Graphical abstract
Highlights
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We investigated the molecular level interaction between HEMA and a collagen model.
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HEMA binding is not detected when demineralized dentin powder is suspended in HEMA solution.
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When the atelocollagen resonance is saturated, no saturation is propagated to HEMA.
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Saturation transfer difference NMR study indicates no interaction between HEMA and atelocollagen.
Abstract
Objective
2-Hydroxyethylmethacrylate (HEMA) diffuses in wet dentin and promotes adhesion during dentin priming and bonding. We have investigated the molecular level interaction between HEMA and a collagen model by using saturation transfer difference (STD) NMR.
Methods
The binding of HEMA to collagen was preliminarily investigated by suspending demineralized human dentin powders in a 4 mM HEMA solution for 1 h and measuring the decrease in the HEMA concentration on a spectrophotometer. The molecular level interaction of HEMA with atelocollagen, which was used as a collagen model, was investigated by STD-NMR spectroscopy.
Results
The HEMA concentration in the suspension did not change, indicating that HEMA did not bind to dentin collagen. This was confirmed by STD-NMR; when the atelocollagen resonance was saturated, no saturation was propagated to HEMA and no STD signals were detected.
Significance
The HEMA protons were not near the atelocollagen surface, indicating HEMA did not interact with atelocollagen. The collagen fibrils may be surrounded by water molecules in dentin/bond interfaces, which prevent the direct HEMA binding interaction.
1
Introduction
2-Hydroxyethyl methacrylate (HEMA) is a neutral hydrophilic monomer. This monomer is utilized in hydrogel products, such as soft contact lenses, and in polymer binders for controlled drug release, absorbents for body fluids, and lubricious coatings . HEMA is used as a comonomer or solvent with other monomers in dental adhesive systems to control hydrophobicity and promote adhesion to dental substrates . In the 1990s, it was assumed that HEMA allowed other monomers to penetrate etched dentin, resulting in bonding at the resin/dentin interface . Because HEMA is a small, hydrophilic monomer, it enhances the wetting of dentin and promotes dentin bonding . Despite the popularity of HEMA in dental adhesives, the interaction of HEMA with the dentin substrate had not been investigated on a molecular level until Nishiyama et al. conducted 13 C NMR studies. They proposed that a hydrogen bond forms between the ester carbonyl group in HEMA and the dentinal collagen, and that this hydrogen-bonded HEMA species promotes the hybridization of the adhesive resin with the dentinal collagens, and thus enhances the dentin bonding at the resin/dentin interface .
In contrast, Pashley et al. have used a macro-model of the dentinal collagen matrix to measure the permeability of demineralized dentin to HEMA. The HEMA uptake of dried, collapsed demineralized dentin matrix was low, and it depended on the degree of hydration of the demineralized dentin matrices and its expansion. They concluded that under their test conditions, HEMA did not bind to collagen.
Thus, the function and interaction of HEMA with the collagen matrix is disputed and not fully understood. It is therefore important to provide a micro-model interpretation of the molecular level interactions of HEMA with collagen, and for this purpose, STD-NMR is a promising method. This method has been widely used in drug delivery studies for screening ligands that bind to proteins and for determining the binding epitopes of the ligands . In STD-NMR, a resonance belonging to the protein receptor is selectively saturated. If the ligand binds to the protein, the saturation propagates from the selected receptor protons to other receptor protons via spin diffusion, and then the saturation is transferred to the binding component by cross relaxation at the ligand/receptor interface. The binding moieties of the ligands having the strongest interactions with the protein show the most intense STD signals, whereas those having weak interactions show weak STD signals. This allows the ligand binding epitope to be mapped.
In this study, we have investigated the macro-level binding of HEMA to collagen fibrils, and the molecular level binding of HEMA to atelocollagen using STD-NMR.
2
Materials and methods
2.1
Spectrophometrical quantification on HEMA interaction with dentin collagen
HEMA was purchased from Wako Pure Chemical Industries Ltd., Japan. The HEMA binding experiments were performed in neutral or acidic conditions. The aqueous 4 mM HEMA solution was prepared by dissolving absolute HEMA in distilled water (pH 7.0). The acidic 4 mM HEMA solution was prepared by dissolving a DMSO solution of HEMA in a buffer solution, which contained 50 mM acetic acid, 150 mM NaCl, 5 mM CaCl 2 , and 0.02% NaN 3 in H 2 O (pH 4.0, adjusted with NaOH). The buffer was also used in the NMR measurements because the pH allowed the atelocollagen to dissolve.
Twelve freshly extracted unerupted third molars were obtained from patients and used according to the guidelines of the Ethics Committee of the Graduate School and Hospital, Tokyo Medical and Dental University. The pulp, periodontal ligament, enamel, and cementum were manually removed, and the remaining dentinal fragments were pulverized under liquid N 2 in a cryogenic mill (CertiPrep 6750, SPEX CertiPrep, USA) into particles with diameters of less than 75 μm. The pulverized dentin was demineralized with 0.5 M EDTA (pH 7.4) for 10 days at 4 °C, thoroughly washed with distilled water and lyophilized. Six aliquots (5 mg) of demineralized dentin samples were allocated to each HEMA solution ( n = 6). The first absorbance was measured immediately when the dentin sample was transferred to HEMA solution (1 mL) by UV–vis spectrophotometer (NanoDrop 1000, Thermo Fisher Scientific Inc., USA). The concentration of HEMA was determined from its absorbance at 215 nm using a standard HEMA curve. The samples were then incubated at 37 °C for 1 h with tumbling at 10 rpm. Preliminary studies showed that the HEMA binding after incubation for 24 h was similar to the binding after 1 h; therefore, the samples were incubated for 1 h. After 1 h incubation, the tubes were centrifuged at 3000 rpm for 5 min, and the concentration of HEMA in the supernatant was measure. The t -tests with a significance level of 0.05 were performed for statistical comparisons of the HEMA concentration before and after the 1 h incubation using a statistical software package (SigmaStat Version 16.0, SPSS, USA).
2.2
STD-NMR experiment on HEMA interaction with atelocollagen
Highly purified type I atelocollagen powder from bovine dermis (CLP-01, Koken Co. Ltd., Japan) was used as the collagen type I tri-helix model. Atelocollagen is obtained by digesting the telopeptide ends of the non-helical regions in collagen molecules with an appropriate enzymatic treatment. Atelocollagen retains its triple-helix structure and its crosslinks, and its solubility is greater than that of collagen . Therefore, atelocollagen was a suitable collagen model for the STD-NMR study. The HEMA ligand was dissolved in deuterated dimethyl sulfoxide ( d 6 -DMSO) to 20 mM. The atelocollagen powder was dissolved using an acidic buffer solution that contained 50 mM d 4 -acetic acid, 150 mM NaCl, 5 mM CaCl 2 , and 0.02% NaN 3 in D 2 O (pD 4.0, adjusted with NaOD). The saturated solution of atelocollagen was diluted fourfold with the buffer solution to reduce viscosity. The HEMA ligand solution was added to the atelocollagen solution and its final concentration was adjusted to 4 mM for the STD experiments. The concentration of d 6 -DMSO was 20% in the final sample solution. The NMR experiments were performed on 600 and 800 MHz spectrometers equipped with a cryogenic probe (Bruker BioSpin Corporation, USA) at 298 K. The resonance assignment of HEMA was accomplished by using a conventional set of 2D NMR spectra: 1 H– 1 H COZY, TOCSY with an 80 ms mixing time, NOESY with a 400 ms mixing time, 1 H– 13 C HMBC, and 1 H– 13 C HMQC. For the STD experiments , the selective saturation of the atelocollagen receptor protein was achieved by a train of 50 ms Gaussian pulses, yielding a total saturation time of 2 s. The on-resonance irradiation of atelocollagen was performed at chemical shifts of 1.36 and 0.86 ppm, and the off-resonance irradiation was set at 40 ppm, where no HEMA or atelocollagen signals were present. The STD spectrum was produced by subtracting the on- and off-resonance spectrum. The total scan number in the STD experiments was 512 with 16,384 time domain data points. The spectra were processed using Topspin (Bruker BioSpin Corporation, USA) or NMRPipe and were analyzed using Topspin or NMRView .
2
Materials and methods
2.1
Spectrophometrical quantification on HEMA interaction with dentin collagen
HEMA was purchased from Wako Pure Chemical Industries Ltd., Japan. The HEMA binding experiments were performed in neutral or acidic conditions. The aqueous 4 mM HEMA solution was prepared by dissolving absolute HEMA in distilled water (pH 7.0). The acidic 4 mM HEMA solution was prepared by dissolving a DMSO solution of HEMA in a buffer solution, which contained 50 mM acetic acid, 150 mM NaCl, 5 mM CaCl 2 , and 0.02% NaN 3 in H 2 O (pH 4.0, adjusted with NaOH). The buffer was also used in the NMR measurements because the pH allowed the atelocollagen to dissolve.
Twelve freshly extracted unerupted third molars were obtained from patients and used according to the guidelines of the Ethics Committee of the Graduate School and Hospital, Tokyo Medical and Dental University. The pulp, periodontal ligament, enamel, and cementum were manually removed, and the remaining dentinal fragments were pulverized under liquid N 2 in a cryogenic mill (CertiPrep 6750, SPEX CertiPrep, USA) into particles with diameters of less than 75 μm. The pulverized dentin was demineralized with 0.5 M EDTA (pH 7.4) for 10 days at 4 °C, thoroughly washed with distilled water and lyophilized. Six aliquots (5 mg) of demineralized dentin samples were allocated to each HEMA solution ( n = 6). The first absorbance was measured immediately when the dentin sample was transferred to HEMA solution (1 mL) by UV–vis spectrophotometer (NanoDrop 1000, Thermo Fisher Scientific Inc., USA). The concentration of HEMA was determined from its absorbance at 215 nm using a standard HEMA curve. The samples were then incubated at 37 °C for 1 h with tumbling at 10 rpm. Preliminary studies showed that the HEMA binding after incubation for 24 h was similar to the binding after 1 h; therefore, the samples were incubated for 1 h. After 1 h incubation, the tubes were centrifuged at 3000 rpm for 5 min, and the concentration of HEMA in the supernatant was measure. The t -tests with a significance level of 0.05 were performed for statistical comparisons of the HEMA concentration before and after the 1 h incubation using a statistical software package (SigmaStat Version 16.0, SPSS, USA).
2.2
STD-NMR experiment on HEMA interaction with atelocollagen
Highly purified type I atelocollagen powder from bovine dermis (CLP-01, Koken Co. Ltd., Japan) was used as the collagen type I tri-helix model. Atelocollagen is obtained by digesting the telopeptide ends of the non-helical regions in collagen molecules with an appropriate enzymatic treatment. Atelocollagen retains its triple-helix structure and its crosslinks, and its solubility is greater than that of collagen . Therefore, atelocollagen was a suitable collagen model for the STD-NMR study. The HEMA ligand was dissolved in deuterated dimethyl sulfoxide ( d 6 -DMSO) to 20 mM. The atelocollagen powder was dissolved using an acidic buffer solution that contained 50 mM d 4 -acetic acid, 150 mM NaCl, 5 mM CaCl 2 , and 0.02% NaN 3 in D 2 O (pD 4.0, adjusted with NaOD). The saturated solution of atelocollagen was diluted fourfold with the buffer solution to reduce viscosity. The HEMA ligand solution was added to the atelocollagen solution and its final concentration was adjusted to 4 mM for the STD experiments. The concentration of d 6 -DMSO was 20% in the final sample solution. The NMR experiments were performed on 600 and 800 MHz spectrometers equipped with a cryogenic probe (Bruker BioSpin Corporation, USA) at 298 K. The resonance assignment of HEMA was accomplished by using a conventional set of 2D NMR spectra: 1 H– 1 H COZY, TOCSY with an 80 ms mixing time, NOESY with a 400 ms mixing time, 1 H– 13 C HMBC, and 1 H– 13 C HMQC. For the STD experiments , the selective saturation of the atelocollagen receptor protein was achieved by a train of 50 ms Gaussian pulses, yielding a total saturation time of 2 s. The on-resonance irradiation of atelocollagen was performed at chemical shifts of 1.36 and 0.86 ppm, and the off-resonance irradiation was set at 40 ppm, where no HEMA or atelocollagen signals were present. The STD spectrum was produced by subtracting the on- and off-resonance spectrum. The total scan number in the STD experiments was 512 with 16,384 time domain data points. The spectra were processed using Topspin (Bruker BioSpin Corporation, USA) or NMRPipe and were analyzed using Topspin or NMRView .
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