Abstract
Objectives
To identify proteoglycans (PGs) and collagen fibrils (CF) within human dentin by means of a dual immunofluorescent labeling technique and to investigate the monomer infiltration of two etch-and-rinse adhesives to tosyl-phenylalanine chloromethyl-ketone (TPCK)-treated trypsin (TRY)-pretreated dentin.
Methods
Thirty-micrometer sections of middle coronal dentin were obtained and etched with 37% phosphoric acid gel for 15 s. After preconditioning with or without TRY digestion, the sections were subjected to dual immunofluorescent labeling and observed with a confocal laser scanning microscope (CLSM). Demineralized dentin matrixes treated with or without TRY were observed with field emission scanning electron microscope (FE-SEM). Two etch-and-rinse adhesives, Adper Single Bond 2 (SB) and Prime & Bond NT (PBNT), were applied to the dentin surfaces that were pretreated with or without TRY. The thickness of the hybrid layers was evaluated under confocal micro-Raman spectroscopy and analyzed with a two-way ANOVA.
Results
Green and red fluorescence was used to represent the PGs and the CF that were colocalized in the same section with different distributions. PGs were localized in the lumens of the dentin tubules and in peritubular dentin, while the type-I collagen fibrils were localized in intertubular dentin and peritubular dentin. After preconditioning with TRY digestion, the red fluorescence decreased or disappeared, the organic filaments in the lumens of the dentin tubules disappeared, the tubules were enlarged, and the hybrid layer thickness for adhesives bonded to the TRY-pretreated dentin surfaces were significantly increased ( p < 0.001 for both SB and PBNT).
Significance
The dual immunofluorescence labeling methodology can be used to study the human dentin matrix without decalcifying the entire dentin fragment. Proteoglycans were localized in the lumens of the dentin tubules and in peritubular dentin, which could depress the infiltration of the adhesive resin monomers. The use of TRY digestion increased the thickness of the hybrid layer created by the tested two-step etch-and-rinse adhesive.
1
Introduction
Dentin bonding techniques have generally been used in operative dentistry and to restore teeth using resin-based materials. However, insufficient dentin bonding and poor durability of adhesive interfaces frequently cause recurrent caries and marginal discoloration, leading to the failure of the restoration . To achieve higher bond strength and improve the longevity of dentin bonding, it is important to know the mechanism of the adhesive technique. The formation of an adequate and non-porous hybrid layer is currently accepted as the principal bonding mechanisms of dentin adhesives. Complete penetration of liquid resin monomers into the nano-porosities of the demineralized dentin matrix and dentin tubules is considered to be a pre-requisite for high bond strength and durability of the adhesive interface . To minimize the collagen collapse due to excessive air-drying, the wet-bonding technique relies on residual rinsing water left on the dentin surface after the etching-rinsing-drying steps. Indeed, the degree of substrate hydration determines the size of the interfibrillar spaces within the demineralized dentin matrix, which ultimately contributes to the proper impregnation of the monomers into the collagen network. Proteoglycans (PGs) are believed to play an important role in determining the interfibrillar collagen spaces due to their intrinsic ability to bind water .
PGs exist in the extracellular matrix of many connective tissues. PGs and phosphoproteins are the main constituents of the noncollagenous dentin matrix . PGs are macromolecules composed of a protein core, to which one or more anionic glycosaminoglycan (GAG) chains are attached. Glycosaminoglycans are linear chains of repeating disaccharides that bind covalently to the protein core and extend into the spaces of the collagen fibrils . In the dentin matrix, there are two main families of PGs with different GAG side chains, keratan sulfate (KS) and chondroitin sulfate (CS) , of which the latter is a main component. Sulphation of the amino sugars in the GAG chains confers a highly negative charge to the linear chain molecules; therefore, GAGs may contribute to the ability of PGs to bind and organize water molecules in the wet bonding technique and play an important role in regulating the architecture of the demineralized dentin matrix .
However, the role of dentin proteoglycans in the bonding procedure remains unclear. Observing the distribution of PGs in physiological conditions of sound dentin tissue and identifying alterations in their distribution after removal may be useful in improving our understanding of their role in the etch-and rinse wet bonding technique. Several studies have been conducted to examine the effect of proteoglycans on the demineralized dentin matrix or dentin bond strength . These studies have found that PGs exist in human dentin, and the bond strengths were affected after the PGs were removed; however, the bond strength data were found to be increased or decreased, which was not align with prior reports, and did not provide a clear description of the distribution of PGs in the full thickness of the demineralized dentin matrix after etching or of the effect of PGs on the penetration of adhesive resin monomers. Thus, the objectives of this study were to determine the localization of proteoglycan in dentin using a dual immunofluorescence labeling technique in conjunction with confocal laser scanning microscopy (CLSM) and evaluate the effects of removing the proteoglycans on the hybrid layer formation. In order to completely remove the PGs, TRY enzyme was employed to selectively cleave the core protein of proteoglycans. Once the proteoglycan protein core is digested, the GAG side chains are disrupted and vanished after rinsing. We hypothesized that TRY digestion would remove the proteoglycans from the demineralized dentin matrix and subsequently affect the hybrid layer formation of the two-step etch-rinse adhesive system.
2
Materials and methods
2.1
Tooth collection
Non-carious, sound human third molars were collected after obtaining the patients’ informed consent, as approved by the Ethic Committee for Human Studies of the Fourth Military Medical University.
2.2
Immunofluorescence analysis
Four middle coronal dentin fragments above the pulp cavity were obtained using a low-speed cutting saw (SYJ-150; MTI Corp., Shenyang, China) under water cooling. The non-decalcified dentin fragments were embedded in methylmethacrylate and immersed in a water bath for 24 h at 37 °C. After polymerization of the resin, 30-μm sections were cut using a microtome (SP1600; Leica, Bensheim, Germany) under water cooling and attached to polyester slides (Bowen, Xi’an, China) with cyanoacrylate glue (Loctite, Henkel Corp., Avon, OH, USA). Four sections were obtained from the same dentin fragment ( N = 4 × 4). The dentin sections were then etched with 37% phosphoric acid (PA) gel (Vericom Co., Ltd., Korea) for 15 s and rinsed with distilled water to remove all acids. To identify the alterations of demineralized dentin matrix after removal of PGs, the sections from each tooth were divided into two groups ( n = 8). One group was digested using tosylphenylalanyl chloromethyl ketone (TPCK)-treated trypsin (1 mg/mL, Sigma–Aldrich, St. Louis, MO, USA) with 0.2 M bicarbonate solution at pH 7.9 (TRY) for 48 h in a 37 °C water bath under constant stirring, and the solutions were exchanged after 24 h. The other group was stored in deionized water under the same conditions without digestion, and this group was defined as the control group.
After rinsing with distilled water for 5 min, all sections were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 6 h at room temperature and washed with PBS (0.01 M, pH 7.2) three times for 5-min intervals. The sections were then incubated with normal goat serum (Boster, Wuhan, China) for 30 min at 37 °C to block nonspecific staining and subsequently incubated overnight at 4 °C with the following two primary antibodies: a monoclonal antibody directed against type I collagen (mouse IgG, Sigma–Aldrich, St. Louis, MO, USA, Clone Col-1, lot 059K4824) and a monoclonal antibody directed against chondroitin sulfate (mouse IgM, Sigma–Aldrich, St. Louis, MO, USA, Clone CS-56, lot 071 M4864). After rinsing with PBS (0.01 M, pH 7.2) three times, the sections were subjected to fluorescent double-labeling with FITC-conjugated goat anti-mouse IgG (Bioss, Beijing; China) and RBITC-conjugated goat anti-mouse IgM (Bioss, Beijing; China) in a moist box for 30 min at 37 °C. All of the antibodies were diluted in PBS (0.01 M, pH 7.2) with the suggested dilution and utilized away from light sources. After the last rinse, the sections were coverslipped with a special mounting medium (Beyotime, Beijing, China) to prevent the fluorophores from quenching. All of the samples were viewed by CLSM (FV1000; Olympus, Japan) using the following parameters: excitation/emission wavelengths of 488/519 nm for FITC and 559/591 nm for RBITC. Two stacks of optical z -axis section images (green and red channels) 1-μm apart were obtained for each field of view. The blank control group was incubated overnight with an equivalent volume of PBS (0.01 M, pH 7.2) without the primary antibodies and then incubated with the two secondary antibodies. To confirm the specificity of the secondary antibodies, the following two crossover trials were performed: (a) sections were incubated with the anti-type I collagen (mouse IgG) and then with the RBITC-conjugated goat anti-mouse IgM or (b) sections were incubated with the antibody anti-chondroitin sulfate (mouse IgM) and then incubated with the FITC-conjugated goat anti-mouse IgG. To avoid the non-specificity binding of the primary antibodies to the minerals, sections without acid etching were incubated with primary antibodies and then with secondary antibodies.
2.3
FE-SEM analysis
Three mid-coronal dentin disks with 1-mm thickness were obtained and sectioned into two equal halves. All the halves were etched with 37% phosphoric acid (PA) gel for 15 s and rinsed thoroughly with deionized water. One-half from each tooth was digested with TRY, while the other half was kept in deionized water (the same operating procedures as in the immunofluorescence analysis). All the specimens were then fixed in 2.5% glutaraldehyde for 6 h at room temperature and dehydrated in ascending grades of ethanol, immersed in hexamethyldisilazane (HMDS), air dried, mounted on aluminum stubs and subsequently sputter-coated with gold and examined using a field-emission scanning electron microscope (FE-SEM) (S-4800, Hitachi, Tokyo, Japan) operating at 5-kV accelerating voltage.
2.4
Raman spectroscopy analysis
Ten flat mid-coronal dentin surfaces were prepared perpendicular to the longitudinal axis of each tooth using a low-speed cutting saw (SYJ-150; MTI Corp., Shenyang, China) under water irrigation. The surfaces were polished with 600-grit silicon carbide papers under running water to create a standardized smear layer and sequentially sectioned into two equal halves. The surfaces were then etched with 37% phosphoric acid (PA) gel for 15 s. One-half from each tooth was digested with TRY (TRY group), while the other half was kept in deionized water (control group). The digestion procedures were performed as in the immunofluorescence analysis. Each group was then randomly divided into two subgroups and bonded with Adper Single Bond 2 (SB) (3 M ESPE, St. Paul, MN, USA) and Prime & Bond NT (PBNT) (Dentsply DeTrey, Konstanz, Germany) strictly according to the manufacturers’ instructions ( Table 1 ) for the conventional wet-bonding technique. Following the bonding procedure, the surfaces were built up with Filtek Z250 resin composite (3 M ESPE, St. Paul, MN, USA) to a height of 1 mm and light-cured for 30 s.
| Adhesives | Composition [lot number] | Steps of application |
|---|---|---|
| Adper Single Bond 2 (3 M ESPE, St. Paul, MN, USA) | Bis-GMA; polyalkenoic acid copolymer; dimethacrylate; HEMA; CQ; ethanol; water [N281497] | Etch for 15 s. Rinse with water and keep the dentin surface moist. Immediately after blotting, apply 2–3 consecutive coats of the adhesive for 15 s with gentle agitation using a fully saturated applicator. Dry gently for 5 s. Light-cure for 10 s. |
| Prime & Bond NT (Dentsply DeTrey, Konstanz, Germany) | PENTA, UDMA, T-resin, D-resin, silica nanofiller, initiators, stabilizer, cetylamine hydrofluoride, acetone [1203000616] | Etch for 15 s. Rinse with water and keep the surface moist. Saturate the dentin surface with ample amounts of the adhesive and leave the surface undisturbed for 20 s. Blow air gently for 5 s. Light-cure for 10 s. |
After storage in deionized water at 37 °C for 24 h, each specimen was sectioned perpendicular to the adhesive-dentine surface into 1-mm thick sections. Two samples above the pulp cavity were chosen for testing. Confocal micro-Raman spectroscopy was performed using HR 800 (HORIBA Jobin Yvon, France). The micro-Raman spectrometer was first calibrated for zero and then for coefficient values using a silicon sample. The samples were analyzed using the following micro-Raman parameters: 17 mW HeNe laser with 633 nm wavelength, lateral ( XY ) spatial resolution <1 μm, spectral resolution ∼5 cm −1 , spectral region 800–1800 cm −1 and magnification ×100. The accumulation time per spectrum was 10 s, and two spectra were taken per point. Two-dimensional mapping was performed over 20-μm × 20-μm areas across the adhesive-dentin interface at 1-μm intervals in both X and Y directions using a computer-controlled XY stage. Two mappings were performed on each sample at random sites. The reference spectra were previously taken from an unaffected dentin sample and adhesive samples cured on glass slides. Data acquisition and analysis were performed with the dedicated software Lab Spec 4.18 (HORIBA Jobin Yvon). Statistical analysis was performed using SPSS 13.0 software package (SPSS Inc., Chicago, IL, USA). A two-way ANOVA with Tukey’s multiple comparison post-test was used to analyze the differences in HL thickness between the two adhesives and preconditioning treatments. Statistical significance was set in advance at α = 0.05.
2
Materials and methods
2.1
Tooth collection
Non-carious, sound human third molars were collected after obtaining the patients’ informed consent, as approved by the Ethic Committee for Human Studies of the Fourth Military Medical University.
2.2
Immunofluorescence analysis
Four middle coronal dentin fragments above the pulp cavity were obtained using a low-speed cutting saw (SYJ-150; MTI Corp., Shenyang, China) under water cooling. The non-decalcified dentin fragments were embedded in methylmethacrylate and immersed in a water bath for 24 h at 37 °C. After polymerization of the resin, 30-μm sections were cut using a microtome (SP1600; Leica, Bensheim, Germany) under water cooling and attached to polyester slides (Bowen, Xi’an, China) with cyanoacrylate glue (Loctite, Henkel Corp., Avon, OH, USA). Four sections were obtained from the same dentin fragment ( N = 4 × 4). The dentin sections were then etched with 37% phosphoric acid (PA) gel (Vericom Co., Ltd., Korea) for 15 s and rinsed with distilled water to remove all acids. To identify the alterations of demineralized dentin matrix after removal of PGs, the sections from each tooth were divided into two groups ( n = 8). One group was digested using tosylphenylalanyl chloromethyl ketone (TPCK)-treated trypsin (1 mg/mL, Sigma–Aldrich, St. Louis, MO, USA) with 0.2 M bicarbonate solution at pH 7.9 (TRY) for 48 h in a 37 °C water bath under constant stirring, and the solutions were exchanged after 24 h. The other group was stored in deionized water under the same conditions without digestion, and this group was defined as the control group.
After rinsing with distilled water for 5 min, all sections were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 6 h at room temperature and washed with PBS (0.01 M, pH 7.2) three times for 5-min intervals. The sections were then incubated with normal goat serum (Boster, Wuhan, China) for 30 min at 37 °C to block nonspecific staining and subsequently incubated overnight at 4 °C with the following two primary antibodies: a monoclonal antibody directed against type I collagen (mouse IgG, Sigma–Aldrich, St. Louis, MO, USA, Clone Col-1, lot 059K4824) and a monoclonal antibody directed against chondroitin sulfate (mouse IgM, Sigma–Aldrich, St. Louis, MO, USA, Clone CS-56, lot 071 M4864). After rinsing with PBS (0.01 M, pH 7.2) three times, the sections were subjected to fluorescent double-labeling with FITC-conjugated goat anti-mouse IgG (Bioss, Beijing; China) and RBITC-conjugated goat anti-mouse IgM (Bioss, Beijing; China) in a moist box for 30 min at 37 °C. All of the antibodies were diluted in PBS (0.01 M, pH 7.2) with the suggested dilution and utilized away from light sources. After the last rinse, the sections were coverslipped with a special mounting medium (Beyotime, Beijing, China) to prevent the fluorophores from quenching. All of the samples were viewed by CLSM (FV1000; Olympus, Japan) using the following parameters: excitation/emission wavelengths of 488/519 nm for FITC and 559/591 nm for RBITC. Two stacks of optical z -axis section images (green and red channels) 1-μm apart were obtained for each field of view. The blank control group was incubated overnight with an equivalent volume of PBS (0.01 M, pH 7.2) without the primary antibodies and then incubated with the two secondary antibodies. To confirm the specificity of the secondary antibodies, the following two crossover trials were performed: (a) sections were incubated with the anti-type I collagen (mouse IgG) and then with the RBITC-conjugated goat anti-mouse IgM or (b) sections were incubated with the antibody anti-chondroitin sulfate (mouse IgM) and then incubated with the FITC-conjugated goat anti-mouse IgG. To avoid the non-specificity binding of the primary antibodies to the minerals, sections without acid etching were incubated with primary antibodies and then with secondary antibodies.
2.3
FE-SEM analysis
Three mid-coronal dentin disks with 1-mm thickness were obtained and sectioned into two equal halves. All the halves were etched with 37% phosphoric acid (PA) gel for 15 s and rinsed thoroughly with deionized water. One-half from each tooth was digested with TRY, while the other half was kept in deionized water (the same operating procedures as in the immunofluorescence analysis). All the specimens were then fixed in 2.5% glutaraldehyde for 6 h at room temperature and dehydrated in ascending grades of ethanol, immersed in hexamethyldisilazane (HMDS), air dried, mounted on aluminum stubs and subsequently sputter-coated with gold and examined using a field-emission scanning electron microscope (FE-SEM) (S-4800, Hitachi, Tokyo, Japan) operating at 5-kV accelerating voltage.
2.4
Raman spectroscopy analysis
Ten flat mid-coronal dentin surfaces were prepared perpendicular to the longitudinal axis of each tooth using a low-speed cutting saw (SYJ-150; MTI Corp., Shenyang, China) under water irrigation. The surfaces were polished with 600-grit silicon carbide papers under running water to create a standardized smear layer and sequentially sectioned into two equal halves. The surfaces were then etched with 37% phosphoric acid (PA) gel for 15 s. One-half from each tooth was digested with TRY (TRY group), while the other half was kept in deionized water (control group). The digestion procedures were performed as in the immunofluorescence analysis. Each group was then randomly divided into two subgroups and bonded with Adper Single Bond 2 (SB) (3 M ESPE, St. Paul, MN, USA) and Prime & Bond NT (PBNT) (Dentsply DeTrey, Konstanz, Germany) strictly according to the manufacturers’ instructions ( Table 1 ) for the conventional wet-bonding technique. Following the bonding procedure, the surfaces were built up with Filtek Z250 resin composite (3 M ESPE, St. Paul, MN, USA) to a height of 1 mm and light-cured for 30 s.
