Graphical abstract
Abstract
Objective
To study the combined effect of simulated occlusal loading and plaque-derived biofilm on the interfacial integrity of dental composite restorations, and to explore whether the effects are modulated by the incorporation of sucrose.
Methods
MOD-class-II restorations were prepared in third molars. Half of the specimens (n = 27) were subjected to 200,000 cycles of mechanical loading using an artificial oral environment (ART). Then, both groups of specimens (fatigued and non-fatigued) were divided into three subgroups for testing in CDC-reactors under the following conditions: no biofilm (Control), biofilm with no sucrose (BNS) and biofilm pulsed with sucrose (BWS). BNS and BWS reactors were incubated with a multispecies inoculum from a single plaque donor whereas the control reactor was not. The BWS reactor was pulsed with sucrose five times a day. The biofilm challenges were repeated sequentially for 12 weeks. pH was recorded for each run. Specimens were examined for demineralization with micro-CT and load capacity by fast fracture test.
Results
Demineralization next to the restorations was only detectable in BWS teeth. Fracture loads were significantly reduced by the concomitant presence of biofilm and sucrose, regardless of whether cyclic mechanical loading was applied. Cyclic loading reduced fracture loads under all reactor conditions, but the reduction was not statistically significant.
Conclusions
Sucrose pulsing was required to induce biofilm-mediated degradation of the adhesive interface.
We have presented a comprehensive and clinically relevant model to study the effects of mechanical loading and microbial challenge on the interfacial integrity of dental restorations.
1
Introduction
During their lifetime, dental composite restorations are exposed to an array of environmental factors involved in the breakdown of the adhesive interface between the restorative material and the dental tissues. For instance, when the polymerization shrinkage stress of a dental composite exceeds the interfacial bond strength, delamination and gap formation at the interface occurs . These gaps are susceptible to infiltration by oral fluids, acids, metabolites and colonization by oral bacteria . Even if the initial shrinkage stress is not sufficiently high to cause delamination, the mechanical, chemical and biological stresses from oral functions can contribute to interfacial degradation . Ultimately, the loss of the marginal sealing facilitates oral bacteria accumulation at the interface, which may lead to secondary caries around composite restorations.
Masticatory forces from normal chewing and para-functional habits can impose great stress at the adhesive interface. Although the mechanical degradation of dental composite materials has been largely characterized , much less is known about the effect of mechanical challenge on the interfacial integrity of dental restorations. The few studies published so far show that cyclic loading leads to mechanical weakening of the adhesive interface regardless of the fatigue configuration (i.e. tensile, bending or compression) . Moreover, similar to the fracture strength of monolithic materials, an inverse correlation between number of loading cycles and bond strength values has been shown . These data suggest that cyclic mechanical loading can affect the integrity of the bond irrespective of the initial bond strength of the adhesive interface. Thus, fatigue is relevant when exploring interfacial degradation processes.
In addition to mechanical challenge, biological and chemical agents present in oral fluids and food can affect dental composite restorations. Research by Santerre and coworkers demonstrated that esterase activity of saliva promotes the breakdown of condensation bonds present in dental composite polymers . Those studies focused on the composite material, however, biological degradation effects on the adhesive interface remain largely unknown. A recent study showed that exposure of model restorations to esterases increased the amount of microbial leakage along the interface , suggesting that biological degradation could contribute to the interfacial breakdown.
Oral biofilms have been proposed to have a role in the degradation of the interface . Recent data demonstrated that Streptococcus mutans , an oral pathogen, possesses esterase activity capable of degrading dental composites and dental adhesives implying a potential microbial mechanism for degrading the adhesive interface.
In the mouth, oral biofilms produce organic acids by fermenting carbohydrates from diet , thus promoting the demineralization of hard dental tissues . In addition, oral bacteria secrete a large array of enzymes and other metabolic products that might degrade the adhesive interface. To gain insight into the role of oral bacteria in the degradation process, we previously investigated the effect of a multi-species oral microcosm biofilm on the interfacial integrity of model dental restorations . In that model, dentin-composite disks prepared with two different restorative systems were exposed to oral biofilms, and then tested under diametral compression. During the microbial challenge, half of the specimens were exposed to a sucrose-pulsed biofilm to explore the contribution of sucrose as an environmental factor. A reduction in the debonding load was observed in both groups exposed to biofilm compared to the control (no biofilm), although only the reduction seen in the sucrose-fed biofilm group was statistically significant.
Most of the factors involved in interfacial breakdown are commonly studied in isolation. In the oral cavity, multiple challenging factors may be present at the same time, and they might contribute synergistically to the degradation process. The aim of this study was to examine the combined effect of mechanical loading and microbial challenge on the interfacial integrity of composite restorations placed in extracted teeth, and to explore whether the effects of those challenges were modulated by environmental factors, such as sucrose.
2
Materials and methods
2.1
Specimen preparation
De-identified extracted human third molars were collected from oral surgery clinics at the University of Minnesota and in the Minneapolis/Saint Paul metropolitan area. Only teeth free of caries, fractures and cracks were included. The molars were cleaned of soft tissues and hard deposits (when present), and stored in 1% thymol solution. Mesio-occlusal-distal (MOD) class II cavities were prepared using a modified flat end tapered burs (SS White Burs Inc., Lakewood, NY, USA). The dimensions of the cavities were ∼2.5 mm wide and ∼2.5 mm deep for the occlusal box, and ∼3 mm wide and ∼1.5 mm deep for the proximal box. All teeth were restored using Adper™ Single Bond Plus adhesive system (SB) and Z100™ Restorative (Z100) system (3M ESPE, St. Paul, MN, USA) ( Table 1 ). Briefly, 35% phosphoric acid (Scotchbond etchant, 3M ESPE, St. Paul, MN, USA) was applied to dentin and enamel for 15 s and rinsed with water. Gentle air-drying was used to avoid collagen collapse in dentin. Two consecutive coatings of SB were rubbed on the internal walls of the preparation and polymerized for 20s. The teeth were restored with 2 mm thick increments of Z100. Each layer was polymerized for 20 s with an Elipar™ S10 curing light (3M ESPE, St. Paul, MN, USA) operated at 1200 mW/cm The samples were polished after 24 h of water storage. A finishing diamond stone was used to remove excess material from the restoration margin before polishing was conducted with Progloss™ One Step Composite Polishers (Kerr Corporation, Orange, CA, USA). A total of 54 MOD class-II composite restorations were made.
| Product | Composition | Batch number |
|---|---|---|
| Z100™ Restorative | Silane treated ceramic, triethylene glycol dimethacrylate (TEGDMA), bisphenol a diglycidyl ether dimethacrylate (BISGMA), 2-benzotriazolyl-4-methylphenol. | N649950 |
| Adper Single Bond Plus | Ethyl alcohol, silane treated silica (nanofiller), bisphenol a diglycidyl ether dimethacrylate (BISGMA), 2-hydroxyethyl methacrylate(HEMA), glycerol 1,3-dimethacrylate, copolymer of acrylic and itaconic acids, water, diurethane dimethacrylate (UDMA) diphenyliodonium hexafluorophosphate, ethyl 4-dimethyl aminobenzoate (EDMAB). | N561025 |
2.2
Fatigue (chewing simulation)
Half of the specimens (n = 27) were mounted in Teflon rings with self-curing acrylic resin (Dentsply Caulk, Milford, DE, USA). Each mounted specimen was positioned in the mandibular chamber of an artificial oral environment (ART), developed by the Minnesota Dental Research Center for Biomaterials and Biomechanics ( Fig. 1 ). A 6 mm diameter steatite bead attached to the upper arm was used as the antagonist. The target loading point was located in the functional cusp of the third molars. The artificial mouth was set to simulate 200,000 chewing cycles with a maximum load of 50N and an excursion of 0.8 mm at the contact point . Throughout the duration of the mechanical challenge the specimens were submerged in deionized water at room temperature. The cyclic load was constantly monitored during the loading process.
2.3
Frozen multispecies biofilm stocks
Frozen multispecies biofilm stocks from multiple subjects were made and stored during the course of previous studies . Briefly, plaque and saliva samples were collected from children with mixed dentitions (6–12 years of age) determined to be at high risk for caries. Plaque was retrieved from the margin of dental restorations. Whole saliva was collected by expectoration. Those sampling protocols were approved by the University of Minnesota Institutional Review Board; detailed descriptions are available in our previous publications .
This study design called for 12 sequential weeks of biofilm exposure. Our resources did not allow us to run stocks from multiple subjects. A pair of stocks from a single subject (781) were selected to provide the inoculums for the biofilm challenge in this study. One stock had been grown without sucrose (781NS), whereas the other had been pulsed with sucrose five times a day (781WS). The original plaque sample for subject 781 was collected from the margin of a composite restoration with active secondary caries. Detailed information on the taxonomic composition of the 781plaque sample, and the taxonomic and metaproteomic composition of the 781NS and 781WS biofilms can be found in the Supplementary files that accompany a recent article .
2.4
In-vitro biofilm model
Before biofilm exposure, all the restored teeth (fatigued and non-fatigued) were coated with color-coded acid resistant nail varnish, leaving only the composite and the restoration margin exposed. The nail varnish was allowed to dry in a HEPA-filtered chamber, to minimize the likelihood of contamination. The two groups of specimens were further divided into three subgroups according to the biofilm growth condition in the experiment: Control (no biofilm), biofilm with no sucrose (BNS), and biofilm with sucrose (BWS). This defined six different groups ( Table 2 ).
| Control reactor (no biofilm) |
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| BNS reactor |
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| BWS reactor |
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All of the following steps likewise were performed inside the HEPA enclosure. The color-coded samples were immersed in 75% ethanol for 1 min, air-dried, and then mounted in customized sample holders designed to fit in the sampling rods of the CDC reactor. The tooth-holder combinations were immersed in 75% ethanol for 1 min, and air-dried as before. They were then mounted in autoclaved sampling rods and the areas unprotected by nail varnish (composite and tooth interfaces) were coated with 30 μl of filtered-sterilized saliva (originally collected from subject 781 and stored at −80 °C). The specimens allocated to the BNS group were then inoculated with 30 μl of 781NS biofilm stock re-suspended in anaerobic transport media. The BWS group was inoculated with a corresponding suspension of the 781WS stock.
The rods with non-inoculated specimens (9 Fatigue and 9 No-fatigue) were inserted into the Control reactor, which contained 350 ml of sterile basal mucin medium (BMM) with 0.02% sodium azide added to inhibit microbial growth during the experiment ( Fig. 2 a and b). The rest of the samples were allocated in the same manner to the BNS and BWS reactors, which each contained an initial volume of 350 ml sterile BMM without sodium azide ( Fig. 2 a and b). A ∼24-h incubation was carried out in all reactors at 37 °C under constant shear (125 rpm) but with no media flow. This allowed bacteria to attach to the restoration surface (in the case of the inoculated specimens). After incubation, the BNS and BWS reactors were connected to the nutrient carboy and sterile BMM was pumped through the system for 48 h ( Fig. 2 b). The initial rate was set at 17 ml min −1 (125 rpm, 37 °C). In the BNS reactor, the biofilm was grown in the absence of sucrose, while in the BWS reactor sucrose was pulsed five times a day to simulate daily food intake. This was done by adding 42 ml of 40% sucrose (for a final concentration of 5%) into the reactor at intervals of 2 h. No sucrose pulsing was conducted at night. The flow rate was set at 20 ml min −1 during the second day of sucrose pulsing to avoid biofouling and to prevent plugging of the efflux tubing. BMM was not pumped through the Control reactor, in order to minimize the volume of sodium azide-containing hazardous waste produced. However, it was stirred continuously at 37 °C. After 48 h the systems were taken down; the samples were cleaned by rinsing and sonication, disinfected with 75% ethanol, and stored in sterile phosphate-buffered saline at 4 °C until the next biofilm challenge. Twelve sequential cycles of biofilm challenge with the same inoculum were conducted for the BNS and BWS teeth. During the same period, the Control teeth were exposed to fresh sterile BMM with sodium azide, as described above. Between each experimental cycle all instruments and containers used were disinfected with 10% bleach for 30 min, rinsed with water, washed with detergent, rinsed with water again and finally autoclaved prior to the next cycle. A fresh layer of nail varnish was applied before each run and the samples then were disinfected saliva-coated and inoculated as described above.
All reactors were incubated under aerobic conditions, to simulate the natural succession of supragingival plaque. Data published elsewhere had previously shown that anaerobic species were able to survive in this system, presumably due to oxygen consumption by facultative species .
2.5
pH recording
To continuously measure the pH changes in the medium, the CDC vessel’s lid was modified to fit an autoclavable pH electrode. The pH was recorded every 15 min throughout the 72-h period of experimentation.
2.6
Demineralization assessment using micro-CT
After 12 sequential biofilm challenges, three specimens from each group were scanned for demineralization around the interface using an X-ray micro-computed tomography (micro-CT) machine (XT H 225, Nikon Metrology, Brighton, MI, USA). The teeth were mounted with acrylic resin in Teflon rings and scanned with the following operational parameters: 90 kV, 90 μA, 720 projections and 4 frames per projection. 3D spatial reconstructions were done with CT Pro 3D (Nikon Metrology, Brighton, MI, USA) and visualized with VG Studio MAX 2.1 (Volume Graphics GmbH, Heidelberg, Germany). For the demineralization analysis, three different landmarks per sample were assessed: occlusal margin, proximal wall and gingival margin. For each chosen region, nine attenuation coefficient profiles were retrieved extending from the surface of the enamel lesion to the deeper sound enamel. The data from the three representative specimens were averaged and converted to mineral percentage profiles for each group.
2.7
Fracture test
Following the biofilm challenge, the restored teeth (including the scanned samples) were disinfected, and the protective layer of nail varnish was removed with a clean spatula. The teeth were then mounted in Teflon rings with acrylic resin with the occlusal plane facing up. Each specimen was fixed in the lower plate of a universal test machine (858 Mini Bionix II, MTS, MN, USA) and loaded until fracture with a stainless-steel hemispherical loading head (6 mm in diameter). Loading was applied in a stroke-control mode at a cross-head speed of 0.1 mm min −1 , and the maximum load achieved was taken to be the fracture load. The test specimens then were scanned with micro-CT to identify the fracture mode. Scanning parameters were: 90 kV, 90 μA, 720 projections and 2 frames per projection.
2.8
Statistical analysis
The mean fracture loads from the different groups were compared using two-way analysis of variance (ANOVA) with biofilm growth condition (Control, BNS, BWS) and fatigue (Fatigue, No Fatigue) as the between-group factors. Multiple comparisons among the different conditions were conducted with Bonferroni post hoc test at alpha level of 0.05.
2
Materials and methods
2.1
Specimen preparation
De-identified extracted human third molars were collected from oral surgery clinics at the University of Minnesota and in the Minneapolis/Saint Paul metropolitan area. Only teeth free of caries, fractures and cracks were included. The molars were cleaned of soft tissues and hard deposits (when present), and stored in 1% thymol solution. Mesio-occlusal-distal (MOD) class II cavities were prepared using a modified flat end tapered burs (SS White Burs Inc., Lakewood, NY, USA). The dimensions of the cavities were ∼2.5 mm wide and ∼2.5 mm deep for the occlusal box, and ∼3 mm wide and ∼1.5 mm deep for the proximal box. All teeth were restored using Adper™ Single Bond Plus adhesive system (SB) and Z100™ Restorative (Z100) system (3M ESPE, St. Paul, MN, USA) ( Table 1 ). Briefly, 35% phosphoric acid (Scotchbond etchant, 3M ESPE, St. Paul, MN, USA) was applied to dentin and enamel for 15 s and rinsed with water. Gentle air-drying was used to avoid collagen collapse in dentin. Two consecutive coatings of SB were rubbed on the internal walls of the preparation and polymerized for 20s. The teeth were restored with 2 mm thick increments of Z100. Each layer was polymerized for 20 s with an Elipar™ S10 curing light (3M ESPE, St. Paul, MN, USA) operated at 1200 mW/cm The samples were polished after 24 h of water storage. A finishing diamond stone was used to remove excess material from the restoration margin before polishing was conducted with Progloss™ One Step Composite Polishers (Kerr Corporation, Orange, CA, USA). A total of 54 MOD class-II composite restorations were made.
| Product | Composition | Batch number |
|---|---|---|
| Z100™ Restorative | Silane treated ceramic, triethylene glycol dimethacrylate (TEGDMA), bisphenol a diglycidyl ether dimethacrylate (BISGMA), 2-benzotriazolyl-4-methylphenol. | N649950 |
| Adper Single Bond Plus | Ethyl alcohol, silane treated silica (nanofiller), bisphenol a diglycidyl ether dimethacrylate (BISGMA), 2-hydroxyethyl methacrylate(HEMA), glycerol 1,3-dimethacrylate, copolymer of acrylic and itaconic acids, water, diurethane dimethacrylate (UDMA) diphenyliodonium hexafluorophosphate, ethyl 4-dimethyl aminobenzoate (EDMAB). | N561025 |
2.2
Fatigue (chewing simulation)
Half of the specimens (n = 27) were mounted in Teflon rings with self-curing acrylic resin (Dentsply Caulk, Milford, DE, USA). Each mounted specimen was positioned in the mandibular chamber of an artificial oral environment (ART), developed by the Minnesota Dental Research Center for Biomaterials and Biomechanics ( Fig. 1 ). A 6 mm diameter steatite bead attached to the upper arm was used as the antagonist. The target loading point was located in the functional cusp of the third molars. The artificial mouth was set to simulate 200,000 chewing cycles with a maximum load of 50N and an excursion of 0.8 mm at the contact point . Throughout the duration of the mechanical challenge the specimens were submerged in deionized water at room temperature. The cyclic load was constantly monitored during the loading process.
