This study assessed a 6 month resin/dentin bond’s durability and cytotoxic effect of Zn-doped model dentin adhesives. The mechanical and physicochemical properties were also tested.
A model etch-and-rinse single-bottle adhesive was formulated (55 wt.% Bis-GMA, 45 wt.% HEMA, 0.5 wt.% CQ, 0.5 wt.% DMAEMA) and Zinc methacrylate (Zn-Mt) or ZnO nanoparticles (ZnOn) were added to the model’s adhesive, resulting in three groups: Group Control (control model adhesive); Group Zn-Mt (1 wt.% Zn-Mt incorporated to adhesive) and Group ZnOn (1 wt.% ZnOn incorporated to adhesive). The microtensile bond strength (mTBS) was assessed after 24 h or 6 months in water storage. Mechanical properties (diametral tensile strength/DTS, flexural strength/FS, flexural modulus/FM, resilience modulus/RM, and compressive strength/CS) and physicochemical properties (polymerization shrinkage/PS, contact angle/CA, water sorption/WS, and water solubility/WS) were also tested. Cytotoxicity was evaluated with SRB biochemical assay.
No significant difference in the DTS, FS, FM, CS, CA, WS, and WS were found when 1% of ZnOn or Zn-Mt was added to the model dentin adhesive. Group Zn-Mt decreased the RM of adhesive. Groups Zn-Mt and ZnOn decreased the PS of adhesives. Group ZnOn reduced the cytotoxicity of adhesive. Group ZnOn preserved mTBS after 6 months storage without degradation areas as seen by SEM analysis.
The 1 wt.% ZnOn may preserve the integrity of the hybrid layer and may reduce cytotoxicity and polymerization shrinkage of model dentin adhesive. The addition of Zn-Mt to the adhesive had no beneficial effects.
The lack of durable dental adhesives is considered one of the main problems with the use of composite materials in dentistry. Failures of composite restorations are observed mainly at the dentin/adhesive interface. The bonding to dentin is obtained by the formation of a hybrid layer; however, the longevity of the hybrid layer has still been widely questioned as a consequence of hydrolytic degradation of the adhesive interface over time .
Among the various factors that are related to the degradation of the hybrid layer, two factors are highlighted: the hydrolytic degradation over time of the polymer present in the hybrid or adhesive layers , and a poorly infiltrated hybrid layer with unprotect collagen fibers . This unprotected collagen can hydrolyze by host-derived matrix metalloproteinase (MMP) enzymes . Therefore, new strategies are being exhaustively studied with the purpose of increasing the durability of the dentin/bond interface, one of which is the inhibition of MMP’s proteolytic activity within the unprotected collagen fibers .
MMPs require Zn 2+ ions to maintain their proper tertiary structure and functional active sites . On the other hand, Zn can be used to protect collagen from MMPs activity . Previous studies observed the durability of the resin/dentin interface when Zn was added to commercial dental adhesives. Zn can promote subtle conformational in collagenase cleavage sites in collagen molecules that protect collagen from MMP’s activity . The incorporation of Zn in commercial adhesives has also resulted in the formation of apatite crystallites on the collagen fibrils, favoring dentin mineralization , reduce MMPs-mediated collagen degradation , may inhibit dentin demineralization , and may promote enamel remineralization .
However, any change in the chemical composition of adhesive systems involves potential mechanical and physico-chemical failures and biohazards. Therefore, the insertion of Zn in adhesive systems as an alternative to preserve the longevity of the restoration, although having promising results, requires further reviews, due to the possibility of changing adhesive systems’ biological, physico-chemical and mechanical properties, and, consequently, their clinical performance.
The aim of this study was to evaluate a 6 month resin/dentin bond’s durability of Zn-doped etch-and-rinse model dentin adhesives. Mechanical properties (diametral tensile strength, flexural strength, flexural modulus, resilience modulus, and compressive strength) and physicochemical properties (polymerization shrinkage, contact angle, water sorption, and water solubility) were also tested. In vitro cytotoxicity of these adhesives was evaluated on human dental pulp fibroblasts.
This study tested three null hypotheses: (1) the model dentin adhesives tested can achieve similar bond durability to dentin; (2) the storage period does not affect the bonding effectiveness of model dentin adhesives; and (3) the model dentin adhesives tested can achieve similar results for mechanical and physicochemical properties and cytotoxicity.
Material and methods
Model dentin adhesives preparation
A model simplified etch-and-rinse adhesive was formulated through intensive mixing of bisphenol-A diglycidyl ether dimethacrylate (Bis-GMA) and 2-hydroxyethyl methacrylate (HEMA), with a mass ratio of 45:55 (HEMA:Bis-GMA). The photoinitiators used were 0.5 mol% of camphorquinone as a hydrophobic photosensitizer and 0.5 mol% of 2-(dimethylamino) ethyl methacrylate (DMAEMA) as a hydrophilic co-initiator. Model dentin adhesives were prepared in a brown glass vial in the absence of visible light. All materials were obtained from Aldrich Chemical Co. (Milwaukee, WI, USA) .
Zinc methacrylate (Sigma Aldrich) and ZnO nanoparticles (Sigma Aldrich) were added to the control model dentin adhesive at 1, 2, 5, and 10 wt.% concentrations. The Zn-doped model dentin adhesives were shaken using a tube agitator in the dark for 10 min at 2000 rpm.
Degree of conversion (pilot study)
In order to obtain a maximal concentration that did not alter the conversion degree of the Zn-doped model dentin adhesives, the degree of conversion of polymerized Zn-doped adhesives was compared to the control model dentin adhesive. The degree of conversion was determined using Raman spectroscopy. Spectra were collected using a MicroRaman spectrometer (model 2000, Renishaw Engineering, Wotton-under-Edge, UK) with an Argon laser as an excitation source. To determine the degree of conversion, spectra of a droplet of uncured adhesives and polymerized adhesives were acquired over a spectral range of 700–1800 cm −1 . The change of the band height ratios of the aliphatic carbon–carbon double bond (peak at 1640 cm −1 ) and the aromatic C C (peak at 1610 cm −1 ) (phenyl) in both the cured and uncured states was monitored. The degree of conversion was calculated using the following formula based on the decrease in the intensity band ratios before and after light curing (Eq. (1) ):
D C ( % ) = 1 − R cured R uncured × 100
All experiments were carried out in triplicate and the results were averaged. The results were analyzed statistically using one-way analysis of variance (ANOVA) and Tukey’s test (5%).
The degree of conversion for all adhesives was in the range of 87–77%. Zn-Doped adhesives at 2, 5, and 10 wt.% concentrations showed a significantly lower degree of conversion (degree of freedom = 9, F = 124, p = 0.000) as compared to the control adhesive. Table 1 shows Tukey’s results of the degree of conversion of the adhesive polymers.
|Experimental adhesives||Degree of conversion % (SD)|
|Control dentin adhesive||87.25 (3.65)a|
|Zinc methacrylate 1%||86.90 (3.18)a|
|Zinc oxide nanoparticles 1%||86.66 (3.20)a|
|Zinc oxide nanoparticles 2%||81.08 (1.33)b|
|Zinc oxide nanoparticles 5%||80.27 (2.19)b|
|Zinc methacrylate 2%||79.27 (1.72)b|
|Zinc oxide nanoparticles 10%||79.12 (1.79)b|
|Zinc methacrylate 5%||77.71 (1.65)b|
|Zinc oxide nanoparticles 10%||77.62 (1.42)b|
Based on the results of the pilot study ( Table 1 ), it was used three formulations of dentin adhesives: (1) control dentin adhesive (Control adhesive); (2) Zn-doped dentin adhesive with 1 wt.% Zinc methacrylate (Zn-Mt adhesive); and (3) Zn-doped dentin adhesive with 1 wt.% ZnO nanoparticles (ZnOn adhesive).
The three tested model dentin adhesives (Control adhesive, Zn-Mt adhesive, and ZnOn adhesive) were evaluated in vitro for longitudinal microtensile bond strength (mTBS), diametral tensile strength, flexural strength, flexural modulus, resilience modulus, compressive strength, water sorption, water solubility, polymerization shrinkage, contact angle, and cytotoxicity.
Microtensile bond strength (mTBS)
Sixty extracted healthy human molars were used. The Research Ethics Committee of the São Jose dos Campos School of Dentistry – UNESP approved this study. A flat dentin surface was exposed after grinding the occlusal enamel using abrasive water papers (450-grit) under water cooling (Politriz, DP-10, Panambra, São Paulo, SP, Brazil). The smear layers of the dentin surfaces were standardized with 600-grit silicon carbide paper for 30 s under water cooling.
Before the restorative procedures, a solvent of 30 wt.% (99% ethanol, Sigma Aldrich) was mixed into to the model dentin adhesives, using a shaker tube in the dark for 3 min at 2000 rpm.
The dentin surfaces were etched for 15 s with a 37% phosphoric acid gel (CondacAC 37%, FGM Prod. Odont., Ltda, Joinville, SC, Brazil), rinsed, and the excess moisture was removed with absorbent paper. Two layers of model dentin adhesives were applied on the surface actively for 20 s and gently air dried for 10 s. Adhesives were light activated for 20 s with an LED light unit (Demi LED Light Curing System) with a power density of 1000 mW/cm 2 .
Composite build-ups (Filtek Z350, 3 M ESPE, St. Paul, MN, USA) were placed on the bonded surfaces (4 mm high, in 2 increments). Each 2 mm portion was light activated for 40 s. The bonded teeth were stored in distilled water at 37 °C for 24 h.
To obtain six sticks per tooth, the teeth were cut in both “ x ” and “ y ” directions across the bonded interface into parallel sections measuring approximately 1 mm, using a diamond disk (Labcut 1010, Extec Technologies Inc., Enfield, CT, USA) at low speed and under water cooling.
Half of the total sticks from each group were immediately tested for μTBS and the other half were stored in distilled water at 37 °C for 6 months before testing. The water was changed weekly.
The sticks were attached to a microtensile device in a universal testing machine (DL-1000, EMIC, São José dos Pinhais, PR, Brazil), with a 10 kg load cell, at a cross-head speed of 0.5 mm/min, according to ISO 11405 Standard. The bond strength data were expressed in megapascals (MPa).
The failure modes were analyzed with a 20× stereomicroscope (Stemi 2000 – Karl Zeiss, Göttingen, Germany) and were classified as adhesive, mixed, or cohesive in dentin or composite. Only the adhesive and mixed failures were included in the statistical analysis. The mean value for the sticks originating from each tooth was calculated and used for the statistical analysis. Data were analyzed by two-way ANOVA (adhesive and storage time) followed by Tukey test ( α 5%).
Two teeth from each group were used in the Scanning electron microscopy (SEM) analysis according to Marimoto et al. . After bonding and restorative procedures, the teeth were sectioned perpendicularly to the bonding interface and were stored in distilled water at 37 °C for 24 h or 6 months. Then, the sections were polished with 2000 and 4000 mesh sheets. Phosphoric acid etchant was applied for 5 s and rinsed off with water for 10 s. Specimens were dehydrated, sputter-coated with gold-palladium, and the bonding interfaces were examined using SEM.
Flexural strength, modulus of elasticity and resilience modulus
Ten specimens of each group were prepared for the test. Unpolymerized model dentin adhesive was placed on rectangular silicon molds (25 mm long × 2 mm wide × 2 mm high; ISO 4049), which were covered with a Mylar strip and a glass slide. Specimens were light polymerized on the top for 20 s (Demi LED Light Curing System) at 3 different positions (right, middle, and left). Additional light-activation was made for 20 s on the bottom surface. Specimens were stored for 24 h in distilled water at 37 °C and subjected to a three-point bending test using a universal testing machine (DL-1000, EMIC, São José dos Pinhais, PR, Brazil) .
After the specimens had been finished with 1000-grit silicon carbide paper, their final dimensions were measured. Specimens were individually stored in deionized water at 37 °C for 24 h prior to testing. The flexural properties were evaluated by a three-point bending test using the same universal testing machine at a crosshead speed of 1.0 mm/min. Flexural strength was obtained by measuring the load at fracture, and the modulus of elasticity was calculated from the recorded load–deflection curves .
The coefficients of variation for the resilience modulus (in MPa) were calculated from the data of the flexural strength and modulus of elasticity using the formula described in Eq. (2) .
RM = ( FS ) 2 2 x ( ME )
FS is the flexural strength (in MPa), and ME is the modulus of elasticity (in MPa).
Data collected of flexural strength (in MPa), modulus of elasticity (in GPa), and resilience modulus (in MPa) were statistically analyzed using one-way ANOVA and Tukey’s test ( p < 0.05).
Diametral tensile strength
For the diametral tensile strength test, 10 specimens of each group were prepared. Three increments (approximately 2 mm each) of unpolymerized model dentin adhesive was placed in a cylindrical silicon mold (4.0 mm in diameter × 6.0 mm in height), and each increment was light polymerized for 20 s (Demi LED Light Curing System). The third increment was recovered with a Mylar strip and a glass slide. Additional light-activation for 20 s was done on the lateral portions of the cylinder. Specimens were then stored in distilled water at 37 °C for 24 h.
The specimens were tested under a compressive load in the universal testing machine (DL-1000, EMIC, São José dos Pinhais, PR, Brazil) at a crosshead speed of 1.0 mm/min. Load was applied vertically on the lateral portion of the cylinder. Fracture load ( F ) (in N), was recorded and the diametral tensile strength (in MPa) was calculated (Eq. (3) ).
DTS = 2 F π × d × l
Since d is the diameter (4 mm), h is the height (6 mm), and π is 3.1416.
Ten specimens of each model dentin adhesive were prepared using a silicon mold (4.0 mm in diameter × 8.0 mm in height). The silicon mold was filled in four approximately 2.0-mm-thick increments, and each increment was light-activated for 20 s. After insertion of the last increment, a Mylar strip and a glass slide were placed onto the silicon mold and light curing was done. Additional light-activation for 20 s was done on the lateral portions of the cylinder. Specimens were then stored in distilled water at 37 °C for 24 h.
The specimens were tested under a compressive load in compressive strength in the universal testing machine (DL-1000, EMIC) with a crosshead speed of 1 mm/min. The data were obtained in N and converted to MPa using the formula shown in Eq. (4) .
CS = F A
Since CS is the compressive strength (MPa), F is the supported maximum load (N), and A is the cross-sectional area of the specimen (mm 2 ).
Data were tabulated and analyzed statistically using one-way ANOVA and the Tukey test for pairwise comparisons. Significance level was set at 5%.
Water sorption and water solubility
Ten disk-shaped specimens of each model dentin adhesive were fabricated using a silicon mold (6.0 mm in diameter × 2.0 mm in height) (ISO 4049.2000). One increment of unpolymerized model dentin adhesive was placed in the silicon mold, and a Mylar strip and a glass slide were placed onto the silicon mold and light-polymerized for 20 s. Additional light-activation for 20 s was done on the bottom of the specimen. Specimens were stored in a desiccator containing freshly dried silica gel . After 24 h, the specimens were weighed with a precision balance of 0.0001 mg (model Ohaus Adventurer, Barueri, SP, Brazil). This cycle was repeated until a constant mass ( m i ) was obtained. The specimens were immersed in 1 ml of distilled water at 37 °C. Every 24 h, specimens were removed, blotted dry, re-weighed ( m s ) and returned to the water. After 28 days, specimens were again dried inside a desiccator and weighed daily until constant mass was obtained ( m d ) . Water sorption (WS) and water solubility (WL) were calculated using the formula (ISO 2000) shown in Eqs. (5) and (6) respectively.
WS ( % ) = m s − m i m i × 100
WL ( % ) = m i − m d m i × 100