFil Bond Primer (UB) on the tooth were investigated by using liquid-state and solid-state 13 C NMR techniques. The addition of hydroxyapatite or dentine to MB and the addition of dentine to UB resulted in the decrease in the peak intensity of the 13 C NMR peaks attributed to the methacryloxy decyl phosphoric acid, MDP in the MB or 4-methacryloyloxy ethoxy carbonylphthalic acid, 4-MET in the UB. This decrease was because the MDP or 4-MET demineralised the tooth and the calcium salts produced from the MDP or 4-MET were precipitated from the MB or UB solution. The NMR technique is very powerful in evaluating the demineralisation aspects of the tooth by a self-etching primer. However, the calcium salts produced by the MDP or 4-MET on the tooth surface would not facilitate retention in bonding, since these calcium salts were merely deposited on to the surface of the tooth. Q 2005 Published by Elsevier Ltd. Introduction It is well understood that the application of a selfetching primer, consisting of acidic and hydrophilic * Corresponding author. Address: Department of Operative Dentistry, Nihon University School of Dentistry at Matsudo, 870-1 Sakaecho, Nishi 2, Matsudo, Chiba 271-8587, Japan. Tel.: C81 47 360 9360; fax: C81 47 364 6295. E-mail address: kou@mascat.nihon-u.ac.jp (K. Fujita). 0300-5712/$ - see front matter Q 2005 Published by Elsevier Ltd. doi:10.1016/j.jdent.2005.04.005 methacrylate monomers, enhances the bonding at the resin-tooth interface. 1,2 This enhancement is due to the fact that the acidic monomers permeate the smear layer, demineralise the intact enamel and dentine, and prime the enamel and dentine surfaces at the same time. In general, acidic monomers with phosphoric acid or carboxylic acid functional groups are utilised in commercially available self-etching primers. However, the demineralisation mechanisms of these acidic monomer have not yet been identified. In order to understand these mechanisms, chemical analyses have been

2 K. Fujita, N. Nishiyama conducted using electron spectroscopy 3 and nuclear magnetic resonance. 4,5 Yoshida and Nishiyama designed a self-etching primer comprised of NMGly (N-methacryloyl glycine), from which the demineralisation mechanisms by the carboxylic acid in the NMGly on the tooth were elucidated using the 13 C NMR technique. 6 The addition of hydroxyapatite or dentine to the NMGly aqueous solution resulted in a shift of the 13 C NMR peak, that was attributed to the carbonyl carbon for the carboxylic acid of the NMGly to a lower field. This shift was caused by the carboxylic acid of the NMGly demineralising the hydroxyapatite or dentine, as well as to the formation of calcium salt from the NMGly. The amount of demineralisation by the carboxylic acid in the NMGly on the tooth was directly correlated with the bond strength of the resin to both the enamel and dentine. This NMR technique is a very powerful method for investigating the demineralization mechanisms by the acidic monomer in a self-etching primer on the tooth. In this study, the effect of two different types of acidic group in acidic monomers utilised in selfetching primers on the demineralisation of enamel and dentine were examined using liquid-state and solid-state NMR techniques. Materials and methods Materials Clearfil Mega Bond Primer, MB (Lot number: 0261CA, Kuraray Medical Inc., Tokyo, Japan) and UniFil Bond Primer, UB (Lot number: 0203261, GC Corp., Tokyo, Japan) were used. The acidic monomer employed in the MB is methacryloxy decyl phosphoric acid (MDP), a resin monomer with phosphoric acid functional group, and that in the UB is 4-methacryloyloxy ethoxy carbonylphthalic acid (4-MET), a monomer with carboxylic acid functional groups. The hydroxyapatite (HAP 200, Taihei Chemistry, Japan) was used as a model compound for the enamel, since the Ca/P ratio of the hydroxyapatite and it s crystallinity are similar to those of enamel. The dentine particles were prepared as follows 6 : crown dentine from a bovine tooth was crosssectioned by using a diamond cutter under water cooling. The crown dentine was then cut by using an air-turbine with a diamond bur under a stream of cooling water. The dentine particles were obtained by decantation, rinsed with water and then airdried in a thermo-stabilised room at 20 8C for 1 day. Reactivity of the MEGA Bond Primer or UniFil Bond Primer with hydroxyapatite and dentine Deuterium oxide of 0.8 g was added to 4.2 g of MB or UB. Four hundred milligram of hydroxyapatite or dentine particles were then suspended in 5.0 g of the MB or UB solution. The suspension was sonicated for 30 min and then centrifuged. Next, the ph value of the supernatant MB or UB solution was measured. Liquid-state 13 C NMR observation for the supernatant MB or UB solution was then conducted by using an EX 270 spectrometer (JEOL, Tokyo, Japan). The accumulation and repetition times were 800 and 3.8 s, respectively. Hexamethyl-di-siloxane (HMDSO) was used as an external reference. Analyses of the resultant residue of the self-etching primers with hydroxyapatite and dentine The centrifuged residue obtained from the MB and the hydroxyapatite or dentine was dispersed in ethanol. After this suspension was sonicated for 5 min and centrifuged, the supernatant ethanol solution was discarded. These procedures were repeated until no 13 C NMR peaks attributed to the MB components were detected in the NMR spectrum for the supernatant ethanol solution. This process was undertaken to remove any soluble components attributed to the MB. The residues were then airdried in a thermo-stabilised room at 20 8C for 1 day. Solid-state 13 C NMR observation of the residues obtained from the hydroxyapatite or dentine was examined using the EX 270 spectrometer. The contact, repetition and accumulation times were 0.1, 4.0, and 1000 s, respectively. Hexamethylbenzene was used as an external reference. Furthermore, the solid-state NMR observations of the resultant residues obtained from the UB and the hydroxyapatite or dentine were also performed according to the methods previously discussed. The accumulation times were 20,000. Scanning electron microscope (SEM) examination of the tooth surface conditioned by the self-etching primers To obtain a fresh enamel or dentine surface, a bovine tooth was ground with 100-grit and 600-grit silicon carbide papers under water irrigation. Next, the enamel or dentine surface was conditioned with the MB or UB for 60 s, and then air-dried for few seconds. The conditioned teeth were then rinsed

DTD 5 Etching efficacy of self-etching primers 3 with water or ethanol for 30 s. The rinsed teeth were then dehydrated by using 70, 80, 90, and 100 vol.% of ethanol aqueous solutions. After the teeth were immersed in tertiary-butyl alcohol for 1 day, the teeth were freeze-dried under vacuum (Hitachi ES-3200, Tokyo, Japan). They were mounted onto aluminum stubs, and sputter-coated with a platinum palladium alloy. Each specimen was examined at numerous magnifications and tilt angles in a scanning electron microscope (Hitachi S-4500) at 5 kv. Results 13 C NMR peak assignments attributed to acidic and hydrophilic methacrylate monomers utilised in the self-etching primers Fig. 1A shows the liquid-state 13 C NMR spectrum for MB. In order to assign the 13 C NMR peaks detected in the MB spectrum, the 13 C NMR observations for Figure 1 Liquid-state 13 C NMR spectra for the MEGA Bond Primer, MDP and HEMA. Spectrum A is of the Mega Bond Primer. Spectrum B is of the MDP only. Spectrum C is of the HEMA only.

4 K. Fujita, N. Nishiyama the MDP and 2-hydroxyethyl methacrylate, HEMA were conducted, as shown in Fig. 1B and C, respectively. The 13 C NMR peaks attributed to the MDP and HEMA were assigned, and the 13 C NMR peak assignments are summarised in Fig. 1A. Fig. 2A shows the liquid-state 13 C NMR spectrum for UB. The 13 C NMR spectra of the 4-MET and HEMA are shown in Fig. 2B and C, respectively. The 13 C NMR peaks attributed to the 4-MET and HEMA were assigned, 7 and the 13 C NMR peak 1* and 2* were assigned to the methylene carbon that was directly bonded to the hydroxy group and to the methyl carbon of the ethanol, respectively. The 13 C NMR peak assignments are summarised in Fig. 2A. Figure 2 Liquid-state 13 C NMR spectra for the UniFil Bond Primer, 4-MET and HEMA. Spectrum A is of the UniFil Bond Primer. Spectrum B is of the MDP only. Spectrum C is of the HEMA only.

DTD 5 Etching efficacy of self-etching primers 5 Demineralisation mechanisms of the tooth components by the acidic monomers Fig. 3 shows the liquid-state 13 C NMR spectra for MB, before and after the addition of the tooth components. When the hydroxyapatite or dentine powder was suspended in the MB solution, the ph values of the MB solution increased from 1.89 to 3.24 or 3.56, respectively. This increase in the ph values of the supernatant MB solution was due to the formation of an acid base interaction between the phosphoric acid of the MDP and the calcium salts in the tooth. Furthermore, when the intensity in the 13 C NMR peaks assigned to the MDP was compared with the intensity in the 13 C NMR peaks assigned to the HEMA in the original spectrum for MB (Fig. 3A), the addition of hydroxyapatite (Fig. 3B) or dentine (Fig. 3C) resulted in a dramatic decrease in the intensity of the 13 C NMR peaks a f attributed to the MDP by 70.6 or 77.4%, respectively. This decrease in the intensity of the 13 C NMR peaks for the MDP was attributed to the formation of the calcium salt of MDP that precipitated from the MB solution. Fig. 4 shows the liquid-state 13 C NMR spectra for UB, before and after the addition of the tooth Figure 3 Liquid-state 13 C NMR spectra of the Mega Bond Primer, before and after, reacted with the hydroxyapatite or dentine. Spectrum A is before reaction with the tooth. Spectrum B is after reaction with the hydroxyapatite. Spectrum C is after reaction with the dentine.

6 K. Fujita, N. Nishiyama Figure 4 Liquid-state 13 C NMR spectra of the UniFil Bond Primer, before and after, reacted with the hydroxyapatite or dentine. Spectrum A is before reaction with the tooth. Spectrum B is after reaction with the hydroxyapatite. Spectrum C is after reaction with the dentine. components. The addition of hydroxyapatite or dentine to the UB resulted in an increase in the ph value of the supernatant UB solution from 1.82 to 2.63 or from 1.82 to 3.07, respectively. Moreover, when the chemical shift of the 13 C NMR peaks h and i, attributed to the carbonyl carbons for the meta- and para-carboxylic acids were compared with the original UB spectrum, the addition of hydroxyapatite (Fig. 4B) resulted in a shift of the 13 C NMR peaks h and i to the lower field from 169.13 to 169.46 ppm and from 170.47 to 170.84 ppm, respectively. This was caused by the formation of the carboxylic acid salts of 4-MET following its demineralisation of the hydroxyapatite. Unlike MB,

DTD 5 Etching efficacy of self-etching primers 7 however, no decrease in peak intensity of the 4-MET was observed in UB before and after the addition of hydroxyapatite, when compared with the peak intensity for HEMA in the original spectrum (Fig. 4A). Conversely, the addition of the dentine powder to UB (Fig. 4C) resulted in a shift of the 13 C NMR peaks h and i to the lower field from 169.13 to 169.69 ppm and from 170.47 to 171.12 ppm. There was a simultaneous decrease in the intensity of the 13 C NMR peaks attributed to the 4-MET by 38.4%, when compared with the peak intensities for 4-MET against HEMA in the original UB spectrum. Structural analyses of resultant residues obtained from the reaction of self-etching primers with hydroxyapatite or dentine The solid-state 13 C NMR spectra of the resultant residues obtained from MB and hydroxyapatite or Figure 5 Solid-state 13 C NMR spectra of the reactant residue obtained from the Mega Bond Primer and hydroxyapatite or dentine. Solid-state NMR spectrum A is of the hydroxyapatite reactant. Solid-state NMR spectrum B is of the dentine reactant. Liquid state NMR spectrum C is of the MDP.

8 K. Fujita, N. Nishiyama dentine are shown in Fig. 5A and B, respectively. The detected 13 C NMR peaks in these spectra were attributed to the formation of MDP calcium salt. However, the chemical shift of peak g, assigned to the methylene carbon bonded to the phosphate group, as well as the shape of peak f, assigned to the eight methylene carbons as a spacer group, in the MDP calcium salt for the hydroxyapatite reactant, differed from those associated with the dentine reactant. The solid-state 13 C NMR spectra of the reactants obtained from UB and hydroxyapatite or dentine are shown in Fig. 6A and B, respectively. For the UB and hydroxyapatite reactant, 13 C NMR peaks Figure 6 Solid-state 13 C NMR spectra of the reactant residue obtained from the UniFil Bond Primer and hydroxyapatite or dentine. Solid-state NMR spectrum A is of the hydroxyapatite reactant. Solid-state NMR spectrum B is of the dentine reactant. Liquid-state NMR spectrum C is of the 4-MET.

DTD 5 Etching efficacy of self-etching primers 9 attributed to the 4-MET could not be detected in the NMR spectrum (Fig. 6A), unlike those that were seen between UB and dentine (Fig. 6B). Ultrastructural changes after the application of the self-etching primers on enamel and dentine The SEM views of the enamel and the dentine surfaces conditioned with MB and UB followed by rinsing with water are shown in Fig. 7. The micrographs on the left represent those of the control, unetched ground enamel and dentine as a control. When the enamel surface was conditioned with MB, the typical etching patterns attributed to the alignment of the enamel prism were observed (top middle view). In contrast, when the enamel surface was conditioned by the UB, only a blank outline of the enamel prism was observed (top right view). When the dentine surface was conditioned with MB, the dentinal tubules were widened and blocked by precipitates, with the collagen fibrils adjacent to the tubules exposed (bottom middle view). Conversely, when the dentine surface was conditioned with UB, the dentinal tubes were only widened and the precipitate inside of the dentinal tubes was observed. The collagen fibrils were not exposed (bottom right view). These differences were due to the more effective demineralisation of enamel and dentine by the MDP in MB than the 4-MET in UB. Enamel and dentine surfaces that were conditioned with MB and rinsed with ethanol were covered with crystalline deposits of MDP calcium salts that were insoluble in ethanol (Fig. 8). These crystalline deposits were absent when the conditioned surfaces were rinsed with water (Fig. 7). In contrast, crystalline deposits were not observed after rinsing the 4-MET conditioned surfaces with ethanol, as the carboxylic acidic groups in 4-MET were not strong enough to demineralized the tooth components. Discussion In this study, the addition of hydroxyapatite or dentine to MB resulted in a selective decrease in the intensity of the 13 C NMR peaks that were attributed to the phosphoric acid monomer MDP. This decrease in the peak intensity was due to the formation of insoluble calcium salts in the MB solution, after demineralization of the hydroxyapatite and dentine by MDP. The larger decrease in the dentine samples than that for the hydroxyapatite may be attributed to the higher rate of solubilisation of the dentine apatite crystallites. Additional solid-state 13 C NMR confirmed the existence of the calcium salt of MDP. However, Figure 7 SEM views of the enamel and dentine surface, condition by the Mega Bond Primer or UniFil Bond Primer after rinsing with water (5000!). The left views are of the ground enamel and dentine as a control. The middle views are of the ground enamel and dentine conditioned by the MB. The right views are of the ground enamel and dentine conditioned by the UB.

10 K. Fujita, N. Nishiyama Figure 8 Effects of ethanol rinsing on the enamel and dentine surfaces, condition by the Mega Bond Primer. Upper photograph (3000!). Lower photograph (10,000!). the chemical shift of peak g and the shape of peak f obtained from the MDP and hydroxyapatite reactant were different from those obtained with the MDP and dentine reactant. Nishiyama et al. investigated the ph dependency of the 13 C NMR peak for the a-methylene carbon bonded to the phosphorus in the N-methacryloyl-2-aminoethyle phosphonic acid (NMEP) by using the liquid-state 13 C NMR technique. 8,9 The chemical shift of the 13 C NMR peak for the a-methylene carbon was strongly dependent on the ph value of the NMEP solution. When one of the two hydroxy groups bonded to the phosphorus dissociated, R PaO(OH)(O K ), the a-methylene carbon peak shifted to a lower field by 1.44 ppm, when compared with the chemical shift of the 13 C NMR peak for the a-methylene carbon attributed to the undissociated NMEP. Furthermore, when the two hydroxy groups of the phosphonic acid completely dissociated, R PaO(O K ) 2, the a-methylene carbon peak additionally shifted to a lower field by 1.08 ppm. The observed shift differential of peak g between the hydroxyapatite and the dentine reactants suggests that the chemical structure of the calcium salt produced by the MDP is different between the hydroxyapatite and the dentine reactant. Consequently, when the MB was applied to the hydroxyapatite, the predominant species was the MDP calcium hydrogen phosphate, in which one of two hydroxy groups bonded to the phosphorus had reacted with the calcium cation, R O PaO(O- H)(O K [Ca 2C ] 1/2 ). On the contrary, the reactant obtained from the dentine was the calcium phosphate of the MDP, in which two hydroxy groups bonded to the phosphorus had reacted with the calcium cation, R O PZOðO K 2 CaC2 Þ. Unlike MB, when the hydroxyapatite was suspended in UB, decrease in the peak intensity attributed to the 4-MET was not observed in the NMR spectrum of the supernatant UB solution. However, the addition of dentine to the UB resulted in a decrease in the peak intensity for the 4-MET. This decrease was due to the production of the calcium salt by the 4-MET. However, the degree of reduction in the peak intensity for the 4-MET was lower than that observed with the MDP. This is probably attributed to the demineralisation potential of the carboxylic acid of the 4-MET being lower

Etching efficacy of self-etching primers 11 than that of the phosphoric acid of the MDP. The solid-state 13 C NMR spectrum of the resultant residue obtained from the UB and dentine was very noisy and its signal-to-noise ratio was very low. These observations were attributed to the fact that the amount of calcium salt produced by the 4-MET was very low, since demineralisation of the dentine by the carboxylic acid was limited. The chemical structure of the resultant residue obtained from the 4-MET and dentine was not determined, since we were unable to secure sufficient enough data to assign its structure. The results of our chemical analyses were complemented by SEN examination that demonstrated the difference in etching pattern between the MB and UB. There, we rinsed the tooth surfaces, conditioned by the MB and UB, using water or ethanol, to evaluate the degree in the demineralization of the tooth by the MDP and 4-MET or to reconfirm the production of the calcium salt by the MDP and 4-MET, respectively. Therefore, these rinsing procedures are not clinically necessary for the resin restoration by using the self-etching bonding system. The difference in the etching pattern was due to the fact that the etching potential of the tooth by the self-etching primer was strongly dependent on the types of acidic functional group that are present in the selfetching primer. When the enamel or dentine surface conditioned by the MB was rinsed with ethanol, precipitates of the MDP calcium salt were observed on both surfaces. However, the precipitate obtained from the hydroxyapatite appeared amorphous, whereas the precipitate of those that were seen in the dentine appeared in the form of needle-shaped crystallites. These precipitates were easily removed from the enamel or dentine surface by rinsing with water, even though the MDP calcium salts were insoluble in water. This indicates that the MDP calcium salts were merely deposited onto the enamel or dentine surfaces. This result suggests that these calcium salts are unlikely to facilitate adhesion of the resin. Conclusions The application of a self-etching primer to the tooth demineralises the enamel and dentine with the production of calcium salts on the tooth surface. However, the calcium salts from the acidic monomer are unlikely to contribute to the retention of the resin components, since they were merely surface depositions. Acknowledgements Part of this work was supported by a grant-in-aid for Developmental Scientific Research from the Ministry of Education, Science and Culture in Japan (#13470421), and Technology to promote 2001-multidisciplinary research projects (in 2001 2005). References 1. Watanabe I, Nakabayashi N, Pashley DH. Bonding to ground dentin by a phenyl-p self-etching primer. Journal of Dental Research 1994;73:1212 20. 2. Yoshiyama M, Matsuo T, Ebis S, Pashley DH. Regional bond strength of self-etching/self-priming adhesive system. Journal of Dentistry 1998;26:609 16. 3. Van Meerbeek B, Yoshida Y, Inoue S, Vargas M, Abe Y, Fukuda R, et al. Bonding mechanisms and micro-tensile bond strength of a 4-MET-based self-etching adhesive. Journal of Dental Research 2000;79(Special Issue):249 [Abstract 845]. 4. Wolinsky LE, Armstrong RW, Seghi RR. The determination of ionic bonding interactions of N-phenyl glycine and N-(2- hydroxy-3-methacryloxypropyle)-n-phenyl glycine as measured by carbon-13 NMR analysis. Journal of Dental Research 1993;72:72 7. 5. Fujisawa S, Ito S. 1H-NMR studies of the interaction of dental adhesive monomer, 4-META with calcium. Dental Material Journal 1999;18:54 62. 6. Yoshida H, Nishiyama N. Development of self-etching primer comprised of methacrylamide, N-methacryloyl glycine. Biomaterials 2003;24:5203 7. 7. Fujisawa S, Kadoma Y, Komoda Y. Hemoritic activity of a dental adhesive monomer (4-methacryloxyethoxycarbonylphthalic anhydride, 4-META) and its interaction with phospholipid liposomes. Dental Material Journal 1991;9: 18 26. 8. Nishiyama N, Teshima H, Nemoto K, Suzuki K. Bonding mechanisms and bond strength of a Gly-EP self-etching adhesive. Journal of Dental Research 2003;82(Special Issue):B-101 [Abstract 709]. 9. Nishiyama N, Fujita K, Ikemi T, Maeda T, Suzuki K, Nemoro K. Efficacy of varying the NMEP concentration in the NMGly- NMEP self-etching primer on the resin dentin interface. Biomaterials 2005;26(15):2653 61.