test of AH Plus / gutta-percha , EndoREZ and RealSeal systems

test of AH Plus/gutta-percha, EndoREZ and RealSeal systems Alaa Abdul MAHDI1, Victoria BOLAÑOS-CARMONA2, Santiago GONZALEZ-LOPEZ3 1PhD student, Department of Stomatology, School of Dentistry, University of Granada, Granada, Spain. 2PhD, MD, Associate Professor, Department of Pediatric Dentistry, School of Dentistry, University of Granada, Granada, Spain. 3PhD, MD, Associate Professor, Department of Operative Dentistry, School of Dentistry, University of Granada, Granada, Spain. Corresponding address: Santiago Gonzalez-Lopez Faculty of Dentistry, Department of Operative Dentistry Colegio Máximo s/n. Campus Universitario de Cartuja, University of Granada E-18071 Granada Spain Phone: 958243803 Fax: + 34 958240908 e-mail: sglopez@ugr.es O To investigate the bond strength and seal ability produced by AH Plus/guttapercha, EndoREZ and RealSeal systems to root canal dentin. Material and Methods: Sixty extracted single-root human teeth, instrumented manually to size 40, were divided into three groups (n=20) according to the sealer used; G1: AH Plus, G2: EndoREZ, and group was randomly divided into two subgroups according to the tests applied (n=10 for ! "#$ % & ' quantitative evaluation of apical leakage. Four 1-mm-thick slices (cervical and medium level) were obtained from each root sample and a μPush-out test was performed. Failure modes were examined under microscopy at 40x, and a one-way ANOVA was applied to analyze the permeability. Non-parametrical statistics for related (Friedman's and Wilcoxon's rank tests) or unrelated samples (Kruskal-Wallis' and Mann-Whitney's tests) allowed for comparisons of μPush-out strength values among materials at the different levels. Statistical ' + + 3 5 $6 7 & 7 8 & 9 ! < 3 >?"@ BCD +"$ #$% 9 ' ' 3 >I 2=23.93; p<0.001). AH Plus and RealSeal obtained higher bond strength to intraradicular dentin in the medium I 7 < ' + & < 9 global μPush-out bond strength to root canal dentin achieved by AH Plus/gutta-percha, EndoREZ and RealSeal systems.


INTRODUCTION
The fundamental goals of endodontic therapy are to achieve successful cleaning and shaping of root canals and a hermetic apical seal 25 . An ideal root canal sealer should adhere to both dentin and WKH FRUH ¿OOLQJ PDWHULDO 0HWKDFU\ODWH UHVLQEDVHG sealers, based on dentin adhesion technologies, have been developed in an attempt to seal the root canal system more effectively, improving bonding to radicular dentin 26 , but their utilization requires removal of the smear layer and collagen exposure, because retention is largely achieved by micromechanical interlocking between collagen matrix and resin 29 . Ethylenediaminetetraacetic acid (EDTA) has been widely used for this purpose DV WKH ¿QDO LUULJDQW EHIRUH DSSO\LQJ PHWKDFU\ODWH resin-based sealers 5,13 ; however, the results were poor regarding its bond strength adhesion to root dentin, whereas the non-bonding AH Plus sealer presented greater adhesion 7,10,17,18 .
No correlation has been found between apical microleakage and sealer bond strengths to intraradicular dentin in vitro 1,2 . While we assume 2013;21(4):369-75 that preventing effective microleakage is perhaps more important for endodontic application than bond strength 26 , there is no universally accepted method for the evaluation of leakage 33 . Each has its OLPLWDWLRQV EHLQJ JRYHUQHG E\ HOHFWULFDO ¿OWUDWLRQ and/or diffusion laws 24 . Comparisons between different methods point to contradictory results, and, therefore, questionable clinical relevance 12, 24 .
Push-out bond strength testing has become a common method for determining the effectiveness of adhesion between endodontic materials and intraradicular dentin. Zicari, et al. 34 (2008) have correlated the push-out bond strength and sealing DELOLW\ ZLWK WKH ÀXLG ¿OWUDWLRQ PHWKRG RI DGKHVLYH FHPHQWV URXWLQHO\ XVHG IRU ¿EHUSRVW ERQGLQJ EXW to date there is no documented correlation using methacrylate resin-based sealers. We believe that this aspect is very important, because any ¿OWUDWLRQ FDQ K\GURO\WLFDOO\ GHJUDGH WKH DGKHVLYH interface. Hence, the purpose of this in vitro study was to quantitatively assess the bond strength to dentin root canal and the sealing properties of two methacrylate resin-based sealers and their corresponding core points (RealSeal and EndoREZ), then compare them with the gold standard conventional nonbonding AH Plus/gutta-percha. 7KH 3XVKRXW WHVW DQG ÀXLG ¿OWUDWLRQ PHWKRG were used to test the null hypothesis that there is no difference in the bond strength and sealing properties of RealSeal, EndoREZ systems and AH Plus/gutta-percha.

MATERIAL AND METHODS
We used 60 human anterior teeth, recently extracted for periodontal reasons from anonymous subjects under a protocol (nº 11/2011) approved by the Ethics Committee of the School of Dentistry, University of Granada. All teeth had single straight root canals and closed apices and none showed caries lesions or had received restorative or root canal treatment. After extraction, any calculus and soft tissue was removed and they were stored in 0.1% thymol diluted in distilled water at 4°C for use in this study within 3 months following extraction.
The crowns of the teeth were cut perpendicular to the long axis of the root at the cement-enamel junction with a diamond coated disk at slow speed with constant water-cooling. Root canals were instrumented using the step back technique to REWDLQ D ÀDUHG SUHSDUDWLRQ ,Q DOO WHHWK WKH ZRUNLQJ length was determined visually by subtracting 1mm IURP WKH OHQJWK RI D .¿OH 'HQWVSO\ 0DLOOHIHU Ballaigues, Switzerland) at the apical foramen. Biomechanical preparation was performed manually ZLWK .¿OHV XQWLO VL]H $IWHU HDFK VWHS LQ WKH ÀDUH SUHSDUDWLRQ WKH FDQDO ZDV LUULJDWHG ZLWK sodium hypochlorite during 1 min. Then, 17% EDTA ZDV XVHG DV D ¿QDO LUULJDQW IRU PLQ DIWHU D ULQVH of distilled water (1 min) to remove any remnants of the irrigating solutions. The canals were dried using paper points.
)RU WKH JURXS ¿OOHG ZLWK $+ 3OXV WKH VHDOHU was mixed using AH Plus jet mixing system, and WKHQ LQWURGXFHG LQWR WKH URRW FDQDO RUL¿FHV ZLWK WKH intraoral tip. A 40.02 taper master gutta-percha cone was placed into the canal and then the canal ZDV ¿OOHG XS XVLQJ WKH FROG ODWHUDO FRQGHQVDWLRQ technique and accessory gutta-percha points size #25 and #20 dipped in a small amount of sealer.
)RU WKH JURXS ¿OOHG ZLWK 5HDO6HDO 5HDO6HDO Primer (SybronEndo, Glendora, CA, USA) was introduced into the root canal with a paper point (Roeko, Langenau, Germany) soaked with the primer for the apical region. The primer was placed into the middle and coronal part using a micro brush (Microbrush International, Grafton, WI, USA). After 30 seconds, excess primer was removed with paper points. The sealer was dispensed directly from the tip of its auto mix dual-chamber syringe, according to the manufacturer's instructions. A 40.02 taper RealSeal master point was placed into the canal and then additional RealSeal accessory points size #25 and #20 were placed into the canal using the cold lateral condensation technique.
In all specimens, the excess of points was removed with a hot instrument and the remainder was condensed vertically. They were resected at the apex perpendicular to the long axis of the tooth until 10 mm of root length was obtained. Each root was transferred to a test tube (Eppendorf ,EpULFD 0DGULG 6SDLQ DQG VWRUHG LQ D KXPLGL¿HU at 37°C for 24 hours to complete sealer setting. All preparation and obturation procedures were done by one operator. Finally, each sealer group (n=20) was randomly divided into two subgroups according to tests DSSOLHG Q IRU 3XVKRXW WHVW DQG Q IRU ÀXLG

Leakage evaluation
The external root surfaces of subgroups for the ÀXLG ¿OWUDWLRQ WHVW ZHUH FRYHUHG ZLWK D OD\HU RI QDLO YDUQLVK OHDYLQJ WKH FRURQDO DQG DSLFDO RUL¿FHV open. The specimens were attached to a device WR PHDVXUH WKH SHUPHDELOLW\ E\ ÀXLG WUDQVSRUW described by Pashley, et al. 22 (1988). The roots were inserted into a silicon tube (2 mm internal diameter), and attached with cyanoacrylate glue on the outer surface of the tube. A pressure tank of 250 ml of distilled water was placed 100 cm higher than the root to create a pressure of 100 cm H 2 O. A 10 ml micropipette was inserted and attached with cyanoacrylate glue to measure movement of the air bubble introduced into the system with a micro syringe. Fluid pressure was applied from the coronal area in apical direction. 7KH YROXPH RI ÀXLG WUDQVSRUW ZDV PHDVXUHG E\ observing the movement of air bubbles within a 10 ml micropipette at 24 hours. Previously, the system was sealed and checked for 10 min to ensure there was no leakage. The root was covered with water and then covered with a plastic dish to prevent evaporation. Each specimen was attached to the device, and then the system was bled by opening the joint between the water reservoir and the specimen. The air bubble was then aligned with the zero point of the 10 ml scale. Permeability was measured as the distance that the bubble had moved by the end of 24 hours. This PHDVXUHPHQW VKRZHG WKH DPRXQW RI ÀXLG ZKLFK KDG SHUPHDWHG WKURXJK WKH URRW ¿OOLQJ LQ O $ WHVW was done with two additional roots, covering the apex with two layers of nail varnish, to ensure that WKHUH ZDV QR OHDNDJH RU ÀXLG PRYHPHQW DQ\ZKHUH within the device.

Statistical analysis
The normality of the data distribution in all the VWXG\ JURXSV ZDV YHUL¿HG E\ DSSO\LQJ WKH 6KDSLUR Wilk test, and Levene's test was used to explore the homogeneity of variance among groups.
Fluid movement values at 24 hours were compared among the three different sealers and ¿OOLQJ PDWHULDOV E\ PHDQV RI RQHZD\ $129$ DQG Tukey's tests. To

Micro Push-out bond strength
Descriptive statistics of the μPush-out bond strength are given in Table 2. ANOVA for repeated measures demonstrated that root level (F=27.148, p<0.001), and the interaction between the root level and sealer/core PDWHULDO ) S VLJQL¿FDQWO\ DIIHFW WKH μPush-out bond strength to the walls of the root canal. The sealer/core material in itself does not VLJQL¿FDQWO\ LQÀXHQFH WKH 3XVKRXW ERQG VWUHQJWK values (F=2.492; p=0.102).
)RU WKH ZKROH VDPSOH VWDWLVWLFDOO\ VLJQL¿FDQW differences were detected with respect to the root level (Friedman's test, Chi 2 =23.93; p<0.001). Pairwise comparisons gave differences among all levels, with increased μPush-out values from cervical to apical except between M1 and M2 levels.
μPush-out values of the three sealing systems were also compared in each level. Levels C1 and C2 showed no differences in terms of sealer/core materials, but there were differences between sealers at deeper levels: M1 and M2. RealSeal demonstrated greater bond strength than EndoREZ at M1 level (U=21.00; p=0.028). At M2 level, EndoREZ showed a significantly lesser bond strength than AH Plus (U=22.00; p=0.034) and RealSeal (U=13.00; p=0.005) (Figure 1).

Mode of failure
The predominating type of failure for all three sealer/core materials tested was adhesive failure, followed by mixed type failures. For the whole sample, this distribution was similar among the three sealers used (Chi 2 =2.45, p=0.65), and the four root levels (Chi 2 =4.66, p=0.58) ( Table 3).

DISCUSSION
In this study, permeability and μPush-out bond strength were compared among roots treated endodontically with AH Plus, EndoREZ and 5HDO6HDO VHDOHUV DQG URRW FDQDOV ¿OOHG XVLQJ ODWHUDO condensation technique at 24 hours. The results showed that the permeability and global μPush-out bond strength is not affected by the kind of sealer/ core material.
Microleakage studies are most commonly used to measure the ex vivo sealing ability of a URRW FDQDO ¿OOLQJ WKRXJK YDULRXV PHWKRGV KDYH been proposed or recommended for evaluation of leakage 33 ,Q WKLV VWXG\ DQ HQGRGRQWLF ÀXLG transport model was chosen to evaluate the sealing ability of three different root canal sealers, in view of its advantages: the samples are not destroyed, it provides quantitative measurement, and the results are precise, as small volumes can be recorded 15, 19,24 . We used different samples for ÀXLG ¿OWUDWLRQ DQG 3XVKRXW ERQG VWUHQJWK WR EH performed at exactly 24 hours´ time, thus avoiding different degrees of conversion for the sealers. We did not light-cure the coronal portion of the RealSeal sealer as recommend by manufacturer's instruction, because fast-setting light-cured resin sealers SURGXFH UHVWULFWLRQ LQ WKH ÀRZ DQG FRQVHTXHQW defects or weakening of bond strength 9 . Moreover, the slow chemical reaction of methacrylate-based self-etching sealers may reduce shrinkage stress by PHDQV RI SURORQJHG SODVWLF ÀRZ GXULQJ WKH VHWWLQJ time of the material 14 . We held that such behavior ZRXOG DOORZ IRU FRPSOHWH UHVLQ LQ¿OWUDWLRQ LQWR WKH demineralized dentin.
Our results showed that there were no statistically VLJQL¿FDQW GLIIHUHQFHV LQ WKH YDOXHV RI ÀXLG ¿OWUDWLRQ among the three sealers used in the study. It is noteworthy, however, that none of the investigated sealers/core materials achieved a tight seal at the ¿OOLQJFHPHQWURRW LQWHUIDFH D SRLQW DOVR EURXJKW out by Vasconcelos, et al. 32   the outcome of the tests depends on the evaluation method 12, 24 . In the absence of correlation between the ex vivo VHDOLQJ DELOLW\ RI URRW ¿OOHG WHHWK DQG "clinical success" 20 , we believe there is a threshold for microleakage values that would prove clinically relevant 21 .
The adhesion tests of methacrylate bond sealer to dentin have not yet been standardized. The μPush-out test seems to be more reliable because it allows for adequate standardization of the specimens, the absence of premature failures and the variability of data distribution, while supplying a better estimation of the actual bonding effectiveness 11 . In all of the studies reviewed, biomechanical preparation is done with mechanical instrumentation because it is easier to perform and provides a more appropriate standardization of root canals. We used the step back technique and lateral condensation because it is still widely used throughout the world; moreover, there is no study of endodontic sealer adhesion and μPush-out, EHFDXVH WKH VPDOO ¿QDO GLDPHWHU RI WKH FDQDO PDNHV WKH 3XVKRXW WHVW PRUH GLI¿FXOW WR SHUIRUP In this study, we used three totally different kinds of sealers: a conventional nonbonding epoxy resin-EDVHG VHDOHU $+ 3OXV D ¿UVW JHQHUDWLRQ QRQDFLGLF diurethane dimethacrylate and triethyleneglycol dimethacrylate, EndoREZ; and a second generation sealer, RealSeal, based on adhesive technology with a self-etching primer and in association with a thermoplastic synthetic polyester polymer-based URRW FDQDO ¿OOLQJ PDWHULDO ,W VKRXOG EH QRWHG WKDW the conventional nonbonding AH Plus/gutta-percha URRW ¿OOLQJ ZDV HTXDO WR 5HDO6HDO DQG (QGR5(= These discouraging results are in line with those of Ungor, et al. 31 (2006), who found that the Epiphany/ Resilon combination was not superior to the AH Plus/gutta-percha combination. Still, most studies report that AH Plus sealer presents greater adhesion to dentin than RealSeal, regardless of root canal wall treatment 3,7,18 . Clinicians need to be aware that methacrylate resin-based sealers did not meet expectations regarding adhesion to root dentin, and, at this point in their development, there are QR FOHDU EHQH¿WV LQ WKHLU XVH 26 .
The μPush-out bond strength results were VLJQL¿FDQWO\ LQÀXHQFHG E\ GHQWLQ ORFDWLRQ $+ 3OXV and RealSeal obtained higher values in the deeper slices, while EndoREZ showed the lowest values, regardless of location. All sealers exhibited their low 3XVKRXW ERQG VWUHQJWK YDOXHV LQ WZR VXSHU¿FLDO slices. This can be explained by the presence of oxygen, inhibiting sealer setting and producing a layer with low polymerization.
Because we adopted a clinical approach, we were not able to ascertain whether an increase of the adhesive strength to the apex will remain in the most apical sections. Manual instrumentation until 40 ISO width produce very limited widening of the canal in the apical section, making it impossible to perform μPush-out tests without values having a frictional component with the canal walls. Previous authors have tested apices, but no realistic enlargements were made regarding canal preparation with drills for a post 2 or using a tapered diamond bur 18 .
The values of the μPush-out test are very low, consistent with results of other studies using the same methodology, indicating that the bonding of two methacrylate resin-based sealers and root dentin is much weaker than with resin-dentin bonds, [25][26][27][28][29][30] . Furthermore, the adhesive failures between sealer/dentin interface clearly suggest an inadequate level of adhesion between sealer and dentin in terms of bond strength 2,17,27 . This may EH GXH WR WKH GLI¿FXOWLHV LQ WHVWLQJ PDWHULDOV ZLWK great plasticity, such as gutta-percha, RealSeal and EndoREZ points; in contrast, when the test was done with sealer and no core material, values were higher 1,2 , suggesting failure may be traced to the sealer/material interface. In a root canal WKHUH LV D KLJKO\ XQIDYRUDEOH FRQ¿JXUDWLRQ IDFWRU (ratio of bonded to unbounded resin surfaces) 6 that contributes to maximizing the polymerization stress of resin-based materials along the root canal walls; this may even exceed the bond strength of dentin adhesives to dentin, resulting in gap formation along the surfaces 4,6,8 . Along these lines, Souza, et al. 28 (2012) report that there is a correspondence between the presence of gaps and microleakage.
Despite the material´s relatively low bond strength to root dentin, it may be effective in preventing microleakage 16 . The main problem is that this low μPush-out bond strength is accompanied by ÀXLG ¿OWUDWLRQ 7KH DGKHVLRQ RI UHVLQ VHDOHU WR JXWWD percha or core seems to play an important role in microleakage prevention, since it does not have to be at the sealer-dentin interface. Microleakage can affect the bonding of sealers to dentin by SODVWLFL]DWLRQ ÀXLGV DUH DEVRUEHG E\ UHVLQV 8 and hydrolysis due to water entry in the interface. In addition, collagen degradation may occur due to host-derived matrix metalloproteinases (MMPs) in dentin that are slowly released over time when self-etching adhesives are used 23 . RealSeal may therefore adversely affect the longevity of bonded URRW FDQDO ¿OOLQJV E\ DFFHOHUDWLQJ GHJUDGDWLRQ RI WKH ERQG WKURXJK WKH PRYHPHQW RI ÀXLG EHWZHHQ WKH hybrid layer and unaffected dentin 8,23 .
This study reveals that global μPush-out bond strength and permeability were not affected by different sealer/core materials: this leads us to partially accept the null hypothesis, because in the two deeper slices, RealSeal and AH Plus achieved higher μPush-out bond strength than EndoREZ. :H FDQQRW FRQ¿UP DQ\ FOHDU UHODWLRQVKLS EHWZHHQ