Abstract
Objective
Dentinal MMPs have been claimed to contribute to the auto-degradation of collagen fibrils within incompletely resin-infiltrated hybrid layers and their inhibition may, therefore, slow the degradation of hybrid layer. This study aimed to determine the contribution of a synthetic MMPs inhibitor (galardin) to the proteolytic activity of dentinal MMPs and to the morphological and mechanical features of hybrid layers after aging.
Methods
Dentin powder obtained from human molars was treated with galardin or chlorhexidine digluconate and zymographically analyzed. Microtensile bond strength was also evaluated in extracted human teeth. Exposed dentin was etched with 35% phosphoric acid and specimens were assigned to (1) pre-treatment with galardin as additional primer for 30 s and (2) no pre-treatment. A two-step etch-and-rinse adhesive (Adper Scotchbond 1XT, 3M ESPE) was then applied in accordance with manufacturer’s instructions and resin composite build-ups were created. Specimens were immediately tested for their microtensile bond strength or stored in artificial saliva for 12 months prior to being tested. Data were evaluated by two-way ANOVA and Tukey’s tests ( α = 0.05). Additional specimens were prepared for interfacial nanoleakage analysis under light microscopy and TEM, quantified by two independent observers and statistically analyzed ( χ 2 test, α = 0.05).
Results
The inhibitory effect of galardin on dentinal MMPs was confirmed by zymographic analysis, as complete inhibition of both MMP-2 and -9 was observed. The use of galardin had no effect on immediate bond strength, while it significantly decreased bond degradation after 1 year ( p < 0.05). Interfacial nanoleakage expression after aging revealed reduced silver deposits in galardin-treated specimens compared to controls ( p < 0.05).
Conclusions
This study confirmed that the proteolytic activity of dentinal MMPs was inhibited by the use of galardin in a therapeutic primer. Galardin also partially preserved the mechanical integrity of the hybrid layer created by a two-step etch-and-rinse adhesive after artificial aging.
1
Introduction
Formation of a perfect resin-infiltrated hybrid layer, composed of collagen fibrils embedded by methacrylate-based resins, has been thought as essential to provide durable and successful adhesion to human dentin. The breakdown of resin-bonded interfaces has been directly related with the loss of stability of the hydrophilic resin components that comprises the hybrid layers . Nevertheless, the clinical implications of the integrity of dentin matrices within in vivo hybrid layers have been increasingly demonstrated as being fundamental for the loss of longevity of resin-bonded restorations .
The outstanding resistance of the collagenous dentin matrix against thermal and proteolytic disruption has been attributed to the high degree of intermolecular cross-linking and tight mechanical weave of this specialized connective tissue . However, great attention to the potential proteolytic activity of dentin has been raised since complexed and active forms of matrix metalloproteinases (MMPs) were identified in either non-mineralized or mineralized compartments of human dentin matrices . MMPs belong to a group of zinc- and calcium-dependent enzymes that have been shown to be able to cleave native collagenous tissues at neutral pH in the metabolism of all connective tissues .
Dentin matrix has shown to contain at least four MMPs: the stromelysin-1 (MMP-3) , the true collagenase (MMP-8) and the gelatinases A and B (MMP-2 and MMP-9 respectively) . These host-derived proteases are thought to play an important role in numerous physiological and pathological processes occurring in dentin, including the degradation of collagen fibrils that are exposed by suboptimally infiltrated dental adhesive systems after acid etching .
Collagenolytic/gelatinolytic activity of human dentin matrices has recently shown to be suppressed by a non-specific protease inhibitor, chlorhexidine digluconate (CHX) , at two concentrations: 0.2% (2.2 mM) and 2% (22 mM) and this inhibition has reported to be beneficial in the preservation of the hybrid layer both in vivo and in vitro .
Although the use of CHX as antiproteolytic primer for dentin matrix preservation has shown to be extremely convenient due to its commercial availability as mouthrinse agent (at 0.12–0.2%) and cavity disinfectant (at 2%), it is quite reasonable to speculate that specific MMP-inhibitors, which normally exert inhibitory activity under extremely low concentrations, may be as or even more effective than CHX in increasing the durability of hybrid layers.
Galardin is a synthetic MMP-inhibitor with potent activity against MMP-1, -2, -3, -8 and -9 and its action was firstly reported in the 90s by Grobelny et al. . Galardin has a collagen-like backbone to facilitate binding to the active site of MMPs and a hydroxamate structure ( R –CO–NH–OH, where R is an organic residue) which chelates the zinc ion located in the catalytic domain of MMPs . Galardin, also known as GM6001 or Ilomastat, has been used to selectively inhibit MMP-2, -3, -8 and -9 that have already been demonstrated to be present in human dentin matrix .
Thus, the aim of this study was to test the efficacy of galardin on the morphological and mechanical preservation of aged hybrid layers. It was hypothesized that: (1) the gelatinolytic activity of phosphoric acid demineralized dentin is similarly inhibited by CHX and galardin and (2) galardin can preserve the morphological and mechanical integrity of aged hybrid layers.
2
Materials and methods
Sixty recently extracted, non-carious human molars were collected after the patients’ informed consent had been obtained under a protocol reviewed and approved by the Ethic Committee for Human Studies of the University of Trieste.
2.1
Zymographic analysis (gelatinolytic activity of phosphoric acid demineralized dentin)
Reagents were purchased from Sigma Chemical (St. Louis, MO, USA) unless otherwise specified.
Twenty teeth were selected for this part of the study. Enamel and residual pulp tissue of these teeth were removed and dentin powder was obtained by pulverizing the liquid nitrogen-frozen dentin with a steel mortar/pestle (Reimiller, Reggio Emilia, Italy) in accordance with Mazzoni et al. . Five 1 g aliquots of dentin powder were obtained and assigned to one of the following treatments: Group 1: untreated mineralized dentin powder, Group 2: dentin powder partially demineralized with 1% phosphoric acid for ( Table 1 ) 10 min at 4 °C, Group 3: dentin powder partially demineralized with 1% phosphoric acid for 10 min at 4 °C then incubated with 0.2 mM galardin (water solution) for 30 min and Groups 4 and 5: dentin powder partially demineralized with 1% phosphoric for 10 min then treated, respectively, with 2.2 mM (0.2%) and 22 mM (2%) CHX water solution for 30 min. All specimens were then rinsed with 1 mL of distilled water (5 times), then re-suspended for 24 h in 4 mL extraction buffer: 50 mM Tris–HCl pH 6, containing 5 mM CaCl 2 , 100 mM NaCl, 0.1% Triton X-100, 0.1% non-ionic detergent P-40, 0.1 mM ZnCl 2 , 0.02% NaN 3 and EDTA-free protease inhibitor cocktail (Roche Diagnostics GmbH, Germany). Specimens were centrifuged at 14,000 rpm (Eppendorf, Minispin Plus, Hamburg, Germany), supernatants were collected and protein content was precipitated with 25% trichloroacetic acid (TCA) at 4 °C. TCA precipitates were re-solubilized in loading buffer pH 8.8 containing Trizma and 2% sodium dodecyl sulfate (SDS) in water.
| Adhesive | Composition | Mode of application |
|---|---|---|
| Adper Scotchbond 1XT | Etching | Etching 15 s |
| 35% phosphoric acid | Water rinsing 30 s | |
| Primer/bond | Dentine blot drying | |
| 2-Hydroxyethylmethacrylate (HEMA) | Adhesive application | |
| Polyalkenoic acid copolymer | ||
| Bis-phenol A diglycidylmethacrylate (Bis-GMA) | ||
| Water | ||
| Ethanol | ||
| Silica particles |
Total protein concentration of mineralized and partially demineralized dentin extracts was determined using the Bradford assay. Proteins were electrophorized under non-reducing conditions on SDS-polyacrylamide gels copolymerized with 2 g/L gelatin (porcine skin). Activation of gelatinase proforms was achieved with 2 mM p-aminophenylmercuric acetate (APMA) for 1 h at 37 °C and then the SDS-polyacrylamide gels were incubated for 24 h at 37 °C in zymography buffer (CaCl 2 , NaCl and Tris–HCl, pH 8.0). Gels were stained in 0.2% Coomassie Brilliant Blue R-250 and destained in destaining buffer (50% methanol, 10% acetic acid, 40% water).
Control zymograms were incubated in the presence of 5 mM EDTA and 2 mM 1,10-phenanthroline to inhibit gelatinases.
2.2
Microtensile bond strength test
Twenty-eight of the selected teeth were used in this part of the study. Flat surfaces of middle/deep dentin were exposed with a slow speed diamond saw (Micromet, Bologna, Italy). Smear layer-covered dentin surfaces were etched with 35% phosphoric acid for 15 s (etching gel, 3 M ESPE), rinsed and surfaces were blot-dried according to the wet bonding technique. Specimens were randomly assigned to the following treatments ( N = 14). Groups 1: acid-etched dentin surfaces were treated with 0.2 mM galardin (water solution) for 30 s, blot-dried and bonded with SB1XT, Group 2: received no pre-treatment before SB1XT application (control). SB1XT was applied in accordance with manufacturer’s instructions and light-cured. Resin composite build-ups were created with Filtek Z250 (3M ESPE, St. Paul, MN, USA).
Resin–dentin sticks with cross-sectional area of approximately 0.9 mm 2 were obtained in accordance with the non-trimming technique . Each stick was measured and recorded for bond strength calculation. Sticks were divided in two equal groups and either stored at 37 °C for 24 h ( T 0 ) or for 1 year ( T 1yr ) in artificial saliva prepared in accordance with Pashley et al. , but without protease inhibitors. Sticks were stressed until failure with a simplified universal testing machine at a crosshead speed of 1 mm/min (Bisco Inc., Schaumburg, IL, USA). Failure modes were evaluated as described by Breschi et al. .
The number of prematurely debonded sticks per each tested group was also recorded, but not included in the statistical analysis since all premature failures occurred during the cutting procedure, which was performed at time zero and did not exceed 3% of the total number of tested specimens. As values were normally distributed (Kolmogorov–Smirnof test), data were analyzed with a two-way ANOVA (tested variables were: the presence of galardin, time of storage) and Tukey’s post hoc tests. To analyze the effect of galardin on fracture modes, mixed and dentin cohesive failures were combined when performing the statistical analysis. Wilcoxon Signed Ranks Test was used to analyze the differences in failure modes between T 0 and T 1yr for each group, and Kruskal–Wallis test was used to compare the fracture modes between the groups within each time points. Statistical significance was set at α = 0.05.
2.3
Nanoleakage evaluation
The remaining twelve teeth ( N = 3/Group) were prepared and bonded as previously described for nanoleakage evaluation. Resin-bonded specimens were then vertically cut into 1 mm thick slabs to expose the bonded surfaces, which were further submitted to storage in artificial saliva at 37 °C for two different times: T 0 (24 h) and T 1yr (1 year). After storage (24 h or 1 year), the resin-bonded interfaces were immersed in 50 wt.% ammoniacal AgNO 3 solution according to the protocol described by Tay et al. , thoroughly rinsed in distilled water, and immersed in photodeveloping solution. The silver-impregnated specimens were fixed, dehydrated, embedded in epoxy resin (Epon 812; Fluka, Buchs, Switzerland), and processed for light microscopy (LM) in accordance with Saboia et al. .
Briefly, specimens were fixed on glass slides using cyanoacrylate glue, flattened with 600, 800, 1200 and 2400 grit SiC paper under running water (LS2; Remet) and stained with 0.5% acid fuchsine for 15 min, finally analyzed by light microscopy (Nikon E 800; Nikon, Tokyo, Japan). All interface images were obtained at a magnification of 100× and the amount of silver tracer precipitation along the interface (i.e., the degree of interfacial nanoleakage) was scored on a scale of 0–4 by two observers in accordance with Saboia et al. , i.e. interfacial nanoleakage was scored based on the percentage of the adhesive surface showing silver nitrate deposition: 0, no nanoleakage; 1, <25% with nanoleakage; 2, 25 ≤ 50% with nanoleakage; 3, 50 ≤ 75% with nanoleakage; 4, >75% with nanoleakage ( Table 2 ). Intra-examiner reliability was assessed by the Kappa test ( K = 0.84). Statistical differences among score groups (i.e. number of specimens falling within each score category) were analyzed using the χ 2 test ( p < 0.05).
| Bonding strategy | Bond strength | Failure Mode (%) | ||||
|---|---|---|---|---|---|---|
| A | CD | CC | M | |||
| GL + SB1XT | Immediate | 44.1 ± 7.3 (7) a | 30 A | 5 B | 20 A | 55 C |
| [1/140] | ||||||
| 1 year | 32.4 ± 6.6 (7) b | 26 A | 7 B | 18 A | 49 C | |
| [4/144] | ||||||
| SB1XT control | Immediate | 41.4 ± 5.9 (7) a | 20 A | 7 B | 28 A | 45 C |
| [3/152] | ||||||
| 1 year | 22.6 ± 5.4 (7) c | 24 A | 5 B | 19 A | 52 C | |
| [1/149] | ||||||
Additional embedded specimens were processed for nanoleakage analysis under TEM in accordance with Suppa et al. and examined under TEM (Philips CM-10, Eindhoven, The Netherlands) operating at 70 kV.
2
Materials and methods
Sixty recently extracted, non-carious human molars were collected after the patients’ informed consent had been obtained under a protocol reviewed and approved by the Ethic Committee for Human Studies of the University of Trieste.
2.1
Zymographic analysis (gelatinolytic activity of phosphoric acid demineralized dentin)
Reagents were purchased from Sigma Chemical (St. Louis, MO, USA) unless otherwise specified.
Twenty teeth were selected for this part of the study. Enamel and residual pulp tissue of these teeth were removed and dentin powder was obtained by pulverizing the liquid nitrogen-frozen dentin with a steel mortar/pestle (Reimiller, Reggio Emilia, Italy) in accordance with Mazzoni et al. . Five 1 g aliquots of dentin powder were obtained and assigned to one of the following treatments: Group 1: untreated mineralized dentin powder, Group 2: dentin powder partially demineralized with 1% phosphoric acid for ( Table 1 ) 10 min at 4 °C, Group 3: dentin powder partially demineralized with 1% phosphoric acid for 10 min at 4 °C then incubated with 0.2 mM galardin (water solution) for 30 min and Groups 4 and 5: dentin powder partially demineralized with 1% phosphoric for 10 min then treated, respectively, with 2.2 mM (0.2%) and 22 mM (2%) CHX water solution for 30 min. All specimens were then rinsed with 1 mL of distilled water (5 times), then re-suspended for 24 h in 4 mL extraction buffer: 50 mM Tris–HCl pH 6, containing 5 mM CaCl 2 , 100 mM NaCl, 0.1% Triton X-100, 0.1% non-ionic detergent P-40, 0.1 mM ZnCl 2 , 0.02% NaN 3 and EDTA-free protease inhibitor cocktail (Roche Diagnostics GmbH, Germany). Specimens were centrifuged at 14,000 rpm (Eppendorf, Minispin Plus, Hamburg, Germany), supernatants were collected and protein content was precipitated with 25% trichloroacetic acid (TCA) at 4 °C. TCA precipitates were re-solubilized in loading buffer pH 8.8 containing Trizma and 2% sodium dodecyl sulfate (SDS) in water.
