Degradation of dentin-bonded interfaces treated with collagen cross-linking agents in a cariogenic oral environment: An in situstudy

Abstract

Objectives

To evaluate the effect of treatment using collagen cross-linking agents as primer on resin–dentin bond interfaces subjected to cariogenic oral environment (COE).

Methods

Each of forty human teeth had two cavities (4 × 4 × 1.5 mm) prepared within enamel margins. These cavities were acid-etched and treated by the primers containing one of the following treatment agents (6.5% proanthocyanidins, 0.1% riboflavin-UVA activated light, 5% glutaraldehyde or distilled water as a control group). After that the cavities were bonded and restored with resin composite. One restoration for each tooth was tested immediately (IM) and another was included in an intra-oral palatal device that was placed in each mouth of ten adult volunteers for 14 days in COE. After 14 days, the teeth were removed and each restoration was sectioned to obtain a slice for Knoop microhardness (KHN) and resin–dentin bonded sticks for microtensile bond strength (μTBS) and nanoleakage (NL) evaluation. Data were evaluated by two-way ANOVA and Tukey’s tests ( α = 0.05).

Results

After 14 days in a COE, the KHN was reduced for all groups, except for the glutaraldehyde group; however, the proanthocyanidins group retained the highest KHN in IM and after COE ( p < 0.05). The μTBS was not reduced after COE for the proanthocyanidins and glutaraldehyde groups, however only the proanthocyanidins treatment did not increase the NL after COE ( p > 0.05).

Conclusion

The in situ study model seems to be a suitable short-term methodology to investigate the degradation of the bonding interfaces under a more realistic condition. Under COE, the proanthocyanidins and glutaraldehyde treatments produced stable interfaces that are worth further clinical investigation.

Introduction

The hybrid structure formed during the dental bonding procedure occurs by demineralization of the dentin surface, followed by infiltration and subsequent polymerization of monomers around the collagen fibrils . Therefore, to achieve effective and stable bonding, the preservation of dentin collagen is critical, since collagen represents the major organic component of the dentin matrix.

Unfortunately this is not an easy task. During bonding procedures, the demineralized collagen fibrils are not completely infiltrated by resin monomers, and these denuded collagen fibrils are more prone to degradation. Fluctuations of acidity produced by different pHs of foods and drinks as well as that induced by bacterial acids may increase the amount of exposed organic matrix to be broken-down by bacterially derived enzymes. Additionally, host-derived enzymes such as matrix metalloproteinases (MMPs) and cysteine cathepsins present in the dentin matrix and in the gingival crevicular fluid, also play a role on resin–dentin bond degradation.

The role of these host-derived proteases in the breakdown of the collagen matrices during the pathogenesis of dentin caries , periodontal disease and degradation of resin–dentin bonded interfaces has already been demonstrated . Measures that enhance dentin resistance against collagenolytic activities have great potential for improving the longevity of the dentin bonding. In this context, collagen cross-linking agents have been investigated as dentin biomodifiers.

These crosslinking agents can interact with various extracellular matrix components inducing increases in the mechanical properties of the tissue, decreasing the biodegradation rates and possibly inducing mineral nucleation , which make them a promising solution for preservation of resin–dentin bonded interfaces .

However, most of the studies that support the benefits of cross-linking agents are performed in laboratories, where the challenging conditions of the oral environment are barely reproduced. Although randomized clinical studies are the best study design to evaluate both the performance and longevity of restorative materials, they are time demanding, costly and dependent on the approval by a local Ethics Committee. Under this scenario, the conduction of in situ studies may gather important information to the field, as it resembles the challenging clinical conditions that resin–dentin interfaces are prone better than in vitro studies. In situ studies may be considered as an intermediate stage between in vitro and clinical studies. Therefore, the aim of this study was to investigate the degradation of resin–dentin interfaces treated by different collagen cross-linking agents after in situ cariogenic challenge, using microhardness, microtensile bond strength and nanoleakage. The test null hypotheses were that after 14 days of exposure to an intra oral cariogenic environment, the Knoop hardness, μTBS and nanoleakage of resin–dentin interfaces treated by different collagen cross-linking agents did not change.

Material and methods

The study protocol was approved by the Local Ethics Committee Review Board under protocol number 314.563. Ten healthy adult volunteers (aged 21–30 years, female and male) were selected according to the following inclusion criteria: good general and oral health and normal salivary flow rate. Participants that took antibiotics for the last 2 months before the experiment or wearing prosthesis or orthodontic devices were not included in this study. All volunteers agreed to participate and signed an informed written consent.

A total of forty extracted, non-erupted human third molars were used. The teeth were collected after obtaining the patients’ informed consent under a protocol approved by the previously described Ethics Committee previously described. Teeth free from cracks or any other kinds of structural defects were selected. The teeth were disinfected by storage in 10% buffered formalin solution, pH 7, for 7 days and stored in distilled water for up to 2 months after extraction.

Experimental design

This in situ , split-mouth study was designed for accumulation of a plaque-like biofilm on the restorations in a high cariogenic challenge promoted by sucrose exposure. This protocol was performed for 14 days. The factors under evaluation were: (1) three different collagen cross-linking agents (proanthocyanidins from grape seed extract, UVA-activated riboflavin, glutaraldehyde and distilled water as control); and (2) evaluation time—2 levels (immediate and 14 days after degradation in a cariogenic oral environment). Then, a total of eight experimental conditions were tested.

Teeth preparation and bonding procedures

Microtensile bond strength (μTBS), microhardness and nanoleakage evaluation

Flat superficial enamel surfaces were exposed on each tooth after wet grinding the occlusal, buccal and lingual enamel on # 180-grit SiC paper. On each tooth, two dental blocks (6 × 6 × 3 mm) were obtained from the buccal and lingual surfaces. In each dental block, a standardized rectangular cavity was prepared (4 mm wide, 4 mm long, and 1.5 mm deep) with a carbide bur (# 330, KG Sorensen Ind. & Com. Ltda, Barueri, SP, Brazil), so that the axial wall was located in dentin and the thickness of enamel border ranged from 0.3 to 0.5 mm ( Fig. 1 ). Teeth were then randomized by lottery and distributed within the different levels of the cross-linking agents ( n = 10 specimens per group), so that the two cavities from the same tooth could be evaluated at 0 day and after 14 days in a paired design to reduce the intra-tooth variability.

Fig. 1
Representation of experimental design used in this study. CT: control group; PA: proanthocyanidin group; RB: riboflavina group and GA: glutaraldehyde group.

All cavities were etched with 35% phosphoric acid gel for 15 s, water rinsed (30 s), air-dried (5 s) and kept slightly moist (details in Table 1 ). After that, the different collagen cross-linking primers ( Table 1 ) were applied for 60 s under agitation. The riboflavin group was irradiated with UVA-light for 2 min (Philips, Hamburg, Germany; λ = 370 nm at 3 mW/cm 2 ) after the priming step with the cross-linking agent . Following, the adhesive system was applied according to the manufacturer’s instructions ( Table 1 ) and the cavities were incrementally filled with a resin composite (Z250, 3M ESPE, Shade A3, batch number N549511) and with each increment light cured for 40 s. The light curing procedures were performed using a LED light curing unit (Radii Cal, SDI, Bayswater, Victoria, Australia) set at 1200 mW/cm 2 . One of the cavity restorations from each tooth was tested immediately while the other one was placed in a palatal appliance for in situ challenge.

Table 1
Description of products, composition and application mode.
Product (Company) Composition Application mode
Scotchbond etchant
(3 M ESPE, St. Paul, USA)
batch number
N261433
Phosphoric acid 35%, water and poly (vinyl alcohol). Application on dentin surface. Wait 15 s. Rinsing for 10 s. Blot excess water using a cotton pellet
Single Bond Plus
(3 M ESPE, St. Paul, USA)
batch number N531785
Ethyl alcohol, BisGMA, silane treated silica (nanofiller), HEMA, copolymer of acrylic and itaconic acids, glycerol 1,3-dimethacrylate, water, UDMA, diphenyliodonium hexafluorophosphate, EDMAB. After treatment according the experimental groups, application 2 consecutive coats of adhesive for 15 s with gentle agitation using a fully saturated applicator. Gently air thin for 5 s to evaporate solvent. Light-cure for 10 s
Proanthocyanidin (PA) primer
(Mega Natural Gold, Madera, USA)
Batch number 05592502-01
Proanthocyanidin-Grape seed extract 6.5% weight, deionized water. After acid etching step, application for 60 s with gentle agitation using a fully satured applicator. Gently air-drier for 5 s and kept slightly moist the surface
Riboflavin (RB) primer
(Fisher Scientific GmbH, Schwerte, Germany)
Batch number 070046
Riboflavin 0.1% weight, deionized water. After acid etching step, application for 60 s with gentle agitation using a fully satured applicator. After that, irradiation using UVA-light for 2 min (Philips, Hamburg, Germany; λ = 370 nm at 3 mW/cm 2 ). Gently air-drier for 5 s and kept slightly moist the surface
Glutaraldehyde (GA) primer
(Fisher Scientific GmbH, Schwerte, Germany)
Batch number 186852
Glutaraldehyde 5% weight, deionized water. After acid etching step, application for 60 s with gentle agitation using a fully satured applicator. Gently air-drier for 5 s and kept slightly moist the surface
Control Group (CT) Distilled water After acid etching step, application for 60 s with gentle agitation using a fully satured applicator. Gently air-drier for 5 s and kept slightly moist the surface
BisGMA: bisphenol a diglycidyl ether dimethacrylate, HEMA: 2-hydroxyethyl methacrylate. UDMA: diurethane dimethacrylate, EDMAB: ethyl 4-dimethyl aminobenzoate.

Palatal device preparation

For each volunteer, acrylic custom-made palatal devices were made with four sites (6.5 × 6.5 × 4 mm) in which the dental blocks from each group were positioned and fixed with wax ( Fig. 1 ). To allow for plaque accumulation and for protection from mechanical disturbance, a plastic mesh was fixed to the acrylic resin, leaving a 1 mm space from the surface of the specimen . Within each side of the palatal device, the positions of the specimens were randomly determined by lottery.

Intra-oral phase

During a 1-week lead-in period, and throughout the entire experimental phase, the volunteers brushed their teeth with a non-fluoride silica-based dentifrice formulation (Fleming, Ponta Grossa, PR, Brazil) prepared for this study. To provide a cariogenic challenge in all four specimens, each of the volunteers was instructed to remove the device and drip in a 20% sucrose solution (Fleming) onto all blocks four times a day (8 and 11 am and 3:30 and 7 pm) during 14 days . Five minutes later, the device was re-inserted in the mouth.

All volunteers consumed city fluoridated water (0.6–0.8 mg F/l) and foods prepared with it. No restriction was made with regard to diet of the volunteers. They were instructed to wear the intraoral devices the whole time for 14 consecutive days, removing them only for dental hygiene and during the meals. The appliances were extra-orally brushed, except the restorations, and the volunteers were asked to brush carefully over the palatal area, to avoid disturbing the biofilm covering the mesh. They were asked to brush their teeth and appliance for up to 5 min. On the 15th day of the oral phase, around the 12 h after the last application of the sucrose solution, the volunteers stopped wearing the intraoral devices. The restorations were then removed from the dental appliances and washed in tap water.

From this point on, both the restorations aged in the oral environment as well as the ones performed for immediate bonding were prepared in a similar manner. Each of the restorations was longitudinally sectioned to obtain a thin slice from the resin–dentin specimen for cross-sectional Knoop microhardness. The remaining of the restoration was used for resin–dentin μTBS and nanoleakage evaluation.

Cross-sectional microhardness

The thin restoration slice was embedded in acrylic resin with the cut surface being exposed, which receiving subsequent flattening and polishing with 1000, 1500, 2000, and 2500-grit SiC papers and 1 and 0.25 μm diamond pastes (Buehler, Lake Bluff, IL, USA) using a polish cloth. After ultrasonic cleaning, cross-sectional microhardness measurements were made in dentin with a microhardness tester (HMV-2, Shimadzu, Tokyo, Japan) equipped with a Knoop indenter (KHN) under a 15 g load for 5 s. Three lines of three indentations on each specimen were made, one line being 20 μm distant from the restoration margin and the other two lines being 100 and 200 μm distant, respectively. The indentations were made at the following depths from the enamel–dentin junction: 5, 15, and 25 μm .

Microtensile bond strength evaluation

The remaining part of the restoration was longitudinally sectioned in both “ x ” and “ y ” directions across the bonded interface with a diamond saw. This procedure was performed to obtain resin–dentin sticks with a cross-sectional area of approximately 0.8 mm 2 . The cross-sectional area of each stick was measured with a digital caliper (Absolute Digimatic, Mitutoyo, Tokyo, Japan) to the nearest 0.01 mm. Each bonded stick was attached to a jig for microtensile testing with cyanoacrylate resin (Super Bonder Gel, Loctite, São Paulo, Brazil) and subjected to a tensile force in a universal testing machine (Model 5565, Instron, Canton, OH, USA) at a crosshead speed of 0.5 mm/min. The failure modes were evaluated under stereomicroscopy at 100× magnification and classified as cohesive (within dentin or resin composite), adhesive (failure at resin/dentin interface), or adhesive/mixed (failure at resin/dentin interface with partial cohesive failure of the neighboring substrates). Approximately 15–20 resin-bonded sticks could be obtained per tooth including the pre-test failures. Usually 13–18 resin-bonded sticks were tested in this method.

Nanoleakage evaluation

Two resin-bonded sticks from each restoration, not used for microtensile testing, were randomly selected for nanoleakage evaluation. The sticks were immersed in 50 wt% ammoniacal silver nitrate solution in total darkness for 24 h. Thereafter, they were rinsed thoroughly in distilled water, and immersed in a photo-developing solution for 8 h under fluorescent light to reduce silver ions into metallic silver grains within voids along the bonded interface. Specimens were polished using 1000-, 1500-, 2000- and 2500-grit SiC papers and 1 and 0.25 μm diamond pastes (Buehler Ltd., Lake Bluff, IL, USA) on polishing clothes. They were ultrasonically cleaned, air-dried, mounted on stubs and coated with evaporated carbon (MED 010, Balzers Union, Balzers, Liechtenstein).

The interfaces were observed in a scanning electron microscope (SEM) in the backscattered mode at 12 kV (VEGA 3 TESCAM, Shimadzu, Tokyo, Japan). Three images were taken from each specimen. The first image was obtained in the center of the stick, while the further two were obtained 0.3 mm left and 0.3 mm right from the first picture. A total of six images were obtained per tooth at each period (3 images × 2 resin-bonded sticks). Thus, for each experimental condition, 60 images were evaluated per group (6 images × 10 teeth) . A blinded author to the experimental conditions took the pictures. The relative percentage of silver nitrate uptake within the hybrid layer was measured in all pictures using the ImageTool 3.0 software (Department of Dental Diagnostic Science, University of Texas Health Science Center, San Antonio, USA).

Statistical analysis

The microhardness (KHN), μTBS (MPa) and nanoleakage (%) data from the same experimental unit (tooth) were averaged for statistical purposes at each storage time interval. The resin-bonded sticks with premature and cohesive failures were not included in the tooth mean due to their low frequency of occurrence in this experiment.

The Kolmogorov–Smirnov test was employed to assess whether the data from each test (microhardness, μTBS and nanoleakage) followed a normal distribution. Barlett’s test was performed to determine if the assumption of equal variances was valid. After observing the data normality and equality of the variances, the data from microhardness (KHN), μTBS (MPa) and nanoleakage (%) were subjected to two-way repeated measures ANOVA (cross-linking agents and evaluation time) and Tukey’s test for pair wise comparisons ( α = 0.05).

Material and methods

The study protocol was approved by the Local Ethics Committee Review Board under protocol number 314.563. Ten healthy adult volunteers (aged 21–30 years, female and male) were selected according to the following inclusion criteria: good general and oral health and normal salivary flow rate. Participants that took antibiotics for the last 2 months before the experiment or wearing prosthesis or orthodontic devices were not included in this study. All volunteers agreed to participate and signed an informed written consent.

A total of forty extracted, non-erupted human third molars were used. The teeth were collected after obtaining the patients’ informed consent under a protocol approved by the previously described Ethics Committee previously described. Teeth free from cracks or any other kinds of structural defects were selected. The teeth were disinfected by storage in 10% buffered formalin solution, pH 7, for 7 days and stored in distilled water for up to 2 months after extraction.

Experimental design

This in situ , split-mouth study was designed for accumulation of a plaque-like biofilm on the restorations in a high cariogenic challenge promoted by sucrose exposure. This protocol was performed for 14 days. The factors under evaluation were: (1) three different collagen cross-linking agents (proanthocyanidins from grape seed extract, UVA-activated riboflavin, glutaraldehyde and distilled water as control); and (2) evaluation time—2 levels (immediate and 14 days after degradation in a cariogenic oral environment). Then, a total of eight experimental conditions were tested.

Teeth preparation and bonding procedures

Microtensile bond strength (μTBS), microhardness and nanoleakage evaluation

Flat superficial enamel surfaces were exposed on each tooth after wet grinding the occlusal, buccal and lingual enamel on # 180-grit SiC paper. On each tooth, two dental blocks (6 × 6 × 3 mm) were obtained from the buccal and lingual surfaces. In each dental block, a standardized rectangular cavity was prepared (4 mm wide, 4 mm long, and 1.5 mm deep) with a carbide bur (# 330, KG Sorensen Ind. & Com. Ltda, Barueri, SP, Brazil), so that the axial wall was located in dentin and the thickness of enamel border ranged from 0.3 to 0.5 mm ( Fig. 1 ). Teeth were then randomized by lottery and distributed within the different levels of the cross-linking agents ( n = 10 specimens per group), so that the two cavities from the same tooth could be evaluated at 0 day and after 14 days in a paired design to reduce the intra-tooth variability.

Fig. 1
Representation of experimental design used in this study. CT: control group; PA: proanthocyanidin group; RB: riboflavina group and GA: glutaraldehyde group.

All cavities were etched with 35% phosphoric acid gel for 15 s, water rinsed (30 s), air-dried (5 s) and kept slightly moist (details in Table 1 ). After that, the different collagen cross-linking primers ( Table 1 ) were applied for 60 s under agitation. The riboflavin group was irradiated with UVA-light for 2 min (Philips, Hamburg, Germany; λ = 370 nm at 3 mW/cm 2 ) after the priming step with the cross-linking agent . Following, the adhesive system was applied according to the manufacturer’s instructions ( Table 1 ) and the cavities were incrementally filled with a resin composite (Z250, 3M ESPE, Shade A3, batch number N549511) and with each increment light cured for 40 s. The light curing procedures were performed using a LED light curing unit (Radii Cal, SDI, Bayswater, Victoria, Australia) set at 1200 mW/cm 2 . One of the cavity restorations from each tooth was tested immediately while the other one was placed in a palatal appliance for in situ challenge.

Table 1
Description of products, composition and application mode.
Product (Company) Composition Application mode
Scotchbond etchant
(3 M ESPE, St. Paul, USA)
batch number
N261433
Phosphoric acid 35%, water and poly (vinyl alcohol). Application on dentin surface. Wait 15 s. Rinsing for 10 s. Blot excess water using a cotton pellet
Single Bond Plus
(3 M ESPE, St. Paul, USA)
batch number N531785
Ethyl alcohol, BisGMA, silane treated silica (nanofiller), HEMA, copolymer of acrylic and itaconic acids, glycerol 1,3-dimethacrylate, water, UDMA, diphenyliodonium hexafluorophosphate, EDMAB. After treatment according the experimental groups, application 2 consecutive coats of adhesive for 15 s with gentle agitation using a fully saturated applicator. Gently air thin for 5 s to evaporate solvent. Light-cure for 10 s
Proanthocyanidin (PA) primer
(Mega Natural Gold, Madera, USA)
Batch number 05592502-01
Proanthocyanidin-Grape seed extract 6.5% weight, deionized water. After acid etching step, application for 60 s with gentle agitation using a fully satured applicator. Gently air-drier for 5 s and kept slightly moist the surface
Riboflavin (RB) primer
(Fisher Scientific GmbH, Schwerte, Germany)
Batch number 070046
Riboflavin 0.1% weight, deionized water. After acid etching step, application for 60 s with gentle agitation using a fully satured applicator. After that, irradiation using UVA-light for 2 min (Philips, Hamburg, Germany; λ = 370 nm at 3 mW/cm 2 ). Gently air-drier for 5 s and kept slightly moist the surface
Glutaraldehyde (GA) primer
(Fisher Scientific GmbH, Schwerte, Germany)
Batch number 186852
Glutaraldehyde 5% weight, deionized water. After acid etching step, application for 60 s with gentle agitation using a fully satured applicator. Gently air-drier for 5 s and kept slightly moist the surface
Control Group (CT) Distilled water After acid etching step, application for 60 s with gentle agitation using a fully satured applicator. Gently air-drier for 5 s and kept slightly moist the surface
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Jun 19, 2018 | Posted by in General Dentistry | Comments Off on Degradation of dentin-bonded interfaces treated with collagen cross-linking agents in a cariogenic oral environment: An in situstudy
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