Highlights
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The use of different experimental primers on dentin–resin restorations is proposed.
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Experimental primers significantly reduced the enzymatic activity at the dentin–resin interface.
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Proanthocyanidins from enriched-grape seed extract, chlorhexidine and doxycycline inactivated rMMPs-2 and 9.
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Increased enzymatic activity took place under simulated cyclic loading.
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Cyclic loading did not have a significant effect on the resin–dentin bond strength.
Abstract
Objective
To evaluate the effect of experimental primers (chlorhexidine, enriched mixture of proanthocyanidins, and doxycycline) on the adhesive properties and gelatinolytic activity at dentin–resin interfaces of occlusal Class I restorations.
Methods
The inactivation of enzymes by the experimental primers was assessed by fluorescence assay and gelatin zymography. To assess the adhesive properties, occlusal Class I cavities were prepared in sound human molars, etched with phosphoric acid and restored with one of the primers and an etch-and-rinse adhesive system (Adper Single Bond Plus—3M ESPE). After the restorative procedures, specimens were divided into two subgroups ( n = 6) consisting of storage in incubation buffer or axial cyclic loading at 50 N and 1,000,000 cycles. Then, the specimens were sectioned and slices were assigned to in situ zymography assay and microtensile bond strength (TBS) test.
Results
Fluorescence assay and gelatin zymography revealed that the experimental primers inactivated rMMPs. In situ zymography (2-way ANOVA, Tukey, p < 0.05) showed that cyclic loading increased the gelatinolytic activity at the resin–dentin interface and the experimental primers decreased the gelatinolytic activity at the adhesive interface. The experimental primers had no significant effects on dentin–adhesive bond strengths with or without cyclic loading (2-way ANOVA, p > 0.05).
Significance
The use of experimental primers impaired the enzymatic activity at the dentin–adhesive interface after cyclic loading and the activity of rMMPs. Cyclic loading did not have a significant effect on the bond strength.
1
Introduction
Chemical and technical advances in adhesive materials/techniques have improved the performance of resin composite restorations . However, there are still challenges that must be overcome, mostly those associated with the stability of dentin–resin composite interfaces . The dentin matrix degradation along with the breakdown of hydrophilic contemporary etch-and-rinse adhesives are believed to be the two major causes of suboptimal adhesive interface over time . Non-collagenous components, such as endogenous enzymes play key biological role as mediators of cellular interactions in the maturation and mineralization of dentin and also in the in vitro breakdown of organic dentin matrix in intimate contact with adhesive interfaces. Specifically a group of endogenous zinc/calcium-dependent matrix metalloproteinases (MMPs) degrades extracellular matrix components including collagen in its native and denatured forms .
Poorly resin infiltrated collagen fibrils are susceptible to enzymatic degradation mediated by endogenous proteases . Such proteases are activated during surface conditioning as shown by high enzymatic activities at the bottom of the hybrid layer . The most well investigated synthetic agent to successfully inactivate endogenous proteases at the dentin–adhesive interface is chlorhexidine digluconate (CHX) . Other synthetic inactivators of endogenous proteases with fewer outcomes include tetracycline , carbodiimide and galardin . Doxycycline (DOXY) is a tetracycline semi-synthetic analog, which is considered the most potent and non-selective MMPs inactivating agent among tetracyclines . Encapsulation and sustained short term effect of DOXY from a nanotube-modified dentin adhesive has recently shown promising outcomes .
The use of plant derived compounds to preserve the dentin–adhesive interface is an attractive and potent alternative to synthetic agents . Proanthocyanidins (PAC) are known antioxidant and collagen cross-linking agent with vast biological and functional activities . Certain grape seed extracts (GSE) are main sources of PAC shown to enhance the mechanical properties and reduce biodegradation rates of demineralized dentin by multi-interaction with dentin matrix components, including type I collagen , proteoglycans and endogenous proteases . Isolation of highly bioactive compounds of GSE has recently shown promising results for future design of a standardized clinical intervention material.
In this context, the use of protease inactivators in the demineralized dentin as a pretreatment before resin infiltration appears to be a logical approach for extending the longevity of resin composite restorations . The inactivation of MMPs by experimental primers may increase the functional stability of dentin–adhesive interfaces . However the effectiveness of such primers under simulated oral conditions is still not well known. The aim of this study was to evaluate the effect of different experimental primers on the enzymatic activity and adhesive properties of dentin–resin interfaces from occlusal Class I restorations under simulated cyclic loading. The null hypotheses tested were that (1) there would be no difference among the anti-proteolytic action of the experimental primers on MMPs activities and on gelatinolytic activity at the hybrid layer (2) there would be no difference in the dentin–adhesive bond strength, regardless of the use of experimental primers and simulated cyclic loading.
2
Material and methods
2.1
Preparation of experimental primers
Three experimental primers were prepared as follow: (i) oligomeric proanthocyanidin enriched grape seed extract (e-GSE) obtained by a solvent partitioning protocol previously published and prepared at 15% w/v concentration in buffer solution (20 mM HEPES pH 7.4); (ii) primer of Doxycycline Hydrochloride (DOXY – Fisher Scientific – New Jersey, NJ, USA) at 3% w/v in buffer solution; (iii) Chlorhexidine digluconate (CHX) primer prepared by dilution of stock solution (20% CHX, Sigma; St. Louis, MO, USA) to 0.2% CHX in distilled water. HEPES buffer solution was used as negative control primer. The primers were freshly prepared and the pH adjusted to 7.2 using NaOH at room temperature.
2.2
rMMP-2 activity—fluorescence assay
The gelatinolytic activity of rMMP-2 (Human MMP-2, recombinant, 10 μg/mL, AnaSpec, Fremont, CA, USA) incubated with the experimental primers was assayed according to the protocol described by Tay et al. , using EnzChek Gelatinolytic/Collagenolytic Assay Kit (D-12054, Molecular Probes, Eugene, OR, USA). Primers concentrations were 0.2% CHX, 0.65% e-GSE and 3% DOXY. Enzyme activation with 4-aminophenylmercuric acetate (APMA) was done previously for one hour at 37 °C . The fluorescent cleavage products were read in a 96-well fluorescent plate reader (Victor X5, PerkinElmer, Waltham, MA, USA), operated with an absorption maxima at 495 nm and fluorescence emission maxima at 515 nm. Fluorescence measurements were taken at 0 (baseline), 1 h and 2 h incubation at 37 °C and data were expressed in percentage of enzyme inactivation. All analyses were carried out in triplicate and included positive control gelatinase standards as well as reagent blanks.
2.3
Gelatinolytic activity of rMMP-2 and -9—zymography assay
The rMMP-2 (AnaSpec) and -9 (Human MMP-9, recombinant, catalytic domain, AnaSpec) enzymes incubated with experimental primers for 1 h at 37 °C were subjected to electrophoresis under non-reducing conditions in 10% SDS-polyacrylamide gels copolymerized with 0.1% gelatin from porcine skin (Sigma–Aldrich, St. Louis, MO, USA), as previously described . Activation of gelatinase proforms was done with 2 mM of APMA at 37 °C for 1 h. After electrophoresis, gels were washed in 2% Triton-X 100 (Sigma–Aldrich, St. Louis, MO, EUA) with agitation and then incubated for 24 h at 37 °C in enzyme incubation buffer (Tris–HCl 50 mM, CaCl 2 5 mM and ZnCl 2 1 μM). Negative control zymogram was incubated in the same buffer with presence of 2 mM 1,10-phenanthroline. Then, gels were stained in 0.1% Coomassie Brilliant Blue R-250 (Bio-Rad, Laboratories Inc., CA, USA), de-stained and scanned by an imaging system Odyssey CLx (LI-COR, NE, USA) with white light exposure. The quantification of gelatinolytic activity was carried out in ImageJ software (NIH, Frederick, MD, USA) and expressed as percentage of inactivation considering MMP-2 and MMP-9 positive control bands as 100%.
2.4
Dental restorative procedures and simulated cyclic loading
A total of 48 freshly extracted human sound molars stored at −20 °C were selected (University of Illinois at Chicago IRB approved protocol # 2011-0132). Roots were cleaned, embedded in acrylic resin and the occlusal surfaces were ground flat 5 mm above the cemento-enamel junction. Occlusal Class I preparations were done using a #245 carbide bur (Regular Carbide Burs—Dentsply, Des Plaines, IL, USA) in a high-speed water cooled handpiece to final dimensions of 5 mm length, 4 mm width and 3 mm depth.
A total-etch bonding protocol was selected using 35% phosphoric acid (3M ESPE, St. Paul, MN, USA) for 15 s as, followed by active application of Adper Single Bond Plus (3M ESPE, St. Paul, MN, USA—N561025) on the cavity surfaces for 15 s. Then, gentle air jets were applied to remove the excess solvent, and the adhesive was light cured for 20 s using a halogen light unit (Optilux 501—Halogen Curing Light, Kerr Corporation, Orange, CA, USA G 830 mW/cm 2 ). Experimental primers were used after the acid etching step as follow: Control (20 mM HEPES), 15% e-GSE or 3% DOXY 3% primers were applied to the preparations for 60 s, rinsed for 60 s and the excess water removed with absorbent paper (Kimwipes, Kimberly Clark, Irving, TX, USA); or 0.2% CHX primer applied to the preparation for 60 s followed by removal of excess primer with absorbent paper.
The cavity preparations were filled with two 1.5 mm thick horizontal increments of Filtek Supreme Ultra resin composite (3M ESPE, St. Paul, MN, USA—N566267) and each increment was light-cured for 20 s (Optilux 501-Kerr Corp., Orange, CA, USA). The specimens were stored in distilled water at 37° C for 24 h and then divided into two subgroups ( n = 6): storage in incubation buffer (5 mM HEPES, 2.5 mM CaCl 2 , 0.3 mM NaN 3 , 0.05 mM ZnCl 2 pH 7.4) at 37° C for 9 days; or simulated cyclic loading (Coil—Cycler, Proto-tech, at 50 N load, 1.5 Hz frequency, and 1 × 10 6 cycles for 9 days). After cyclic loading, sections from each tooth were obtained as follows, and assigned to in situ zymography assay and microtensile bond strength test.
2.4.1
In situ zymography at the dentin–resin interface
Restored teeth were cut vertically into 0.6-mm-thick slices to expose the adhesive interfaces using a slow-speed saw under water cooling (Isomet 1000, Buehler, Lake Bluff, IL, USA) and further manually polished (Buehler) to obtain 500-μm-thick specimens. In situ zymography was performed with quenched fluorescein-conjugated gelatin as MMPs substrate (EnzChek Gelatinolytic/Collagenolytic Assay Kit, D-12054, Molecular Probes, Eugene, OR, USA). A 1.0 mg/mL stock solution of fluorescein-labeled gelatin was diluted 1:8 in buffer (NaCl 150 mM, CaCl2 5 mM, Tris–HCl 50 mM, pH 8.0). An 80-μL quantity of the fluorescent gelatin mixture was placed on top of each slab and covered with a coverslip (Microscope Cover Slips, Rochester Scientific Co., Inc., Rochester, NY, USA). The hydrolysis of the quenched fluorescein-conjugated gelatin substrate, indicative of endogenous gelatinolytic enzyme activity, was assessed by examination under a fluorescence microscope in 40× magnification (Leica DMI 6000, Buffalo Grove, IL, USA) after 2 h incubation in humidified chamber at 37 °C . Images were acquired along the adhesive interface of all specimens, and then three representative images of each group were selected for software analysis with ImageJ (NIH, Frederick, MD, USA). The intrinsic fluorescence was expressed as relative fluorescence units (RFU). The data did not meet the criteria of equality of variance across groups (Levene’s test, p < 0.001). The results were statistically compared using ANOVA and Games-Howell post hoc tests ( α = 0.05).
2.4.2
Dentin–resin microtensile bond strength (TBS)
The occlusal Class I restorations ( n = 6) were sectioned perpendicular to the adhesive–dentin interface into dentin–resin specimens (0.8 × 0.8 mm; 5 per tooth) using a slow-speed diamond saw under water cooling (Buehler—Series 15 LC Diamond, Buehler, Lake Buff, IL, USA). The specimens were fixed with cyanoacrylate glue (Loctite Super Glue—Gel Control, Rocky Hill, CT, USA) to a jig, which was mounted on a microtensile tester machine (Bisco Inc., Schaumburg, IL, USA) and subjected to a tensile force at 1 mm/min. Each tooth was considered as a statistical unit. The data did not meet the assumption of equality of variance across groups (Levene’s test, p < 0.001). The results were statistically subjected to ANOVA ( α = 0.05).
2
Material and methods
2.1
Preparation of experimental primers
Three experimental primers were prepared as follow: (i) oligomeric proanthocyanidin enriched grape seed extract (e-GSE) obtained by a solvent partitioning protocol previously published and prepared at 15% w/v concentration in buffer solution (20 mM HEPES pH 7.4); (ii) primer of Doxycycline Hydrochloride (DOXY – Fisher Scientific – New Jersey, NJ, USA) at 3% w/v in buffer solution; (iii) Chlorhexidine digluconate (CHX) primer prepared by dilution of stock solution (20% CHX, Sigma; St. Louis, MO, USA) to 0.2% CHX in distilled water. HEPES buffer solution was used as negative control primer. The primers were freshly prepared and the pH adjusted to 7.2 using NaOH at room temperature.
2.2
rMMP-2 activity—fluorescence assay
The gelatinolytic activity of rMMP-2 (Human MMP-2, recombinant, 10 μg/mL, AnaSpec, Fremont, CA, USA) incubated with the experimental primers was assayed according to the protocol described by Tay et al. , using EnzChek Gelatinolytic/Collagenolytic Assay Kit (D-12054, Molecular Probes, Eugene, OR, USA). Primers concentrations were 0.2% CHX, 0.65% e-GSE and 3% DOXY. Enzyme activation with 4-aminophenylmercuric acetate (APMA) was done previously for one hour at 37 °C . The fluorescent cleavage products were read in a 96-well fluorescent plate reader (Victor X5, PerkinElmer, Waltham, MA, USA), operated with an absorption maxima at 495 nm and fluorescence emission maxima at 515 nm. Fluorescence measurements were taken at 0 (baseline), 1 h and 2 h incubation at 37 °C and data were expressed in percentage of enzyme inactivation. All analyses were carried out in triplicate and included positive control gelatinase standards as well as reagent blanks.
2.3
Gelatinolytic activity of rMMP-2 and -9—zymography assay
The rMMP-2 (AnaSpec) and -9 (Human MMP-9, recombinant, catalytic domain, AnaSpec) enzymes incubated with experimental primers for 1 h at 37 °C were subjected to electrophoresis under non-reducing conditions in 10% SDS-polyacrylamide gels copolymerized with 0.1% gelatin from porcine skin (Sigma–Aldrich, St. Louis, MO, USA), as previously described . Activation of gelatinase proforms was done with 2 mM of APMA at 37 °C for 1 h. After electrophoresis, gels were washed in 2% Triton-X 100 (Sigma–Aldrich, St. Louis, MO, EUA) with agitation and then incubated for 24 h at 37 °C in enzyme incubation buffer (Tris–HCl 50 mM, CaCl 2 5 mM and ZnCl 2 1 μM). Negative control zymogram was incubated in the same buffer with presence of 2 mM 1,10-phenanthroline. Then, gels were stained in 0.1% Coomassie Brilliant Blue R-250 (Bio-Rad, Laboratories Inc., CA, USA), de-stained and scanned by an imaging system Odyssey CLx (LI-COR, NE, USA) with white light exposure. The quantification of gelatinolytic activity was carried out in ImageJ software (NIH, Frederick, MD, USA) and expressed as percentage of inactivation considering MMP-2 and MMP-9 positive control bands as 100%.
2.4
Dental restorative procedures and simulated cyclic loading
A total of 48 freshly extracted human sound molars stored at −20 °C were selected (University of Illinois at Chicago IRB approved protocol # 2011-0132). Roots were cleaned, embedded in acrylic resin and the occlusal surfaces were ground flat 5 mm above the cemento-enamel junction. Occlusal Class I preparations were done using a #245 carbide bur (Regular Carbide Burs—Dentsply, Des Plaines, IL, USA) in a high-speed water cooled handpiece to final dimensions of 5 mm length, 4 mm width and 3 mm depth.
A total-etch bonding protocol was selected using 35% phosphoric acid (3M ESPE, St. Paul, MN, USA) for 15 s as, followed by active application of Adper Single Bond Plus (3M ESPE, St. Paul, MN, USA—N561025) on the cavity surfaces for 15 s. Then, gentle air jets were applied to remove the excess solvent, and the adhesive was light cured for 20 s using a halogen light unit (Optilux 501—Halogen Curing Light, Kerr Corporation, Orange, CA, USA G 830 mW/cm 2 ). Experimental primers were used after the acid etching step as follow: Control (20 mM HEPES), 15% e-GSE or 3% DOXY 3% primers were applied to the preparations for 60 s, rinsed for 60 s and the excess water removed with absorbent paper (Kimwipes, Kimberly Clark, Irving, TX, USA); or 0.2% CHX primer applied to the preparation for 60 s followed by removal of excess primer with absorbent paper.
The cavity preparations were filled with two 1.5 mm thick horizontal increments of Filtek Supreme Ultra resin composite (3M ESPE, St. Paul, MN, USA—N566267) and each increment was light-cured for 20 s (Optilux 501-Kerr Corp., Orange, CA, USA). The specimens were stored in distilled water at 37° C for 24 h and then divided into two subgroups ( n = 6): storage in incubation buffer (5 mM HEPES, 2.5 mM CaCl 2 , 0.3 mM NaN 3 , 0.05 mM ZnCl 2 pH 7.4) at 37° C for 9 days; or simulated cyclic loading (Coil—Cycler, Proto-tech, at 50 N load, 1.5 Hz frequency, and 1 × 10 6 cycles for 9 days). After cyclic loading, sections from each tooth were obtained as follows, and assigned to in situ zymography assay and microtensile bond strength test.
2.4.1
In situ zymography at the dentin–resin interface
Restored teeth were cut vertically into 0.6-mm-thick slices to expose the adhesive interfaces using a slow-speed saw under water cooling (Isomet 1000, Buehler, Lake Bluff, IL, USA) and further manually polished (Buehler) to obtain 500-μm-thick specimens. In situ zymography was performed with quenched fluorescein-conjugated gelatin as MMPs substrate (EnzChek Gelatinolytic/Collagenolytic Assay Kit, D-12054, Molecular Probes, Eugene, OR, USA). A 1.0 mg/mL stock solution of fluorescein-labeled gelatin was diluted 1:8 in buffer (NaCl 150 mM, CaCl2 5 mM, Tris–HCl 50 mM, pH 8.0). An 80-μL quantity of the fluorescent gelatin mixture was placed on top of each slab and covered with a coverslip (Microscope Cover Slips, Rochester Scientific Co., Inc., Rochester, NY, USA). The hydrolysis of the quenched fluorescein-conjugated gelatin substrate, indicative of endogenous gelatinolytic enzyme activity, was assessed by examination under a fluorescence microscope in 40× magnification (Leica DMI 6000, Buffalo Grove, IL, USA) after 2 h incubation in humidified chamber at 37 °C . Images were acquired along the adhesive interface of all specimens, and then three representative images of each group were selected for software analysis with ImageJ (NIH, Frederick, MD, USA). The intrinsic fluorescence was expressed as relative fluorescence units (RFU). The data did not meet the criteria of equality of variance across groups (Levene’s test, p < 0.001). The results were statistically compared using ANOVA and Games-Howell post hoc tests ( α = 0.05).
2.4.2
Dentin–resin microtensile bond strength (TBS)
The occlusal Class I restorations ( n = 6) were sectioned perpendicular to the adhesive–dentin interface into dentin–resin specimens (0.8 × 0.8 mm; 5 per tooth) using a slow-speed diamond saw under water cooling (Buehler—Series 15 LC Diamond, Buehler, Lake Buff, IL, USA). The specimens were fixed with cyanoacrylate glue (Loctite Super Glue—Gel Control, Rocky Hill, CT, USA) to a jig, which was mounted on a microtensile tester machine (Bisco Inc., Schaumburg, IL, USA) and subjected to a tensile force at 1 mm/min. Each tooth was considered as a statistical unit. The data did not meet the assumption of equality of variance across groups (Levene’s test, p < 0.001). The results were statistically subjected to ANOVA ( α = 0.05).
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