- Email: [email protected]
Evaluation of pH and calcium ion release of Acroseal sealer in comparison with Apexit and Sealapex sealers
Evaluation of pH and calcium ion release of Acroseal sealer in comparison with Apexit and Sealapex sealers Ayce Unverdi Eldeniz, DDS, PhD,a Ali Erdemir, DDS, PhD,b Firuze Kurtoglu, DVM, PhD,c and Timur Esener, DDS, PhD,d Konya, Kirikkale, and Istanbul, Turkey UNIVERSITY OF SELCUK AND UNIVERSITY OF KIRIKKALE
Objective. This is an in vitro assessment of pH level and calcium ion release exhibited by 3 calcium hydroxide– based root canal sealers—Sealapex, Apexit, and Acroseal. Study design. The materials were prepared according to the manufacturers’ instructions and placed in 1 cm long and 4 mm diameter tubes. The tubes were then immersed in a glass flask containing 10 mL bidistilled water (n ⫽ 15), which was sealed and stored at 37°C before the materials had set. The control group contained bidistilled water with empty tubes (n ⫽ 12). At predetermined time intervals (24 h, 96 h, and 7, 15, and 28 days) the pH of the bidistilled water was tested with a pH meter and for released calcium by using spectrophotometry. The data were statistically analyzed using 1-way analysis of variance for the comparison of the materials at each time point. If the difference was significant, individual comparisons were performed by Tukey multiple comparisons test (␣ ⫽ .05). Results. Sealapex produced higher pH and released significantly higher calcium amounts than the other 2 sealers at all periods (P ⬍ .05). Apexit showed higher calcium release than Acroseal at the end of 15 days (P ⬍ .05). There was no significant difference in the pH between Apexit and Acroseal (P ⬎ .05). Conclusion. The new Acroseal sealer presented the least calcium ion release and pH than Sealapex and less calcium ion release than Apexit sealer. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103:e86-e91)
Successful root canal treatment requires proper cleaning and shaping of the root canal, as well as hermetic sealing of the canal space with an inert, dimensionally stable, and biologically compatible material.1 Therefore, the search continues for an endodontic sealer that fulfills the requirements for the ideal physicochemical and biologic properties. Many different root canal filling materials have been advocated through the years. Because these materials may be in direct contact with periapical tissues for a prolonged period of time, their biocompatibility is of primary importance. The use of improved “biologic” sealers based on calcium hydroxide has been proposed for permanent sealing of the root canal system.2 Calcium hydroxide– containing compounds have an excellent bactericidal action because of their high pH,3 mediate degradation of bacterial lipopolysaccharides,4 induce healing by hard tissue formation,5 and control inflammatory root resorption.6
a
Assistant Professor, Department of Endodontics, Faculty of Dentistry, University of Selcuk. b Assistant Professor, Department of Endodontics, Faculty of Dentistry, University of Kirikkale. c Professor, Department of Biochemistry, Faculty of Veterinary Medicine, University of Selcuk. d Clinician, Kadikoy, Istanbul. Received for publication Aug 9, 2005; returned for revision Oct 15, 2006; accepted for publication Oct 18, 2006. 1079-2104/$ - see front matter © 2007 Mosby, Inc. All rights reserved. doi:10.1016/j.tripleo.2006.10.018
e86
Consequently, calcium hydroxide has been widely used and recommended in root canal therapy. One of the first commercially available endodontic sealers containing calcium hydroxide was Sealapex. Investigations assessing biologic responses to Sealapex supported the advantages of the presence of the calcium hydroxide in the composition of sealers.7 Endodontic sealers containing calcium hydroxide in their formulations therefore became commercially available; among them were Apexit, CRCS, and Sealer 26. Recently, a new calcium hydroxide– based sealer, Acroseal, has been introduced to the market. Sealers containing calcium hydroxide in their composition will only perform their biologic and microbiologic action if calcium and hydroxyl ion release occurs.8 Increased pH has been shown to be bactericidal as it changes the environment of inflammatory tissue in the periapical region.9 The diffusion of hydroxyl ions from the root canal raises the pH at the surface of the root adjacent to the periodontal tissues, thereby possibly interfering with osteoclastic activity,10 and promotes a state of alkalinity in adjacent tissues, a condition that favors repair.10 It has also been shown that pH level affects experimental wound healing of human fibroblasts in vitro.11 In addition to an alkaline pH, calcium release is important because abundance of calcium ions in tissue participates in the activation of calcium-dependant adenosine triphosphatase.12 Calcium reacts with tissue
OOOOE Volume 103, Number 3
Eldeniz et al. e87
Table I. Sealers tested Sealer; manufacturer
Lot no.
Apexit; Ivoclar Vivadent, Schaan, Liechtenstein
B06053
Acroseal; Septodont, Saint Maur des Fosses, France
Catalyst M4 098, Base M3 190
Sealapex; Kerr, Romulus, MI
3-1016
Base
Activator
Calcium hydroxide 32%, hydrogenized Trimethylhexanedioldisalicylate 25%, bismuth colophony 32%, silicon dioxide 8%, carbonate 18%, bismuth oxide 18%, silicon calcium oxide 6%, zinc oxide 6%, dioxide 15%, 1-3,butanedioldisalicylate tricalciumphosphate 3%, zinc 11%, tricalcium phosphate 5%, zinc stearate 3% stearate 2% Calcium hydroxide, DGEBA Glycyrrhetic acid (enoxolone) methenamine; (diglycidyl ether of bisphenol A); radiopaque excipient radiopaque excipient Mixed sealer: calcium hydroxide 24%, barium sulfate 20%, zinc oxide 7%, sub-micron silica 4%, barium sulfate 20%, titanium dioxide 2%, zinc stearate 1%
carbonic gas to form calcium carbonate crystals, which serve as a nucleus for calcification, and thus favors mineralization,8,12 and calcium is needed for cell migration and differentiation.13 It has also been demonstrated that calcium ions react with carbon dioxide and reduce the source of respiration for anaerobic bacteria.8,14 The mere presence of calcium hydroxide in a dental material does not ensure that it will become available as calcium and hydroxyl ions to exert its mentioned therapeutic effects. One study reported that the release of calcium and hydroxyl ions from the calcium hydroxide– containing sealers may be variable,15 and this could be attributable to differences in the disintegration rate of the sealers as a consequence of their composition.15 It was also demonstrated that the acidity of cements and sealers changed considerably over time as a result of solubility of these materials.16 Therefore different time periods should be used in comparing different brands of calcium hydroxide– based sealers’ calcium and hydroxyl ion releases. Because studies involving the pH and calcium ion release of calcium hydroxide– based sealers are relatively scarce and no study was found regarding the pH and calcium ion release of the new calcium hydroxide– based Acroseal sealer in the literature, the objective of this in vitro study was to replicate previous pH and calcium ion release results provided by 2 conventional calcium hydroxide– based sealers, Sealapex and Apexit, and then compare them with the new data for Acroseal sealer at different time intervals.
open ends that were 4 mm in diameter. A total of 15 samples were used for each sealer. Separate mixing of test sealers were done before filling the individual tubes. Each sealer was placed in 5 mL plastic syringes (Ayset Enjektör, Adana, Turkey) and syringed into the PVC tubes. All tubes with the inserted material were found to have approximately the same weight, 0.135 ⫾ 0.005 g. Each tube was placed in a glass flask containing 10 mL bidistilled water (pH 7.0) before the sealer had set. The control group contained bidistilled water with empty tubes (n ⫽ 12). After sample immersion, glass flasks were hermetically sealed with rubber caps to attenuate any effects of external environmental factors and maintained at 37°C during all experimental periods. A pH meter (Knick, Berlin, Germany) was calibrated with known standard pH solutions of 4.7 and 10. The pH level of the solution was determined at 24 h, 96 h, and 7, 15, and 28 days after mixing. The solution was homogenized by a magnetic stirrer before each measurement after the removal of sealer tubes, and the tubes were moved to new flasks with fresh bidistilled water for the next measurement. Each sample was tested twice, and the mean value for each compound was recorded. After each measurement, the mean and standard deviation of the pH values of each material at each time point were calculated. The data were statistically analyzed using 1-way analysis of variance for the comparison of the materials at each time point. If the difference was significant, individual comparisons were performed by Tukey multiple comparisons test (␣ ⫽ .05).
MATERIAL AND METHODS Table I shows the 3 different calcium hydroxide– based sealers used in this study.
Analysis of calcium ion release The procedure before measurement of calcium ion release was the same as that for pH analysis. Tubes of the same diameter and length were immersed in 10 mL bidistilled water and evaluated for calcium ion release at the same time intervals. A total of 15 samples were used for each material. Before each measurement, the solution was homogenized by a magnetic stirrer to
Determination of pH The materials were mixed according to the manufacturers’ instructions and then placed in 1 cm long PVC tubes (Deutsch & Neumann, Berlin, Germany) with
e88
Eldeniz et al.
Fig. 1. Mean pH levels and standard deviations for 3 sealers at 5 different times.
OOOOE March 2007
Fig. 3. Mean calcium ion release and standard deviations for 3 sealers at 5 different times.
Fig. 2. Total increase in pH levels for the various times. Fig. 4. Total calcium ion release for the various times.
ensure uniform Ca2⫹ distribution, and the tubes were moved to new flasks with fresh bidistilled water for the next measurement. The amount of calcium released was measured using a calcium measurement reagent (DDS calcium arsenazo III; DDS Diagnostic Systems Co., Istanbul, Turkey). Ten microliters of solution was obtained from the flask and mixed with 10 L of a calcium measurement reagent in another glass flask. The calcium measurement reagent changes the color of the solution according to the calcium content (purplish red color). The color changes of the samples were measured with a spectrophotometer (UV2100 UV-VIS; Shimadzu Corporation, Kyoto, Japan) at 650 nm. Data obtained was calculated as mg/dL. To prevent the possible interference of phosphates and alkaline metals, all glassware was prewashed with 5% nitric acid. One-way analysis of variance for the comparison of materials at each time point was applied and followed by Tukey multiple comparisons test for individual comparisons if the difference between the materials was significant (␣ ⫽ .05). RESULTS The mean pH values and standard deviations recorded for the materials at the different experimental
periods tested are plotted in Fig. 1 and cumulative increases in pH are shown in Fig. 2 for the various times. In this study the measured pH of original distilled water used was 7, and no noticeable change was recorded in the controls over the experimental period. Sealapex tended to display a higher alkalizing potential than the other 2 sealers at all periods of evaluation (P ⬍ .05). No significant difference was observed between Apexit and Acroseal during all experimental periods (P ⬎ .05). Figure 3 shows the mean calcium ion release and standard deviations provided by the sealers as a function of time and Fig. 4 shows the cumulative calcium ion release for the various times. At 24 h, the Sealapex group had the highest calcium ion level than the other 2 (P ⬍ .05), which were not significantly different from each other. These results were also obtained for the 96 h and 7 day measurements of calcium ion level. At 15 days and 28 days the Sealapex group still had higher calcium ion levels than Apexit and Acroseal groups, but the Acroseal group had significantly lower calcium levels than the Apexit group (P ⬍ .05).
OOOOE Volume 103, Number 3
DISCUSSION A well established method of placing sealers in plastic tubes and immersing them in glass flasks for varying amounts of time was used.17-19 Although this method does not imitate clinical conditions, attempts to closely do so have resulted in complicated models that are difficult to reproduce and sometimes even to interpret. This method offers the advantages of simplicity, replication of the results, and time economy, so that in vitro comparisons between different materials can be easily achieved. Recently, Duarte et al.17 reported a method of sealer placement for evaluation of pH and calcium ion release of root canal sealers. They placed the sealers in 1 cm long and 4 mm diameter tubes. The method used in our investigation followed their descriptions. We acknowledge that this sample size is hardly representative of a root canal, which is in most cases less than 1 mm in diameter; nevertheless, we decided to use this method to be better able to compare our results to already published work. Some authors have placed the material inside root canals to use more clinically relevant substrate.10,20 However, special care should be taken when using teeth, because of potential differences in size of the apical foramina and anatomic variations, because microscopic examinations of root canals show that they are irregular and complex systems with many cul-desacs, fins, and lateral canals. Additionally, numerous dentinal tubules open onto the root canal surface.21 Different results could have been obtained when these variables were not considered. Bidistilled water with a pH 7 was used as a solvent to detect the pH levels and Ca2⫹ ion concentrations of the samples not only to follow previous studies10,15 but also because saline or a buffered liquid22 may have affected different materials in a dissimilar way, because dissociation of calcium hydroxide is dependent on the constitution, quantity, and buffering environment of the surrounding medium.20 In addition, bidistilled water was preferred because the reagent used for Ca2⫹ detection is highly sensitive to saline, and with other solvents subtle error can interfere with the results. The quantity of the medium in the flasks is important, and it was chosen for convenience. It was sufficient for conducting the tests and permitted extraction of most of the available calcium hydroxide. A smaller quantity would not have resulted in complete exhaustion of extractable calcium hydroxide in a reasonable number of transfers.22 A larger quantity of water would have extracted the available ions too rapidly, making it impossible to grade the sealers and to determine which sealer has increased solubility over the long term. The original intention of this study was to evaluate
Eldeniz et al. e89
the release of calcium and hydroxyl ions from freshly mixed materials during their setting period and after their setting at the end of 28 days. Although Sealapex has a slower setting time than Acroseal and Apexit, no attempt was made to modify the procedure to imitate the clinical use. On the basis of findings of previous studies,22 which indicated that not all of the calcium hydroxide present in the sample was available for extraction, it was judged irrelevant to evaluate the materials by correlating the amount of ions released with the weight of the tubes or their sealer content. We observed that the calcium hydroxide– based sealer Sealapex started to show better calcium and hydroxyl ion release values than Apexit and Acroseal from the outset and continued to do so to the end of the experimental period. These data are in accordance with the results of earlier reports.9,15,17 One reason for the differences between sealers may be related to differences in the percentage of extractable calcium hydroxide in the content of various calcium hydroxide– based sealers or to the intrinsic properties of the material which may lead to different chemical reactions and interference calcium and hydroxyl ion release and their solubility properties as a consequence of the hydrophilic/ hydrophobic nature of their matrix.23 Apexit has 5 times more zinc stearate, which is known to be highly hydrophobic and thus prevents an ingress of water, than Sealapex, and as a result has low solubility24 and less ionization in water.9,17,24 In contrast, it has been shown that set Sealapex has a poorly formed matrix25 together with low dimensional stability26 and demonstrates a very high water absorption.25 It was theorized that this porous material permits marked ingress of water over time which promotes continuous reaction between powder and binder.25 This degradation of the material could be the cause of the high solubility of Sealapex and supports our findings about the higher release of calcium and hydroxyl ions from that material. Acroseal showed the least amount of calcium and hydroxyl ion release. This may be explained by the relative insolubility of its ingredients. It contains diglycidyl ether of bisphenol A and methenamine, which are known epoxy compounds and are also found in the structure of AH 26 and Sealer 26. The sealer AH 26 is reported to be less soluble than Apexit and Sealapex in water.24 Sealer 26 also contains calcium hydroxide in its formulation, and previous reports showed that Sealapex demonstrated higher calcium ion release and higher pH than Sealer 26.17 This confirms our findings that an epoxy matrix having calcium hydroxide– based sealers have less ion liberation as a result of less solubility. The need for calcium hydroxide– based sealers to be
e90
OOOOE March 2007
Eldeniz et al.
soluble to liberate hydroxyl and calcium ions to better display their therapeutic effects brings to mind the question of whether this jeopardizes their sealing ability with time compared with other sealers as a possible consequence of gaps and voids formed along the sealerdentin or the sealer– gutta-percha interface.26 We believe it to be minimal, as demonstrated by previous in vitro leakage studies demonstrating that they provide a seal equal to or superior to the zinc oxide– eugenol sealers27-29 and comparable to resin based sealers.30 As a consequence of the limited surface area of the sealer that is exposed to the tissue fluids and owing to the demonstrated apical closure of teeth with hard tissue formation31-32 when calcium hydroxide– based sealers are used, the problematic dissolution of these sealers from the root canals is limited. In the light of published literature, it can be concluded that the high solubility property of Sealapex is beneficial from both a physicochemical and a biologic viewpoint compared with other calcium hydroxide based sealers, because the release of more calcium ions into the tissue as well as the higher pH may lead this sealer to exhibit more potent antibacterial effect after some time33 and may support more rapid healing of apical periodontitis,34 probably by eliminating the bacteria with macrophage activation and cell differentiation35 and inducing biologic sealing of the root apex by the formation of mineralized tissue.31-32,36 Besides these important properties of Sealapex, clinically the result of the present study is very important in that as tissue fluids circulate in the periapical region, this could induce continuous and sustained release of ions from Sealapex and explains why Sealapex in partial pulpectomy cases at the dentinocemental junction had equal success rate as calcium hydroxide with 70% closure and better success rate than calcium hydroxide in total pulpectomy cases (33.3%).37 Additional investigation is needed to attempt to correlate the release of ions from new and conventional calcium hydroxide– based sealers with the histologic results of usage tests with the same sealers in animals. CONCLUSIONS On the basis of the results obtained, it may be concluded that the new Acroseal sealer presented the least calcium ion release and pH compared with Sealapex and less calcium ion release than Apexit sealer. The authors thank research assistant Pinar Peker (Selcuk University, Veterinary Medicine Faculty, Department of Biochemistry) for her help in the calcium ion analysis, and senior scientist I. Eystein Ruyter (NIOM-Nordic Institute of Dental Materials, Haslum, Norway) for useful advice and discussion with regard to this study.
REFERENCES 1. Beltes P, Koulaouzidou E, Kotoula V, Kortsaris AH. In vitro evaluation of the cytotoxicity of calcium hydroxide– based root canal sealers. Endod Dent Traumatol 1991;11:245-9. 2. Manhart MJ. The calcium hydroxide method of endodontic sealing. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1982;54:219-24. 3. Byström A, Claesson R, Sundqvist G. The antibacterial effect of camphorated paramonochlorophenol, camphorated phenol and calcium hydroxide in the treatment of infected root canals. Endod Dent Traumatol 1985;1:170-5. 4. Safavi KE, Nichols FC. Effect of calcium hydroxide on bacterial lipopolysaccharide. J Endod 1993;19:76-8. 5. Holland R, Mello W, Nery MJ, Bernabe PFE, Souza V. Reaction of human periapical tissue to pulp extirpation and immediate root canal filling with calcium hydroxide. J Endod 1977;3:63-7. 6. Tronstad L. Root resorption— etiology, terminology and clinical manifestations. Endod Dent Traumatol 1988;4:241-52. 7. Tagger M, Tagger E. Periapical reactions to calcium hydroxidecontaining sealers and AH 26 in monkeys. Endod Dent Traumatol 1989;5:139-46. 8. Estrela C, Sydney GB, Bammann LL, Felippe O Jr. Mechanism of action of calcium and hydroxyl ions of calcium hydroxide on tissue and bacteria. Brazil Dent J 1995;6:85-90. 9. Hosoya N, Takahashi G, Arai T, Nakamura J. Calcium concentration and pH of the periapical environment after applying calcium hydroxide into root canals in vitro. J Endod 2001; 27:343-346. 10. Tronstad L, Andreasen JO, Hasselgren G, Kristerson L, Riis I. pH changes in dental tissues after root canal filling with calcium hydroxide. J Endod 1981;7:17-21. 11. Lengheden A, Jansson L. pH effects on experimental wound healing of human fibroblasts in vitro. Eur J Oral Sci 1995;103:148-55. 12. Seux D, Couble ML, Hartmann DJ, Gauthier JP, Magloire H. Odontoblast-like cytodifferentiation of human dental pulp cells in vitro in the presence of calcium hydroxide– containing cement. Arch Oral Biol 1991;36:117-28. 13. Schroder U. Effects of calcium hydroxide– containing pulp-capping agents on pulp cell migration, proliferation, and differentiation. J Dent Res 1985;64:541-8. 14. Kontakiotis G, Nakou M, Georgopoulou M. In vitro study of the indirect action of calcium hydroxide on the anaerobic flora of the root canal. Int Endod J 1995;28:285-9. 15. Tagger M, Tagger E, Kfir A. Release of calcium and hydroxyl ions from set endodontic sealers containing calcium hydroxide. J Endod 1988;14:588-91. 16. Denli N, Eskitascioglu M. pH changes of different cements. Ankara Univ Hekim Fak Derg 1990;17:57-60. 17. Duarte MA, Demarchi AC, Giaxa MH, Kuga MC, Fraga SC, de Souza LC. Evaluation of pH and calcium ion release of three root canal sealers. J Endod 2000;26:389-90. 18. Duarte MA, Demarchi AC, Yamashita JC, Kuga MC, Fraga Sde C. pH and calcium ion release of 2 root-end filling materials. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003; 95:345-7. 19. Duarte MAH, de O Demarchi AC, de Moraes IG. Determination of pH and calcium ion release provided by pure and calcium hydroxide– containing AH Plus. Int Endod J 2004;37:42-45. 20. Ardeshna SM, Qualtrough AJ, Worthington HV. An in vitro comparison of pH changes in root dentine following canal dressing with calcium hydroxide points and a conventional calcium hydroxide paste. Int Endod J 2002;35:239-44. 21. Torabinejad M, Khademi AA, Babagoli J, Cho Y, Johnson WB, Bozhilov K, et al. A new solution for the removal of the smear layer. J Endod 2003;29:170-5.
OOOOE Volume 103, Number 3 22. Forsten L, Söderling E. The alkaline and antibacterial effect of seven Ca(OH)2 liners in vitro. Acta Odontol Scand 1984;42: 93-8. 23. Schäfer E, Zandbiglari T. Solubility of root-canal sealers in water and artificial saliva. Int Endod J 2003;36:660-9. 24. Siqueira FJ Jr, Fraga RC, Garcia PF. Evaluation of sealing ability, pH and flow rate of three calcium hydroxide– based sealers. Endod Dent Traumatol 1995;11:225-8. 25. Caicedo R, von Fraunhofer JA. The properties of endodontic sealer cements. J Endod 1988;14:527-34. 26. Ørstavik D, Nordahl I, Tibballs JE. Dimensional change following setting of root canal sealer materials. Dent Mater 2001;17:512-9. 27. Chailertvanitkul P, Saunders WP, Mackenzie D. An assessment of microbial coronal leakage in teeth root filled with gutta-percha and three different sealers. Int Endod J 1996;29:387-92. 28. Chailertvanitkul P, Saunders WP, Mackenzie D. Coronal leakage of obturated root canals after long-term storage using a polymicrobial marker. J Endod 1997;23:610-3. 29. Haikel Y, Wittenmeyer W, Bateman G, Bentaleb A, Allemann C. A new method for the quantitative analysis of endodontic microleakage. J Endod 1999;25:172-7. 30. 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. 31. Sonat B, Dalat D, Günhan O. Periapical tissue reaction to root fillings with Sealapex. Int Endod J 1990;23:46-52.
Eldeniz et al. e91 32. Berbert FL, Leonardo MR, Silva LA, Tanomaru Filho M, Bramante CM. Influence of root canal dressings and sealers on repair of apical periodontitis after endodontic treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;93:184-9. 33. Fuss Z, Weiss EI, Shalhav M. Antibacterial activity of calcium hydroxide-containing endodontic sealers on Enterococcus faecalis in vitro. Int End J;30:397-402. 34. Waltimo T, Boiesen J, Eriksen HM, Orstavik D. Clinical performance of 3 endodontic sealers. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;92:89-92. 35. Silva LA, Leonardo MR, Faccioli LH, Figueiredo F. Inflammatory response to calcium hydroxide based root canal sealers. J Endod 1997;23:86-90. 36. Tanomaru Filho M, Leonardo MR, Silva LA, Utrilla LS. Effect of different root canal sealers on repair of teeth with chronic periradicular periodontitis. Int End J 1998;31:85-9. 37. Holland R, de Souza V. Ability of a new calcium hydroxide root canal filling material to induce hard tissue formation. J Endod 1985;11:535-43. Reprint requests: Ayce Unverdi Eldeniz Department of Endodontics Faculty of Dentistry Selcuk University Konya, Turkey [email protected]
Objective. This is an in vitro assessment of pH level and calcium ion release exhibited by 3 calcium hydroxide– based root canal sealers—Sealapex, Apexit, and Acroseal. Study design. The materials were prepared according to the manufacturers’ instructions and placed in 1 cm long and 4 mm diameter tubes. The tubes were then immersed in a glass flask containing 10 mL bidistilled water (n ⫽ 15), which was sealed and stored at 37°C before the materials had set. The control group contained bidistilled water with empty tubes (n ⫽ 12). At predetermined time intervals (24 h, 96 h, and 7, 15, and 28 days) the pH of the bidistilled water was tested with a pH meter and for released calcium by using spectrophotometry. The data were statistically analyzed using 1-way analysis of variance for the comparison of the materials at each time point. If the difference was significant, individual comparisons were performed by Tukey multiple comparisons test (␣ ⫽ .05). Results. Sealapex produced higher pH and released significantly higher calcium amounts than the other 2 sealers at all periods (P ⬍ .05). Apexit showed higher calcium release than Acroseal at the end of 15 days (P ⬍ .05). There was no significant difference in the pH between Apexit and Acroseal (P ⬎ .05). Conclusion. The new Acroseal sealer presented the least calcium ion release and pH than Sealapex and less calcium ion release than Apexit sealer. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103:e86-e91)
Successful root canal treatment requires proper cleaning and shaping of the root canal, as well as hermetic sealing of the canal space with an inert, dimensionally stable, and biologically compatible material.1 Therefore, the search continues for an endodontic sealer that fulfills the requirements for the ideal physicochemical and biologic properties. Many different root canal filling materials have been advocated through the years. Because these materials may be in direct contact with periapical tissues for a prolonged period of time, their biocompatibility is of primary importance. The use of improved “biologic” sealers based on calcium hydroxide has been proposed for permanent sealing of the root canal system.2 Calcium hydroxide– containing compounds have an excellent bactericidal action because of their high pH,3 mediate degradation of bacterial lipopolysaccharides,4 induce healing by hard tissue formation,5 and control inflammatory root resorption.6
a
Assistant Professor, Department of Endodontics, Faculty of Dentistry, University of Selcuk. b Assistant Professor, Department of Endodontics, Faculty of Dentistry, University of Kirikkale. c Professor, Department of Biochemistry, Faculty of Veterinary Medicine, University of Selcuk. d Clinician, Kadikoy, Istanbul. Received for publication Aug 9, 2005; returned for revision Oct 15, 2006; accepted for publication Oct 18, 2006. 1079-2104/$ - see front matter © 2007 Mosby, Inc. All rights reserved. doi:10.1016/j.tripleo.2006.10.018
e86
Consequently, calcium hydroxide has been widely used and recommended in root canal therapy. One of the first commercially available endodontic sealers containing calcium hydroxide was Sealapex. Investigations assessing biologic responses to Sealapex supported the advantages of the presence of the calcium hydroxide in the composition of sealers.7 Endodontic sealers containing calcium hydroxide in their formulations therefore became commercially available; among them were Apexit, CRCS, and Sealer 26. Recently, a new calcium hydroxide– based sealer, Acroseal, has been introduced to the market. Sealers containing calcium hydroxide in their composition will only perform their biologic and microbiologic action if calcium and hydroxyl ion release occurs.8 Increased pH has been shown to be bactericidal as it changes the environment of inflammatory tissue in the periapical region.9 The diffusion of hydroxyl ions from the root canal raises the pH at the surface of the root adjacent to the periodontal tissues, thereby possibly interfering with osteoclastic activity,10 and promotes a state of alkalinity in adjacent tissues, a condition that favors repair.10 It has also been shown that pH level affects experimental wound healing of human fibroblasts in vitro.11 In addition to an alkaline pH, calcium release is important because abundance of calcium ions in tissue participates in the activation of calcium-dependant adenosine triphosphatase.12 Calcium reacts with tissue
OOOOE Volume 103, Number 3
Eldeniz et al. e87
Table I. Sealers tested Sealer; manufacturer
Lot no.
Apexit; Ivoclar Vivadent, Schaan, Liechtenstein
B06053
Acroseal; Septodont, Saint Maur des Fosses, France
Catalyst M4 098, Base M3 190
Sealapex; Kerr, Romulus, MI
3-1016
Base
Activator
Calcium hydroxide 32%, hydrogenized Trimethylhexanedioldisalicylate 25%, bismuth colophony 32%, silicon dioxide 8%, carbonate 18%, bismuth oxide 18%, silicon calcium oxide 6%, zinc oxide 6%, dioxide 15%, 1-3,butanedioldisalicylate tricalciumphosphate 3%, zinc 11%, tricalcium phosphate 5%, zinc stearate 3% stearate 2% Calcium hydroxide, DGEBA Glycyrrhetic acid (enoxolone) methenamine; (diglycidyl ether of bisphenol A); radiopaque excipient radiopaque excipient Mixed sealer: calcium hydroxide 24%, barium sulfate 20%, zinc oxide 7%, sub-micron silica 4%, barium sulfate 20%, titanium dioxide 2%, zinc stearate 1%
carbonic gas to form calcium carbonate crystals, which serve as a nucleus for calcification, and thus favors mineralization,8,12 and calcium is needed for cell migration and differentiation.13 It has also been demonstrated that calcium ions react with carbon dioxide and reduce the source of respiration for anaerobic bacteria.8,14 The mere presence of calcium hydroxide in a dental material does not ensure that it will become available as calcium and hydroxyl ions to exert its mentioned therapeutic effects. One study reported that the release of calcium and hydroxyl ions from the calcium hydroxide– containing sealers may be variable,15 and this could be attributable to differences in the disintegration rate of the sealers as a consequence of their composition.15 It was also demonstrated that the acidity of cements and sealers changed considerably over time as a result of solubility of these materials.16 Therefore different time periods should be used in comparing different brands of calcium hydroxide– based sealers’ calcium and hydroxyl ion releases. Because studies involving the pH and calcium ion release of calcium hydroxide– based sealers are relatively scarce and no study was found regarding the pH and calcium ion release of the new calcium hydroxide– based Acroseal sealer in the literature, the objective of this in vitro study was to replicate previous pH and calcium ion release results provided by 2 conventional calcium hydroxide– based sealers, Sealapex and Apexit, and then compare them with the new data for Acroseal sealer at different time intervals.
open ends that were 4 mm in diameter. A total of 15 samples were used for each sealer. Separate mixing of test sealers were done before filling the individual tubes. Each sealer was placed in 5 mL plastic syringes (Ayset Enjektör, Adana, Turkey) and syringed into the PVC tubes. All tubes with the inserted material were found to have approximately the same weight, 0.135 ⫾ 0.005 g. Each tube was placed in a glass flask containing 10 mL bidistilled water (pH 7.0) before the sealer had set. The control group contained bidistilled water with empty tubes (n ⫽ 12). After sample immersion, glass flasks were hermetically sealed with rubber caps to attenuate any effects of external environmental factors and maintained at 37°C during all experimental periods. A pH meter (Knick, Berlin, Germany) was calibrated with known standard pH solutions of 4.7 and 10. The pH level of the solution was determined at 24 h, 96 h, and 7, 15, and 28 days after mixing. The solution was homogenized by a magnetic stirrer before each measurement after the removal of sealer tubes, and the tubes were moved to new flasks with fresh bidistilled water for the next measurement. Each sample was tested twice, and the mean value for each compound was recorded. After each measurement, the mean and standard deviation of the pH values of each material at each time point were calculated. The data were statistically analyzed using 1-way analysis of variance for the comparison of the materials at each time point. If the difference was significant, individual comparisons were performed by Tukey multiple comparisons test (␣ ⫽ .05).
MATERIAL AND METHODS Table I shows the 3 different calcium hydroxide– based sealers used in this study.
Analysis of calcium ion release The procedure before measurement of calcium ion release was the same as that for pH analysis. Tubes of the same diameter and length were immersed in 10 mL bidistilled water and evaluated for calcium ion release at the same time intervals. A total of 15 samples were used for each material. Before each measurement, the solution was homogenized by a magnetic stirrer to
Determination of pH The materials were mixed according to the manufacturers’ instructions and then placed in 1 cm long PVC tubes (Deutsch & Neumann, Berlin, Germany) with
e88
Eldeniz et al.
Fig. 1. Mean pH levels and standard deviations for 3 sealers at 5 different times.
OOOOE March 2007
Fig. 3. Mean calcium ion release and standard deviations for 3 sealers at 5 different times.
Fig. 2. Total increase in pH levels for the various times. Fig. 4. Total calcium ion release for the various times.
ensure uniform Ca2⫹ distribution, and the tubes were moved to new flasks with fresh bidistilled water for the next measurement. The amount of calcium released was measured using a calcium measurement reagent (DDS calcium arsenazo III; DDS Diagnostic Systems Co., Istanbul, Turkey). Ten microliters of solution was obtained from the flask and mixed with 10 L of a calcium measurement reagent in another glass flask. The calcium measurement reagent changes the color of the solution according to the calcium content (purplish red color). The color changes of the samples were measured with a spectrophotometer (UV2100 UV-VIS; Shimadzu Corporation, Kyoto, Japan) at 650 nm. Data obtained was calculated as mg/dL. To prevent the possible interference of phosphates and alkaline metals, all glassware was prewashed with 5% nitric acid. One-way analysis of variance for the comparison of materials at each time point was applied and followed by Tukey multiple comparisons test for individual comparisons if the difference between the materials was significant (␣ ⫽ .05). RESULTS The mean pH values and standard deviations recorded for the materials at the different experimental
periods tested are plotted in Fig. 1 and cumulative increases in pH are shown in Fig. 2 for the various times. In this study the measured pH of original distilled water used was 7, and no noticeable change was recorded in the controls over the experimental period. Sealapex tended to display a higher alkalizing potential than the other 2 sealers at all periods of evaluation (P ⬍ .05). No significant difference was observed between Apexit and Acroseal during all experimental periods (P ⬎ .05). Figure 3 shows the mean calcium ion release and standard deviations provided by the sealers as a function of time and Fig. 4 shows the cumulative calcium ion release for the various times. At 24 h, the Sealapex group had the highest calcium ion level than the other 2 (P ⬍ .05), which were not significantly different from each other. These results were also obtained for the 96 h and 7 day measurements of calcium ion level. At 15 days and 28 days the Sealapex group still had higher calcium ion levels than Apexit and Acroseal groups, but the Acroseal group had significantly lower calcium levels than the Apexit group (P ⬍ .05).
OOOOE Volume 103, Number 3
DISCUSSION A well established method of placing sealers in plastic tubes and immersing them in glass flasks for varying amounts of time was used.17-19 Although this method does not imitate clinical conditions, attempts to closely do so have resulted in complicated models that are difficult to reproduce and sometimes even to interpret. This method offers the advantages of simplicity, replication of the results, and time economy, so that in vitro comparisons between different materials can be easily achieved. Recently, Duarte et al.17 reported a method of sealer placement for evaluation of pH and calcium ion release of root canal sealers. They placed the sealers in 1 cm long and 4 mm diameter tubes. The method used in our investigation followed their descriptions. We acknowledge that this sample size is hardly representative of a root canal, which is in most cases less than 1 mm in diameter; nevertheless, we decided to use this method to be better able to compare our results to already published work. Some authors have placed the material inside root canals to use more clinically relevant substrate.10,20 However, special care should be taken when using teeth, because of potential differences in size of the apical foramina and anatomic variations, because microscopic examinations of root canals show that they are irregular and complex systems with many cul-desacs, fins, and lateral canals. Additionally, numerous dentinal tubules open onto the root canal surface.21 Different results could have been obtained when these variables were not considered. Bidistilled water with a pH 7 was used as a solvent to detect the pH levels and Ca2⫹ ion concentrations of the samples not only to follow previous studies10,15 but also because saline or a buffered liquid22 may have affected different materials in a dissimilar way, because dissociation of calcium hydroxide is dependent on the constitution, quantity, and buffering environment of the surrounding medium.20 In addition, bidistilled water was preferred because the reagent used for Ca2⫹ detection is highly sensitive to saline, and with other solvents subtle error can interfere with the results. The quantity of the medium in the flasks is important, and it was chosen for convenience. It was sufficient for conducting the tests and permitted extraction of most of the available calcium hydroxide. A smaller quantity would not have resulted in complete exhaustion of extractable calcium hydroxide in a reasonable number of transfers.22 A larger quantity of water would have extracted the available ions too rapidly, making it impossible to grade the sealers and to determine which sealer has increased solubility over the long term. The original intention of this study was to evaluate
Eldeniz et al. e89
the release of calcium and hydroxyl ions from freshly mixed materials during their setting period and after their setting at the end of 28 days. Although Sealapex has a slower setting time than Acroseal and Apexit, no attempt was made to modify the procedure to imitate the clinical use. On the basis of findings of previous studies,22 which indicated that not all of the calcium hydroxide present in the sample was available for extraction, it was judged irrelevant to evaluate the materials by correlating the amount of ions released with the weight of the tubes or their sealer content. We observed that the calcium hydroxide– based sealer Sealapex started to show better calcium and hydroxyl ion release values than Apexit and Acroseal from the outset and continued to do so to the end of the experimental period. These data are in accordance with the results of earlier reports.9,15,17 One reason for the differences between sealers may be related to differences in the percentage of extractable calcium hydroxide in the content of various calcium hydroxide– based sealers or to the intrinsic properties of the material which may lead to different chemical reactions and interference calcium and hydroxyl ion release and their solubility properties as a consequence of the hydrophilic/ hydrophobic nature of their matrix.23 Apexit has 5 times more zinc stearate, which is known to be highly hydrophobic and thus prevents an ingress of water, than Sealapex, and as a result has low solubility24 and less ionization in water.9,17,24 In contrast, it has been shown that set Sealapex has a poorly formed matrix25 together with low dimensional stability26 and demonstrates a very high water absorption.25 It was theorized that this porous material permits marked ingress of water over time which promotes continuous reaction between powder and binder.25 This degradation of the material could be the cause of the high solubility of Sealapex and supports our findings about the higher release of calcium and hydroxyl ions from that material. Acroseal showed the least amount of calcium and hydroxyl ion release. This may be explained by the relative insolubility of its ingredients. It contains diglycidyl ether of bisphenol A and methenamine, which are known epoxy compounds and are also found in the structure of AH 26 and Sealer 26. The sealer AH 26 is reported to be less soluble than Apexit and Sealapex in water.24 Sealer 26 also contains calcium hydroxide in its formulation, and previous reports showed that Sealapex demonstrated higher calcium ion release and higher pH than Sealer 26.17 This confirms our findings that an epoxy matrix having calcium hydroxide– based sealers have less ion liberation as a result of less solubility. The need for calcium hydroxide– based sealers to be
e90
OOOOE March 2007
Eldeniz et al.
soluble to liberate hydroxyl and calcium ions to better display their therapeutic effects brings to mind the question of whether this jeopardizes their sealing ability with time compared with other sealers as a possible consequence of gaps and voids formed along the sealerdentin or the sealer– gutta-percha interface.26 We believe it to be minimal, as demonstrated by previous in vitro leakage studies demonstrating that they provide a seal equal to or superior to the zinc oxide– eugenol sealers27-29 and comparable to resin based sealers.30 As a consequence of the limited surface area of the sealer that is exposed to the tissue fluids and owing to the demonstrated apical closure of teeth with hard tissue formation31-32 when calcium hydroxide– based sealers are used, the problematic dissolution of these sealers from the root canals is limited. In the light of published literature, it can be concluded that the high solubility property of Sealapex is beneficial from both a physicochemical and a biologic viewpoint compared with other calcium hydroxide based sealers, because the release of more calcium ions into the tissue as well as the higher pH may lead this sealer to exhibit more potent antibacterial effect after some time33 and may support more rapid healing of apical periodontitis,34 probably by eliminating the bacteria with macrophage activation and cell differentiation35 and inducing biologic sealing of the root apex by the formation of mineralized tissue.31-32,36 Besides these important properties of Sealapex, clinically the result of the present study is very important in that as tissue fluids circulate in the periapical region, this could induce continuous and sustained release of ions from Sealapex and explains why Sealapex in partial pulpectomy cases at the dentinocemental junction had equal success rate as calcium hydroxide with 70% closure and better success rate than calcium hydroxide in total pulpectomy cases (33.3%).37 Additional investigation is needed to attempt to correlate the release of ions from new and conventional calcium hydroxide– based sealers with the histologic results of usage tests with the same sealers in animals. CONCLUSIONS On the basis of the results obtained, it may be concluded that the new Acroseal sealer presented the least calcium ion release and pH compared with Sealapex and less calcium ion release than Apexit sealer. The authors thank research assistant Pinar Peker (Selcuk University, Veterinary Medicine Faculty, Department of Biochemistry) for her help in the calcium ion analysis, and senior scientist I. Eystein Ruyter (NIOM-Nordic Institute of Dental Materials, Haslum, Norway) for useful advice and discussion with regard to this study.
REFERENCES 1. Beltes P, Koulaouzidou E, Kotoula V, Kortsaris AH. In vitro evaluation of the cytotoxicity of calcium hydroxide– based root canal sealers. Endod Dent Traumatol 1991;11:245-9. 2. Manhart MJ. The calcium hydroxide method of endodontic sealing. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1982;54:219-24. 3. Byström A, Claesson R, Sundqvist G. The antibacterial effect of camphorated paramonochlorophenol, camphorated phenol and calcium hydroxide in the treatment of infected root canals. Endod Dent Traumatol 1985;1:170-5. 4. Safavi KE, Nichols FC. Effect of calcium hydroxide on bacterial lipopolysaccharide. J Endod 1993;19:76-8. 5. Holland R, Mello W, Nery MJ, Bernabe PFE, Souza V. Reaction of human periapical tissue to pulp extirpation and immediate root canal filling with calcium hydroxide. J Endod 1977;3:63-7. 6. Tronstad L. Root resorption— etiology, terminology and clinical manifestations. Endod Dent Traumatol 1988;4:241-52. 7. Tagger M, Tagger E. Periapical reactions to calcium hydroxidecontaining sealers and AH 26 in monkeys. Endod Dent Traumatol 1989;5:139-46. 8. Estrela C, Sydney GB, Bammann LL, Felippe O Jr. Mechanism of action of calcium and hydroxyl ions of calcium hydroxide on tissue and bacteria. Brazil Dent J 1995;6:85-90. 9. Hosoya N, Takahashi G, Arai T, Nakamura J. Calcium concentration and pH of the periapical environment after applying calcium hydroxide into root canals in vitro. J Endod 2001; 27:343-346. 10. Tronstad L, Andreasen JO, Hasselgren G, Kristerson L, Riis I. pH changes in dental tissues after root canal filling with calcium hydroxide. J Endod 1981;7:17-21. 11. Lengheden A, Jansson L. pH effects on experimental wound healing of human fibroblasts in vitro. Eur J Oral Sci 1995;103:148-55. 12. Seux D, Couble ML, Hartmann DJ, Gauthier JP, Magloire H. Odontoblast-like cytodifferentiation of human dental pulp cells in vitro in the presence of calcium hydroxide– containing cement. Arch Oral Biol 1991;36:117-28. 13. Schroder U. Effects of calcium hydroxide– containing pulp-capping agents on pulp cell migration, proliferation, and differentiation. J Dent Res 1985;64:541-8. 14. Kontakiotis G, Nakou M, Georgopoulou M. In vitro study of the indirect action of calcium hydroxide on the anaerobic flora of the root canal. Int Endod J 1995;28:285-9. 15. Tagger M, Tagger E, Kfir A. Release of calcium and hydroxyl ions from set endodontic sealers containing calcium hydroxide. J Endod 1988;14:588-91. 16. Denli N, Eskitascioglu M. pH changes of different cements. Ankara Univ Hekim Fak Derg 1990;17:57-60. 17. Duarte MA, Demarchi AC, Giaxa MH, Kuga MC, Fraga SC, de Souza LC. Evaluation of pH and calcium ion release of three root canal sealers. J Endod 2000;26:389-90. 18. Duarte MA, Demarchi AC, Yamashita JC, Kuga MC, Fraga Sde C. pH and calcium ion release of 2 root-end filling materials. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003; 95:345-7. 19. Duarte MAH, de O Demarchi AC, de Moraes IG. Determination of pH and calcium ion release provided by pure and calcium hydroxide– containing AH Plus. Int Endod J 2004;37:42-45. 20. Ardeshna SM, Qualtrough AJ, Worthington HV. An in vitro comparison of pH changes in root dentine following canal dressing with calcium hydroxide points and a conventional calcium hydroxide paste. Int Endod J 2002;35:239-44. 21. Torabinejad M, Khademi AA, Babagoli J, Cho Y, Johnson WB, Bozhilov K, et al. A new solution for the removal of the smear layer. J Endod 2003;29:170-5.
OOOOE Volume 103, Number 3 22. Forsten L, Söderling E. The alkaline and antibacterial effect of seven Ca(OH)2 liners in vitro. Acta Odontol Scand 1984;42: 93-8. 23. Schäfer E, Zandbiglari T. Solubility of root-canal sealers in water and artificial saliva. Int Endod J 2003;36:660-9. 24. Siqueira FJ Jr, Fraga RC, Garcia PF. Evaluation of sealing ability, pH and flow rate of three calcium hydroxide– based sealers. Endod Dent Traumatol 1995;11:225-8. 25. Caicedo R, von Fraunhofer JA. The properties of endodontic sealer cements. J Endod 1988;14:527-34. 26. Ørstavik D, Nordahl I, Tibballs JE. Dimensional change following setting of root canal sealer materials. Dent Mater 2001;17:512-9. 27. Chailertvanitkul P, Saunders WP, Mackenzie D. An assessment of microbial coronal leakage in teeth root filled with gutta-percha and three different sealers. Int Endod J 1996;29:387-92. 28. Chailertvanitkul P, Saunders WP, Mackenzie D. Coronal leakage of obturated root canals after long-term storage using a polymicrobial marker. J Endod 1997;23:610-3. 29. Haikel Y, Wittenmeyer W, Bateman G, Bentaleb A, Allemann C. A new method for the quantitative analysis of endodontic microleakage. J Endod 1999;25:172-7. 30. 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. 31. Sonat B, Dalat D, Günhan O. Periapical tissue reaction to root fillings with Sealapex. Int Endod J 1990;23:46-52.
Eldeniz et al. e91 32. Berbert FL, Leonardo MR, Silva LA, Tanomaru Filho M, Bramante CM. Influence of root canal dressings and sealers on repair of apical periodontitis after endodontic treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;93:184-9. 33. Fuss Z, Weiss EI, Shalhav M. Antibacterial activity of calcium hydroxide-containing endodontic sealers on Enterococcus faecalis in vitro. Int End J;30:397-402. 34. Waltimo T, Boiesen J, Eriksen HM, Orstavik D. Clinical performance of 3 endodontic sealers. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;92:89-92. 35. Silva LA, Leonardo MR, Faccioli LH, Figueiredo F. Inflammatory response to calcium hydroxide based root canal sealers. J Endod 1997;23:86-90. 36. Tanomaru Filho M, Leonardo MR, Silva LA, Utrilla LS. Effect of different root canal sealers on repair of teeth with chronic periradicular periodontitis. Int End J 1998;31:85-9. 37. Holland R, de Souza V. Ability of a new calcium hydroxide root canal filling material to induce hard tissue formation. J Endod 1985;11:535-43. Reprint requests: Ayce Unverdi Eldeniz Department of Endodontics Faculty of Dentistry Selcuk University Konya, Turkey [email protected]