Influence of ethanol on dentin roughness , surface free energy , and interaction between AH Plus and root dentin

This study aimed to evaluate the influence of different ethanol concentrations on dentin roughness, surface free energy, and contact angle between AH Plus and the root canal dentin. One hundred human maxillary anterior teeth were split longitudinally and 200 dentin specimens were polished to make the surface flatter and smoother. An acrylic bar was positioned between two dentin specimens and impression material was added to create a block, simulating an instrumented root canal space. Specimens were removed from the mold and cleaned in an ultrasonic bath for 10 min. Thereafter, dentin specimens were divided into four groups (n = 50) according to the drying methods used: a) wet: vacuum only, b) paper points: vacuum + absorbent paper points, c) 70% alcohol: 70% alcohol (1 min) + vacuum + absorbent paper points, and d) 100% alcohol: 100% alcohol (1 min) + vacuum + absorbent paper points. A rugosimeter and a goniometer were used to verify the roughness (Ra) and to measure the surface free energy and the contact angle between the AH Plus sealer and the root canal dentin. ANOVA and Tukey tests (α = 0.05) were used for statistical analysis. The 70% and 100% ethanol groups showed significantly decreased roughness as well as increased surface free energy in the root canal dentin when compared to the wet and paper point groups. In addition, ethanol significantly reduced the contact angle between the AH Plus sealer and the root canal dentin. Ethanol solutions (70% and 100%) provide better wettability of AH Plus sealer on dentin surfaces.


Introduction
Obturation is supposed to provide a three-dimensional filling of the root canal system, 1,2 preventing bacterial infiltration from the oral cavity and from periradicular tissues. 1,3,4Filling materials can fill root canal wall irregularities, apical ramifications, and dentinal tubules, 5,6 and entomb irritants that were not removed during chemomechanical preparation. 7,8Thus, the physicochemical properties of the filling material and dentin wall characteristics may affect root canal sealing 9,10 and treatment outcomes.
Wettability is an important thermomechanical property 11 that represents the interactions between solids and liquids.It is strongly dependent on dentin roughness 12 and on surface free energy. 13The surface with a lower contact angle (greater surface free energy) presents high wettability.In solids with high surface free energy, the sealer spreads and interacts better, creating a low contact angle. 14,15entin surface wettability can be changed by using ethanol solutions at different concentrations.Osorio et al. 16 and Pei et al. 17 verified a significant decrease in dentin roughness after all dentin specimens were etched with phosphoric acid and rinsed with water.Based on that, the authors suggested that ethanol might offer potential benefits to root dentin bonding with a hydrophobic adhesive.
Additional ethanol application had been previously proposed -in a technique called ethanol wetbonding -in order to lure hydrophobic monomers into demineralized dentin collagen matrices. 18,19here is a higher compatibility between ethanolsaturated dentin and hydrophobic resin monomers, preventing collagen shrinkage and allowing for higher impregnation. 20This technique has been shown to produce adhesive interfaces with higher bond strength, reduced interface nanoleakage, and increased stability over time when compared to the "water-wet bonding technique." 21,22n endodontics, it is suggested that a final rinse of dentin walls with different substances 23 or with ethanol may alter root dentin properties, enhancing endodontic sealer penetration into dentinal tubules. 24,25he interaction between a hydrophobic sealer and a surface with higher hydrophobic characteristics would provide a low contact angle, higher sealer penetration, greater mechanical interlock into the tubules, retention, sealing ability and, consequently, in vivo antibacterial effectiveness. 7,26owever, further investigation is needed to evaluate the role of physicochemical properties of dentinal surfaces in ethanol application.Therefore, the aim of this study was to evaluate the influence of different ethanol concentrations on dentin roughness, surface free energy, and contact angle between the AH Plus sealer and dentin walls.The null hypothesis tested was that no ethanol concentration would interfere with dentin surface roughness, surface free energy, and interaction between the AH Plus sealer and the root canal dentin.

Methodology Specimen preparation
After approval by the Research Ethics Committee of the Piracicaba Dental School, University of Campinas -FOP-UNICAMP (process no.029/2013), one hundred recently extracted human maxillary anterior teeth were selected.All teeth were cleaned with an ultrasonic scaler, washed with saline solution, and stored in 0.2% thymol at 4°C.The tooth crowns were removed using a 0.3-mm saw microtome (Isomet 1000, Buehler, Lake Bluff, IL, USA).Each root was sectioned longitudinally into two parallel dentin slices with a rotary diamond disk 7020 (KG Sorensen, Cotia, São Paulo, Brazil) at low speed under cool water.An acrylic bar (4 x 4 x 20 mm) was positioned between two dentin specimens and impression material was added to create a block, simulating an instrumented root canal space (Figure 1) and standardizing irrigant volumes.The specimens were removed from the mold and cleaned in an ultrasonic bath for 10 min.The samples were polished with wet 400-, 600-and 1200-grit silicon carbide (SiC) abrasive paper (Carbimet Disc Set, no.305178180, Buehler, Coventry, UK) to make the surface flatter and smoother, according to Hu et al. 11 All samples were treated with 17% ethylenediaminetetraacetic acid (EDTA) for 1 min and this procedure was repeated three times.A final rinse with 5 mL of distilled water was performed.
Two hundred dentin samples were randomly divided into four groups (n=50) according to the following drying methods: Group 1 -drying only with a capillary tip attached to a vacuum adapter (Ultradent Products Inc, South Jordan, USA) for 5 s; Group 2 -drying with a capillary tip attached to a vacuum adapter (5 s), followed by two absorbent paper points (Endo Points Industrial Amazônica, Manacapuru, Brazil); Group 3 -treatment with 70% alcohol (Prolink Indústria Química Ltda., São José do Rio Preto, Brazil) for 1 min, followed by capillary tip attachment to a vacuum adapter (5 s) and two absorbent paper points; Group 4, the same procedures used in Group 3, but the treatment was performed with 100% alcohol (Prolink Indústria Química Ltda., São José do Rio Preto, Brazil) for 1 min.

Roughness measurements
Forty dentin samples (n = 10, each group) were individually positioned in a Surfcorder SE 1700 profilometer (Kosaka Lab, Tokyo, Japan) to verify the roughness (Ra) of the root canal dentin subjected to drying methods.Three readings were performed, in the same middle area on each surface using a stylus tip (0.5 μm in diameter).The maximum track length of each reading was 1.25 mm, using a 0.25mm cutoff filter.The mean and standard deviation of Ra were determined.The Ra parameter describes the overall roughness of a surface and can be defined as the arithmetic mean of all absolute distances of the roughness profile from the centerline within the measuring length.

Surface free energy measurements
A Ramé-hart goniometer (Ramé-hart Instrument Co., Netcong, USA) was used to measure the contact angle between dentin and the following solutions: water (polar), bromonaphthalene (nonpolar), and formamide (polar).The solutions were used to determine the dispersive/polar components and the dentin total free energy by means of the Ramé-hart DROP Image Standard software (Ramé-hart Instrument Co., Netcong, USA).A total of 120 dentin samples were used (30 samples per group and 10 samples per solution).One drop of liquid was measured in each sample.Thirty measurements were made in each drop and the arithmetic mean was then calculated.Based on the data on the three solutions above, the Ramé-hart  3 Braz.Oral Res.2018;32:e33 software was able to measure the surface free energy of the different experimental groups.

Contact angle measurements
The same Ramé-hart goniometer software (Ramé-Hart Instrument Co, Netcong, USA) was used to measure the contact angle between dried dentin surfaces and a hydrophobic endodontic sealer, AH Plus (Dentsply, Petropolis, RJ, Brazil).The sealer was manipulated according to the manufacturer's instructions.A total of 40 samples were used (10 samples per group).A drop of the sealer (0.1 mL) was deposited onto dentin surfaces with a 0.5-mL BD ultrafine syringe and a 20 x 0.55-mm needle (24G) (Becton Dickinson, Franklin Lakes, USA). 23Three drops of the sealer were evaluated for each treatment.Images of the drop were analyzed to provide the values of contact angles.Thirty measurements were made in each sample as recommended by Ramé-Hart Instrument Co.The data were computed with the Origin Pro 70 software (Origin Lab Northampton, USA).

Data analysis
Since the normality assumptions of the data were valid, the data were analyzed with one-way analysis of variance (ANOVA) and Tukey tests, with statistical significance preset at α = 0.05.

Results
Treatment with the ethanol solutions led to decreased roughness and increased surface free energy in the root canal dentin, when compared to the wet and paper point groups (Figures 2 and 3).There was no difference between different ethanol concentrations or between the wet and paper point groups.Lower contact angles between AH Plus and the dentin surface were obtained on surfaces treated with 70% and 100% ethanol (Figure 4).Again, no significant difference was found between ethanol concentrations or between the wet and paper points groups.

Discussion
Ethanol solutions showed a considerable amount of influence on the physicochemical properties of  dentin, leading to the rejection of the null hypothesis.Regardless of the concentration, ethanol caused changes in dentin roughness (decreased) and in surface free energy (increased).Consequently, the contact angles between the endodontic sealer and the dentin surface also changed (decreased).Previous studies 24,25 have evaluated the influence of ethanol on these physicochemical properties and found a better penetration of irrigant solution or endodontic sealer into the dentinal tubules and into small spaces of the root canal system.Pei et al. 17 found lower roughness in dentin treated with stepwise ethanol application, when compared to water-wet dentin.Our results concur with this previous study and show a decrease in roughness and an increase in surface free energy in samples treated with a final rinse of 100% and 70% ethanol.These results can be explained by the ability of ethanol solutions to promote a partial chemical dehydration of demineralized collagen fibers and contraction of dentin collagen network diameters, 16 thereby increasing interfibrillar spaces 27 and favoring hydrophobic material embedment. 19Pei et al. 17 also pointed out that ethanol solution may reduce the intrinsic wetness of the root dentin.
The ethanol wet-bonding technique combines the reduction of polarity of the collagen network with the low polarity of highly hydrophobic resins, since hydrophobic monomers can better infiltrate into the ethanol-saturated demineralized dentin.The association between the ethanol wet-bonding technique and hydrophobic resin blends improves adhesion to the dentinal substrate, thus increasing the longevity of the bonding interface.It is already known that these hydrophobic resin blends have higher stiffness and stability than do hydrophilic ones. 22,28,29,30,31Ethanol wet-bonding can promote the infiltration of hydrophobic dimethacrylate resins into interfibrillar spaces and dentinal tubules to improve stability of resin-dentin interfaces in vitro. 32herefore, as epoxy resin binds to collagen, 33 especially in demineralized dentin, it is suggested that in the presence of alcohol instead of water, some hydrophobic materials, such as AH Plus, may have improved flow into dentinal tubules and infiltration into collagen interfibrillar spaces.
Nagas et al. 9 concluded that the degree of residual moisture can affect the adhesion of root canal sealers to root dentin and suggested that slight moisture would be advantageous to the tested sealers.Taken altogether, their results seem to be completely different from ours.However, Nagas et al. 9 used a 95% ethanol final rinse and stored the roots at 37°C to ensure complete dryness, leading the specimens to extreme dehydration.The moisture conditions tested by these authors are quite close to the drying protocols used by us, but in the ethanol groups, water was replaced with ethanol.Thus, the collagen network was filled with residual ethanol or at least with water/ethanol mixture, preventing its collapse 17 and probably favoring hydrophobic material infiltration.It is important to emphasize that ethanol evaporates more quickly than water. 34herefore, dentin physicochemical tests or even root canal filling should be performed right after the drying protocol to prevent ethanol evaporation and collapse of the collagen network.
The influence of alcoholic solution on periapical tissues should also be considered.A few studies have demonstrated this relationship.Oguntebi et al. 35 evaluated the effect of 95% ethanol on rat teeth with periapical lesions.After daily injections of 0.1 to 0.2 mL of alcoholic solution for 6 weeks, inflammatory cells were found in the apical periodontal ligament.Yesilsoy et al. 36 evaluated the cytotoxic effects of root canal irrigants and found that an 11.6% alcoholic solution in contact with subcutaneous tissue of an in vivo animal model (guinea pig) at different times (2 h, 2 days, and 2 weeks) did not cause inflammatory reactions.Further research should focus on the in vivo assessment of the effect of alcoholic solutions at different concentrations and time intervals on apical periodontal ligament structures.
Taken together, the results showed that 70% and 100% ethanol can improve wettability of AH Plus sealer on the dentin surface, reducing AH Plus contact angles and favoring the interaction between this sealer and the root canal dentin.Further studies should be performed to evaluate whether ethanol solutions could enhance endodontic sealer penetration into dentinal tubules.

Conclusion
Ethanol solutions (70% and 100%) enhance dentin characteristics (roughness and surface free energy), thereby providing better wettability of AH Plus sealer on dentin surfaces.

Figure 1 .
Figure 1.Representative design of dentin sample preparation using impression material in order of fabrication.1. Longitudinal sectioning of the root with a rotary diamond disk, 2. Dentin samples, 3. Polished samples, 4. Acrylic bar between samples, 5. Preparation and placement of impression material, 6. Removal of excess impression material, wax, and acrylic bar, 7. Final samples embedded in impression material and covered with a white sealing tape.