EFFECTIVENESS OF GRAPE SEED EXTRACT-BASED INTRACANAL DRESSINGS AGAINST Enterococcus Faecalis AND ITS INFLUENCE ON DENTIN MICROHARDNESS AND BOND STRENGTH OF FILLING MATERIAL EFICÁCIA DE MEDICAMENTOS INTRACANAIS À BASE DE EXTRATO DE SEMENTE DE UVA CONTRA O Enterococcus Faecalis E SUA INFLUÊNCIA NA MICRO-DUREZA DA DENTINA E NA RESISTÊNCIA DE UNIÃO DO MATERIAL OBTURADOR

The aim of this study was to evaluate the effectiveness of grape seed extract (GSE)based intracanal dressings against Enterococcus faecalis (E. faecalis) and its influence on dentin microhardness and bond strength of the filling material. The root canals of 126 human teeth were distributed into three test groups: antimicrobial activity (60 teeth), dentin microhardness (30 teeth) and bond strength (36 teeth). In all three groups, specimens were subdivided into six groups, according to intracanal dressing protocols: G1 – distilled water (DW); G2 – 2% chlorhexidine gel (CHX); G3 – calcium hydroxide (Ca[OH]2)+DW; G4 – GSE+DW; G5 – Ca(OH)2+CHX; G6 – GSE+CHX. The counting of colony-forming units (CFUs), the Vickers microhardness tester and the push-out test were performed to evaluate the antimicrobial activity, dentin microhardness and bond strength, respectively. Specific statistical analysis was performed for each evaluation (α=5%). The greatest bacterial reduction was observed in G5 (Ca[OH]2+CHX) and G6 (GSE+CHX) (p<0.05). There was no statistically significant difference among groups in the dentin microhardness evaluation (p<0.05). The highest bond strength in the immediate evaluation was observed in G4 (GSE+DW) and G6 (GSE+CHX), whereas the highest bond strength after 12 months of storage was observed in G2 (CHX), G3 (Ca[OH]2+DW), G4 (GSE+DW), and G6 (GSE+CHX) (p<0.05). After the storage period, bond strength was increased in G2 (CHX) and G3 (Ca[OH]2+DW), and remained unchanged in G4 (GSE+DW) and G6 (GSE+CHX) (p<0.05). GSE-based intracanal dressings have antimicrobial potential against E. faecalis, have no influence in dentin microhardness and preserve the high bond strength of filling materials for root dentin over time.


INTRODUCTION
Conventional chemo-mechanical preparation by using irrigants and endodontic instruments does not promote complete neutralization of microorganisms during endodontic therapy (SOUZA et al., 2018). This occurs due to the presence of resistant bacteria in the endodontic microbiota that colonize root canals and persist after decontamination procedures (TENNERT et al., 2014). Furthermore, due to the anatomical complexity of the root canal system, microorganisms, inorganic and organic components cannot always be reached by chemical auxiliary substances, and some areas of the main root canal are also not reached by endodontic instruments (VAUDT et al., 2009). Thus, additional antimicrobial strategies are necessary, such as intracanal dressings, in order to aid in the decontamination of the root canal system.
Currently, there are a growing number of studies testing natural substances in the endodontic field, since literature has shown antimicrobial potential against endodontic pathogens for these substances, such as grape seed extract (GSE) (CECCHIN et al., 2015;SOUZA et al., 2017). GSE represents a natural compound, which has proanthocyanidins (PACs) in its composition. PACs are recognized to present therapeutic activities, providing antioxidant, antimutagenic and antibacterial effects (FURIGA; ROQUES; BADET, 2014). In addition, PACs are known to enhance the dentine biomechanical properties and biostability . However, previous studies have reported the use of GSE as chemical auxiliary substance or final decontamination protocol (CECCHIN et al., 2015;SOUZA et al., 2017). There are no studies in literature regarding the use of GSE as intracanal dressing, evaluating the antimicrobial effectiveness and the influence on dentin mechanical properties and bond strength of the filling material.
The purpose of this study was to evaluate, in vitro, the effectiveness of GSE-based intracanal dressings against E. faecalis and its influence on dentin microhardness and bond strength of filling materials. The present hypotheses were that GSEbased intracanal dressings (i) promote effective reduction of E. faecalis, (ii) do not interfere in dentin microhardness, and (iii) improve the bond strength of the filling material.

Ethical aspects
This study was approved by the local ethics committee (Passo Fundo, RS, Brazil) (protocol 2.033.198).

Specimen acquisition and preparation
One hundred and twenty six extracted single-rooted human teeth were obtained from the Biobank of the School of Dentistry University of Passo Fundo. Only teeth with a single canal, complete root apex, similar buccal-lingual / mesiodistal dentin thickness and absence of root curvature were included in the present study. The teeth that did not have these characteristics were not included in the present study. Dental crowns were sectioned at the cement-enamel junction using a rotating diamond saw so that all roots remained with length of 15 mm. The cervical third was prepared using Largo drill #3 (Dentsply-Maillefer, Ballaigues, Switzerland). The working length was established by inserting a K-file #10 (Dentsply-Maillefer) into the canal until its tip was visualized at the apical foramen. From this measurement, 1 mm was subtracted, thus establishing the working length. Root canals were enlarged to the working length using the ProTaper system (Dentsply Tulsa Dental Specialties, Johnson City, TN, USA), following the sequence S1 to F3. ProTaper files were used in a 16:1 reduction handpiece with controlled torque under constant rotation of 300 rpm, inserted into the root canal in the crown-apex direction, performing a smooth digital back-and-forth movement. The irrigant solution was distilled water (DW) (Natupharma, Passo Fundo, RS, Brazil), which was replaced with each change of instrument. Firstly, 1 mL of DW was inserted into the root canal with extravasation to root canal entrance. The S1 file worked until the WL and after that irrigation with 5 mL of DW was performed. This step was repeated with S2, F1, F2 and F3 files, totalizing 30 mL of DW that were used in each canal of each specimen.
After instrumentation, root canals were irrigated with 3 mL of 17% EDTA (Natupharma, Passo Fundo, RS, Brazil), which remained for 1 minute into the root canal in order to remove the smear layer. Then, root canals were irrigated with 5 mL of DW again and dried using suction cannula. The 126 roots were randomly divided into three experimental test groups, as follows: 60 roots were used for antimicrobial activity evaluation, 30 roots for microhardness evaluation and 36 roots for bond strength evaluation. The sample size for each method was based in previous studies that also evaluated antimicrobial activity of antimicrobial strategies (SOUZA et al., 2017) and its influence on dentin microhardness (LYSAGHT; DEBELLIS, 1969) and bond strength (CECCHIN et al., 2015).

Antimicrobial activity evaluation
The 60 roots had its apical foramen sealed with composite resin. Each root was fixed with Putty-C Silicone for Impression (Silon2APS -Dentsply, Petrópolis, RJ, Brazil) in a plastic micro-tube (Axygen Inc, Union City, CA, USA), so that it remained upright with the cervical portion facing upwards. Then, all roots were sterilized at 120 °C in autoclave (Dabi Atlante, Ribeirão Preto, SP, Brazil) for 30 min. After sterilization, six teeth were randomly submitted to sterilization control. Sterile paper point #30 (Tanari, Manucapuru, AM, Brazil) was placed in contact with the root canal walls for 30 seconds and individually transported to plastic microtube containing 1 ml of 0.9% saline solution (Basso, Caxias do Sul, RS, Brazil). The material was homogenized and an aliquot of 100 µL of saline solution was cultivated on blood agar after five minutes. Samples were incubated at 37 ºC for 48 hours, in order to verify bacterial growth. Samples did not show any bacterial growth.
Subsequently, a 100-μL aliquot of E. faecalis culture (ATCC 19433) was inoculated into the root canal of each specimen. The remaining volume was completely filled with sterile brainheart-infusion (BHI) broth (Acumedia -Neogen, Lansing, MI, USA). The E. faecalis culture was maintained for 14 days, promoting bacterial growth. The BHI broth was replaced every 48 hours. Once a week, an aliquot of BHI obtained from six teeth randomly selected was submitted to Gram staining and cultured on blood agar, followed by catalase and esculin tests, in order to verify the absence of contamination with other microorganisms. This fact was detected, obtaining pure of E. faecalis culture after 14 days.
After contamination, the 60 specimens were irrigated with 5 mL of DW and randomly divided into six groups (n=10) according to the intracanal dressing: G1 -DW (negative control); G2 -2% chlorhexidine gel (CHX); G3 -Ca(OH) 2 +DW; G4 -GSE+DW; G5 -Ca(OH) 2 +CHX; and G6 -GSE+CHX. The first specimen was chosen randomly from all specimens and assigned to group 1, the second specimen to group 2, and so on to group 6. The distribution was repeated until each group presented the complete number of specimens.
In G1 (DW) and G2 (CHX), the intracanal dressing was inserted with a 3 mL disposable syringe (Descarpack, São Paulo, SP, Brazil) and a 30 gauge needle (Navi-Tip -Ultradent, South Jordan, UT, USA), until the root canal was completely filled. In G3 (Ca[OH] 2 +DW) and G4 (GSE+DW), the intracanal dressing was obtained from mixing 0.1 g of the test substance powder and 100 µl of DW. After the paste was obtained, root canals were completely filled with the respective intracanal dressing using Lentulo drill (Dentsply-Maillefer, Ballaigues, Switzerland), which was positioned 3 mm short of the working length. In G5 (Ca[OH] 2 +CHX) and G6 (GSE+CHX), the intracanal dressing was obtained from mixing 0.1 g of the test substance powder and 100 µl of CHX. After the paste was obtained, root canals were completely filled with the respective intracanal dressing in the same way as that described for G3 and G4.
The 60 specimens were sealed with provisional restorative material (Cavitec -Caitthec, Rio do Sul, SC, Brazil) and stored for 14 days at 37 o C under humidity. After 14 days, the provisional restoration and intracanal dressing were removed by using #1012 drill (Dentsply-Maillefer, Ballaigues, Switzerland) and irrigation with 10 mL of DW respectively.
Microbiological analysis was performed in two stages: initial sample (S1) -after contamination and before intracanal dressing protocols; and final sample (S2) -after 14 days of storage and removal of intracanal dressing protocols. In both stages, root canals were filled with sterile saline solution. Sterile K-file #30 (Dentsply-Maillefer) was inserted at the working length, promoting contact with the root canal walls for 30 seconds. Sterile absorbent paper point #30 (Tanari) was placed into the root canal in the same way for 30 seconds, after which, it was transferred to tube containing 450 μL of sterile saline solution. The material was homogenized and diluted to 1×10 -3 . Aliquots of 100 μL of the solution and all dilutions were cultivated on the blood agar surface in duplicate. Samples were incubated for 18-24 h at 37 C. The number of colony-forming units (CFUs) was then counted on plates.
The effectiveness of intracanal dressing protocols was analyzed according to the percentage reduction of E. faecalis from initial (S1) and final (S2) samples. All procedures were performed under aseptic conditions in a laminar flow chamber.

Microhardness evaluation
After obtaining specimens and removal of dental crowns, two longitudinal grooves were produced in the external root surface of each of the 30 roots without reaching the canal space, using a diamond disc. Chemo-mechanical preparation was performed as previously described in the antimicrobial activity evaluation. The 30 specimens were randomly divided into six groups (5 roots per group) and submitted to the same intracanal dressing protocols, as previously described in the antimicrobial activity evaluation.
After 14 days, the provisional restoration and intracanal dressing were removed using #1012 drill (Dentsply-Maillefer) and irrigation with 10 mL of DW respectively. Roots were split into two halves with hammer and chisel, providing 2 halves of each root and resulting in 10 specimens per group (n=10).
Roots were embedded in self-curing acrylic resin held in plastic rings. The coronal portion of each segment was ground with carborundum disks (300, 600, and 1200 grade) under cooling and then polished using diamond paste. Microhardness measurements were performed on each section at 500 µm from the pulp-dentin interface, and two other indentations were made at distance of 200 µm from each other. At each depth, one indentation was made using 10-g load perpendicularly oriented to the indentation surface for 15 seconds. Measurements were performed with Knoop Microhardness Tester (HMV 2000 -Shimadzu, Kyoto, Japan). The dentin microhardness value for each specimen was obtained as the average of the results for three indentations.

Bond strength evaluation
After obtaining specimens and removal of dental crowns, chemo-mechanical preparation was performed in each one of 36 remaining roots, as previously described in the antimicrobial activity evaluation. The 36 specimens were randomly divided into six groups (6 roots per group) and submitted to the same intracanal dressing protocols, as previously described in the antimicrobial activity evaluation. After 14 days, provisional restoration and intracanal dressing were removed using #1012 drill (Dentsply-Maillefer) and irrigation with 10 mL of DW respectively. Then, root canals were dried with suction cannula and absorbent paper points.
Root canals were filled by lateral compaction using gutta-percha points and AH Plus endodontic sealer (Dentsply Tulsa Dental Specialties). Excess filling material was removed by cutting with #2 heated plugger (SS White Duflex, Rio de Janeiro, RJ, Brazil). The 6 specimens of each group were randomly divided into 2 subgroups (3 roots per subgroup), according to their storage period: 21 days and 12 months of storage, being immersed in DW at 37 o C.
In both evaluation periods, each root was transversely sectioned from the root canal entrance into 1-mm-thick discs in a metallographic cutter with diamond disk at speed of 350 rpm under cooling. The first disc was discarded and the next five root discs were selected from each root, totaling 15 specimens per group (n=3×5=15). Each disc was submitted to the push-out test in a mechanical testing machine (EMIC DL 2000 -São José dos Pinhais, PR, Brazil) at speed of 1 mm/min, until displacement of the filling material. Care was taken to ensure that the contact between the punch tip and the filling material occurred over the most extended area as possible to avoid the notching effect of the punch tip on the filling material surface. Furthermore, the punch tip was centralized in the root canal and positioned to contact only the filling material without stressing the surrounding root canal walls. The force required to displace the material from the root canal was recorded in newtons (N). To express the bond strength in megapascals, the load at failure recorded in N was divided by the area (mm 2 ) of the filling material interface. To calculate the bonding area, the formula π(R + r) [(h) 2 +(Rr) 2 ] 0,5 was used, where "R" represents the coronal root canal radius, "r" the apical root canal radius, and "h" the disc thickness. Debonded specimens were measured under 20x magnification with stereoscope to classify the failure pattern into three types: 1: adhesive failure (between the dentin and the filling material, presenting absence of filling material on the root canal walls); 2: cohesive failure (presence of filling material on the root canal walls); 3: mixed (failures 1 and 2 may be observed) (figure 1).  Bacterial reduction and dentin microhardness values were statically analyzed by one-way analysis of variance (ANOVA), followed by post-hoc Tukey test. Bond strength values were statically analyzed by Kruskall-Wallis test, followed by post-hoc Student-Newman-Keuls test. The failure mode distribution was evaluated by the chisquare test. All tests were set at 5% significance level. Data were analyzed using Stat Plus AnalystSoft Inc. version 6.0 (Vancouver, BC, Canada).

RESULTS
The mean and standard deviation of E. faecalis percentage reduction and dentin microhardness values after intracanal dressing protocols (table 1). The greatest bacterial reduction was observed in G5 (Ca[OH] 2 +CHX) and G6 (GSE+CHX) (p<0.05). There was no statistically significant difference among groups in the dentin microhardness evaluation (p<0.05). The bond strength median and percentile values after intracanal dressing protocols and storage periods (table 2). The highest bond strength value in the immediate evaluation was observed in G4 (GSE+DW) and G6 (GSE+CHX), whereas the highest bond strength value after 12 months of storage was observed in G2 (CHX), G3 (Ca[OH] 2 +DW), G4 (GSE+DW), and G6 (GSE+CHX) (p<0.05). Regarding the failure pattern, high number of adhesive failures was observed in the immediate evaluation and cohesive failures after 12 months of storage, regardless of intracanal dressing protocol (p<0.05). * Data are presented as median and percentiles. Different superscript index letters represent statistically significant differences in the column; different lower script index letters represent statistically significant differences in the row (p<0.05); ** DW = distilled water; CHX = 2% chlorhexidine gel; Ca(OH) 2 = calcium hydroxide; GSE = grape seed extract; MPa = megapascals.

DISCUSSION
The use of intracanal dressings results in improved microbiological and structural status of the root canal system (VERA et al., 2012), and adequate tests are necessary to evaluate their properties. The counting of CFUs provides bacterial quantification of root canals in an acceptable way, allowing calculating the E. faecalis percentage reduction (PETERS; WESSELINK; MOORER, 1995;MATOS et al., 2019). The Knoop test is more sensitive to surface conditions and, due to its longer diagonals, it is less sensitive to measure errors when equal loads are applied (LYSAGHT; DEBELLIS, 1969). The push-out test is compatible with the clinical situation, produces effective power for SOUZA, M. A. et al.
Biosci. J., Uberlândia, v. 36, n. 6, p. 2288-2296, Nov./Dec. 2020 http://dx.doi.org/10.14393/BJ-v36n6a2020-54066 material displacement and induces less stress at the bond interface during disc preparation (HUFFMAN et al., 2009). Thus, the present study used these tests, in order to evaluate the use of GSE-based intracanal dressings against E. faecalis and its influence on dentin microhardness and bond strength of the filling material.
The antimicrobial mechanism of Ca(OH) 2 is known to be dependent on the dissociation and diffusion of hydroxyl ions, elevating pH and inhibiting enzymatic activities (LEONARDO et al., 2006). Previous studies have reported that 14-21 days are necessary to obtain elevated pH and effective antimicrobial activity of Ca(OH) 2 (LEONARDO et al., 2006). In addition, due to microbial resistance, some studies suggested the addition of CHX to Ca(OH) 2 -based intracanal dressings, in order to improve antimicrobial activity (GOMES et al., 2006;SIGNORETTI et al., 2011). 14 days of maintenance in root canals and the addition of CHX to test intracanal dressings were adopted in the present study.
Intracanal dressings have effective action against inflammatory contents of root canals with primary infections (MARTINHO et al., 2018) and primary periodontal lesions with secondary endodontic involvement (DUQUE et al., 2019). The present results showed that GSE-based intracanal dressings (groups G4 and G6) were as effective as Ca(OH) 2 -based intracanal dressings in reducing the count of the E. faecalis, confirming the first hypothesis (i) of the present study. These findings are in accordance with previous studies, where the use of GSE showed effective bacterial reduction in root canals (CECCHIN et al., 2015;SOUZA et al., 2017). GSE is a natural product composed of PACs, which is structurally formed by flavonols. These polyphenolic components present antimicrobial activity against gram-positive bacteria, acting in structural components of the bacterial cell wall (FURIGA; ROQUES; BADET, 2014). It may help to explain the satisfactory results of GSE-based intracanal dressings in the reduction of E. faecalis, after 14 days in the root canal.
CHX is a cationic bisguanide that binds to the cell wall of the microorganism and causes the leakage of intracellular components (DAMETTO et al., 2005). The addition of CHX to test intracanal dressings revealed higher reduction in the count of E. faecalis, being in accordance with previous studies that obtained similar results with the addition of 2% CHX to intracanal dressings (GOMES et al., 2006;SIGNORETTI et al., 2011). The antimicrobial mechanism occurs by the interaction between CHX molecules (positively charged) and bacterial cellular wall (negatively charged). Then, CHX induces changes in cellular osmotic equilibrium, resulting in the precipitation of cytoplasmic content and the death of microorganisms (DAMETTO et al., 2005). Therefore, the antimicrobial properties of test intracanal dressings and CHX may explain the greater reduction in the count of E. faecalis in these groups.
Dentin discs are usually obtained by transversal cuts from the roots of human teeth, in order to investigate the effects of chemical substances on dentin microhardness (SAYIN et al., 2007). In these cases, the dentin surface is covered by the chemical substance and the microhardness is measured after this protocol. However, the intracanal dressing contact is initially with the most superficial dentin layer of the root canal lumen and then diffuses through dentinal tubules (SLUTZKY-GOLDBERG et al., 2004). Thus, the present study adopted the same methodology of Slutzky-Goldberg et al. (2004), where roots are filled with the chemical substance for a period of time, being longitudinally split to measure the microhardness in the pulp-dentin interface. This procedure seems closer to a clinical situation, providing better approach regarding the effects of test intracanal dressings on dentin structure. Ca(OH) 2 -based intracanal dressings can lead to physical changes on dentin structure, since they promote denaturation of the root dentin collagen matrix. It is caused by the low molecular weight and highly alkaline pH of Ca(OH) 2 (LEIENDECKER et al., 2012). On the other hand, the results of present study revealed that GSE-based intracanal dressings did not influence the dentin microhardness values, being similar to control group and Ca(OH) 2 groups, regardless of CHX addition. The second hypothesis (ii) of this study is then confirmed. Extracts rich in PACs improve dentin mechanical properties due to their highly stable interaction with dentinal collagen. It occurs by the formation of hydrogen bonds between the protein amide carbonyl and the phenolic hydroxyl group of the polyphenol associated with covalent and hydrophobic bonds. Covalent-like bonds resulted from this interaction can help preserving the dentin structure (BEDRAN-RUSSO et al., 2014).
According to the results obtained in the immediate evaluation, the highest bond strength values were observed in groups treated with GSEbased intracanal dressings, regardless of CHX addition. After 12 months, bond strength values were maintained in these groups. The third hypothesis (iii) of this study was then confirmed. SOUZA, M. A. et al. Biosci Cecchin et al. (2015) showed that GSE preserves the bond strength of fiber posts to root dentin for 12 months, improving bond strength stability of dentin-adhesive interfaces in root canals. PACs present in its composition preserves the mechanical properties (CECCHIN et al., 2015) and stabilizes the dentin collagen degradation . It contributes to the maintenance of dentinal substrates for satisfactory bond strength of resinbased filling materials and prevents microbial leakage, creating a favorable environment for regeneration in the periapical region .
After 12 months of storage, the bond strength values decreased in the control group, where intracanal dressing protocol was not performed. On the other hand, the bond strength values increased in G2 (CHX) and G3 (Ca[OH] 2 ). The use of CHX does not interfere with collagen present in the organic matrix of the root dentin (MOREIRA et al., 2009). Similarly, Ca(OH) 2 residues have a positive effect on the dislodgement resistance of root canal sealers (AMIN; SEYAM; EL-SAMMAN, 2012). It contributed to increase the bond strength values in these groups. In addition, the present results showed higher number of adhesive failures in the immediate evaluation and cohesive failures after 12 months of storage. AH Plus epoxy-resin-based endodontic sealer has high flowability and long polymerization time. Moreover, cohesion among AH Plus molecules increases the displacement resistance of the filling material in the root dentin over time, providing greater adhesion (SILVA et al., 2016). It helped to explain the present results regarding the failure pattern.

CONCLUSIONS
Despite the limitations of the present study, it was possible to conclude that GSE-based intracanal dressings have antimicrobial potential against E. faecalis, do not influence dentin microhardness and preserve the high bond strength of the filling material to root dentin over time.