ENDODONTOLOGY. Introduction. Original Research ABSTRACT

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1 Original Research A scanning electron microscopic evaluation of the penetration of root canal dentinal tubules by four different endodontic sealers : A zinc oxide eugenol-based sealer, two resin-based sealers and a Polydimethylsiloxane - based sealer : An in vitro study Ashwini Saraf-Dadpe ## A.I. Kamra * # ABSTRACT Aim and Objectives : The purpose of this study is to evaluate and compare the depth of penetration of Tubli- Seal[EWT], AH Plus, EndoREZ and GuttaFlow sealers into the root canal dentinal tubules using a scanning electron microscope. Materials and methods : Hundred extracted permanent human mandibular premolars with a single root canal were used for the purpose of this study. All teeth were prepared using ProTaper Nickel Titanium Rotary System to apical size #30. After removal of smear layer, teeth were randomly divided into five groups of 20 teeth each. Tubli-Seal [EWT], AH Plus, EndoREZ and GuttaFlow were used as sealers in Group I, Group II, Group III and Group IV respectively. In the control i.e. Group V, no sealer was used. All the teeth were obturated with the thermoplasticized gutta-percha technique using the E&Q Plus System. After storage at 37 0 C and 100% humidity for 14 days, the teeth were sectioned at 3, 5 and 7 mm from the root apex to obtain 2 mm thick specimens. The surfaces of the specimen representing the 3 and 5 mm level were observed using a scanning electron microscope (SEM). The maximum depth of sealer penetration into the dentinal tubules was measured for each section in microns. Statistical analysis was performed by using the Mann Whitney test to compare the penetration of each sealer. Results and Conclusion : Within the parameters of the present study it was concluded that the dentinal tubule penetration of sealers is affected by their physical and chemical properties. While resin-based sealers (AH Plus and EndoREZ) showed a greater depth of penetration, zinc oxide eugenol-based sealer (Tubli-Seal [EWT]) showed the least penetration. The penetration of the polydimethylsiloxane-based sealer (GuttaFlow) was intermediate. Key words : Sealers, dentinal tubule penetration Introduction Obturation is an important step of root canal treatment, as it may overcome some of the limitations of cleaning and shaping. Complete eradication of organisms from the canal space is virtually impossible with current instrumentation techniques, irrigation regimens and intracanal medicaments. Purpose of obturation is two-fold. Firstly, it eliminates avenues of leakage from the oral cavity and the periradicular tissues into the root canal system. Secondly, it seals within the root canal system any irritants that cannot be removed during canal cleaning and shaping 1, 2, 3. Although a plethora of materials have been advocated over the past 150 years for root canal obturation, gutta-percha in combination with a sealer is the contemporary material of choice 3. Sealers play a critical role in success of endodontic therapy by * Professor and Head, # Dept of Conservative Dentistry, C.S.M.S.S Dental College, Kanchanwadi, Aurangabad, Maharashtra, ## Dept of Conservative Dentistry, Govt Dental College & Hospital, Aurangabad, 50

2 ASHWINI SARAF-DADPE, A.I.KAMRA eliminating the space between the root canal wall and the core filling material 2. A good sealer should possess low viscosity and good wetting properties which allow for better penetration into canal irregularities and dentinal tubules 4. Penetration of sealer into the dentinal tubules is considered desirable for a number of reasons. It increases the interface between the sealer and dentine thereby improving the sealing ability and retention of the material. The closer approximation of the sealer to the bacteria enhances it s antibacterial effect. Sealer cements may also entomb any residual bacteria either inactivating them or preventing them from repopulating the root canal. Sealer penetration can be affected by various factors including smear layer, dentine permeability, obturation technique as well as physical and chemical properties of the sealer 1,2. A variety of endodontic sealers are available commercially and they are divided into groups according to their chemical composition. While sealers based on zinc oxide eugenol, for example Tubli-Seal [EWT] (SybronEndo, USA) have been used traditionally, resin-based materials such as AH Plus (Dentsply International, York) and EndoREZ (Ultradent, South Jordan) are steadily becoming popular. Newer, siloxane-based sealers like GuttaFlow (Coltene/ Whaledent, Switzerland) are also making their presence felt 3. Table No. 1 Group Sample Size Mean±SD (µm) I ± II ± III ± IV ± Mean Penetration of Sealers at 3mm Level Table No. 2 Group Sample Size Mean±SD (µm) I ± II ± III ± IV ± Mean Penetration of Sealers at 5mm Level Table No. 3 Group I Group II Extremely Group II Significant p< Group III Extremely Significant Group III Significant p= p< p<0.05 Group IV Extremely Extremely p= Significant Significant Not p< p< Significant Comparison Between the Groups at 3mm Level Table No. 4 Group I Group II Extremely Group II Significant p< Group III Extremely Significant Group III Significant p= p< p<0.05 Extremely Significant Significant Significant p= p= p< p<0.05 p<0.05 Comparison Between the Groups at 5mm Level 51

3 A SCANNING ELECTRON MICROSCOPIC EVALUATION OF THE PENETRATION OF ROOT CANAL DENTINAL TUBULES BY FOUR DIFFERENT ENDODONTIC SEALERS : A ZINC OXIDE EUGENOL-BASED SEALER, TWO RESIN-BASED SEALERS AND A POLYDIMETHYLSILOXANE-BASED SEALER : AN IN VITRO STUDY There appears to be a converse relation between dentinal tubule penetration of root canal sealer and microleakage of external fluids into the root canal 4. Most current tests for evaluation of sealing ability of root canal filling materials including dye/bacterial leakage have poor reproducibility due to lack of standardization of experimental technique 5,6. In the present study a scanning electron microscope is used to evaluate the penetration of four commonly used sealers: Tubli-Seal [EWT], AH Plus, EndoREZ and GuttaFlow into the dentinal tubules of root canals obturated by thermoplasticized gutta-percha technique. Materials and Method Hundred recently extracted human mandibular permanent premolars with relatively straight single roots were used in this study. For all the selected teeth, a straight-line access was gained and a reproducible glide path was established using #10 and #15 K-files. ProTaper Nickel Titanium Rotary System (Dentsply-Maillefer, France) was used for root canal preparation. Coronal two-thirds of the canal was pre-enlarged using ProTaper Shaping instruments S1 followed by S2 employing a brushcutting action. The working length was established 1 mm short of the apex. Both S1 and S2 were then carried sequentially to full working length. Preparation was finished using ProTaper Finishing instruments F1, F2 and F3 in succession 7. All root canals were prepared to apical size 30. During cleaning and shaping, 15% EDTA gel was used as a lubricant with each instrument. After each instrument use, canal was irrigated with 2 ml of 3% sodium hypochlorite using a 27 gauge needle-syringe, recapitulated with a #10 file and then re-irrigated. Prior to obturation each canal was flushed with 10 ml of 17% aqueous EDTA solution for 60 seconds followed by 10 ml of 1% sodium hypochlorite to remove the smear layer. Normal saline was used for the final rinse. The canals were then dried with sterile paper points. Out of the hundred samples eighty were randomly divided into four experimental groups of twenty teeth each. Groups Group I : Tubli-Seal[EWT] sealer (Zinc oxide eugenol-based sealer) Group II : AH Plus (Epoxy resin-based sealer) Group III: EndoREZ (Methacrylate resin-based sealer) Group IV : GuttaFlow (Polydimethylsiloxane - based sealer) Group V : No sealer used (Control group) The procedure of obturation for each group was as follows: The needle tip of the E&Q gun was prefit in the root canal to ensure that it reaches within 3.5-5mm of the working length and does not bind against the canal walls at that level. Likewise the pluggers were prefit to determine the proper depth of placement for compaction. The smallest and first plugger to be used was selected such that it did not contact the root canal walls at the apical third. Similarly the second and third pluggers were chosen so that they did not wedge at middle and coronal thirds respectively. The sealer was mixed according to the manufacturer s instructions and the root canal was lightly coated with it using a lentulospiral. The E&Q Plus control unit was set to a temperature of C. With the needle tip positioned in the canal at the pre-determined depth, the gutta-percha was 52

4 ASHWINI SARAF-DADPE, A.I.KAMRA injected passively into the root canal for 2-5 seconds until the operator felt the characteristic back pressure of the needle. The needle was then removed and controlled compaction of the softened guttapercha was done using the first prefitted plugger. A second injection then followed without exerting any pressure until the needle was withdrawn 3mm, and the gutta-percha was vertically condensed with the help of the second plugger. A final injection followed and complete obliteration of the canal space was accomplished by the third vertical compaction using the third plugger. For the EndoREZ sealer group, i.e. Group III, following this every sample was light cured for 40 seconds. The coronal access of all samples was sealed with IRM cement after which they were kept in 100% humidity at 37 0 C for 14 days. Later they were sectioned horizontally at 3, 5 and 7 mm from the root apex using a diamond disc under continuous water-cooling to obtain 2 mm thick specimens. The surfaces of the specimen representing the 3 and 5 mm level were then prepared for scanning electron microscopic analysis. The specimens were dehydrated by placing them sequentially in 50%, 75% and 100% ethyl alcohol for 8 hours total and left overnight in a drying chamber maintained at 60ºC.The sections were then sputter coated with a thin gold coating prior to observation using a scanning electron microscope (SEM). The mean values for maximum depth of sealer penetration at 3mm and 5mm levels were measured for each of the experimental groups using a calibrated measuring tool, which was incorporated into the microscope control system. Statistical analysis was performed by using the Mann - Whitney test to compare the penetration of each sealer. Results and Observations The mean maximum penetration of each sealer at 3mm and 5mm level is shown in Table no. 3 and Table no. 4 respectively. The samples in Group V i.e. control group showed no penetration of sealer into the dentinal tubules. Group I consisted of teeth obturated by thermoplasticized gutta-percha technique using Tubli-Seal [EWT] sealer. The mean maximum penetration of this group at 3mm level was 51.4 ± µm and at 5mm level it was 73.3 ± µm. The penetration of Tubli-Seal [EWT] was significantly lesser than the other three groups at both the 3mm and 5mm levels. (Table nos.5 & 6) In Group II, teeth were obturated using the thermoplasticized gutta-percha technique using AH Plus sealer. It showed the deepest penetration among all other groups, at both 3 and 5mm levels with the mean maximum penetration values being ± µm and ± µm respectively. The difference was statistically significant. (Table nos.5 & 6) EndoREZ sealer using thermoplasticized guttapercha technique was used for the obturation of samples in Group III. The mean maximum penetration of this group at 3mm level was ± µm while at 5mm level it was ± µm. In Group IV, the samples were obturated using the GuttaFlow sealer in combination with the thermoplasticized gutta-percha technique. At 3mm level, the mean maximum penetration of the sealer was 409± µm and at 5mm level it was 759.6± µm. 53

5 A SCANNING ELECTRON MICROSCOPIC EVALUATION OF THE PENETRATION OF ROOT CANAL DENTINAL TUBULES BY FOUR DIFFERENT ENDODONTIC SEALERS : A ZINC OXIDE EUGENOL-BASED SEALER, TWO RESIN-BASED SEALERS AND A POLYDIMETHYLSILOXANE-BASED SEALER : AN IN VITRO STUDY Discussion Among the available obturating materials, gutta-percha is by far the most universally used solidcore root canal filling material. Although cold lateral compaction of gutta-percha has been one of the most popular methods of obturating root canals; voids, spreader tracks, spaces between the guttapercha points and sealer pools have been reported by various investigators after obturation by lateral condensation 8. Injection of thermoplasticized guttapercha for obturation has been reported to be superior to lateral condensation in that it is capable of better replication of canal irregularities, exhibits lesser voids and has better homogenicity. The thermoplasticized gutta-percha obturation technique has been used for obturation in this study. The use of a root canal sealer has been advocated as essential with thermoplasticized guttapercha to achieve the best possible seal. Sealers are necessary to seal the space between the dentinal wall and the obturating core interface. In addition, they often have the ability to penetrate areas such as lateral canals and dentinal tubules. This property is highly relevant because the penetration of sealer cements into dentinal tubules increases their surface contact with the root canal dentin thus improving the sealing ability. Sealer plugs inside the dentinal tubules provide a mechanical interlocking, improving the retention of the filling material. Antibacterial active ingredients in the sealers might be more effective in achieving root canal disinfection through a closer contact with isolated bacteria in the tubules. Sealer cements may entomb any residual bacteria in the tubules rendering them harmless. Further, it has been proposed that penetration of the sealer into the dentinal tubules may have a root strengthening effect due to filling of the voids. Thus the ability of a sealer to penetrate dentinal tubules effectively may be one of the factors influencing the choice for selection of sealer during obturation. The present study was undertaken to compare four commonly used sealers viz. Tubli-Seal [EWT], AH Plus, EndoREZ and GuttaFlow, with respect to their penetrability. Group II showed the deepest penetration while Group I showed the least penetration at both 3mm and 5mm levels. The control group i.e. Group V showed no penetration of sealer into the dentinal tubules. 54

6 ASHWINI SARAF-DADPE, A.I.KAMRA A. Comparison between Group I and Group II the Tubli-Seal [EWT] group (Group I) and AH Plus group (Group II) showed that there was a statistically significant difference between Group I and Group II at both 3mm/5mm levels. These findings may be explained on the basis that AH Plus has a good flow, exhibits lesser structural defects, is strongly cross and end to end linked and forms covalent bonds with root dentin collagen. Thus it not only penetrates tubules better but also is more likely to remain intact in the dentinal tubules during the sectioning procedure. In comparison, zinc oxide eugenol-based sealer Tubli-Seal [EWT] may have shown dentinal tubule penetration due to adequate flow and low film thickness. However, compared to epoxy-resin it s consistency is more viscous resulting in numerous pores and vacuoles of large diameters. All these factors may have resulted in low mean maximum penetration values by Tubli-Seal [EWT]. The findings of the present study are in agreement with results obtained by Mamootil et al, Kokkas et al and Kouvas et al. In an SEM evaluation, Mamootil et al. found the mean maximum penetration of epoxy resin-based sealer AH 26 [1337µm] in the middle third of root canal to be higher than that of zinc oxide eugenol-based sealer Pulp Canal Sealer EWT [71µm] at the same level 2. Study is also in agreement with the SEM study by Kokkas et al. in that the resin-based sealer showed greater penetration than the zinc oxide eugenol-based sealer 10. However, our findings differ from the results obtained by Patel et al. who reported higher depths of penetration for Tubliseal into the root canal dentinal tubules. They found the mean maximum penetration in the middle third to be µm and that in the apical third to be 85.50µm 13. B. Comparison between Group I and Group III the Tubli-Seal [EWT] group (Group I) and EndoREZ group (Group III) showed that there was a statistically significant difference between Group I and Group III at both 3mm/5mm levels. The good penetration of EndoREZ can be explained on the basis that it has hydrophilic methacrylate resin monomer additives and low viscosity. Also it shows good canal adaptation. In comparison, as described previously although it has adequate flow and film thickness, Tubli-Seal EWT may show less penetration and retention in dentinal tubules because of poor structural integrity. The findings of the present study are in agreement with results obtained by Mamootil et al and Tay et al. Mamootil et al. found in their SEM study that the mean maximum penetration of methacrylate resin-based sealer EndoREZ [863µm] was higher than that of zinc oxide eugenol-based sealer Pulp Canal Sealer EWT [71µm] 2. In their SEM examination of the effectiveness of EndoREZ sealer in obturating the root canal, Tay et al. reported sealer penetration depths of µm in the middle and coronal sections of the root canals 14. C. Comparison between Group I and Group IV the Tubli-Seal [EWT] group (Group I) and GuttaFlow group (Group IV) showed that there was a statistically significant difference between Group I and Group IV at both 3mm/5mm levels. This finding of the present study may be explained on the basis that GuttaFlow has excellent flow properties. According to the manufacturer because the material is thixotropic, the viscosity diminishes under pressure. This may account for its penetration into the dentinal 55

7 A SCANNING ELECTRON MICROSCOPIC EVALUATION OF THE PENETRATION OF ROOT CANAL DENTINAL TUBULES BY FOUR DIFFERENT ENDODONTIC SEALERS : A ZINC OXIDE EUGENOL-BASED SEALER, TWO RESIN-BASED SEALERS AND A POLYDIMETHYLSILOXANE-BASED SEALER : AN IN VITRO STUDY tubules of the root canal. It has also been suggested that the material flows into the smallest dentinal tubules because of the small particle size (<0.9µm) of the GuttaFlow matrix filler. In addition the material shows good canal adaptation. A review of the endodontic literature does not cite any study comparing the depth of dentinal tubule penetration of polydimethylsiloxane-based sealer GuttaFlow and zinc oxide eugenol-based sealer Tubli-Seal [EWT]. D. Comparison between Group II and Group III Comparison of mean maximum penetration in the AH Plus group (Group II) and EndoREZ group (Group III) showed that there was a statistically significant difference between Group II and Group III at both 3mm/5mm levels. It has been suggested that the dentinal penetration of resin-based sealers may be dependent on the sealer being drawn into the tubules by capillary action. This may explain why AH Plus sealer with a setting time of minimum 8 hours may have shown deeper infiltration than EndoREZ sealer with a full setting time of minutes. Also, the dual cure nature of EndoREZ would mean higher forces of polymerization shrinkage which may challenge the initial resin-dentin adaptation. On the other hand, AH Plus being chemically cured may allow for compensation of polymerization shrinkage and exhibits zero polymerization stress. This difference between the two sealers may partly account for their varied depth of penetration into the dentinal tubules. The findings of the present study are in agreement with results obtained by Mamootil et al, Sevimay and Kalayci and Tay et al 2, 14, 15. E. Comparison between Group II and Group IV the AH Plus group (Group II) and GuttaFlow group (Group IV) showed that there was a statistically significant difference between Group II and Group IV at both 3mm/5mm levels. This may be attributed to the fact that the contact angle of silicone-based sealers like GuttaFlow has been reported to be significantly higher than conventional resin-based sealers. Thus their wettability and capacity to spread over dentin is lower. Also, the film thickness of GuttaFlow is greater than that of AH Plus. This may have adversely affected its penetration into dentinal tubules. A review of the endodontic literature does not cite any study comparing the depth of dentinal tubule penetration of epoxy resin-based sealer AH Plus and polydimethylsiloxane-based sealer GuttaFlow. The penetration depths of AH Plus recorded in this study corroborate the findings of Mamootil et al 2. However, the findings of this study do not agree with the maximum penetration depth value of AH Plus [54.6µm] noted by Kokkas et al. in their study evaluating the influence of smear layer on sealer penetration 10. F. Comparison between Group III and Group IV the EndoREZ group (Group II) and GuttaFlow group (Group IV) showed that there was a statistically significant difference between Group III and Group IV at 5mm level. However at 3mm level, although Group III showed greater penetration than Group IV the difference was not statistically significant. The presence of silicone in the polydimethysiloxane-based GuttaFlow sealer possibly produces high surface tension forces, thus making its spreading on dentin difficult. Also, its film 56

8 ASHWINI SARAF-DADPE, A.I.KAMRA thickness is higher than that of EndoREZ. This may account for the lower dentinal tubule penetration of GuttaFlow compared to that of EndoREZ. A review of the endodontic literature does not cite any study comparing the depth of dentinal tubule penetration of methacrylate resin-based sealer EndoREZ and polydimethylsiloxane-based sealer GuttaFlow. The findings of the present study are in agreement with results obtained by Mamootil et al and Tay et al. regarding the depth of penetration of EndoREZ sealer 2, 14. G. Comparison between 3mm and 5mm levels For all the sealers tested the mean maximum penetration at 5mm level was greater than that at 3mm level of the root canal. This regional disparity may be explained on the basis of anatomic complexities in the apical portion of root canal. This area is characterized by irregular and variable dentinal structure such as areas of resorption, occasional pulp stones, presence of cementum-like tissue and atubular dentin. Also, the tubule size and number reduces in this area. Another possible explanation for this may be the reduced effectiveness of smear layer removal techniques in areas closer to the apex. Regional variation in the depth of tubular penetration has been demonstrated by a number of authors. Weis et al in their study evaluating the effect of obturation technique on sealer cement thickness and dentinal tubule penetration found the greatest penetration at the 5mm level followed by the 3mm level, which was in turn greater than at the 1mm level 9. This finding also concurs with that of Patel et al. who reported mean maximum penetration in the middle third to be µm and that in the apical third to be 85.50µm. 13 Similarly these findings are consistent with that of Sen et al who reported higher sealer penetration in the middle thirds as compared to the apical third of the canal while studying the effect of tubular penetration of root canal sealers on dye microleakage 4. Conclusion Within the parameters of this study the following results were obtained: 1. All the four sealers studied showed dentinal tubule penetration to various depths into the root canal wall. Group II showed the maximum depth of penetration while Group I showed the least penetration at both 3mm and 5mm levels. 2. Statistically significant difference in the depth of sealer penetration was found between all the groups except between Group III and Group IV at the 3mm level. 3. A statistically significant difference was found when the sealer penetration depth of all the groups was compared at the 5mm level. While Group I showed the least penetration and Group II showed the maximum penetration depth; Group III and Group IV showed intermediate penetration depth with the former being more than the later. 4. Penetration depth of the sealer at the 5mm level was more than at the 3mm level for all the groups. Therefore, within the parameters of the present study it can be concluded that the dentinal tubule penetration of sealers is affected by their physical and chemical properties. While resin-based sealers (AH Plus and EndoREZ) showed a greater depth of penetration, zinc oxide eugenol-based sealer (Tubli- 57

9 A SCANNING ELECTRON MICROSCOPIC EVALUATION OF THE PENETRATION OF ROOT CANAL DENTINAL TUBULES BY FOUR DIFFERENT ENDODONTIC SEALERS : A ZINC OXIDE EUGENOL-BASED SEALER, TWO RESIN-BASED SEALERS AND A POLYDIMETHYLSILOXANE-BASED SEALER : AN IN VITRO STUDY Seal [EWT]) showed the least penetration. The penetration of the polydimethylsiloxane-based sealer (GuttaFlow) was intermediate. References : 1. Peters L, Wesselink P, Moorer W. The fate and role of bacteria left in root dentinal tubules. Int Endod J 1995; 28: Mamootil K, Messer H. Penetration of dentinal tubules by endodontic sealer cements in extracted teeth and in vivo. Int Endod J 2007; 40: Gutmann J, Witherspoon D. Obturation of the cleaned and shaped root canal system. In: Cohens S, Burns RC eds. Pathways of the Pulp, 8th edn. St Louis, MO: Mosby: Sen B, Piskin B, Baran N. The effect of tubular penetration of root canal sealers on microleakage. Int Endod J 1996; 29: Al-Ghamdi A, Wennberg A. Testing of sealing ability of endodontic filling materials. Endod Dent Traumatol 1994; 10: Wu M, Wesselink P. Endodontic leakage studies reconsidered. Part I. Methodology, application and relevance. Int Endod J 1993; 26: Ruddle C. The ProTaper technique. Endodontic Topics 2005, 10, Ozok A, Sluis L, Wu M, Wesselink P. Sealing ability of a new polydimethylsiloxane-based root canal filling material. J Endod 2008; 34(2): Weis M, Parashos P, Messer H. Effect of obturation technique on sealer cement thickness and dentinal tubule penetration. Int Endod J 2004; 37: Kokkas A, Boutsiokis A, Vassiliadis L, Stavrianos C. The influence of smear layer on dentinal tubule penetration depth by three different root canal sealers: an in vitro study. J Endodon 2004; 30(2): Oksan T, Akener B, Sen B, Tezel H. The penetration of root canal sealers into dentinal tubules. A scanning electron microscopic study. Int Endod J 1993; 26: Kouvas V, Liolios E, Vassiliadis L, Parissis-Messimeris S, Boutsioukis A. Influence of smear layer on depth of penetration of three endodontic sealers: an SEM study. Endod Dent Traumatol 1998; 14: Patel D, Sheriff M, Ford T, Watson T, Mannocci F. The penetration of RealSeal primer and Tubliseal into root canal dentinal tubules: a confocal microscopic study. Int Endod J 2007; 40: Tay F, Loushine R, Monticelli F, Weller R, Breschi L, Ferrari M, Pashley D. Effectiveness of resin-coated guttapercha cones and a dual-cured, hydrophilic methacrylate resin-based sealer in obturating root canals. J Endod 2005; 31(9): Sevimay S, Kalayci A. Evaluation of the apical sealing ability and adaptation to dentine of two resin-based sealers. J Oral Rehab 2005; 32: