Abstract
Objective
The objective of the study was to determine if zinc-doped etch-and-rinse dentin adhesive may induce therapeutic effects within the resin–dentin interface.
Methods
Human acid-etched dentin was infiltrated with Adper™ Single Bond Plus (SB, 3M ESPE, St. Paul, MN, USA), SB doped with 10 wt.% ZnO nanoparticles (ZnO-SB) or SB doped with 2 wt.% ZnCl 2 (ZnCl 2 -SB). AFM/nanoindentation analysis was performed on fully hydrated specimens to evaluate the nanomechanical properties ( H i : hardness; E i : modulus of elasticity) across the resin–dentin interface after different SBF storage periods (24 h, 1 m, 3 m). Confocal laser microscopy (CLSM) was used to evaluate the ultramorphology and micropermeability at 24 h and 3 m of SBF storage.
Results
SB control specimens exhibited a decrease in H i in the hybrid layer (HL) and bottom of the hybrid layer (BHL) and a decrease in E i in the HL after 3 m of SBF storage, indicating that severe degradation occurred in the control interface. ZnO-SB bonded specimens preserved the initial H i and E i at the HL and BHL subsequent SBF storage; ZnCl 2 -SB bonded specimens showed a decrease in E i , in the HL over time. CLSM analysis confirmed that both Zn-doped adhesives were able to preserve the integrity of the HL.
Significance
Specific formulation of Zn-doped etch-and-rinse adhesives may offer the possibility to maintain the nano-mechanical properties along the dentin-bonded interface by inhibiting dentin MMPs and by protective mineral crystals formation within the resin–dentin interface. Clinical advantages may be expected by preserving and improving the integrity of the hybrid layer when Zn-doped adhesives are employed.
1
Introduction
The two fundamental processes involved in dentin etch and rinse bonding procedures are the dissolution of the mineral phase up to 10 μm of depth and the infiltration of the demineralized collagen matrix with adhesive resin monomers that undergoes complete in situ polymerization ( i.e . the formation of a resin-reinforced or hybrid layer) . The ideal hybrid layer (HL) may be classified as a 3-dimensional polymer/collagen network that provides a continuous and stable link between the bulk adhesive and dentin substrate . However, a high quality and durable HL can be only achieved if the demineralized dentin collagen matrix is fully resin-infiltrated . Unfortunately, this is still conjecture as to the functional significance of a decreasing gradient of resin monomer diffusion within acid-etched dentin that is always attained during bonding procedures, which results in a poorly infiltrated phase both within the HL and at the base of the hybrid layer (BHL) .
These poorly infiltrated HL are thought to be responsible for the degradation and the reduction of the resin–dentin durability . Host-derived matrix-metalloproteinases (MMPs 2, 8, 9, 20), a family of structurally related zinc-dependent endopeptidases that are activated during acid-etching procedures, contribute to the degradation of dentin collagen matrix. They also play an important role in dentin matrix modulation during caries progression and in the collagen degradation of the HL, jeopardizing the longevity of bonded restorations . What is needed is a suitable strategy to increase the longevity of adhesive restorations can be the inhibition of MMPs activity within the demineralized collagen matrix .
Osorio et al. have recently demonstrated that the quality and the longevity of the resin–dentin interface may be increased by using innovative dental adhesives containing zinc within their composition; Zn-doped adhesives have also been shown to induce proteolytic resistance and dentin mineralization . Indeed, zinc may not only protect collagen from MMPs activity, but may also influence signaling pathways and stimulate hard tissue mineralization . Furthermore, the presence of Zn may protect the seed crystallites on collagen fibrils for later dentin remineralization and extend the durability of the resin–dentin interface . The development of innovative bioactive ion-releasing restorative materials with a therapeutic effect on the mineral-depleted sites within the bonded-dentin interface remains one of the main targets of the dental biomaterial research .
The objective of this study was to evaluate the ability of two experimental Zn-doped etch-and-rinse bonding systems to induce therapeutic effects on the bonded-dentin interface after storage in a protein-free simulated body fluid (SBF). This study tested the null hypothesis that the inclusion of ZnO or ZnCl 2 within etch-and-rinse dentin adhesive systems does not protect the mechanical properties of resin–dentin bonds, and does not induce remineralization on the mineral-depleted areas within bonded-dentin interfaces.
2
Materials and methods
2.1
Preparation of adhesive solutions
A self-priming/etch and rinse adhesive system, Adper™ Single Bond Plus (SB, 3M ESPE, St. Paul, MN, USA), was doped using 10 wt.% ZnO (SB-ZnO; Sigma–Aldrich, St. Gillingham, UK) or 2 wt.% ZnCl 2 (SB-ZnCl 2 ; Sigma–Aldrich). The adhesive mixtures were vigorously shaken for 5 min (Vortex Wizard, Ref. 51075; Velp Scientifica, Milan, Italy) in a dark room in order to achieve complete dissolution of ZnCl 2 or dispersion of ZnO particles. Description of the adhesive is provided in Table 1 . The specimens were immersed in simulated body fluid solution (SBFS) for 24 h, 1 month (1 m) and 3 months (3 m) of storage (replaced every 72 h). The SBFS was prepared by dissolving reagent grade NaCl (16.070 g), NaHCO 3 (0.710 g), KCl (0.450 g), K 2 HPO 4 ·3H 2 O (0.462 g), MgCl 2 ·6H 2 O (0.622 g), CaCl 2 (0.584 g) and Na 2 SO 4 (0.144 g) into double-distilled water and buffering at pH 7.4 using tris-hydroxymethylaminomethane [(CH 2 OH) 3 CNH 2 ] (12.236 g) and 1 M hydrochloric acid (HCl) (0–10 mL).
| Product details a | Basic formulation | Mode of application |
|---|---|---|
| Adper Single Bond Plus (SB) (3M ESPE, St Paul, MN, USA) 8PT | Bis-GMA, HEMA, dimethacrylates, ethanol, water, a novel photoinitiator system, a methacrylate functional copolymer of polyacrylic, polyitaconic acids | Etch dentin for 15 s, rinse for 30 s, blot dry, apply 2–3 consecutive coats of adhesive for 20 s, air thin for 20 s, light-cure for 10 s |
| X-Flow™ (Dentsply, Caulk, UK) | Strontium alumino sodium fluorophosphorsilicate glass, di- and multifunctional acrylate and methacrylate resins, DGDMA, highly dispersed silicon dioxide UV stabilizer, ethyl-4-dimethylaminobenzoate camphorquinone, BHT, iron pigments, titanium dioxide | Not applicable |
a Material code, manufacturer details and product batch number.
2.2
Specimen preparation and bonding procedures
Human molars extracted for surgical reasons under a protocol approved by an Institutional Review Board were stored in deionized water (pH 7.1) at 4 °C for no longer than 1 month. Dentin crown specimens were prepared by cutting the roots 1 mm beneath the cemento-enamel junction (CEJ) using a hard tissue microtome (Isomet 11/1180, Buehler, Coventry, UK) equipped with a diamond embedded blade (XL 12205; Benetec Limited, London, UK). The occlusal enamel was then removed with a second parallel cut to expose a middle coronal dentin. A standard and more clinically relevant smear layer was created using 600-grit SiC paper for 30 s under constant water irrigation. A 37% phosphoric acid gel was applied for 15 s and copiously water rinsed for 30 s. The bonding procedures were performed in moist dentin using SB, SB-ZnO or SB-ZnCl 2 ; they were applied within a period of 20 s, the solvent evaporated, and light-cured for 30 s. A flowable resin composite (X-Flow™, Dentsply, Caulk, UK) was placed incrementally in two 1 mm layers and light-cured for 40 s. The detailed application mode is shown in Table 1 .
2.3
Confocal laser scanning microscopy evaluation (CLSM)
Three acid-etched dentin specimens for each group were bonded as previously described with adhesives mixed with 0.2 wt.% Rhodamine B (Rh-B; Sigma–Aldrich). The pulp chamber of the specimens was exposed and in half of the specimens, the pulp chambers were immediately filled with 0.1 wt.% sodium fluorescein (FNS; Sigma–Aldrich) for 3 h while, the other half of each group was immersed in SBF for 3 m and subsequently filled with FNS. The specimens were washed in an ultrasonic bath for 2 min, sectioned into slabs (1.5 mm) and finally polished using 1200-grit SiC. A confocal laser scanning microscope (DM-IRE2 CLSM; Leica, Heidelberg, Germany) equipped with a 63×/1.4 NA oil immersion lens and a 488-nm Argon/Krypton ions laser (Fluorescein excitation) or 568-nm Helium/Neon ions laser (Rhodamine B excitation) that was used to analyze the ultra-morphology and micropermeability along the bonded-dentin interfaces; the emission fluorescence was recorded at 512–538 nm and 585–650 nm, respectively. Reflection and fluorescence optical images were captured 5 μm below the outer surface up to 25 μm depth (1 μm z -step) and converted in topographic single projections using the Leica SP2 CLSM image-processing software (Leica, Heidelberg, Germany) . The configuration of the system was standardized and used at the same level for the entire investigation.
2.4
AFM imaging and nano-indentation
Three resin bonded-dentin specimens for each group were longitudinally sectioned in slabs (thickness: 1.5 mm) and polished through ascending SiC papers from #800 up to #4000-grit. A final polishing procedure was performed using diamond pastes (Buheler-MetaDi, Buheler Ltd., Lake Bluff, IL, USA) through 1 μm down to 0.25 μm. The specimens were treated in ultrasonic bath (Model QS3, Ultrawave Ltd, Cardiff, UK) containing deionized water [pH 7.4] for 5 min at each polishing step. An atomic force microscope (AFM-Nanoscope V, Digital Instruments, Veeco Metrology group, Santa Barbara, CA, USA) equipped with a Triboscope indentor system (Hysitron Inc., Minneapolis, MN) and a Berkovich indenter (tip radius ∼20 nm) was employed for the imaging and nano-indentation processes in a fully hydrated status as recently proposed by Sauro et al. . Briefly, six indentations ( Fig. 1 ) with a load of 4000 nN and a time function of 10 s were performed at different SBF storage periods (24 h, 1 m, 3 m) in a straight line starting from the adhesive layer down to the intertubular dentin in order to evaluate changes in hardness ( H i ) and the modulus of elasticity ( E i ). Three indentation lines 3 ± 1 μm distant each were made in five different mesio-distal positions along the interface. The distance between each indentation was kept constant (5 ± 1 μm) by adjusting the distance intervals steps and the load function . ANOVA was performed including H i or E i as the dependent variable. Resin adhesive type (SB, ZnO-SB, ZnCl 2 -SB) and period of SBF storage (24 h/1 m/3 m) were considered as independent variables. Analysis of interactions was also conducted. Student–Newman–Keuls test was used for multiple comparisons. Statistical analysis was set at a significance level of α = 0.05.
2
Materials and methods
2.1
Preparation of adhesive solutions
A self-priming/etch and rinse adhesive system, Adper™ Single Bond Plus (SB, 3M ESPE, St. Paul, MN, USA), was doped using 10 wt.% ZnO (SB-ZnO; Sigma–Aldrich, St. Gillingham, UK) or 2 wt.% ZnCl 2 (SB-ZnCl 2 ; Sigma–Aldrich). The adhesive mixtures were vigorously shaken for 5 min (Vortex Wizard, Ref. 51075; Velp Scientifica, Milan, Italy) in a dark room in order to achieve complete dissolution of ZnCl 2 or dispersion of ZnO particles. Description of the adhesive is provided in Table 1 . The specimens were immersed in simulated body fluid solution (SBFS) for 24 h, 1 month (1 m) and 3 months (3 m) of storage (replaced every 72 h). The SBFS was prepared by dissolving reagent grade NaCl (16.070 g), NaHCO 3 (0.710 g), KCl (0.450 g), K 2 HPO 4 ·3H 2 O (0.462 g), MgCl 2 ·6H 2 O (0.622 g), CaCl 2 (0.584 g) and Na 2 SO 4 (0.144 g) into double-distilled water and buffering at pH 7.4 using tris-hydroxymethylaminomethane [(CH 2 OH) 3 CNH 2 ] (12.236 g) and 1 M hydrochloric acid (HCl) (0–10 mL).
| Product details a | Basic formulation | Mode of application |
|---|---|---|
| Adper Single Bond Plus (SB) (3M ESPE, St Paul, MN, USA) 8PT | Bis-GMA, HEMA, dimethacrylates, ethanol, water, a novel photoinitiator system, a methacrylate functional copolymer of polyacrylic, polyitaconic acids | Etch dentin for 15 s, rinse for 30 s, blot dry, apply 2–3 consecutive coats of adhesive for 20 s, air thin for 20 s, light-cure for 10 s |
| X-Flow™ (Dentsply, Caulk, UK) | Strontium alumino sodium fluorophosphorsilicate glass, di- and multifunctional acrylate and methacrylate resins, DGDMA, highly dispersed silicon dioxide UV stabilizer, ethyl-4-dimethylaminobenzoate camphorquinone, BHT, iron pigments, titanium dioxide | Not applicable |
a Material code, manufacturer details and product batch number.
2.2
Specimen preparation and bonding procedures
Human molars extracted for surgical reasons under a protocol approved by an Institutional Review Board were stored in deionized water (pH 7.1) at 4 °C for no longer than 1 month. Dentin crown specimens were prepared by cutting the roots 1 mm beneath the cemento-enamel junction (CEJ) using a hard tissue microtome (Isomet 11/1180, Buehler, Coventry, UK) equipped with a diamond embedded blade (XL 12205; Benetec Limited, London, UK). The occlusal enamel was then removed with a second parallel cut to expose a middle coronal dentin. A standard and more clinically relevant smear layer was created using 600-grit SiC paper for 30 s under constant water irrigation. A 37% phosphoric acid gel was applied for 15 s and copiously water rinsed for 30 s. The bonding procedures were performed in moist dentin using SB, SB-ZnO or SB-ZnCl 2 ; they were applied within a period of 20 s, the solvent evaporated, and light-cured for 30 s. A flowable resin composite (X-Flow™, Dentsply, Caulk, UK) was placed incrementally in two 1 mm layers and light-cured for 40 s. The detailed application mode is shown in Table 1 .
2.3
Confocal laser scanning microscopy evaluation (CLSM)
Three acid-etched dentin specimens for each group were bonded as previously described with adhesives mixed with 0.2 wt.% Rhodamine B (Rh-B; Sigma–Aldrich). The pulp chamber of the specimens was exposed and in half of the specimens, the pulp chambers were immediately filled with 0.1 wt.% sodium fluorescein (FNS; Sigma–Aldrich) for 3 h while, the other half of each group was immersed in SBF for 3 m and subsequently filled with FNS. The specimens were washed in an ultrasonic bath for 2 min, sectioned into slabs (1.5 mm) and finally polished using 1200-grit SiC. A confocal laser scanning microscope (DM-IRE2 CLSM; Leica, Heidelberg, Germany) equipped with a 63×/1.4 NA oil immersion lens and a 488-nm Argon/Krypton ions laser (Fluorescein excitation) or 568-nm Helium/Neon ions laser (Rhodamine B excitation) that was used to analyze the ultra-morphology and micropermeability along the bonded-dentin interfaces; the emission fluorescence was recorded at 512–538 nm and 585–650 nm, respectively. Reflection and fluorescence optical images were captured 5 μm below the outer surface up to 25 μm depth (1 μm z -step) and converted in topographic single projections using the Leica SP2 CLSM image-processing software (Leica, Heidelberg, Germany) . The configuration of the system was standardized and used at the same level for the entire investigation.
2.4
AFM imaging and nano-indentation
Three resin bonded-dentin specimens for each group were longitudinally sectioned in slabs (thickness: 1.5 mm) and polished through ascending SiC papers from #800 up to #4000-grit. A final polishing procedure was performed using diamond pastes (Buheler-MetaDi, Buheler Ltd., Lake Bluff, IL, USA) through 1 μm down to 0.25 μm. The specimens were treated in ultrasonic bath (Model QS3, Ultrawave Ltd, Cardiff, UK) containing deionized water [pH 7.4] for 5 min at each polishing step. An atomic force microscope (AFM-Nanoscope V, Digital Instruments, Veeco Metrology group, Santa Barbara, CA, USA) equipped with a Triboscope indentor system (Hysitron Inc., Minneapolis, MN) and a Berkovich indenter (tip radius ∼20 nm) was employed for the imaging and nano-indentation processes in a fully hydrated status as recently proposed by Sauro et al. . Briefly, six indentations ( Fig. 1 ) with a load of 4000 nN and a time function of 10 s were performed at different SBF storage periods (24 h, 1 m, 3 m) in a straight line starting from the adhesive layer down to the intertubular dentin in order to evaluate changes in hardness ( H i ) and the modulus of elasticity ( E i ). Three indentation lines 3 ± 1 μm distant each were made in five different mesio-distal positions along the interface. The distance between each indentation was kept constant (5 ± 1 μm) by adjusting the distance intervals steps and the load function . ANOVA was performed including H i or E i as the dependent variable. Resin adhesive type (SB, ZnO-SB, ZnCl 2 -SB) and period of SBF storage (24 h/1 m/3 m) were considered as independent variables. Analysis of interactions was also conducted. Student–Newman–Keuls test was used for multiple comparisons. Statistical analysis was set at a significance level of α = 0.05.
3
Results
3.1
Confocal laser scanning microscopy evaluation (CLSM)
The confocal microscopy showed severe micropermeability in SB specimens within the poorly resin-infiltrated HL ( Fig. 2 D ) after 24 h, while after 3 m of SBF storage, a clear sign of degradation could be observed within the resin–dentin interface ( Fig. 3 A and B ).
No micropermeability or gap formation was present after CLSM observations, in ZnO-SB, at 24 h ( Fig. 2 B); a well defined HL characterized with fluorescence mode nanoleakage could be observed ( Fig. 2 E). After 3 m of storage in SBF, some porosities were detected at the dentin–resin interface ( Fig. 3 D) in reflection/fluorescence mode. CLSM single projection captured from the same resin–dentin specimen ( Fig. 3 C) showed a clear HL with long resin tags penetrating the dentin tubules.
The resin bonded-dentin interface created with ZnCl 2 -SB did not exhibited any micropermeability signs at 24 h. That is, there were no porosities or nanoleakage ( Fig. 2 C and F). However, after 3 m of SBFS storage ( Fig. 3 E) although a clear and stabilized HL was observed in the CLSM single projection image, the same image, studied in the reflection/fluorescence mode, displayed zones of porosities and nanoleakage ( Fig. 3 F).
3.2
AFM imaging and nano-indentation
The nanomechanical properties ( H i and E i ) of the resin bonded interfaces were influenced by SBF immersion and type of resin ( P < 0.01) ( Table 2 ). Interactions between factors were also significant ( P < 0.001). The first indentation performed on the adhesive layer of SB, ZnO-SB and ZnCl 2 -SB showed no variation either both in H i or E i after prolonged SBF storage (1 m and 3 m) ( Tables 3 and 4 ). Conversely, in SB, both the second indentation values performed at the HL and the third value at the bottom of the hybrid layer (BHL) showed a significant reduction in H i and E i ( P < 0.01); except the BHL preserved the original E i values after prolonged SBFS storage. No significant change was observed along the intertubular dentin in the 4th, 5th and 6th position. The AFM imaging showed a scarcely detectable Berkovic-tip indentation mark on the HL both after 24 h ( Fig. 4 A ) and 3 m of SBF storage ( Fig. 5 A ).
| H i | E i | |||
|---|---|---|---|---|
| F | P | F | P | |
| 1st indentation (adhesive layer) | ||||
| Main effects | 8.05 | <0.001 | 10.05 | <0.001 |
| Resin type | 12.56 | <0.001 | 6.04 | 0.005 |
| Time of immersion | 3.54 | 0.04 | 14.06 | <0.001 |
| 2-Way interactions | 0.22 | 0.93 | 0.76 | 0.56 |
| 2nd indentation (middle of hybrid layer) | ||||
| Main effects | 7.61 | <0.001 | 18.12 | <0.001 |
| Resin type | 5.49 | 0.007 | 23.50 | <0.001 |
| Time of immersion | 9.73 | <0.001 | 12.73 | <0.001 |
| 2-way interactions | 2.82 | 0.04 | 3.60 | 0.01 |
| 3rd indentation (bottom of hybrid layer) | ||||
| Main effects | 3.83 | 0.009 | 21.82 | <0.001 |
| Resin type | 5.54 | 0.007 | 43.30 | <0.001 |
| Time of immersion | 2.12 | 0.132 | 0.34 | 0.71 |
| 2-Way interactions | 3.86 | 0.009 | 0.67 | 0.61 |
| 4th indentation (underlying mineralized dentin) | ||||
| Main effects | 1.39 | 0.25 | 25.89 | <0.001 |
| Resin type | 2.69 | 0.08 | 48.16 | <0.001 |
| Time of immersion | 0.10 | 0.91 | 3.61 | 0.04 |
| 2-Way interactions | 0.24 | 0.91 | 1.39 | 0.25 |
| 5th indentation (underlying mineralized dentin) | ||||
| Main effects | 0.46 | 0.77 | 15.24 | <0.001 |
| Resin type | 0.32 | 0.73 | 30.35 | <0.001 |
| Time of immersion | 0.60 | 0.56 | 0.14 | 0.87 |
| 2-Way interactions | 0.21 | 0.93 | 2.11 | 0.09 |
| 6th indentation (underlying mineralized dentin) | ||||
| Main effects | 4.23 | 0.005 | 2.18 | 0.09 |
| Resin type | 7.467 | 0.002 | 2.91 | 0.07 |
| Time of immersion | 0.98 | 0.38 | 1.44 | 0.25 |
| 2-Way interactions | 1.14 | 0.35 | 0.84 | 0.51 |
| 7th indentation (underlying mineralized dentin) | ||||
| Main effects | 2.79 | 0.04 | 0.71 | 0.59 |
| Resin type | 3.66 | 0.03 | 0.09 | 0.91 |
| Time of immersion | 1.91 | 0.16 | 1.33 | 0.28 |
| 2-Way interactions | 1.35 | 0.27 | 0.35 | 0.84 |
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