|Year : 2015 | Volume
| Issue : 1 | Page : 38-45
Adhesion to pulp chamber dentin: Effect of ethanol-wet bonding technique and proanthocyanidins application
Pallavi Sharma1, Rajni Nagpal1, Shashi Prabha Tyagi1, Udai Pratap Singh1, Naveen Manuja2
1 Department of Conservative Dentistry and Endodontics,Kothiwal Dental College and Research Centre, Moradabad, Uttar Pradesh, India
2 Department of Pedodontics, Kothiwal Dental College and Research Centre, Moradabad, Uttar Pradesh, India
|Date of Web Publication||12-Jan-2015|
Department of Conservative Dentistry and Endodontics, Kothiwal Dental College and Research Centre, Moradabad - 244 001, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Aim: To evaluate the microleakage of a simplified etch-and-rinse adhesive bonded to pulp chamber dentin with water-wet bonding (WWB) or ethanol-wet bonding (EWB) with and without proanthocyanidins (PA) application. Materials and Methods: Total 88 non-carious extracted human molar teeth were sectioned horizontally to expose the pulp chambers 1.5 mm coronal to the cemento-enamel junction. After the pulp tissue extirpation, canal orifices were enlarged and the root ends were sealed. The samples were randomly divided equally into following four groups according to the four bonding techniques performed using Adper Single Bond 2 [SB] adhesive (1) WWB; (2) EWB; (3) WWB and PA application [WWB + PA]; (4) EWB and PA application [EWB + PA]. Composite resin restorations were performed in all the pulp chambers. Total 20 samples from each group were subjected to microleakage evaluation, and two samples per group were assessed under scanning electron microscope for interfacial micromorphology. Results: The least microleakage score was observed in group 2 (EWB) with similar results seen in group 4 (EWB + PA) (P = 0.918). Group 2 (EWB) showed significantly less microleakage than group 1 (WWB; P = 0.002) and group 3 (WWB + PA; P = 0.009). Group 4 (EWB + PA) also depicted significantly reduced microleakage as compared with group 1 (WWB; P = 0.001) and group 3 (WWB + PA; P = 0.003). Conclusion: The use of EWB technique in a clinically relevant simplified dehydration protocol significantly reduced microleakage in simplified etch-and-rinse adhesive, Adper Single Bond 2, bonded to pulp chamber dentin. Application of PA had no significant effect on the microleakage of the adhesive bonded with either WWB or EWB.
Keywords: Etch-and-rinse adhesives, ethanol-wet bonding, proanthocyanidins, pulp chamber dentin
|How to cite this article:|
Sharma P, Nagpal R, Tyagi SP, Singh UP, Manuja N. Adhesion to pulp chamber dentin: Effect of ethanol-wet bonding technique and proanthocyanidins application. Saudi Endod J 2015;5:38-45
|How to cite this URL:|
Sharma P, Nagpal R, Tyagi SP, Singh UP, Manuja N. Adhesion to pulp chamber dentin: Effect of ethanol-wet bonding technique and proanthocyanidins application. Saudi Endod J [serial online] 2015 [cited 2021 Apr 15];5:38-45. Available from: https://www.saudiendodj.com/text.asp?2015/5/1/38/149086
| Introduction|| |
Coronal leakage has been extensively demonstrated as a negative contributor to the prognosis of endodontic treatment. Despite apical leakage being considered an important factor in endodontic failure, more attention is now being focused on procedures performed to achieve an effective coronal seal soon after the completion of root canal therapy.  Restoration of endodontically treated teeth with adhesive restoration permits transmission of functional stresses across the bonded interface to the tooth, with the potential to reinforce weakened tooth structure.  However, as compared with coronal dentin, bonding to pulp chamber dentin is more difficult due to its complicated structure. Ferrari et al. also postulated that adhesion to radicular dentin is more unpredictable than that to coronal dentin. 
Despite significant improvement of adhesive systems, the tooth-adhesive interface is the weakest area of the restoration.  When bonding to dentin, it is generally accepted that a high-quality interface is achieved when adhesive monomers thoroughly infiltrate and encapsulate exposed collagen fibrils, creating the resin-dentin interdiffusion zone known as "hybrid layer".  Therefore, the ultimate goal of resin-dentin bonding procedures is the complete infiltration of adhesive into the interfibrillar spaces and encapsulation of the collagen fibrils till the entire depth of demineralized dentin.
According to Becker et al., when etch-and-rinse adhesives are used with water-wet bonding (WWB) technique, water-saturated dentin matrix is too weak to resist evaporation stress during solvent evaporation leading to a dramatic shrinkage of dentin matrix, smaller interfibrillar spaces and deterioration of resin-dentin adhesion.  Several authors have suggested that it may be impossible for resin monomers to displace all the residual water, resulting in the generation of unprotected or poorly encapsulated collagen fibrils at the base of hybrid layer. 
Moreover, application of simplified etch-and-rinse adhesives to demineralized dentin activates endogenous collagen-bound and unbound matrix metalloproteinases and results in progressive loss of exposed collagen fibrils from the base of hybrid layer. As contemporary adhesives are hydrophilic and permeable to water from the underlying bonded dentin, it leads to plasticization that lowers their mechanical properties.  Some authors have emphasized that, to enhance the durability of resin-dentin bonds, future dentin adhesives should be rendered more hydrophobic, which reduces water sorption and thereby decreases resin plasticization. ,,
Accordingly, the promising concept of ethanol-wet bonding (EWB) has been proposed by Pashley et al., Tay et al., and Sadek et al., in which ethanol instead of water is used to support the demineralized dentin collagen matrix. ,, EWB is an in vitro technique developed for the application of etch-and-rinse adhesives.  There are two versions of the EWB technique for use with experimental hydrophobic adhesives.  In the progressive ethanol replacement, the ethanol saturation is achieved using a series of ascending ethanol concentrations, taking approximately 3-4 min, which defies the principles of user-friendliness and technique simplification. To overcome this obstacle, simplified dehydration protocol with single-step 1-min application of 100% ethanol has been developed so that it can be accomplished within a clinically relevant time frame.  However, it is extremely technique-sensitive and does not completely replace water and therefore may not be successfully used with hydrophobic adhesives. On the other hand, it may be assumed that, contemporary hydrophilic adhesives will be more tolerant to the presence of residual water as compared with hydrophobic adhesives after EWB in a simplified dehydration protocol.  Therefore, alternative version of the simplified EWB technique is to apply hydrophilic adhesives to ethanol-saturated demineralized dentin.
The stability of dentin collagen fibrils is critical for effective resin-dentin bonding. Low biodegradation rates and high mechanical properties of collagen are desirable to increase the durability of adhesive restorations. It has been demonstrated that the application of exogenous bioactive collagen cross-linking agents is helpful to biomodify the structures of collagen fibrils and improve their degradation resistance as well as stabilization. ,
One interestingly possible component of such a system is a class of exogenous bioactive natural phytochemicals and dentin collagen cross-linkers known as proanthocyanidins (PA), which have shown to biomodify the dentin matrix, lower the biodegradation rates, and enhance the mechanical properties and long-term stability of resin-dentin bond in a time-dependent manner, indicating its potential value in restorative dentistry. ,, Various other cross-linking reagents, like formaldehyde, glutaraldehyde, epoxy compounds, genipin, and carbodiimide, have been used, but all have drawbacks such as toxicity, difficult to control cross-linking rates, and instability.  Grape seed and cocoa extracts are well known as rich PA sources, where PA can be readily extracted with regular and nontoxic solvents like water, acetone, and ethanol. 
The collagen cross-linking agents are currently under examination for improvement of the quality and durability of the hybrid layer. Although various studies have investigated the effect of EWB and the role of collagen cross-linkers in dentin bonding individually, but most of these have evaluated the bond strength on the flat coronal dentin. However, pulp chamber dentin presents a different bonding substrate along with a high C-factor. There is no published literature evaluating the effect of EWB technique and PA application on the microleakage of adhesive restorations in the pulp chamber. Therefore, the aim of the study was to evaluate the microleakage of a simplified etch-and-rinse adhesive bonded to pulp chamber dentin with WWB or EWB with and without sequential PA application. The null hypothesis tested was that there is no difference between the microleakage of simplified etch-and-rinse adhesive bonded to pulp chamber dentin with WWB or EWB with and without PA application.
| Materials and Methods|| |
Eighty-eight human molars, freshly extracted due to periodontal reasons, were collected and cleaned and stored in normal saline water and used within 6 months of extraction. Teeth with any caries, cracks, abrasions, attrition, and restorations were excluded from the study. The roof of the pulp chamber was exposed using a carborundum disc (Bego, Germany) horizontally 1.5 mm coronal to the cemento-enamel junction, and roots were sectioned 2 mm apical to the bifurcation with water coolant. Pulp tissue was removed carefully with the help of excavator and broaches (Spirocolorinox, Dentsply Maillefer, Switzerland). The canal orifices were widened with Gates Glidden (Gates drills, Mani, Inc., Tochigi, Japan) drill no. 2-3. Root ends were sealed with amalgam (DPI Alloy, Dental Products of India, Mumbai, India), and the pulp chambers were irrigated with normal saline.
Preparation of 15% PA
After removing the coating from the 95% purity grape seed extract capsules (Zenith Nutrition), PA powder was obtained. Accordingly, 15.79 g powder was dissolved in 84.21 ml of 100% ethanol and distilled water separately at NTP to obtain a 15% PA solution. It was allowed to sediment by centrifugation at 1200 rpm for 15 min. The supernatant fluid was collected in a closed beaker and double filtered through a filter paper.
The teeth were then randomly and equally divided into four groups (n = 22) according to the four bonding techniques to be performed using Adper Single Bond 2 (SB) [3M, ESPE] adhesive. In all the four groups, pulp chambers were acid etched with 37% phosphoric acid for 15 s followed by rinsing and blot drying.
Group 1 (WWB): Water-Wet Bonding - acid-etched dentin surface was bonded with SB according to manufacturer's instructions.
Group 2 (EWB): Ethanol-Wet Bonding - acid-etching followed by chemically dehydration treatment with 100% ethanol for 1 min and gently blot dried before bonding.
Group 3 (WWB + PA): Water-Wet Bonding with Proanthocyanidins Application - acid-etching followed by treatment with 15% PA for 1 min.
Group 4 (EWB + PA): Ethanol-Wet Bonding with Proanthocyanidins Application - acid-etching followed by treatment with 100% ethanol for 1 min and successive application of 15% PA for 1 min.
After this, composite core build-up restorations were done in the pulp chambers of all the samples using a dual cure composite, Core X Flow [Dentsply Caulk, Milford, USA] and light cured at 500 mw/cm 2 by Spectrum 800 [Dentsply, Caulk, Milford, USA] for 20 s according to the manufacturer's instructions. All the samples were then subjected to dye penetration test.
Twenty samples from each group were then coated with two layers of nail polish leaving 1 mm window around composite restored margins and immersed in 2% methylene blue dye for 2 days. They were washed under running water and air dried at room temperature for 24 hours. All 80 samples were later sectioned for evaluation under stereomicroscope (magnification 10x) and were scored as follows: (0 = no leakage, 1 = leakage extending into pulp chamber, 2 = leakage involving pulp floor, and 3 = leakage involving root canal).
Scanning electron microscopic (SEM) evaluation
Two samples per group were used for SEM analysis. The restored samples were sectioned mesiodistally and polished with wet 210 grit SiC paper. Acid-base treatment (6N HCl for 30 s followed by 4% NaOCl for 10 min) was done, and the samples were dehydrated in ascending ethanol concentrations (50%, 75%, and 95% for 20 min each and 100% for 1 h) and then transferred to a critical point dryer for 30 min. The specimens were then gold sputter coated and the surfaces were examined under an SEM.
The microleakage scores obtained were subjected to statistical analysis using Kruskal-Wallis and Mann-Whitney U-tests (SPSS Base 15.0 software) at significance level of P < 0.05.
| Results|| |
The mean microleakage scores of different groups and P values are presented in [Table 1] and [Table 2]. The maximum microleakage scores were found in group 1 (WWB) followed by group 3 (WWB + PA), with no significant difference between them (P = 0.510). Least microleakage was observed in group 4 (EWB + PA) with similar results seen in group 2 (EWB) (P = 0.918). Group 2 (EWB) showed significantly less microleakage than group 1 (WWB; P = 0.002) and group 3 (WWB + PA; P = 0.009). Group 4 (EWB + PA) also depicted significantly reduced microleakage score as compared with group 1 (WWB; P = 0.001) and group 3 (WWB + PA; P = 0.003).
The representative SEM photomicrographs of each group are shown in [Figures 1] [Figure 2] [Figure 3] [Figure 4]. Scanning electron microscopic observation of resin-dentin interfaces demonstrated generalized gap along the entire interface showing poor adaptation after bonding with SB using WWB technique in both group 1 (WWB) and group 3 (WWB + PA) [Figure 1] and [Figure 3]. Ethanol-wet-bonded groups with and without PA (group 2 - EWB and group 4 - EWB + PA) revealed perfect adaptation depicting the absence of interfacial gaps after bonding with SB [Figure 2] and [Figure 4].
|Figure 1: Scanning electron microscopic view depicting generalized gap at resin-dentin interface after bonding with SB using water-wet bonding technique. (Group 1: WWB)|
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|Figure 2: Scanning electron microscopic view depicting perfect interfacial seal after 100% ethanol pre-treatment and bonded with SB. (Group 2: EWB)|
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|Figure 3: Scanning electron microscopic view depicting pooradaptation at resin-dentin interface with SB bonded to 15% PA pretreatedpulp chamber dentin using water-wet bonding technique.(Group 3: WWB + PA)|
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|Figure 4: Scanning electron microscopic view depicting absence of gap and good interfacial adaptation after 100% ethanol and 15% PA pre-treatment of pulp chamber dentin bonded using SB. (Group 4: EWB + PA)|
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| Discussion|| |
In tooth-colored restorative materials, early loss of restoration is no longer a clinical problem. However, marginal leakage and consequential marginal discoloration remains the most frequent reason to replace/repair an adhesive restoration.  Therefore, besides bond strength, testing of marginal sealing effectiveness of adhesives is needed.
In this study, significant reduction in microleakage was observed when adhesive was applied following EWB in a clinically relevant simplified dehydration protocol with 1-min application of 100% ethanol. This may be attributed to the fact that ethanol, is a desirable solvent for most hydrophobic resin monomers, which increases the resin-dentin bond durability as it can slowly replace residual water in the dentin matrix by chemical dehydration, thus preventing collagen hydrolysis, plasticization of resin, and enzymatic degradation of resin-dentin interface.  It produces shrinkage of the collagen fibrils thereby enlarging the interfibrillar spaces and allowing better resin-infiltration and sealing of the demineralized collagen matrix, thus resulting in improved bonding efficacy of contemporary etch-and-rinse adhesives. , Our findings are supported by the study of Sauro et al. who compared hybrid layers created with commercially available etch-and-rinse adhesive using WWB or EWB, and reported significantly less micropermeability of the fluorescent tracer in hybrid layers created with EWB.  Cadenaro et al. reported significantly less permeability of resins bonded to ethanol-saturated dentin as compared with water-saturated dentin.  Shin et al. also observed that wet bonding with ethanol instead of water permitted higher infiltration of hydrophobic BisGMA monomer and better collagen encapsulation thereby improving the quality of resin-dentin interface. 
Li et al. concluded that compared with WWB, EWB could improve the bonding efficacy of contemporary etch-and-rinse adhesives.  And this positive effect of EWB might be influenced by the composition of adhesives. Hosaka et al. reported that EWB increased the resin-dentin bond strength and durability.  Duan et al. found a significant increase in shear bond strength of experimental hydrophobic and commercial hydrophilic adhesives to root dentin when used with EWB technique compared with WWB.  Sauro et al. evaluated the bond strength of resin-dentin interfaces created with adhesives applied on root canal dentin using the WWB or EWB technique.  They reported that EWB technique gave higher bond strength values for all the adhesives tested.
Findings from these studies suggest that EWB technique may help in overcoming the deficiencies associated with contemporary etch-and rinse adhesives, mainly, their inability to replace free and loosely bound water from the intrafibrillar compartments of water-saturated collagen fibrils. Biomimetic remineralization provides an indirect evidence for this phenomenon, as intrafibrillar spaces created by simplified adhesives are amenable to remineralization by apatite crystallites.  Tay and Pashley  reported absence of intrafibrillar remineralization and nanoleakage following EWB technique thus emphasizing that resin can impregnate the intrafibrillar compartment completely if it is saturated with ethanol instead of water.  However, ethanol replacement should be meticulously performed to prevent water-saturated collagen from exposure to air as the surface tension present along the air-collagen interface can easily result in collapse of the collagen matrix and prevent optimal infiltration of the adhesive monomers. During application of the adhesive, dentin matrix should be fully saturated with ethanol.
The properties of dentin-resin interface may be enhanced by the establishment of tissue engineering/biomimetics approaches to improve the intrinsic properties of the bonding substrate i.e. dentin.  This may be achieved with the help of collagen cross-linkers like PA, which are a class of bioflavonoids, natural antioxidants, and matrix metalloproteinases inhibitors that have proven to be safe in different clinical applications and as dietary supplements. It stabilizes the collagen in vivo and in vitro by the primary mechanism of hydrogen bond formation between the protein amide carbonyl and the phenolic hydroxyl.  Treating root dentin with PA-rich grape seed extracts has improved the biodegradation resistance of demineralized root dentin and enhanced the bond strength and durability between resin-based sealer and root dentin after short-term water storage. 
However, incorporation of PA directly into the adhesive may have a deleterious effect on the radical polymerization of adhesive monomers as they are known to possess free radical scavenging property.  While the alternative use of PA, namely, as a primer, could potentially avoid this side-effect, previous studies have reported only clinically unfeasible application times varying from 10 min to 1 h. , Proanthocyanidins can effectively cross-link collagen and improve its biological stability in time periods as short as 10 s. The use of PA is therefore clinically feasible and is a promising approach to improving the durability of current dentin bonding systems. 
However in our study, application of PA did not improve the sealing ability of simplified etch-and-rinse adhesive. Similar results have been reported in other studies that reported no significant difference in immediate resin-dentin bond strength with or without PA application. [, This might be attributed to the shorter duration of application (1 min) or the high C-factor in pulp chamber as opposed to flat surface used in bond strength studies. ,, First, as compared to coronal dentin, the dentin of the pulp chamber is more complex in structure with the presence of collagen-rich predentin, sclerotic dentin, regular and irregular secondary dentin, and several accessory canals. It contains less amount of intertubular dentin due to greater number of dentinal tubules and large tubule diameters.  Second, high C-factor associated with the box shape of the pulp chamber leads to greater polymerization shrinkage stress, thereby negatively affecting the microleakage of the adhesives. Third, adhesion may be influenced by the characteristics of the bonding substrate. Here, the bonding substrate is pulp chamber dentin that is devoid of smear layer as compared to coronal dentinal surfaces that are inevitably covered with smear layer after instrumentation.
Moreover, contrary to our study, some authors found that in vitro application of PA to etched dentin significantly enhanced the immediate dentin bond strength. , However, in their study, PA was applied for 1 hour and 5-10 min, respectively, which might account for the discrepancy from our results. But such application protocols are time consuming and have been judged clinically unrealistic to justify the use of PA, which is not in case of our study.
Although it has been reported that ethanol decreases the dielectric constant of the media, stimulating PA and collagen interactions for longer periods,  no such interaction appeared to be evident in our study when PA was applied to ethanol-saturated dentin matrix during EWB as no significant decrease in microleakage was observed.
Further studies should aim to delineate guidelines for the time, the concentration and the source of PA and development of simplified EWB technique for use with different adhesive systems in order to improve their bonding effectiveness. One shortcoming of grape-seed extract PA treatment is that it stains dentin reddish brown. Future research should be directed toward isolating PA components or synthesizing PA-mimicking compounds that inherit the cross-linking ability of PA while exhibiting less intense color. The present study evaluated the bonding potential to pulp chamber dentin; however, the durability of bonding with simplified adhesives after various pre-treatments needs to be tested in clinical conditions.
| Conclusion|| |
The use of EWB technique in a clinically relevant simplified dehydration protocol significantly reduced microleakage in simplified etch-and-rinse adhesive bonded to pulp chamber dentin. However, application of PA had no significant effect on the microleakage of adhesive bonded with either WWB or EWB.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]
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