Collagen cross-linkers on dentin bonding: Stability of the adhesive interfaces, degree of conversion of the adhesive, cytotoxicity and in situ MMP inhibition

Abstract

Objective

To investigate the effect of collagen cross-links on the stability of adhesive properties, the degree of conversion within the hybrid layer, cytotoxicity and the inhibition potential of the MMPs’ activity.

Methods

The dentin surfaces of human molars were acid-etched and treated with primers containing: 6.5 wt% proanthocyanidin, UVA-activated 0.1 wt% riboflavin, 5 wt% glutaraldehyde and distilled water for 60 s. Following, dentin was bonded with Adper Single Bond Plus and Tetric N-Bond; and restored with resin composite. The samples were sectioned into resin–dentin “sticks” and tested for microtensile bond strength (μTBS) after immediate (IM) and 18-month (18 M) periods. Bonded sticks at each period were used to evaluate nanoleakage and the degree of conversion (DC) under micro-Raman spectroscopy. The enzimatic activity (P1L10 cross-linkers, P1L22 MMPs’ activities) in the hybrid layer was evaluated under confocal microscopy. The culture cell (NIH 3T3 fibroblast cell line) and MTT assay were performed to transdentinal cytotoxicity evaluation. Data from all tests were submitted to appropriate statistical analysis ( α = 0.05).

Results

All cross-linking primers reduced the degradation of μTBS compared with the control group after 18 M ( p > 0.05). The DC was not affected ( p > 0.213). The NL increased after 18 M for all experimental groups, except for proanthocyanidin with Single Bond Plus ( p > 0.05). All of the cross-link agents reduced the MMPs’ activity, although this inhibition was more pronounced by PA. The cytotoxicity assay revealed reduced cell viability only for glutaraldehyde ( p < 0.001).

Significance

Cross-linking primers used in clinically relevant minimized the time degradation of the μTBS without jeopardizing the adhesive polymerization, as well as reduced the collagenolytic activity of MMPs. Glutaraldeyde reduced cell viability significantly and should be avoided for clinical use.

Introduction

The longevity of hybrid layers depends upon the stability of their components, such as collagen fibrils and polymeric chains . However, collagen fibrils are not completely infiltrated by resin monomers when exposed by acid etching , thereby impeding optimal protection against denaturation challenges. Unprotected collagen is more prone to creep and cyclic fatigue rupture after prolonged function. Additionally, these resin-sparse collagen fibrils are also filled with and surrounded by water, which participates in the hydrolysis of resin matrices by esterases and collagen by collagenolytic enzymes .

Increasing the collagen’s resistance against the degradation process may improve the stability of the resin–dentin bonded interface; this was the main purpose of incorporating collagen cross-linkers into the bonding process . Collagen cross-linkers are effective in protecting collagen fibrils from degradation through enhancing the collagen’s chemical and mechanical properties . More recently, their benefits in dentin bonding have been credited to their ability to inhibit the activity of host-derived metalloproteinases . However, literature showing direct evidence of the activity of endogenous dentin MMPs within the hybrid layer after treatment by cross-linkers agents is still scarce.

On the other hand, collagen degradation is only one part of the biodegradation process, and it is not clear how collagen cross-linkers can affect the adhesive properties of the hybrid layer. Collagen cross-linkers showed an inhibitory polymerization effect on dimethacrylates that may impair the achievement of an adequate degree of conversion inside the hybrid layer and jeopardize the bonding effectiveness . Incomplete polymerization of the adhesive monomers has been suggested as one reason for nanoleakage due to the formation of a porous hybridoid structure with reduced sealing ability . All of these factors can also affect the cytotoxicity of an adhesive interface .

Although several collagen cross-linkers, such as glutaraldehyde and proanthocyanidins, have shown therapeutic effects on dentin collagen . This requires their use for prolonged application times (10 min to 4 h), which restricts their clinical applicability. The comparison of different, recently accredited collagen cross-linkers, applied at clinically relevant times, might be useful to select the most effective agent in preventing collagen degradation, while inducing low cytotoxicity, with low polymerization inhibition yet providing stable resin–dentin bond interfaces over time.

Thus, the aim of this study was to evaluate the transdentinal cytotoxicity, the stability of the resin-dentin interfaces by microtensile and nanoleakage tests, the degree of conversion for the adhesive by in situ micro-Raman spectroscopy and the collagenolytic activity of the adhesive interface using in situ zymography, both with and without the incorporation of cross-linkers into the dentin bonding protocol.

Materials and methods

Tooth preparation and experimental design

Seventy-six extracted, caries-free human third molars were used after approval of the Institutional Ethics Committee from the State University of Ponta Grossa, Paraná, Brazil (protocol 314.563). The teeth were stored in 0.5% chloramine solution and used within two months after extraction. A flat dentin surface was exposed after wet-grinding the occlusal enamel using 180-grit SiC paper and 600-grit SiC paper for 60 s.

The dentin of forty teeth was etched for 15 s with 35% phosphoric acid gel (Scotchbond etchant, 3M ESPE, St. Paul, USA, batch number N261433), rinsed with water (30 s), air-dried (5 s) and kept slightly moist. The specimens were then randomly allocated to eight groups according to the combination of the main factors: (1) collagen cross-linking treatment (6.5 wt% proanthocyanidin, ultra-violet activated-0.1 wt% riboflavin, 5 wt% glutaraldehyde and distilled water [control group]) and (2) two etch-and-rinse adhesive systems (Adper Single Bond Plus [SB] and Tetric N-Bond [TN], as detailed in Table 1 ). A total of five teeth were employed per experimental group.

Table 1
Description of all experimental groups, composition and application mode.
Product (company) Composition Application mode
Proanthocyanidin (PA) primer
(Mega Natural Gold, Madera, USA)
Batch number 05592502-01
Proanthocyanidin-grape seed extract 6.5% weight, 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
Riboflavin (RB) primer
(Fisher Scientific GmbH, Schwerte, Germany)
Batch number 070046
Riboflavin 0.1% weight, distilled 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. 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, 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
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
Single Bond Plus (SB)
(3M 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
Tetric N-Bond (TN)
(Ivoclar Vivadent AG,Schaan, Liechtenstein)
Batch number L50568
Phosphonic acid acrylate, HEMA, BisGMA, UDMA, ethanol, nanofiller, catalysts and stabilizer 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
BisGMA : bisphenol a diglycidyl ether dimethacrylate; HEMA : 2-hydroxyethyl methacrylate; UDMA : diurethane dimethacrylate; EDMAB : ethyl 4-dimethyl aminobenzoate.

The acid-etched dentin surfaces were primed according to the experimental groups ( Table 1 ). For the riboflavin (RB), the dentin surfaces were further exposed to ultraviolet light for 2 min with a UV lamp (Philips, Hamburg, Germany; λ = 370 nm at 3 mW/cm 2 ) before air-drying . The light-curing steps were performed using an LED (Radii Cal, SDI, Bayswater, Victoria, Australia; 1200 mW/cm 2 ). Resin composite build-ups (Z250, 3M ESPE, Shade A3, batch number N549511) were incrementally constructed, and each portion was light-cured (40 s). The bonded teeth were then stored for 24 h in distilled water at 37 °C.

Then, the specimens were longitudinally sectioned in both the mesiodistal and buccolingual directions across the bonded interface in a cutting machine (Buehler, Lake Bluff, USA), to obtain resin–dentin sticks (1 mm 2 ). The number of premature failures per tooth during specimen preparation was recorded. The cross-sectional area of each stick was measured with a digital caliper (Absolute Digimatic, Mitutoyo, Tokyo, Japan) to the nearest 0.01 mm.

Resin–dentin microtensile bond strength (μTBS)

For this test, 40 teeth ( n = 5 teeth per group) previously restored were used. 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 (Kratos, São Paulo, SP, Brazil) at 0.5 mm/min. The failure modes were evaluated under stereomicroscopy at 40× magnification and classified as cohesive adhesive or adhesive/mixed.

Nanoleakage evaluation (NL)

Two resin-bonded sticks from each tooth at each storage period (not tested in μTBS) were randomly selected. The specimens were immersed in ammoniacal 50 wt% silver nitrate solution in darkness for 24 h. Then, they were rinsed thoroughly in distilled water and photo-developed (8 h) under fluorescent light to reduce the silver ions into metallic silver grains. The specimens were polished down until 2500-grit SiC paper and 1 and 0.25 μm diamond paste (Buehler Ltd., Lake Bluff, IL, USA). They were ultrasonically cleaned, air-dried, mounted on stubs and coated with 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 of each specimen: the first image was in the center of the stick, while the next two were obtained 0.3 mm left and right from the first picture, respectively .

A total of six images were obtained per tooth at each period (3 images × 2 bonded sticks). A total of 30 images were obtained per group (6 images × 5 teeth) by a blinded author. We measured the relative percentages of NL within the adhesive and hybrid layers with the ImageTool 3.0 software (University of Texas Health Science Center, San Antonio, USA), as described earlier .

In situ degree of conversion (DC) within adhesive/hybrid layers

Two resin–dentin sticks were randomly selected from the immediate period and used to evaluate the DC immediately after sectioning. The sticks were wet polished using 1500- and 2000-grit SiC paper. The specimens were ultrasonically cleaned for 10 min and positioned into micro-Raman equipment (Senterra spectrophotometer Bruker, Ettlingen, Baden-Württemberg, Germany), which was first calibrated for zero and then for coefficient values using a silicon sample. The samples were analyzed using a 20 mW Neon laser with 532 nm wavelength, spatial resolution of 3 μm, spectral resolution 5 cm −1 , accumulation time of 30 s with 5 co-additions and 100× magnification (Olympus microscope, London, UK) with a 1-μm beam diameter. Spectra were obtained at the dentin-adhesive interface, at three random sites (per bonded stick) within intertubular-infiltrated dentin. Spectra of uncured adhesives were taken as references. Post-processing of the spectra was performed using the Opus Spectroscopy Software version 6.5. The ratio of double-bond content of monomer to polymer in the adhesive was calculated according to the following formula: DC (%) = (1 − [ R cured/ R uncured]) × 100, where ‘ R ’ is the ratio of aliphatic and aromatic peak intensities at 1639 cm −1 and 1609 cm −1 in cured and uncured adhesives.

In situ zymography

A dye-quenched MMP probe based on gelatin was prepared by means of a fluorescein isothiocyanate (FITC) hypersaturated gelatin. 5 mg of FITC was dissolved in 2 ml 0.1M sodium carbonate/bicarbonate buffer (pH 9.0, Sigma Aldrich, Milwaukee, USA). This reactant was added dropwise to a 1 mg/ml gelatin solution in the dark and was incubated at room temperature for 2 h. The reacted FITC–gelatin conjugate was isolated from unbound FITC by means of a G-25M Sephadex column. The fluorescein-to-protein ratio of >15 was confirmed from absorbance readings at 495 nm and 280 nm, respectively. This MMP-substrate confocal dye was dissolved (0.3 wt%) in distilled water and actively applied for 60 s onto the phosphoric acid-etched dentin before the bonding agents and cross-linking primers were applied.

Three teeth per group ( n = 3) were bonded as previously described and cut into resin–dentin slabs; their interfaces were observed by confocal laser scanning microscopy (CLSM), similarly to a previous study . The specimens were examined using a CLSM (Leica SP5 CLSM, Heidelberg, Germany) equipped with a 63×/1.4 NA oil immersion lens using 468-nm laser illumination. The z -stack scans (one at each micrometer up to 20 μm below the surface) were compiled into single projections. Each resin–dentin interface was entirely characterized, and images representing the MMP-activity observed along the bonded interfaces were captured.

Cytotoxicity evaluation: cell culture and MTT assay

Twenty-four teeth were used in this test. Dentin disks with a thickness of 0.6 mm were obtained from the mid-coronal dentin of each tooth using a cutting machine (Isomet 1000, Buehler, Lake Bluff, USA). The disks were carefully examined with a stereomicroscope at 40× to confirm the absence of enamel and defects resulting from pulp horn projections. Then, the occlusal sides of the disks were manually finished with wet 320-grit silicon carbide paper to reach a final thickness of 0.5 mm (Mitutoyo South Americana Ltd., Suzano, SP, Brazil). Afterwards, a smear layer was produced with 600-grit SiC on both sides of the disks and immediately removed by 0.5 M EDTA (pH 7.4) for 60 s. After abundant rinsing with deionized water, the dentin’s permeability was measured through a hydraulic conductance device to permit a homogenous distribution of the dentin disks among the experimental groups ( n = 6 disks per group). The dentin disks were positioned in metallic devices and autoclaved (20 min/121° C). The occlusal surfaces of the dentin disks were etched with 35% phosphoric acid (15 s), carefully rinsed with deionized water (10 s) and blot dried with sterile cotton pellets.

The NIH/3T3 fibroblast cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and 1% penicillin/streptomycin (10,000 U/100 μg/ml) at 37 °C with 5% CO 2 in a humidified atmosphere. Briefly, 4 × 10 4 cells were added to each well of a 24-well plate. After 24 h, the medium was removed; the cells were washed twice with phosphate buffer solution (PBS). DMEM containing 1% fetal bovine serum and trans-well chambers were added into each well. The dentin disks were carefully added, and 10 μl of each cross-linking primer was applied onto the occlusal surface. For the riboflavin group, the UVA-light was irradiated by a lamp, as previously described, using a 10.4 mm tip that completely covered the culture well (24-well format). This protocol followed Bouillaguet et al. to prevent modifications to the cell mitochondrial activity.

The trans-well was removed and the cells were washed with phosphate-buffered saline (PBS) 24 h later. MTT cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co., St. Louis, USA) according to the method of Tada et al. with some modifications. MTT solution was added (1.0 mg/ml), and cell viability was then assessed in a colorimetric assay using mitochondrial dehydrogenase activity in active mitochondria to form purple formazan. The absorbance of each well was read at 570 nm using a plate reader (EL808B, BioTech Instruments Inc., Winooski, VT, USA). Cell viability was expressed as the percentage of optical values in the treated samples versus the concurrent control (no treatment), considered as 100%. All of the experiments were performed in triplicate.

Statistical analysis

The μTBS (MPa) and NL (%) from the same experimental unit were averaged for statistical purposes at each storage time. The bonded sticks with premature and cohesive failures were not included in the tooth mean, due to their low frequency in the experiment.

The Kolmogorov–Smirnov test was employed to assess whether the data from each test (μTBS, NL, DC and cytotoxicity) followed a normal distribution. Barlett’s test was performed to determine if the assumption of equal variances was valid. After observing the data’s normality and equality of the variances, the data from the μTBS (MPa) and NL (%) of each adhesive were subjected to a two-way repeated measure ANOVA (on solutions and storage time). The data from the DC (%) and cytotoxicity (%) of each adhesive were subjected to a one-way ANOVA (on solutions). For all of the test Tukey’s test was used for pairwise comparisons ( α = 0.05).

Materials and methods

Tooth preparation and experimental design

Seventy-six extracted, caries-free human third molars were used after approval of the Institutional Ethics Committee from the State University of Ponta Grossa, Paraná, Brazil (protocol 314.563). The teeth were stored in 0.5% chloramine solution and used within two months after extraction. A flat dentin surface was exposed after wet-grinding the occlusal enamel using 180-grit SiC paper and 600-grit SiC paper for 60 s.

The dentin of forty teeth was etched for 15 s with 35% phosphoric acid gel (Scotchbond etchant, 3M ESPE, St. Paul, USA, batch number N261433), rinsed with water (30 s), air-dried (5 s) and kept slightly moist. The specimens were then randomly allocated to eight groups according to the combination of the main factors: (1) collagen cross-linking treatment (6.5 wt% proanthocyanidin, ultra-violet activated-0.1 wt% riboflavin, 5 wt% glutaraldehyde and distilled water [control group]) and (2) two etch-and-rinse adhesive systems (Adper Single Bond Plus [SB] and Tetric N-Bond [TN], as detailed in Table 1 ). A total of five teeth were employed per experimental group.

Table 1
Description of all experimental groups, composition and application mode.
Product (company) Composition Application mode
Proanthocyanidin (PA) primer
(Mega Natural Gold, Madera, USA)
Batch number 05592502-01
Proanthocyanidin-grape seed extract 6.5% weight, 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
Riboflavin (RB) primer
(Fisher Scientific GmbH, Schwerte, Germany)
Batch number 070046
Riboflavin 0.1% weight, distilled 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. 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, 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
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
Single Bond Plus (SB)
(3M 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
Tetric N-Bond (TN)
(Ivoclar Vivadent AG,Schaan, Liechtenstein)
Batch number L50568
Phosphonic acid acrylate, HEMA, BisGMA, UDMA, ethanol, nanofiller, catalysts and stabilizer 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
BisGMA : bisphenol a diglycidyl ether dimethacrylate; HEMA : 2-hydroxyethyl methacrylate; UDMA : diurethane dimethacrylate; EDMAB : ethyl 4-dimethyl aminobenzoate.

The acid-etched dentin surfaces were primed according to the experimental groups ( Table 1 ). For the riboflavin (RB), the dentin surfaces were further exposed to ultraviolet light for 2 min with a UV lamp (Philips, Hamburg, Germany; λ = 370 nm at 3 mW/cm 2 ) before air-drying . The light-curing steps were performed using an LED (Radii Cal, SDI, Bayswater, Victoria, Australia; 1200 mW/cm 2 ). Resin composite build-ups (Z250, 3M ESPE, Shade A3, batch number N549511) were incrementally constructed, and each portion was light-cured (40 s). The bonded teeth were then stored for 24 h in distilled water at 37 °C.

Then, the specimens were longitudinally sectioned in both the mesiodistal and buccolingual directions across the bonded interface in a cutting machine (Buehler, Lake Bluff, USA), to obtain resin–dentin sticks (1 mm 2 ). The number of premature failures per tooth during specimen preparation was recorded. The cross-sectional area of each stick was measured with a digital caliper (Absolute Digimatic, Mitutoyo, Tokyo, Japan) to the nearest 0.01 mm.

Resin–dentin microtensile bond strength (μTBS)

For this test, 40 teeth ( n = 5 teeth per group) previously restored were used. 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 (Kratos, São Paulo, SP, Brazil) at 0.5 mm/min. The failure modes were evaluated under stereomicroscopy at 40× magnification and classified as cohesive adhesive or adhesive/mixed.

Nanoleakage evaluation (NL)

Two resin-bonded sticks from each tooth at each storage period (not tested in μTBS) were randomly selected. The specimens were immersed in ammoniacal 50 wt% silver nitrate solution in darkness for 24 h. Then, they were rinsed thoroughly in distilled water and photo-developed (8 h) under fluorescent light to reduce the silver ions into metallic silver grains. The specimens were polished down until 2500-grit SiC paper and 1 and 0.25 μm diamond paste (Buehler Ltd., Lake Bluff, IL, USA). They were ultrasonically cleaned, air-dried, mounted on stubs and coated with 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 of each specimen: the first image was in the center of the stick, while the next two were obtained 0.3 mm left and right from the first picture, respectively .

A total of six images were obtained per tooth at each period (3 images × 2 bonded sticks). A total of 30 images were obtained per group (6 images × 5 teeth) by a blinded author. We measured the relative percentages of NL within the adhesive and hybrid layers with the ImageTool 3.0 software (University of Texas Health Science Center, San Antonio, USA), as described earlier .

In situ degree of conversion (DC) within adhesive/hybrid layers

Two resin–dentin sticks were randomly selected from the immediate period and used to evaluate the DC immediately after sectioning. The sticks were wet polished using 1500- and 2000-grit SiC paper. The specimens were ultrasonically cleaned for 10 min and positioned into micro-Raman equipment (Senterra spectrophotometer Bruker, Ettlingen, Baden-Württemberg, Germany), which was first calibrated for zero and then for coefficient values using a silicon sample. The samples were analyzed using a 20 mW Neon laser with 532 nm wavelength, spatial resolution of 3 μm, spectral resolution 5 cm −1 , accumulation time of 30 s with 5 co-additions and 100× magnification (Olympus microscope, London, UK) with a 1-μm beam diameter. Spectra were obtained at the dentin-adhesive interface, at three random sites (per bonded stick) within intertubular-infiltrated dentin. Spectra of uncured adhesives were taken as references. Post-processing of the spectra was performed using the Opus Spectroscopy Software version 6.5. The ratio of double-bond content of monomer to polymer in the adhesive was calculated according to the following formula: DC (%) = (1 − [ R cured/ R uncured]) × 100, where ‘ R ’ is the ratio of aliphatic and aromatic peak intensities at 1639 cm −1 and 1609 cm −1 in cured and uncured adhesives.

In situ zymography

A dye-quenched MMP probe based on gelatin was prepared by means of a fluorescein isothiocyanate (FITC) hypersaturated gelatin. 5 mg of FITC was dissolved in 2 ml 0.1M sodium carbonate/bicarbonate buffer (pH 9.0, Sigma Aldrich, Milwaukee, USA). This reactant was added dropwise to a 1 mg/ml gelatin solution in the dark and was incubated at room temperature for 2 h. The reacted FITC–gelatin conjugate was isolated from unbound FITC by means of a G-25M Sephadex column. The fluorescein-to-protein ratio of >15 was confirmed from absorbance readings at 495 nm and 280 nm, respectively. This MMP-substrate confocal dye was dissolved (0.3 wt%) in distilled water and actively applied for 60 s onto the phosphoric acid-etched dentin before the bonding agents and cross-linking primers were applied.

Three teeth per group ( n = 3) were bonded as previously described and cut into resin–dentin slabs; their interfaces were observed by confocal laser scanning microscopy (CLSM), similarly to a previous study . The specimens were examined using a CLSM (Leica SP5 CLSM, Heidelberg, Germany) equipped with a 63×/1.4 NA oil immersion lens using 468-nm laser illumination. The z -stack scans (one at each micrometer up to 20 μm below the surface) were compiled into single projections. Each resin–dentin interface was entirely characterized, and images representing the MMP-activity observed along the bonded interfaces were captured.

Cytotoxicity evaluation: cell culture and MTT assay

Twenty-four teeth were used in this test. Dentin disks with a thickness of 0.6 mm were obtained from the mid-coronal dentin of each tooth using a cutting machine (Isomet 1000, Buehler, Lake Bluff, USA). The disks were carefully examined with a stereomicroscope at 40× to confirm the absence of enamel and defects resulting from pulp horn projections. Then, the occlusal sides of the disks were manually finished with wet 320-grit silicon carbide paper to reach a final thickness of 0.5 mm (Mitutoyo South Americana Ltd., Suzano, SP, Brazil). Afterwards, a smear layer was produced with 600-grit SiC on both sides of the disks and immediately removed by 0.5 M EDTA (pH 7.4) for 60 s. After abundant rinsing with deionized water, the dentin’s permeability was measured through a hydraulic conductance device to permit a homogenous distribution of the dentin disks among the experimental groups ( n = 6 disks per group). The dentin disks were positioned in metallic devices and autoclaved (20 min/121° C). The occlusal surfaces of the dentin disks were etched with 35% phosphoric acid (15 s), carefully rinsed with deionized water (10 s) and blot dried with sterile cotton pellets.

The NIH/3T3 fibroblast cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and 1% penicillin/streptomycin (10,000 U/100 μg/ml) at 37 °C with 5% CO 2 in a humidified atmosphere. Briefly, 4 × 10 4 cells were added to each well of a 24-well plate. After 24 h, the medium was removed; the cells were washed twice with phosphate buffer solution (PBS). DMEM containing 1% fetal bovine serum and trans-well chambers were added into each well. The dentin disks were carefully added, and 10 μl of each cross-linking primer was applied onto the occlusal surface. For the riboflavin group, the UVA-light was irradiated by a lamp, as previously described, using a 10.4 mm tip that completely covered the culture well (24-well format). This protocol followed Bouillaguet et al. to prevent modifications to the cell mitochondrial activity.

The trans-well was removed and the cells were washed with phosphate-buffered saline (PBS) 24 h later. MTT cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co., St. Louis, USA) according to the method of Tada et al. with some modifications. MTT solution was added (1.0 mg/ml), and cell viability was then assessed in a colorimetric assay using mitochondrial dehydrogenase activity in active mitochondria to form purple formazan. The absorbance of each well was read at 570 nm using a plate reader (EL808B, BioTech Instruments Inc., Winooski, VT, USA). Cell viability was expressed as the percentage of optical values in the treated samples versus the concurrent control (no treatment), considered as 100%. All of the experiments were performed in triplicate.

Statistical analysis

The μTBS (MPa) and NL (%) from the same experimental unit were averaged for statistical purposes at each storage time. The bonded sticks with premature and cohesive failures were not included in the tooth mean, due to their low frequency in the experiment.

The Kolmogorov–Smirnov test was employed to assess whether the data from each test (μTBS, NL, DC and cytotoxicity) followed a normal distribution. Barlett’s test was performed to determine if the assumption of equal variances was valid. After observing the data’s normality and equality of the variances, the data from the μTBS (MPa) and NL (%) of each adhesive were subjected to a two-way repeated measure ANOVA (on solutions and storage time). The data from the DC (%) and cytotoxicity (%) of each adhesive were subjected to a one-way ANOVA (on solutions). For all of the test Tukey’s test was used for pairwise comparisons ( α = 0.05).

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Nov 23, 2017 | Posted by in Dental Materials | Comments Off on Collagen cross-linkers on dentin bonding: Stability of the adhesive interfaces, degree of conversion of the adhesive, cytotoxicity and in situ MMP inhibition
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