Matrix metalloproteinase inhibitor modulates esterase-catalyzed degradation of resin–dentin interfaces

Highlights

  • Resin–dentin interfacial biodegradation, fracture toughness and morphology.

  • Esterase accelerated the degradation of total-etch resin–dentin interfaces.

  • Mechanical performance deterioration with total-etch but not self-etch adhesives.

  • MMP inhibition modulate resin–dentin interfacial fracture mode changes.

Abstract

Objective

Assess the modulating effect of matrix metalloproteinase (MMP) inhibition on simulated human salivary enzyme (SHSE)-catalyzed degradation of interfacial fracture-toughness (FT) of self-etched and total-etched resin–dentin interfaces.

Methods

Miniature short-rod FT specimens (N = 10/group) containing a resin composite bonded to human dentin, using a self-etch (Easy Bond, EB) or a total-etch (Scotchbond, SB) adhesives, were prepared with and without application of an MMP inhibitor (galardin). Specimens were non-incubated or incubated in phosphate buffered saline (PBS) or SHSE for 7, 30, 90, or 180-days. FT data were obtained using a universal testing machine. Incubation media were analyzed by high performance liquid chromatography (HPLC) for the presence of a 2,2-bis-[4-2(2-hydroxy-3-methacryloxypropoxy)phenyl]-propane (bisGMA)-derived degradation product, bis-hydroxy-propoxy-phenyl-propane (bisHPPP). Fractographic analysis was performed by scanning electron microscopy and image processing software (ImageJ). Statistical analysis was performed by ANOVA and Tukey’s (p < 0.05).

Results

More bisHPPP was detected in SHSE vs. PBS for both adhesive systems (p < 0.05). EB specimens yielded no difference in FT and failed preferentially in the resin after >30-days (p < 0.05). SB specimens yielded lower FT values after 180-days with SHSE ±galardin vs. 0-days/no-galardin (p < 0.05) and failed preferentially in the hybrid-layer after >30-days (p < 0.05). Galardin mildly modulated the change in fracture mode for both systems.

Significance

Esterase-catalyzed degradation of total-etch interfaces is modulated by MMP-inhibition, however, self-etch interfaces possess greater biostability under simulated intra-oral conditions, regardless of MMP inhibition. This could be related to different chemical compositions and/or mode of adhesion.

Introduction

Resin composites are currently the most frequently used dental restorative material , with their long-term clinical success being largely determined by the physical and chemical integrity of the resin–dentin interface . Recent meta-analysis of 569 restoration failures, spanning 12 separate clinical studies, revealed that 73% of direct posterior composite restorations fail due to either fracture or caries within the first 6 years of placement and that failure rates are up to 3 times higher in high-caries-risk individuals (annual failure rate of 4.6% in high-caries-risk patients vs. 1.6% in low-caries-risk patients after 10 years) . Fracture and caries mediated failure of resin–dentin interfacial margins occurs through biochemical degradation, decreasing the structural integrity of the bond leading to interfacial fracture , and the promotion of bacterial microleakage , which provides a potential contributory explanation to the increased rate of recurrent caries around resin composite restorations . This biochemical degradation, or biodegradation, of the resin–dentin interface occurs via dual mechanisms (1) degradation of the restoration’s adhesive resin matrix by salivary and bacterial esterases and (2) degradation of the dentin’s collagen fibers .

Biodegradation of the resin matrices is largely a result of esterase catalyzed hydrolysis of the methacrylate-based monomers comprising the resin matrix. A common monomer is 2,2-bis[4-2(2-hydroxy-3-methacryloxypropoxy)phenyl] propane (bisGMA) . Upon introduction into the oral cavity, salivary esterase activities and certain species of oral bacteria such as Streptococcus mutans , begin to hydrolyze bisGMA’s ester linkages within the adhesive to produce bis-hydroxy-propoxy-phenyl-propane (bisHPPP) .

Biodegradation of the dentin’s collagen fibers, on the other hand, is a result of matrix metalloproteinases (MMPs) hydrolyzing the collagen fibers of demineralized dentin . MMPs are a class of host-derived, zinc-activated, and calcium-dependent endopeptidases which become activated in low pH environments; a result of the acidic etchants or acidic resin monomers used during adhesive application . Human dentin contains MMPs-2, -8, and -9 which are capable of degrading type I collagen fibers . However, the infiltration and encapsulation of demineralized collagen networks by the adhesive resins is thought to immobilize MMPs and thus provide protection against collagen degradation . Owing to the fact that total-etch adhesives require multi-step application, utilizing an acidic conditioner, ​a low viscosity primer, followed by a high viscosity adhesive, whereas self-etch adhesives can simultaneously etch and bond to the dentin, self-etch adhesives are thought to offer better protection against MMP mediated degradation due to higher levels of collagen encapsulation vs. total-etch adhesives . Nevertheless, although not as substantial as what is seen with total-etch adhesives, studies have revealed the presence of nanovoids beneath the hybrid layer (HL) of the self-etch resin–dentin interface in which MMPs mediated degradation is still a concern .

A study by Shokati et al. examined the biomolecular processes leading to failure of the total-etch resin–dentin interface after exposure to salivary esterases. In their in-vitro study, resin–dentin interfacial fracture toughness specimens were incubated in human salivary derived esterase (HSDE) produced from human saliva. A clinically significant reduction in interfacial fracture toughness was observed after 6 months of incubation and although high performance liquid chromatography (HPLC) confirmed the higher degradation capacity of HSDE vs. the phosphate buffered saline (PBS) control, there was still a gradual reduction in interfacial fracture toughness observed in specimens incubated in the control solution. Furthermore, scanning electron microscopy (SEM) analysis showed that the interfacial fracture plane had moved from the top of the HL at 0 days to beneath the HL after 180 days of HSDE incubation. The gradual reduction of interfacial fracture toughness in samples incubated without the presence of esterase activities and the movement of the fracture plane to the unprotected collagen fibers under the HL layer suggest that in addition to salivary esterases, MMPs could be a contributing factor for interfacial biodegradation. The MMP inhibitor galardin, also known as GM6000 or Ilomastat, has been suggested as a specific MMP-inhibitor for preservation of HL .

Building from the framework and protocols presented by Shokati et al. and Breschi et al. , the present study aims to evaluate the biostability of a self-etch adhesive in a simulated intra-oral environment and compare it to that of a total-etch adhesive. Furthermore, the addition of an MMP inhibitor will allow for the exploration of potential synergistic degradative role between oral esterase activities and MMPs in the degradation of the resin–dentin interface for both adhesive systems. It was hypothesized that simulated human salivary enzyme (SHSE) would accelerate the deterioration of the mechanical performance parameters for both composite restoration adhesive systems and that MMP inhibition would have a differential modulating effect on the degradation for the two adhesive systems.

Materials and methods

Specimen preparation

A total of 360 miniature short-rod (Mini-SR) interfacial fracture toughness specimens , with a resin–dentin interface consisting of a chevron-notched shaped resin composite (Filtek™ Z250 Shade A1, Z250, 3M™ ESPE™, St. Paul, MN, USA) bonded to human dentin using either a self-etch adhesive (Adper™ Easy Bond, EB, 3M™ ESPE™, St. Paul, MN, USA) or a total-etch adhesive (Adper™ Scotchbond™ Multi-Purpose Plus, SB, 3M™ ESPE™, St. Paul, MN, USA) were prepared with (+Gal) or without (−Gal) the application of a MMP inhibitor (galardin, USBiological, Swampscott, MA, USA). Specimens (N = 10/group) were prepared for EB and SB adhesive resin groups, +Gal and −Gal dentin treatment groups, PBS and SHSE incubation solution groups, and either 0, 7, 30, 90, or 180 day incubation period groups ( Fig. 1 ).

Fig. 1
Schematic study representation. N = 10 specimens were prepared per group. Non-galardin and galardin treated groups were made for both the total-etch adhesive, Scotchbond, and the self-etch adhesive, Easy Bond.

Specimen fabrication ( Fig. 2 ) followed previously established methods . With randomly selected, fully intact, unrestored, human third molars stored for no longer than 1 month in distilled water at −20 °C (University of Toronto Human Ethics Protocol #25793). A water-cooled low-speed diamond saw (Buehler Ltd., Lake Bluff, IL, USA) and high-speed bur under constant water irrigation were used to prepare two dentin slabs per tooth . Compressed-air (pressure: 32 psi, distance: ≤5 mm (Taskforce Deluxe Portable Dental Unit, Aseptico Inc., Woodinville, WA, USA)) was used to dry the dentin for 2 s, then the adhesive, EB or SB, was applied and photopolymerized according to the manufacturer’s instructions (Sapphire Plus Plasma Arc Curing System (Dent Mat, Santa Maria, CA, USA)). Light intensity maintained above 1730 mW/cm 2 , and was verified using the internal radiometer. For specimens requiring the application of galardin, galardin (0.2 mM) was applied to the dentin for 30 s with a micro-brush (after 30 s excess galardin was blotted dry with delicate wipes), this was done prior to adhesive application for EB specimens and between etchant and primer application for SB specimens. Resin composite was then packed around the prepared dentin within a stainless steel and Teflon ® mold. The resin composite was photopolymerized for 10 s on both their front and back surfaces while in the molds and for an additional 5 s on their top and bottom surfaces once removed from the molds. The front and back surfaces of prepared specimens were polished with increasingly finer grit SiC paper (240, 600, 1200, and 4000-grit) under wet conditions to remove excess composite resin and oxygen inhibited layer to produce a final Mini-SR specimen with a total surface area of 2.8 ± 0.05 cm 2 . Polishing of the composite collar also prepared it for prospective SEM surface degradation observations.

Fig. 2
Mini-SR specimen preparation: (a) dentin cross-sections cut (thickness: 0.75 ± 0.1 mm). (b) Dentin slabs prepared (width: 3.70 ± 0.05 mm, length: 7.00 ± 0.05 mm) and EB adhesive applied to bottom surface (surface area: 25.9 ± 0.54 mm 2 ). (c) First half: slab placed in stainless steel mold which was filled with Z250 composite and photopolymerized. Exposed dentin polished with 600-grit SiC paper under wet conditions before galardin and/or EB application. Second half: specimen placed into a Teflon ® mold with a stainless steel spacer (thickness: 0.25 mm, 45° bevel with long point in contact with dentin surface) exposing a chevron shaped area of dentin (surface area: 1.05 ± 0.01 mm 2 ). Mold cavity packed with Z250 and photopolymerized. (d) Spacer used in the Teflon ® mold creates a chevron shaped composite resin notch, acting as a stress concentrator during tensile loading. (e) Calculation of fracture toughness.

Incubation schedule and media preparation

Prior to incubation, all specimens were pre-incubated with penicillin/streptomycin (15070-063, Gibco, Grand Island, NY, USA) in a 1:10 dilution with PBS (D8662, Sigma, St. Louis, MO, USA) for 48 h to reduce the risk of contamination and to allow for diffusion of leachable unreacted monomers . Specimens were incubated individually (37 °C, pH 7.0) in autoclaved 10 mL amber glass vials containing 3 mL of media. After pre-incubation, specimens were incubated in their designated media: PBS for specimens to be used as a negative control or SHSE for specimens to be tested in an esterase solution replicating the esterase activity levels of the oral cavity .

Protocols for enzyme activity measurements have been described previously . Briefly, SHSE was prepared by mixing cholesterol esterase (CE, COE-313, Lot# 86621, Toyobo Co., Ltd., Osaka, Japan) and pseudocholinesterase (PCE, C7612-6KU, Lot# 078K7015V, Sigma, St. Louis, MO, USA) in PBS in order to obtain a solution possessing 16 units/mL and 0.01 units/mL CE and PCE activity, respectively . Based on stability assays and in order to maintain SHSE activity, media was replaced daily over the first 5 days of incubation and thereafter the media was replaced every 10th day and replenished every 5th day for the remainder of the incubation period.

Samples incubated in PBS followed the same replacement/replenishment schedule as those incubated in SHSE in order to maintain consistency between experimental groups. The antibiotic cocktail of penicillin/streptomycin was maintained at a 1:50 dilution with both incubation groups, all media was sterile filtered via a Millex-GP 0.22 μm syringe filter unit (Millipore, Bedford, MA, USA), and all sample handling was conducted in a biosafety cabinet to maintain aseptic conditions.

Interfacial fracture toughness testing and analysis

After pre-incubation (“0” time point) or incubation in either PBS or SHSE for 7, 30, 90, or 180 days, specimens were individually mounted into a custom test jig on a universal testing machine (Model 8501, Instron ® , Canton, MA, USA) and loaded in tension at an extension rate of 0.5 mm/min until fracture . Interfacial fracture toughness was calculated based on the equation: K IC = PY / D W where P = peak force, Y = minimum stress intensity factor coefficient (determined previously by way of compliance calibration curves ), D = specimen diameter, and W = length of specimen from load line .

Analysis of biochemical degradation by HPLC

Every 10 days throughout the incubation period PBS and SHSE incubation media were replaced with the respective fresh media. Old media was collected for each experimental group and analyzed via a reverse-phase HPLC system (Waters™, Mississauga, ON, Canada) using a gradient mobile phase method, as described previously , to identify and track the presence of the bisGMA-derived biodegradation by-product bisHPPP. The system consisted of a 600E multi-solvent delivery system, a 996 photodiode array, a column (Luna 5 μm C18 4.6 × 250, Phenomenex, Torrance, CA, USA), and Millennium chromatography manager version 2.15 (Waters™, Mississauga, ON, Canada). bisHPPP levels were quantified using a standard curve and ultraviolet spectrum profile produced from known concentrations of commercial bisHPPP (Sigma, St. Louis, MO, USA). Verification of bisHPPP was done with a mass spectrometer (AB Sciex QStar ESI-Qq-TOF, AB Sciex, Concord, ON, Canada) at the SPARC Biocentre (The Hospital for Sick Children, Toronto, ON, Canada).

Surface degradation and fracture analysis by SEM

After interfacial fracture toughness testing, dentin-containing halves of all Mini-SR specimens were prepared for SEM analysis. Specimens were dehydrated in increasing concentrations of ethanol (30, 50, 75, 95, and 100%) prior to being subject to critical-point-drying (Fisons Instruments, CPD 7501, VG Microtech, England) and receiving a coat of platinum (SC515 SEM coating system, Polaron Equipment Ltd., England) .

Using a Hitachi Ltd. Model S-2500 SEM with the accelerating voltage set to 10 kV specimens were examined (1) along the surface of the composite resin collar to obtain representative images depicting the effects of PBS vs. SHSE incubation and (2) within the fracture site with the aid of the computer imaging software ImageJ (National Institutes of Health, Bethesda, MD, USA). Fracture surfaces were examined in their entirety between ×100 and ×5000 original magnification. Locations of resin fracture vs. HL fracture were mapped out on a ×50 original magnification image the entire fracture site. Using ImageJ the ratio of pixels, and therefore the percentage of fracture surface area, representing resin fracture vs. HL fracture was calculated.

Statistical analysis

Conducting analysis of interfacial fracture toughness and fracture morphology separately and independently between the type of adhesive used, specimen groups were created such that each group possessed a time point (0, 7, 30, 90, or 180 days), dentin treatment (−Gal or +Gal), and type of incubation media (PBS or SHSE) resulting in 18 total groups for each adhesive. Using SPSS software (IBM, Markham, ON, Canada), one-way analysis of variance (ANOVA) and Tukey’s multiple comparison post-hoc tests were used to determine if there was any statistical significance between these 18 groups. These groups were considered the independent variable whereas the interfacial fracture toughness values or fracture morphology percentages acted as dependent variables. The confidence interval was set at 95%.

Materials and methods

Specimen preparation

A total of 360 miniature short-rod (Mini-SR) interfacial fracture toughness specimens , with a resin–dentin interface consisting of a chevron-notched shaped resin composite (Filtek™ Z250 Shade A1, Z250, 3M™ ESPE™, St. Paul, MN, USA) bonded to human dentin using either a self-etch adhesive (Adper™ Easy Bond, EB, 3M™ ESPE™, St. Paul, MN, USA) or a total-etch adhesive (Adper™ Scotchbond™ Multi-Purpose Plus, SB, 3M™ ESPE™, St. Paul, MN, USA) were prepared with (+Gal) or without (−Gal) the application of a MMP inhibitor (galardin, USBiological, Swampscott, MA, USA). Specimens (N = 10/group) were prepared for EB and SB adhesive resin groups, +Gal and −Gal dentin treatment groups, PBS and SHSE incubation solution groups, and either 0, 7, 30, 90, or 180 day incubation period groups ( Fig. 1 ).

Fig. 1
Schematic study representation. N = 10 specimens were prepared per group. Non-galardin and galardin treated groups were made for both the total-etch adhesive, Scotchbond, and the self-etch adhesive, Easy Bond.

Specimen fabrication ( Fig. 2 ) followed previously established methods . With randomly selected, fully intact, unrestored, human third molars stored for no longer than 1 month in distilled water at −20 °C (University of Toronto Human Ethics Protocol #25793). A water-cooled low-speed diamond saw (Buehler Ltd., Lake Bluff, IL, USA) and high-speed bur under constant water irrigation were used to prepare two dentin slabs per tooth . Compressed-air (pressure: 32 psi, distance: ≤5 mm (Taskforce Deluxe Portable Dental Unit, Aseptico Inc., Woodinville, WA, USA)) was used to dry the dentin for 2 s, then the adhesive, EB or SB, was applied and photopolymerized according to the manufacturer’s instructions (Sapphire Plus Plasma Arc Curing System (Dent Mat, Santa Maria, CA, USA)). Light intensity maintained above 1730 mW/cm 2 , and was verified using the internal radiometer. For specimens requiring the application of galardin, galardin (0.2 mM) was applied to the dentin for 30 s with a micro-brush (after 30 s excess galardin was blotted dry with delicate wipes), this was done prior to adhesive application for EB specimens and between etchant and primer application for SB specimens. Resin composite was then packed around the prepared dentin within a stainless steel and Teflon ® mold. The resin composite was photopolymerized for 10 s on both their front and back surfaces while in the molds and for an additional 5 s on their top and bottom surfaces once removed from the molds. The front and back surfaces of prepared specimens were polished with increasingly finer grit SiC paper (240, 600, 1200, and 4000-grit) under wet conditions to remove excess composite resin and oxygen inhibited layer to produce a final Mini-SR specimen with a total surface area of 2.8 ± 0.05 cm 2 . Polishing of the composite collar also prepared it for prospective SEM surface degradation observations.

Fig. 2
Mini-SR specimen preparation: (a) dentin cross-sections cut (thickness: 0.75 ± 0.1 mm). (b) Dentin slabs prepared (width: 3.70 ± 0.05 mm, length: 7.00 ± 0.05 mm) and EB adhesive applied to bottom surface (surface area: 25.9 ± 0.54 mm 2 ). (c) First half: slab placed in stainless steel mold which was filled with Z250 composite and photopolymerized. Exposed dentin polished with 600-grit SiC paper under wet conditions before galardin and/or EB application. Second half: specimen placed into a Teflon ® mold with a stainless steel spacer (thickness: 0.25 mm, 45° bevel with long point in contact with dentin surface) exposing a chevron shaped area of dentin (surface area: 1.05 ± 0.01 mm 2 ). Mold cavity packed with Z250 and photopolymerized. (d) Spacer used in the Teflon ® mold creates a chevron shaped composite resin notch, acting as a stress concentrator during tensile loading. (e) Calculation of fracture toughness.
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Nov 23, 2017 | Posted by in Dental Materials | Comments Off on Matrix metalloproteinase inhibitor modulates esterase-catalyzed degradation of resin–dentin interfaces
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