- Email: [email protected]
Similar Glucose Leakage Pattern on Smear-covered, EDTA-treated and BioPure MTAD–treated Dentin
Basic Research—Technology
Similar Glucose Leakage Pattern on Smear-covered, EDTA-treated and BioPure MTAD–treated Dentin Gustavo De-Deus, DDS, MS,*† Juliana Soares, DDS,† Fernanda Leal, DDS,† Aderval S. Luna, PhD,‡ Sandra Fidel, DDS, MS, PhD,† and Rivail Antonio Sergio Fidel, DDS, MS, PhD† Abstract This study assessed the effect of BioPure MTAD on the sealing ability on the basis of the filtration rate of glucose along the root fillings. Both 17% ethylenediaminetetraacetic acid (EDTA) and smear-covered dentin were used as reference patterns to compare the results. Sixty-four human upper incisors were prepared with different irrigation protocols (n ⫽ 18): G1, NaOCl; G2, NaOCl ⫹ EDTA; and G3, NaOCl ⫹ BioPure MTAD. The teeth were obturated and then prepared in a doublechamber apparatus to evaluate the glucose leakage along the root fillings. The amount of glucose leakage was quantified with spectrophotometry at 7, 14, 21, 28, 35, 42, 49, and 56 days. Leakage existed in every sample and was variable in all of the experimental groups, and Kruskal-Wallis H test results showed that there were no significant differences among the experimental groups (P ⬎ .05). The smear-covered dentin did not allow more glucose leakage than EDTA-treated and BioPure MTAD–treated dentin. (J Endod 2008;34: 459 – 462)
Key Words Chelating solution, final rinse, glucose, irrigation, leakage, MTAD, smear layer
From the *Department of Endodontics, Veiga de Almeida University; and †Department of Endodontics and ‡Department of Analytical Chemistry, Rio de Janeiro State University (UERJ), Rio de Janeiro, RJ, Brazil. Address requests for reprints to Prof Gustavo De-Deus, R. Desembargador Renato Tavares, 11, ap.102, Ipanema, Rio de Janeiro, RJ, 22411-060, Brazil. E-mail address: endogus@ gmail.com. 0099-2399/$0 - see front matter Copyright © 2008 by the American Association of Endodontists. doi:10.1016/j.joen.2008.01.005
JOE — Volume 34, Number 4, April 2008
D
espite the controversy regarding the effect of a smear layer on treatment outcomes, most endodontic clinicians and researchers still think that it is prudent to remove the smear layer in teeth with infected root canals, allowing the disinfection of the root dentin walls (1). Laboratory studies have documented the importance of the smear removal in improving the adaptation of obturation materials to dentin walls (2, 3). Although the importance of the smear-free dentin has been a controversial topic (4 – 6), recently a well-conducted meta-analysis concluded that the procedures to remove the smear layer improved the fluid-tight seal of the root fillings (7). Moreover, the recent introduction of a bonding technology for root fillings emphasized the necessity of knowing the status of the dentin before inserting the filling material (8, 9). Recently, BioPure MTAD (Dentsply/Tulsa, Tulsa, OK) was introduced, representing an innovative approach for the simultaneous removal of the endodontic smear layer (10, 11) and disinfection of the root canals (12). BioPure MTAD contains a mixture of a tetracycline isomer, citric acid, and a detergent. A major difference between the 2 materials is that the doxycycline present in BioPure MTAD has a high binding affinity to dentin, allowing for an extended antibacterial effect (10). A new and interesting method for evaluating the quality of the fluid seal was recently developed and referred to as glucose leakage model (GLM) (13). Since its introduction, the GLM has been used by some studies (14 –17), in which its advantages were well-addressed. In addition, the GLM was accepted by an established leakage research group as an evolution of the reliable fluid transport method (14). The GLM potentially represents an advance in improving the conclusions of laboratory leakage studies. Therefore, the present study aimed to assess the effect of BioPure MTAD on sealing ability, on the basis of the filtration rate of glucose along the root fillings. Both 17% ethylenediaminetetraacetic acid (EDTA) and smear-covered dentin were used as reference patterns to compare the results. The null hypotheses tested were (1) that there are no differences in the ability to seal dentin in teeth irrigated with either BioPure MTAD or 17% EDTA and (2) that there is no effect of the smear-removal procedures on reducing the glucose leakage.
Materials and Methods Sample Preparation Sixty-four well-preserved, extracted human upper central incisors, with straight roots and that were 20 ⫾ 1 mm in length, were selected from the tooth bank of Rio de Janeiro State University. The teeth were disinfected in 0.5% chloramine-T, stored in distilled water at 4°C, and used within 6 months after extraction. Standard access cavities were made in 59 teeth. The apical patency of each canal was confirmed, and a step-back flaring technique was then performed at 1-mm increments with numbers 2– 6 Gates-Glidden burs (Dentsply Maillefer, Ballaigues, Switzerland) and K-Flexofiles, creating a 0.02 mm ⫻ 1 䡠 mm taper (14). The use of different irrigation protocols resulted in 3 experimental groups with 18 specimens each (G1, G2, and G3). Five additional teeth with intact crowns were used as the negative group, and 5 teeth that were not obturated served as positive control. Both experimental and control groups were randomly created by using a computer algorithm (http://www.random.org). In G1, the canals were irrigated between each filing with 1 mL of freshly prepared 5.25% NaOCl for 1 minute, with no final flush to remove the smear layer. The specimens
Leakage on BioPure MTAD–treated Dentin
459
Basic Research—Technology of G2 were prepared similarly to those of G1, with the exception that the smear layer was removed in all specimens with a final flush of 5 mL of 17% EDTA (pH 7.7; Herpo Ltd, Petrópolis, RJ, Brazil) for 5 minutes (1, 5, 6). In G3, 5 mL of BioPure MTAD (pH 2.15) was used as a final rinse for 5 minutes. All root canals in G3 were previously irrigated with 1.3% NaOCl, as recommended by the manufacturer (8 –10). The prepared teeth were filled by the same operator with the lateral condensation technique and Grossman sealer (Endofill; Herpo Ltd). A size 40 file was used to pick up a measured amount of sealer (20 L), which was determined by the use of fixed-volume semiautomatic micropipette. The sealer was placed into the canal while rotating the tooth counterclockwise. A size 55 standardized gutta-percha master cone (Diadent, Chongchong Buk Do, Korea) was placed in the canal up to the full working length. Lateral compaction was achieved in each canal by using 8 accessory gutta-percha cones (Diadent). The crowns of teeth were removed, leaving roots that were 12 mm in length. The filled roots were stored at 37oC and 100% humidity for 7 days to allow setting of the sealer.
Assembled Double-chamber and Glucose Leakage Measuring The specimens of the negative group control were completely coated with nail varnish. Two coats of nail varnish were applied on the external surface of all experimental specimens, except for the area 3 mm of the apical end. The teeth were placed into a device designed to measure glucose leakage (13) (Fig. 1). The teeth were individually inserted into a silicone tube (Ø 0.5 ⫻ 1.5 mm), with the apex (3 mm) protruding through the end. The silicone tube was connected to a plastic tube with a length of at least 15 mm. The assembly was then placed in a sterile 10-mL glass flask with a screw cap. The glass flask contained 7 mL of 0.2% NaN3 so that the root samples were immersed in the solution. NaN3 was used to inhibit the growth of microorganisms that might influence the readings through the decomposition of glucose (14). All of the interfaces were sealed with cyanoacrylate adhesive. Five milliliters of 1 mol glucose solution (pH 7.0; density, 1.09 ⫻ 103 g/L; viscosity, 1.18 ⫻ 10⫺3 Pa 䡠 s at 37°C) containing 0.2% NaN3 was injected into the plastic tube at a level 14 cm higher than the top of the sample, creating a hydrostatic pressure of 1.5 kPa (15 cm H2O). The atmospheric pressure absolute viscosity measurement of the glucose solution was carried out by Hoppler viscosimeter, and liquid density was determined by picnometer method. The model was transferred to an incubator that provided 100% humidity at 37°C for the duration of the experiment (14). A 10-L aliquot of solution was drawn from the glass bottle by using a micropipette at 7, 14, 21, 28, 35, 42, 49, and 56 days. After drawing the sample, 10 L of fresh 0.2% NaN3 was added to the glass bottle reservoir to maintain a constant volume of 7 mL. The sample was then analyzed with a glucose kit (Labtest, Lagoa Santa, SP, Brazil) in a UV-VIS spectrophotometer (Camspec M 330; Camspec Ltd, Cambridge, UK) following a kinetic assay at 505-nm wavelength. The lowest and the highest glucose levels for which the glucose assay is believed to be accurate are 0.34 and 26.7 mmol/L. Therefore, only the values between these ranges were considered. Data Presentation and Statistical Analysis Data are presented as mmol/L of glucose. The preliminary analysis of the raw pooled data did not show a normal distribution (KolmogorovSmirnov test). Further time-dot statistical analysis was performed by using Kruskal-Wallis H test, with the level of significance set at P ⬍ .05.
Results No glucose leakage was recorded for the negative control groups. The positive control group had substantial glucose leakage from the first 460
De-Deus et al.
Figure 1. Set-up of the GLM.
day, with the leakage values increasing over time up to 21 days, when all of the samples had maximum leakage (21 mmol/L). The results from the control groups confirmed the experimental model. The graph in Fig. 2 displays glucose leakage over time. The averages of accumulative glucose leakage were 0.50 ⫾ 0.23 for NaOCl (G1), 0.64 ⫾ 0.31 for MTAD (G2), and 0.88 ⫾ 0.38 for EDTA (G3). The Kruskal-Wallis H test results showed that there were no significant differences among the experimental groups for all experimental times (P ⬎ .05).
Discussion The effect of the smear layer on sealing ability has been evaluated by a variety of laboratory-based experimental models including dye leakage, fluid transport, and bacterial penetration methods. The present study used the recent and innovative glucose leakage research set-up, in which a glucose solution was used as tracer. Glucose has some positive points, such as low molecular weight (180 d), hydrophilicity, is chemically stable, and also serves as a bacteria nutrient. Xu et al (13) stated that if glucose could enter the canal from the oral cavity, bacteria that might survive root canal preparation and obturation could multiply and
JOE — Volume 34, Number 4, April 2008
Basic Research—Technology
Figure 2. Time evolution of glucose leakage. Data points are the average from all of the samples.
potentially lead to periapical inflammation. Therefore, the use of the GLM was thought to be more clinically relevant than other tests (13). Moreover, the present model is quantitative, nondestructive, non– labor-intensive, and a time-saving experiment. It is worth mentioning that the coronal low pressure used in the study could help rule out entrapped air or fluid and seemed to be sufficient for a device with high sensitivity (13, 18). Shemesh et al (14) suggested that the GLM can be seen as a further development of the fluid transportation concept as a measure of the passage of fluid along root fillings, subjecting them to constant pressure. The present results showed no differences in samples irrigated by 17% EDTA or BioPure MTAD or with only 5.25% NaOCl. Therefore, both null hypotheses were accepted because a similar fluid seal pattern was obtained with either dentin status and irrigation protocol used. Recently, a large number of reports have been focused on the properties of BioPure MTAD. The quick and self-limiting smear removal ability of BioPure MTAD has been documented lately (11). Furthermore, unlike EDTA and citric acid, minimal erosion of intraradicular dentin has been reported when BioPure MTAD was used (19). The consequences of dentin matrix destruction remain undefined, although Park et al (20) speculated that increased coronal leakage in EDTAtreated samples might be caused by the erosive property of EDTA. The present results do not agree with this hypothesis, because there was no difference found between EDTA-treated and BioPure MTAD–treated dentin. The results of some previous studies are in line with the present results (20, 21), including a recent well-designed study (14); smearfree dentin did not improve the sealing of the apical 4 mm of the root fillings. However, the present findings are inconsistent with some studies that have shown a fluid-tight seal improvement in smear-free dentin (22, 23). It has been conjectured that the smear layer provides an environment that might facilitate leakage as a result of its heterogeneity, low density, and weak attachment to the dentin (24 –26). Alternatively, the reduction in leakage in smear-free dentin can be attributed to the penetration of the sealer into the dentinal tubules and to better sealer adhesion to the cleaned dentin. On the other hand, some studies have
JOE — Volume 34, Number 4, April 2008
reported that smear removal procedures did not have a significant effect on microleakage (27, 28). Finally, Timpawat et al (29) reported that the removal of the smear layer had adverse effects on microleakage. A new meta-analysis provided a good summary of the confusing background regarding this issue (7). Considering only the results obtained by the reliable and current leakage methods, the following conclusions can be made; 4 of 7 fluid filtration studies and 2 of 6 bacterial leakage studies indicated a significant difference in favor of the smear-free samples. These conflicting results are not new in the endodontic leakage literature and might be attributable to differences in the types of sealer, obturation techniques, and the method of producing the smear layer. Moreover, the limitations of the ex vivo studies must be stressed, such as the subjectivity, low reproducibility, and unreliable statistical results. As a consequence, there is a lack of clear-cut conclusions. Under the conditions of the present ex vivo evaluation, the following conclusions can be drawn: (1) the smear-covered dentin did not allow more glucose leakage than EDTA-treated and BioPure MTAD– treated dentin, and (2) the glucose model represents a useful method for performing quantitative leakage assessments.
References 1. Hülsmann M, Heckendorff M, Lennon A. Chelating agents in root canal treatment: mode of action and indications for their use. Int Endod J 2005;36:810 –30. 2. Sen BH, Piskin B, Baran N. The effect of tubular penetration of root canal sealers on dye microleakage. Int Endod J 1996;29:23– 8. 3. De-Deus G, Gurgel-Filho ED, Maniglia-Ferreira C, Coutinho-Filho T. The influence of filling technique on depth of tubule penetration by root canal sealer: a study using light microscopy and digital image processing. Aust Endod J 2004;30:23– 8. 4. Cergneux M. The influence of smear layer on the sealing ability of canal obturation. Int Endod J 1987;20:228 –32. 5. Zehnder M. Root canal irrigants. J Endod 2006;32:389 –98. 6. Torabinejad M, Handysides R, Khademi AA, Bakland LK. Clinical implications of the smear layer in endodontics: a review. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:658 – 66. 7. Shahravan A, Haghdoost AA, Adl A, Rahimi H, Shadifar F. Effect of smear layer on sealing ability of canal obturation: a systematic review and meta-analysis. J Endod 2007;33:96 –105. 8. Garcia-Godoy F, Loushine RJ, Itthagarun A, et al. Application of biologically-oriented dentin bonding principles to the use of endodontic irrigants. Am J Dent 2005;18:281–90.
Leakage on BioPure MTAD–treated Dentin
461
Basic Research—Technology 9. Tay FR, Hosoya Y, Loushine RJ, Pashley DH, Weller RN, Low DC. Ultrastructure of intraradicular dentin after irrigation with BioPure MTAD: II—the consequence of obturation with an epoxy resin-based sealer. J Endod 2006;32:473–7. 10. Torabinejad M, Khademi AA, Babagoli J, et al. A new solution for the removal of the smear layer. J Endod 2003;29:170 –5. 11. De-Deus G, Reis C, Fidel S, Fidel R, Sidnei P. Dentin demineralization when subjected to BioPure MTAD: a longitudinal and quantitative assessment. J Endod 2007;33:1364 – 8. 12. Torabinejad M, Shabahang S, Aprecio RM, Kettering JD. The antimicrobial effect of MTAD: an in vitro investigation. J Endod 2003;29:400 –3. 13. Xu Q, Fan MW, Fan B, Cheung GS, Hu HL. A new quantitative method using glucose for analysis of endodontic leakage. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;99:107–11. 14. Shemesh H, Wu MK, Wesselink PR. Leakage along apical root fillings with and without smear layer using two different leakage models: a two-month longitudinal ex vivo study. Int Endod 2006;39:968 –76. 15. van der Sluis LW, Shemesh H, Wu MK, Wesselink PR. An evaluation of the influence of passive ultrasonic irrigation on the seal of root canal fillings. Int Endod J 2007;40:356 – 61. 16. Zou L, Liu J, Yin SH, et al. Effect of placement of calcium sulphate when used for the repair of furcation perforations on the seal produced by a resin-based material. Int Endod J 2007;40:100 –5. 17. Xu Q, Ling J, Cheung GS, Hu Y. A quantitative evaluation of sealing ability of 4 obturation techniques by using a glucose leakage test. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;104:e109 –13. 18. Pommel L, Camps J. Effects of pressure and measurement time on the fluid filtration method in endodontics. J Endod 2001;27:256 – 8.
462
De-Deus et al.
19. Torabinejad M, Cho Y, Khademi AA, Bakland LK, Shabahang S. The effect of various concentrations of sodium hypochlorite on the ability of BioPure MTAD to remove the smear layer. J Endod 2003;29:233–9. 20. Park DS, Torabinejad M, Shabahang S. The effect of MTAD on the coronal leakage of obturated root canals. J Endod 2004;30:890 –2. 21. Ghoddusi J, Rohani A, Rashed T, Ghaziani P, Akbari M. An evaluation of microbial leakage after using MTAD as a final irrigation. J Endod 2007;33:173– 6. 22. von Fraunhofer JA, Fagundes DK, McDonald NJ, Dumsha TC. The effect of root canal preparation on microleakage within endodontically treated teeth: an in vitro study. Int Endod J 2000;33:355– 60. 23. Çobankara FK, Adanır N, Belli S. Evaluation of the influence of smear layer on the apical and coronal sealing ability of two sealers. J Endod 2004;30:406 –9. 24. Clark-Holke D, Drake D, Walton R, Rivera E, Guthmiller JM. Bacterial penetration through canals of endodontically treated teeth in the presence or absence of the smear layer. J Dent 2003;31:275– 81. 25. Czonstkowsky M, Wilson EG, Holstein FA. The smear layer in endodontics. Dent Clin North Am 1990;34:13–25. 26. Brånnström M. Smear layer: pathological and treatment considerations. Operative Dentistry Supplement 1984;3:35– 42. 27. Madison S, Krell KV. Comparison of ethylenediamine tetraacetic acid and sodium hypochlorite on the apical seal of endodontically treated teeth. J Endod 1984; 10:499 –503. 28. Evans JT, Simon JH. Evaluation of the apical seal produced by injected thermoplasticized gutta-percha in the absence of smear layer and root canal sealer. J Endod 1986;12:100 –7. 29. Timpawat S, Amornchat C, Trisuwan WR. Bacterial coronal leakage after obturation with three root canal sealers. J Endod 2001;27:36 –9.
JOE — Volume 34, Number 4, April 2008
Similar Glucose Leakage Pattern on Smear-covered, EDTA-treated and BioPure MTAD–treated Dentin Gustavo De-Deus, DDS, MS,*† Juliana Soares, DDS,† Fernanda Leal, DDS,† Aderval S. Luna, PhD,‡ Sandra Fidel, DDS, MS, PhD,† and Rivail Antonio Sergio Fidel, DDS, MS, PhD† Abstract This study assessed the effect of BioPure MTAD on the sealing ability on the basis of the filtration rate of glucose along the root fillings. Both 17% ethylenediaminetetraacetic acid (EDTA) and smear-covered dentin were used as reference patterns to compare the results. Sixty-four human upper incisors were prepared with different irrigation protocols (n ⫽ 18): G1, NaOCl; G2, NaOCl ⫹ EDTA; and G3, NaOCl ⫹ BioPure MTAD. The teeth were obturated and then prepared in a doublechamber apparatus to evaluate the glucose leakage along the root fillings. The amount of glucose leakage was quantified with spectrophotometry at 7, 14, 21, 28, 35, 42, 49, and 56 days. Leakage existed in every sample and was variable in all of the experimental groups, and Kruskal-Wallis H test results showed that there were no significant differences among the experimental groups (P ⬎ .05). The smear-covered dentin did not allow more glucose leakage than EDTA-treated and BioPure MTAD–treated dentin. (J Endod 2008;34: 459 – 462)
Key Words Chelating solution, final rinse, glucose, irrigation, leakage, MTAD, smear layer
From the *Department of Endodontics, Veiga de Almeida University; and †Department of Endodontics and ‡Department of Analytical Chemistry, Rio de Janeiro State University (UERJ), Rio de Janeiro, RJ, Brazil. Address requests for reprints to Prof Gustavo De-Deus, R. Desembargador Renato Tavares, 11, ap.102, Ipanema, Rio de Janeiro, RJ, 22411-060, Brazil. E-mail address: endogus@ gmail.com. 0099-2399/$0 - see front matter Copyright © 2008 by the American Association of Endodontists. doi:10.1016/j.joen.2008.01.005
JOE — Volume 34, Number 4, April 2008
D
espite the controversy regarding the effect of a smear layer on treatment outcomes, most endodontic clinicians and researchers still think that it is prudent to remove the smear layer in teeth with infected root canals, allowing the disinfection of the root dentin walls (1). Laboratory studies have documented the importance of the smear removal in improving the adaptation of obturation materials to dentin walls (2, 3). Although the importance of the smear-free dentin has been a controversial topic (4 – 6), recently a well-conducted meta-analysis concluded that the procedures to remove the smear layer improved the fluid-tight seal of the root fillings (7). Moreover, the recent introduction of a bonding technology for root fillings emphasized the necessity of knowing the status of the dentin before inserting the filling material (8, 9). Recently, BioPure MTAD (Dentsply/Tulsa, Tulsa, OK) was introduced, representing an innovative approach for the simultaneous removal of the endodontic smear layer (10, 11) and disinfection of the root canals (12). BioPure MTAD contains a mixture of a tetracycline isomer, citric acid, and a detergent. A major difference between the 2 materials is that the doxycycline present in BioPure MTAD has a high binding affinity to dentin, allowing for an extended antibacterial effect (10). A new and interesting method for evaluating the quality of the fluid seal was recently developed and referred to as glucose leakage model (GLM) (13). Since its introduction, the GLM has been used by some studies (14 –17), in which its advantages were well-addressed. In addition, the GLM was accepted by an established leakage research group as an evolution of the reliable fluid transport method (14). The GLM potentially represents an advance in improving the conclusions of laboratory leakage studies. Therefore, the present study aimed to assess the effect of BioPure MTAD on sealing ability, on the basis of the filtration rate of glucose along the root fillings. Both 17% ethylenediaminetetraacetic acid (EDTA) and smear-covered dentin were used as reference patterns to compare the results. The null hypotheses tested were (1) that there are no differences in the ability to seal dentin in teeth irrigated with either BioPure MTAD or 17% EDTA and (2) that there is no effect of the smear-removal procedures on reducing the glucose leakage.
Materials and Methods Sample Preparation Sixty-four well-preserved, extracted human upper central incisors, with straight roots and that were 20 ⫾ 1 mm in length, were selected from the tooth bank of Rio de Janeiro State University. The teeth were disinfected in 0.5% chloramine-T, stored in distilled water at 4°C, and used within 6 months after extraction. Standard access cavities were made in 59 teeth. The apical patency of each canal was confirmed, and a step-back flaring technique was then performed at 1-mm increments with numbers 2– 6 Gates-Glidden burs (Dentsply Maillefer, Ballaigues, Switzerland) and K-Flexofiles, creating a 0.02 mm ⫻ 1 䡠 mm taper (14). The use of different irrigation protocols resulted in 3 experimental groups with 18 specimens each (G1, G2, and G3). Five additional teeth with intact crowns were used as the negative group, and 5 teeth that were not obturated served as positive control. Both experimental and control groups were randomly created by using a computer algorithm (http://www.random.org). In G1, the canals were irrigated between each filing with 1 mL of freshly prepared 5.25% NaOCl for 1 minute, with no final flush to remove the smear layer. The specimens
Leakage on BioPure MTAD–treated Dentin
459
Basic Research—Technology of G2 were prepared similarly to those of G1, with the exception that the smear layer was removed in all specimens with a final flush of 5 mL of 17% EDTA (pH 7.7; Herpo Ltd, Petrópolis, RJ, Brazil) for 5 minutes (1, 5, 6). In G3, 5 mL of BioPure MTAD (pH 2.15) was used as a final rinse for 5 minutes. All root canals in G3 were previously irrigated with 1.3% NaOCl, as recommended by the manufacturer (8 –10). The prepared teeth were filled by the same operator with the lateral condensation technique and Grossman sealer (Endofill; Herpo Ltd). A size 40 file was used to pick up a measured amount of sealer (20 L), which was determined by the use of fixed-volume semiautomatic micropipette. The sealer was placed into the canal while rotating the tooth counterclockwise. A size 55 standardized gutta-percha master cone (Diadent, Chongchong Buk Do, Korea) was placed in the canal up to the full working length. Lateral compaction was achieved in each canal by using 8 accessory gutta-percha cones (Diadent). The crowns of teeth were removed, leaving roots that were 12 mm in length. The filled roots were stored at 37oC and 100% humidity for 7 days to allow setting of the sealer.
Assembled Double-chamber and Glucose Leakage Measuring The specimens of the negative group control were completely coated with nail varnish. Two coats of nail varnish were applied on the external surface of all experimental specimens, except for the area 3 mm of the apical end. The teeth were placed into a device designed to measure glucose leakage (13) (Fig. 1). The teeth were individually inserted into a silicone tube (Ø 0.5 ⫻ 1.5 mm), with the apex (3 mm) protruding through the end. The silicone tube was connected to a plastic tube with a length of at least 15 mm. The assembly was then placed in a sterile 10-mL glass flask with a screw cap. The glass flask contained 7 mL of 0.2% NaN3 so that the root samples were immersed in the solution. NaN3 was used to inhibit the growth of microorganisms that might influence the readings through the decomposition of glucose (14). All of the interfaces were sealed with cyanoacrylate adhesive. Five milliliters of 1 mol glucose solution (pH 7.0; density, 1.09 ⫻ 103 g/L; viscosity, 1.18 ⫻ 10⫺3 Pa 䡠 s at 37°C) containing 0.2% NaN3 was injected into the plastic tube at a level 14 cm higher than the top of the sample, creating a hydrostatic pressure of 1.5 kPa (15 cm H2O). The atmospheric pressure absolute viscosity measurement of the glucose solution was carried out by Hoppler viscosimeter, and liquid density was determined by picnometer method. The model was transferred to an incubator that provided 100% humidity at 37°C for the duration of the experiment (14). A 10-L aliquot of solution was drawn from the glass bottle by using a micropipette at 7, 14, 21, 28, 35, 42, 49, and 56 days. After drawing the sample, 10 L of fresh 0.2% NaN3 was added to the glass bottle reservoir to maintain a constant volume of 7 mL. The sample was then analyzed with a glucose kit (Labtest, Lagoa Santa, SP, Brazil) in a UV-VIS spectrophotometer (Camspec M 330; Camspec Ltd, Cambridge, UK) following a kinetic assay at 505-nm wavelength. The lowest and the highest glucose levels for which the glucose assay is believed to be accurate are 0.34 and 26.7 mmol/L. Therefore, only the values between these ranges were considered. Data Presentation and Statistical Analysis Data are presented as mmol/L of glucose. The preliminary analysis of the raw pooled data did not show a normal distribution (KolmogorovSmirnov test). Further time-dot statistical analysis was performed by using Kruskal-Wallis H test, with the level of significance set at P ⬍ .05.
Results No glucose leakage was recorded for the negative control groups. The positive control group had substantial glucose leakage from the first 460
De-Deus et al.
Figure 1. Set-up of the GLM.
day, with the leakage values increasing over time up to 21 days, when all of the samples had maximum leakage (21 mmol/L). The results from the control groups confirmed the experimental model. The graph in Fig. 2 displays glucose leakage over time. The averages of accumulative glucose leakage were 0.50 ⫾ 0.23 for NaOCl (G1), 0.64 ⫾ 0.31 for MTAD (G2), and 0.88 ⫾ 0.38 for EDTA (G3). The Kruskal-Wallis H test results showed that there were no significant differences among the experimental groups for all experimental times (P ⬎ .05).
Discussion The effect of the smear layer on sealing ability has been evaluated by a variety of laboratory-based experimental models including dye leakage, fluid transport, and bacterial penetration methods. The present study used the recent and innovative glucose leakage research set-up, in which a glucose solution was used as tracer. Glucose has some positive points, such as low molecular weight (180 d), hydrophilicity, is chemically stable, and also serves as a bacteria nutrient. Xu et al (13) stated that if glucose could enter the canal from the oral cavity, bacteria that might survive root canal preparation and obturation could multiply and
JOE — Volume 34, Number 4, April 2008
Basic Research—Technology
Figure 2. Time evolution of glucose leakage. Data points are the average from all of the samples.
potentially lead to periapical inflammation. Therefore, the use of the GLM was thought to be more clinically relevant than other tests (13). Moreover, the present model is quantitative, nondestructive, non– labor-intensive, and a time-saving experiment. It is worth mentioning that the coronal low pressure used in the study could help rule out entrapped air or fluid and seemed to be sufficient for a device with high sensitivity (13, 18). Shemesh et al (14) suggested that the GLM can be seen as a further development of the fluid transportation concept as a measure of the passage of fluid along root fillings, subjecting them to constant pressure. The present results showed no differences in samples irrigated by 17% EDTA or BioPure MTAD or with only 5.25% NaOCl. Therefore, both null hypotheses were accepted because a similar fluid seal pattern was obtained with either dentin status and irrigation protocol used. Recently, a large number of reports have been focused on the properties of BioPure MTAD. The quick and self-limiting smear removal ability of BioPure MTAD has been documented lately (11). Furthermore, unlike EDTA and citric acid, minimal erosion of intraradicular dentin has been reported when BioPure MTAD was used (19). The consequences of dentin matrix destruction remain undefined, although Park et al (20) speculated that increased coronal leakage in EDTAtreated samples might be caused by the erosive property of EDTA. The present results do not agree with this hypothesis, because there was no difference found between EDTA-treated and BioPure MTAD–treated dentin. The results of some previous studies are in line with the present results (20, 21), including a recent well-designed study (14); smearfree dentin did not improve the sealing of the apical 4 mm of the root fillings. However, the present findings are inconsistent with some studies that have shown a fluid-tight seal improvement in smear-free dentin (22, 23). It has been conjectured that the smear layer provides an environment that might facilitate leakage as a result of its heterogeneity, low density, and weak attachment to the dentin (24 –26). Alternatively, the reduction in leakage in smear-free dentin can be attributed to the penetration of the sealer into the dentinal tubules and to better sealer adhesion to the cleaned dentin. On the other hand, some studies have
JOE — Volume 34, Number 4, April 2008
reported that smear removal procedures did not have a significant effect on microleakage (27, 28). Finally, Timpawat et al (29) reported that the removal of the smear layer had adverse effects on microleakage. A new meta-analysis provided a good summary of the confusing background regarding this issue (7). Considering only the results obtained by the reliable and current leakage methods, the following conclusions can be made; 4 of 7 fluid filtration studies and 2 of 6 bacterial leakage studies indicated a significant difference in favor of the smear-free samples. These conflicting results are not new in the endodontic leakage literature and might be attributable to differences in the types of sealer, obturation techniques, and the method of producing the smear layer. Moreover, the limitations of the ex vivo studies must be stressed, such as the subjectivity, low reproducibility, and unreliable statistical results. As a consequence, there is a lack of clear-cut conclusions. Under the conditions of the present ex vivo evaluation, the following conclusions can be drawn: (1) the smear-covered dentin did not allow more glucose leakage than EDTA-treated and BioPure MTAD– treated dentin, and (2) the glucose model represents a useful method for performing quantitative leakage assessments.
References 1. Hülsmann M, Heckendorff M, Lennon A. Chelating agents in root canal treatment: mode of action and indications for their use. Int Endod J 2005;36:810 –30. 2. Sen BH, Piskin B, Baran N. The effect of tubular penetration of root canal sealers on dye microleakage. Int Endod J 1996;29:23– 8. 3. De-Deus G, Gurgel-Filho ED, Maniglia-Ferreira C, Coutinho-Filho T. The influence of filling technique on depth of tubule penetration by root canal sealer: a study using light microscopy and digital image processing. Aust Endod J 2004;30:23– 8. 4. Cergneux M. The influence of smear layer on the sealing ability of canal obturation. Int Endod J 1987;20:228 –32. 5. Zehnder M. Root canal irrigants. J Endod 2006;32:389 –98. 6. Torabinejad M, Handysides R, Khademi AA, Bakland LK. Clinical implications of the smear layer in endodontics: a review. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:658 – 66. 7. Shahravan A, Haghdoost AA, Adl A, Rahimi H, Shadifar F. Effect of smear layer on sealing ability of canal obturation: a systematic review and meta-analysis. J Endod 2007;33:96 –105. 8. Garcia-Godoy F, Loushine RJ, Itthagarun A, et al. Application of biologically-oriented dentin bonding principles to the use of endodontic irrigants. Am J Dent 2005;18:281–90.
Leakage on BioPure MTAD–treated Dentin
461
Basic Research—Technology 9. Tay FR, Hosoya Y, Loushine RJ, Pashley DH, Weller RN, Low DC. Ultrastructure of intraradicular dentin after irrigation with BioPure MTAD: II—the consequence of obturation with an epoxy resin-based sealer. J Endod 2006;32:473–7. 10. Torabinejad M, Khademi AA, Babagoli J, et al. A new solution for the removal of the smear layer. J Endod 2003;29:170 –5. 11. De-Deus G, Reis C, Fidel S, Fidel R, Sidnei P. Dentin demineralization when subjected to BioPure MTAD: a longitudinal and quantitative assessment. J Endod 2007;33:1364 – 8. 12. Torabinejad M, Shabahang S, Aprecio RM, Kettering JD. The antimicrobial effect of MTAD: an in vitro investigation. J Endod 2003;29:400 –3. 13. Xu Q, Fan MW, Fan B, Cheung GS, Hu HL. A new quantitative method using glucose for analysis of endodontic leakage. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;99:107–11. 14. Shemesh H, Wu MK, Wesselink PR. Leakage along apical root fillings with and without smear layer using two different leakage models: a two-month longitudinal ex vivo study. Int Endod 2006;39:968 –76. 15. van der Sluis LW, Shemesh H, Wu MK, Wesselink PR. An evaluation of the influence of passive ultrasonic irrigation on the seal of root canal fillings. Int Endod J 2007;40:356 – 61. 16. Zou L, Liu J, Yin SH, et al. Effect of placement of calcium sulphate when used for the repair of furcation perforations on the seal produced by a resin-based material. Int Endod J 2007;40:100 –5. 17. Xu Q, Ling J, Cheung GS, Hu Y. A quantitative evaluation of sealing ability of 4 obturation techniques by using a glucose leakage test. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;104:e109 –13. 18. Pommel L, Camps J. Effects of pressure and measurement time on the fluid filtration method in endodontics. J Endod 2001;27:256 – 8.
462
De-Deus et al.
19. Torabinejad M, Cho Y, Khademi AA, Bakland LK, Shabahang S. The effect of various concentrations of sodium hypochlorite on the ability of BioPure MTAD to remove the smear layer. J Endod 2003;29:233–9. 20. Park DS, Torabinejad M, Shabahang S. The effect of MTAD on the coronal leakage of obturated root canals. J Endod 2004;30:890 –2. 21. Ghoddusi J, Rohani A, Rashed T, Ghaziani P, Akbari M. An evaluation of microbial leakage after using MTAD as a final irrigation. J Endod 2007;33:173– 6. 22. von Fraunhofer JA, Fagundes DK, McDonald NJ, Dumsha TC. The effect of root canal preparation on microleakage within endodontically treated teeth: an in vitro study. Int Endod J 2000;33:355– 60. 23. Çobankara FK, Adanır N, Belli S. Evaluation of the influence of smear layer on the apical and coronal sealing ability of two sealers. J Endod 2004;30:406 –9. 24. Clark-Holke D, Drake D, Walton R, Rivera E, Guthmiller JM. Bacterial penetration through canals of endodontically treated teeth in the presence or absence of the smear layer. J Dent 2003;31:275– 81. 25. Czonstkowsky M, Wilson EG, Holstein FA. The smear layer in endodontics. Dent Clin North Am 1990;34:13–25. 26. Brånnström M. Smear layer: pathological and treatment considerations. Operative Dentistry Supplement 1984;3:35– 42. 27. Madison S, Krell KV. Comparison of ethylenediamine tetraacetic acid and sodium hypochlorite on the apical seal of endodontically treated teeth. J Endod 1984; 10:499 –503. 28. Evans JT, Simon JH. Evaluation of the apical seal produced by injected thermoplasticized gutta-percha in the absence of smear layer and root canal sealer. J Endod 1986;12:100 –7. 29. Timpawat S, Amornchat C, Trisuwan WR. Bacterial coronal leakage after obturation with three root canal sealers. J Endod 2001;27:36 –9.
JOE — Volume 34, Number 4, April 2008