Effect of dentin biomodification techniques on the stability of the bonded interface.

AIM: The aim of this study is to evaluate the effect of different bonding techniques ethanol wet bonding and dimethyl sulfoxide (DMSO) wet bonding and a novel collagen cross-linker Quercetin application on the durability of resin-dentin bond and observe the bonded interface under the scanning electron microscope (SEM).
MATERIALS AND METHODS: For shear bond strength testing, flat coronal dentin surfaces were prepared on 110 extracted human molars. Teeth were randomly divided into five experimental groups according to different surface pretreatments techniques. Group A was control group without any surface pretreatment. In Group B, ethanol wet bonding pretreatment was done before the application of adhesive. In Group C, DMSO wet bonding was done before the application of adhesive and in Groups D and E, Quercetin along with ethanol and Quercetin along with DMSO pretreatment, respectively, were done before adhesive application. Composite restorations were placed in all the samples. Twenty samples from each group were subjected to immediate and delayed (9 months) shear bond strength evaluation. In addition, two samples per group were subjected to the scanning electron microscopic analysis for the observation of resin-dentin interface. STATISTICAL ANALYSIS: Data collected were subjected to the statistical analysis using the one-way analysis of variance and post hoc Tukey's test at a significance level of P < 0.05.
RESULTS: Dentin pretreatment with all the techniques resulted in significantly higher resin-dentin bond strength after 9 months storage with DMSO group showing the highest bond strength values.
CONCLUSION: It can be concluded that these biomodification techniques can improve the durability of the resin-dentin bond. Copyright:

INTRODUCTION

Dentin is a complex mineralized tissue in which organic components are embedded within mineral crystallites. The crosslinking of collagen contributes to the tensile properties of the dentine matrix.[1] In dentin bonding, contemporary dental adhesive systems rely on formation of the hybrid layer, a biocomposite-containing dentin collagen and polymerized resin adhesive. However, loss of dentin-bonded interface integrity and bond strength is commonly seen after aging both in vitro and in vivo.[2] It has been established that the infiltration of collagen by the adhesive is incomplete since its penetration capacity is lower than the depth of conditioning of the etching agent. In addition, removing residual water in the dentin matrix is difficult.[3] Both of these are reasons why a portion of collagen remains unprotected, which results in the activation of endogenous proteases, called extracellular matrix metalloproteinases (MMPs) and cysteine cathepsins (CTs), present in dentin. As collagenolytic enzymes, MMPs and CTs hydrolyze the organic matrix of demineralized dentin, an event that triggers hybrid layer degradation.[45] Several approaches to improve long-term bonding have been developed and tested. These include inhibition of dentinal endogenous proteases, MMPs and CTs, thought to be responsible for degrading the hybrid layer collagenous matrix, and improved penetration and impregnation of the adhesive monomers into ethanol-saturated exposed dentin matrix.[67] However, despite promising results in in vitro research, clinically feasible and routinely applicable methods to improve long-term bonding are still lacking.

Traditional water-wet-bonding technique has been advanced to improve initial bond strength of etch-and-rinse adhesives, as water is an excellent solvent to re-expand collapsed demineralized dentin matrices before resin infiltration.[8] However, excess water often causes suboptimal polymerization of infiltrated resin monomers. In addition, water is not a proper solvent for resin monomers, as their miscibility is limited in the water, resulting in the phase separations of hydrophobic resins.[8] In this context, ethanol wet-bonding was introduced as a proof of concept by Tay et al. to address a sound solution for improving resin-dentin bond durability in 2007.[9] It involves gradual replacement of water present in interfibrillar spaces with increasing concentrations of ethanol and subsequently replacing ethanol with hydrophobic primers and resins.[8]

The so-called ethanol wet-bonding technique presents satisfactory results when used in incremental concentrations in experimental hydrophobic adhesives.[10] However, this method is time-consuming and impractical for the clinical applications. Dimethyl sulfoxide (DMSO) is a polar aprotic solvent and can dissolve polar and nonpolar compounds. It is an excellent solvent and can fully dissolve most monomers in dental adhesives. Its amphiphilic, dipolar aprotic nature may make it the ideal penetration enhancer for medical purposes.[11] DMSO is known to be capable of dissociating the highly cross-linked dentin collagen into a discrete fibril network which may contribute to the infiltration of monomer into the etched dentin matrix.[6] Therefore, exploration of the effect of DMSO on dentin bonding is a promising endeavor. DMSO wet bonding has been tested by Stape et al.[12] In that study, a 50% (v/v) DMSO aqueous solution performed well in terms of improving the strength of composite-dentin bonds. However, its mechanism is still unknown, especially compared to traditional water and ethanol wet bonding techniques. Pretreatment of acid-etched dentin with DMSO may improve long-term bond preservation.[613]

Naturally derived collagen crosslinkers with high antibacterial abilities for dentin bonding have also been widely explored.[1415] Quercetin is the most common flavonol in the diet. Quercetin belongs to the flavonol group and is commonly found in onions, apples, tea, and red wine.[16] As a result of its cross-linking properties, Quercetin has been shown to enhance the mechanical properties and thermal denaturation temperature of the extracelluar matrix of heart valves.[17] Quercetin also possesses multiple functions, including antioxidative, anticarcinogenic, anti-inflammatory, anti-aggregatory, and vasodilating effects.[15] Quercetin downregulates MMP 2 and 9 protein expression in prostate cancer cells and elicits significant antibacterial effects on Gram-positive and Gram-negative bacteria.[1819]

However, the effect of these bonding techniques in association with Quercetin on the resin-dentin bond durability of different adhesive systems still needs to be investigated. Therefore, the aim of the study was to evaluate the effect of different bonding techniques (Ethanol wet bonding, DMSO wet bonding) and a novel collagen cross-linker Quercetin application on the durability of resin-dentin bond.

MATERIALS AND METHODS

The study was conducted after taking ethical approval from Institutional Ethical and Review Board (IERB) with reference No. KDCRC/IERB/10/2018/19. One hundred ten freshly extracted human molars were collected. Teeth with any caries, cracks, abrasions, attrition, and restorations were excluded from the study. The teeth were thoroughly cleaned using ultrasonic scaler followed by thorough cleaning with pumice slurry and rubber prophylaxis cup rotating at slow speed in the contra-angled micromotor handpiece and stored in 0.1% thymol in distilled water at room temperature till commencing the experiment.

In order to prepare 1.0 wt% Quercetin in ethanol solution, 0.5 g of Quercetin powder (Sigma–Aldrich, St. Louis, MO, USA) was dissolved into 250 ml of pure ethanol under water-bath heating at 37°C. For preparation of 1.0 wt% Quercetin in DMSO solution, 0.05 g of Quercetin powder (Sigma–Aldrich, St. Louis, MO, USA) was directly dissolved into 5 ml of DMSO (Sigma Aldrich, St. Louis, MO, USA). For preparing of 50% v/v DMSO solution, 1 ml of DMSO (Sigma Aldrich, St. Louis, MO, USA) was directly mixed with 1 ml of distilled water to prepare 50% v/v DMSO solution.

Teeth were randomly divided into five experimental groups according to five different bio modification techniques. All the samples were subjected to acid etching procedure with Scotch bond multipurpose etchant (3M, ESPE), for 15 s followed by rinsing with water for 10 s and blot dried.

Group A: Water wet bonding

Acid etching followed by the application of distilled water for 1 min and gently blot drying before the application of a 2-step etch and rinse adhesive (Scotch bond multipurpose etchant-3M, ESPE).

Group B: Ethanol wet bonding

Acid etching followed by chemical dehydration treatment with 100% ethanol for 1 min and gently blot drying before the application of the adhesive.

Group C: Dimethyl sulfoxide wet bonding

Acid etching followed by the application of 50% DMSO solution (v/v) for 1 min and gently blot drying before the application of the adhesive.

Group D: Quercetin + ethanol

Application of Quercetin ethanol solution (1%wt) on acid-etched dentin surface for 1 min and gently blot dried with filter paper before application of the adhesive.

Group E: Quercetin + dimethyl sulfoxide

Application of Quercetin DMSO solution (1%wt) on acid-etched dentin surface for 1 min and gently blot dried with filter paper before the application of the adhesive.

Transparent plastic tubes (TYGON laboratory tubing, Saint Gobain, Akron, OH, USA) with 3 mm in diameter and 2 mm in height and thickness of 0.5 mm were precut and placed perpendicular to the previously etched, pretreated and bonded dentin surface. A nanohybrid resin composite (Filtek Z350 XT, Body Shade A2; 3M ESPE Dental Products) was filled into the precut tubes. Each bonded specimen was light-cured for 20 s using quartz-tungsten-halogen light curing unit (Spectrum 800, Dentsply Caulk, Milford, DE, USA) at a light intensity of 600 mW/cm2. The plastic tubes were gently cut and carefully removed with a number 11 surgical blade after polymerization.

Half of the specimens (immediate testing group) were then stored in distilled water at 37°C for 24 h for the completion of polymerization before immediate testing and scanning electron microscopic (SEM) analysis. The remaining half samples from each group (delayed testing group) were stored in artificial saliva (Wet Mouth, ICPA Heath Products Ltd.) at 37°C in an incubator for 9 months before shear bond strength and SEM evaluation. Artificial saliva (pH 7.11) was composed of 0.5% w/v sodium carboxymethyl cellulose, 30% w/v glycerin, and flavored base. 0.2 percent sodium azide (pH 7.31) was further added to prevent bacterial growth. This was confirmed through the maintenance of the clear color of artificial saliva during the storage period. Storage solution was changed every 2 weeks.

Each group was evaluated for shear bond strength at two different time periods: At 24 h (immediate) (I) and at 9 months (delayed) (D) after storage in artificial saliva.

Shear bond strength testing

Half samples from each group were subjected to immediate (24 h) shear bond strength testing in a universal testing machine (Instron, ADMET, Enkay Enterprises, New Delhi) using the corresponding computer software. The specimens were placed and stabilized by the jig, while a straight knife-edge rod (2.0 mm) was applied at the tooth restoration interface at a cross-head speed of 0.5 mm/min. Load was applied until restoration failure. The load at failure was converted to shear bond strength evaluation (MPa) by dividing the load by surface area of the specimen. Other half samples from the group were subjected to shear bond strength evaluation; with same parameters, software and equipment; after retrieval from storage in artificial saliva for 9 months.

All the debonded samples were analyzed under stereomicroscope at ×10 magnification and to define the location of bond failure, categorized as adhesive (a), cohesive (c), and mixed (m).

Scanning electron microscopic evaluation

Two samples from each group were sectioned through the coronal and root portion to obtain dentin blocks. Same pretreatment and bonding procedures were followed as for Shear bond strength (SBS) sample preparation. Composite restoration was done covering all the visible dentin surface. Half the samples were tested immediately (24 h) and the other half stored for 9 months prior to the SEM study. The specimens were fixed in 10% formalin for 24 h and decalcified in 6 N HCl for 30 s, rinsed in distilled water and deproteinized by 10-min immersion in 1% NaOCl, and rinsed in distilled water. After acid base treatment, the specimens were subjected to dehydration in ascending grades of ethanol up to 100% (25% for 20 min, 50% for 20 min, 75% for 20 min, 95% for 30 min and 100% for 60 min), then transferred to a critical point dryer for 30 min. The specimens were then gold sputter coated in gold sputtering unit and then resin–dentin interfacial adaptation was observed under a SEM (LEO 430, England).

Statistical analysis

Values obtained from the shear bond strength were then subjected to the statistical analysis using parametric tests at a significance level of P ≤ 0.05. Mean and standard deviations were calculated for each group. The statistical analysis on the shear bond strengths was done using Statistical Package for the Social Sciences (SPSS software version 20.0; SPSS Inc., Chicago, IL, USA). The values were represented in number (%) and mean ± standard deviation. The statistical tools used were, Tukey's honestly significant difference test, and one-way analysis of variance (ANOVA) (multivariate assessment), Independent-t-test (appendix).

RESULTS

Data were normally distributed as tested using the Shaperio–Wilk W test (P value was more than 0.05). Therefore, the analysis was performed using the parametric test “one way ANOVA test” (for comparing more than two groups). Level of statistical significance was set at P < 0.05. Post hoc Tukey's test was used for pairwise comparison of subgroups. Paired t-test was used for intragroup comparison: Immediate and delayed.

SBS

Mean shear bond strength values of different groups at two different time periods are presented in Table 1. DMSO treated group (Group C) showed the highest 24 h shear bond strength values among all the groups, which was significantly higher than the control group [Table 1]. Quercetin treated specimens (Group D and E) showed significantly higher SBS values at 24 h when compared to the control group, but there was no significant difference between the two groups. Storing the samples for 9 months in artificial saliva significantly decreased the shear bond strength within each of the control and the pretreated groups. However, when compared to the control group, the shear bond strength values of all the pretreated groups were significantly higher at 9 months with DMSO treated group showing the highest bond strength values.

Table 1

Comparison of immediate and delayed bond strength among five groups

Group	Immediate mean	SD	Delayed mean	SD	P b
Group A (C)	35.791a	12.778	22.997d	6.318	0.004* (significant)
Group B (EWB)	57.769b	12.671	42.005e	6.498	0.003* (significant)
Group C (DWB)	71.421c	7.936	49.894e	9.130	0.001* (significant)
Group D (Q + E)	54.256b	11.970	39.939e	8.911	0.031* (significant)
Group E (Q + D)	53.874b	11.148	40.855e	8.970	0.019* (significant)
Pa	0.0001* (significant)		0.0001* (significant)

*Significance of relationship at P<0.05, aP: One-way ANOVA test, bP: Paired t-test, cb,ac,ab,ad,be,ce,deSignificant, ac,bb,eeNonsignificant. SD: Standard deviation, C: Control, EWB: Ethanol wet bonding, DWB: DMSO Wet Bonding, Q + E: Quercetin + Ethanol, Q+D: Quercetin + DMSO

Fractographical analysis

The effect of different dentin pre-treatment and different time intervals on the distribution of failure pattern was compared using the Chi-square test, and results are summarized in Figure 1. Most of the failures encountered were mixed in all the groups tested immediately (P = 0.917). At 9 months, an increase in the number of adhesive failures was observed in all the groups with greater increase in the control group than the experimental groups which still continued to show predominantly mixed failures SEM.

Figure 1

Failure patterns of the experimental groups

In scanning electron micrographs [Figures 2-6], a good interfacial adaptation was seen in all the immediate groups. Among the delayed groups, interfacial gap was observed only in the control group.

Figure 2

Group A (control): Immediate (a), delayed (b)

Figure 6

Group E (Quercetin + dimethyl sulfoxide): Immediate (a), delayed (b)

Group B (EWB): Immediate (a), delayed (b)

Group C (DWB): Immediate (a), delayed (b)

Group D (Quercetin + Ethanol): Immediate (a), delayed (b)

DISCUSSION

This study was conducted to evaluate the effect of different bonding techniques such as ethanol wet bonding and DMSO wet bonding and a novel collagen cross-linker Quercetin along with Ethanol or DMSO, application on the durability of resin-dentin bond.

In our study, we have used simplified ethanol wet bonding technique as used by Yesilyurt et al. and Li et al., 100% ethanol was used for 60 s to replace rinse water from acid-etched dentin.[2021] Our results are in accordance with the previous studies as ethanol wet bonding significantly improved delayed bond strength as compared to the control group water wet bonding (WWB).[2223] This can be attributed to the fact that ethanol can replace rinse water after acid-etching, thus lowering hydrophilicity of matrices thereby, stabilizing matrices and promoting resin infiltration. Further, since most of hydrophobic monomers are mixable in ethanol and not in water, bonding to dentin with hydrophobic adhesives with reduced water adsorption and increased durability could be achieved.[8]

In this study, 50% DMSO was used as used in previous studies (Stape et al. and Guo et al.) as they found better results with this concentration.[1013] In our study, it was found that DMSO significantly improved both immediate and delayed bond strength that is also in accordance with previous studies as that of Sharafeddin et al. and Tjäderhane et al.[624]

The reason for increased immediate bond strength can be because DMSO increases the permeability of collagen matrix and improves resin penetration. DMSO possesses a high dielectric constant combined with low surface energy and a capacity to solvate polymers and adhesives.[25] DMSO is an ideal solvent to facilitate radical polymerization reactions such as are used in dental adhesion.[26] To date, DMSO pretreatment has been shown to have positive effects on dentin bonds strength and its durability.

Natural collagen cross linkers are also widely studied these days, Quercetin being one of them. Quercetin could inhibit the activity of MMP-2 and MMP-9 in PC-3 cells.[26] Quercetin can also cross-link with collagen to decrease the formation of water canals by resisting collagenase attack and strengthen its stability.[27] According to Haslam, polyphenols, such as quercetin, exhibit an amphiphilic property that combines the hydrophobic characteristic provided by its planar aromatic nucleus and the hydrophilic characteristic contributed by its polar hydroxyl groups.[28] The hydrophobic forces of polyphenols drive them into the “holes” or “gap zones” in the collagen fiber, causing the former become embedded in the collagen structure. The hydroxyl group interacts with the proline residues of collagen through hydrogen bonding, providing a secondary interaction that helps to stabilize the complex.

In our study, we have used Quercetin with ethanol as well as DMSO to see the additive effect of penetration enhancers along with collagen cross linkers. Quercetin along with ethanol has been used by researchers like Li et al., still there are very less studies available to compare its effects.[21] However, Quercetin with DMSO has not been used before in any study so no comparisons could be made. We found that both Quercetin groups had significantly higher bond strength as compared to the control (WWB) group but when compared to the DMSO group the bond strength values were lower in both immediate and delayed groups.

Being a natural cross-linker, quercetin has been widely accepted for its biocompatibility and safety. A review even suggested that daily consumption of quercetin at dietary intake levels (200–500 mg day_1) would not produce adverse health effects.[29] Furthermore, when used in dentin bonding, only one or two drops of quercetin/ethanol solution are brushed on the dentin surface. It may be assumed that this tiny amount of solution would not display significant cytotoxicity.

Due to the versatile performance of quercetin, it has a great potential in dental application. However, there are concerns that as an antioxidant, the high concentration of quercetin might possibly inhibit the polymerization of the adhesive and decrease the bonding strength.[19] Regarding quercetin, it can be speculated that it could exert long-term anti-bacterial ability, not only due to its cross-linking effect with dentin collagen, but also its barely soluble character, limiting its leaching rate into saliva.[21]

Our study is not in alignment with the study of Gotti et al. who reported that Quercetin maintained the bond strength over the long-term but decreased the immediate bond strength.[30] Another study conducted by Porto et al. who used various concentrations of Quercetin solution found that at 100% concentration, microtensile bond strength decreased after 120 days where as at concentrations of 250%, 500%, and 1000% the delayed bond strength increased.[31] Hence, we suggest further studies be conducted so that more reliable conclusions could be drawn.

CONCLUSION

Within the limitations of this study, it can be concluded that all the altered bonding techniques (i.e., Ethanol Wet Bonding, DMSO Wet Bonding and Quercetin treatments) resulted in significantly higher delayed shear bond strength value than the control. Further research is needed to explore the possibility of adding quercetin into dental adhesives and to evaluate the stability of adhesive/dentin interface after long-term function.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.