- Email: [email protected]
Initial In Vitro Biological Response to Contemporary Endodontic Sealers
Basic Research—Technology
Initial In Vitro Biological Response to Contemporary Endodontic Sealers Serge Bouillaguet DMD, PD,* John C. Wataha, DMD, PhD,† Franklin R. Tay, DMD, PhD,‡ Martha G. Brackett, DDS, MSD,§ and Petra E. Lockwood† Abstract The objective of this study was to evaluate the cytotoxicity of three endodontic sealers (AH Plus/MailleferDentsply, Epiphany/Pentron, GuttaFlow, ColteneWhaledent). Materials were mixed according to the manufacturer instructions and packed into Teflon molds (10 ⫻ 1 mm). For cytotoxicity testing (MTT method), the specimens were placed in contact with cultured cells, then evaluated at two subsequent time points (24 or 72 h). In addition to testing the mixed materials, 5 l of primer liquid (GuttaFlow and Epiphany) and resin solvents (HEMA, ethanol, sterile water, or acetone) were added directly in culture for 24 and 72 h. The results showed that most materials pose significant cytotoxic risks and that cytotoxicity generally increased with time. At 72 h, GuttaFlow became significantly less toxic than AH Plus, Epiphany sealer, and Resilon. The current results support the need to continue to develop better endodontic sealers that combine the excellent sealing and bonding properties of resins with acceptable biological properties for endodontic applications. (J Endod 2006;32:989 –992)
Key Words Biocompatibility, cell-culture, endodontics, MTT, resilon
From the *Department of Cariology and Endodontology, University of Geneva, Geneva, Switzerland; †Department of Oral Biology and Maxillofacial Pathology, ‡Department of Endodontics, §Department of Oral Rehabilitation, Medical College of Georgia, Augusta, Georgia. Address requests for reprints to Dr. Serge Bouillaguet, Department of Cariology and Endodontology, School of Dental Medicine, University of Geneva, 19 rue B. Menn 1205, Geneva, Switzerland. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright © 2006 by the American Association of Endodontists. doi:10.1016/j.joen.2006.05.006
JOE — Volume 32, Number 10, October 2006
F
or many years, gutta-percha has been used as a core material with zinc oxide eugenolbased or calcium hydroxide-based sealers to obturate the root canal system during endodontic therapy (1). However, these sealers shrink upon setting and dissolve over time, which compromise the quality and life expectancy of the apical seal (2, 3). None of these combinations of materials bond to root dentin and prevent apical leakage in root-end fills (4, 5). Thus, with current treatments, the sealing of the dentin provided by the coronal restoration is more important than the apical seal of the root-end fill (6, 7). Recently, endodontic sealers have been developed to improve the sealing and bonding to root dentin. These improvements rely on the incorporation of resin monomers into the sealer, or application of resins during a distinct conditioning step (primer). Other strategies have focused on substitutes for gutta-percha that bond to the root dentin, thereby establishing a so called “monoblock obturation” (8). AH plus is an epoxy-based endodontic sealer which can be used with threedimensional (3D) gutta-percha compaction techniques. AH plus exhibits very low shrinkage during setting and long-term dimensional stability (9, 10). Although interesting anti-bacterial properties have been reported for AH plus, its sealing ability remains controversial (11). More recently, a thermoplastic synthetic material based on polyester (copolymer of polycaprolactone and urethane dimethacrylate) has been developed for use as an endodontic root filling material (Resilon). Resilon replaces gutta-percha points while providing the same handling properties and improved bonding characteristics to the sealer. The Epiphany obturating system (Pentron) uses Resilon points and is bonded to root dentin via a dual curing resin-based sealer (12). Recent research indicates that endodontically treated teeth filled with Resilon exhibit higher resistance to fracture and better sealing than endodontically treated teeth filled with gutta-percha and conventional sealers (13). Based on a different approach, a new material (GuttaFlow) that contains a mixture of gutta-percha powder and polydimethylsiloxane has been introduced on the market. The gutta-percha powder has nanometer-sized silver particles added to act as a preservative. The manufacturer claims that GuttaFlow slightly expands (0.2%) during setting that results in an improved apical seal of the root end fill. The major advantages of this material are that it flows at room temperature and that a primer can be used to increase its wettability to radicular dentin. Although endodontic sealers are designed to be used only within the root canal during endodontic therapy, they are frequently extruded through the apical constriction (14). Thus, they are often placed in intimate contact with the periapical tissues for extended periods of time. It is generally accepted that the biocompatibility of endodontic sealers is critical to clinical success of endodontic therapy (15). Whereas the cytotoxicity of conventional endodontic sealers has been well documented (16, 17), little is known about the toxicity of newer endodontic sealers such as AHPlus, Epiphany, or GuttaFlow. Therefore, the purpose of the current study was to evaluate the early biological response of these new endodontic sealers. Cytotoxic responses were assessed in cell-culture. The hypothesis of this study was that the composition, formulation, and chemistry of these sealers affect their biological properties.
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Basic Research—Technology TABLE 1. Composition of the materials and their manufactures Materials
Components
AH Plus Dentsply/Maillefer, Konstanz, Germany
Component A: Epoxy resin, calcium tungstate, zirconium oxide, aerosil, iron oxide
GuttaFlow Coltene-whaledent Langenau, Germany
GuttaFlow Primer: Heptane
Epiphany Pentron Clinical Technologies, LLC, Wallingford, CT
Epiphany sealer: BisGMA, ethoxylated BisGMA UDMA, Hydrophilic monomers Fillers: calcium hydroxide, barium sulfate, barium glass, silica
Component B: Adamantane amine, N,N-Dibenzyl-5-oxanonane, TCD-Diamine, Calcium tungstate, Zirconium oxide, Aerosil GuttaFlow: Polydimethylsiloxane, silicone, paraffin oil, platinum catalyst, zirconium dioxide, nano-silver gutta-percha powder Epiphany points: polymers of polyester bioactive glass, bismuth oxychloride, barium sulfate
Materials and Methods
Results
Three endodontic sealers and their primer-adhesives were evaluated (Table 1). Materials were prepared under aseptic conditions to limit the risks of biological contamination. In addition to testing the mixed-materials, 5 l of primer liquid of GuttaFlow and Epiphany were added directly to cultures (see below). HEMA, ethanol, sterile water and acetone (all USP grade) were used as controls for these experiments. Solid materials that required mixing were prepared according to the instructions given by the manufacturers. AH Plus pastes A and B were hand-mixed for 20 seconds to obtain a homogeneous consistency; GuttaFlow caps were activated and vibrated for 15 seconds using a dental amalgamator, and the two components of the Epiphany sealer were mixed using the automix tip of the dual chamber syringe and polymerized. The Epiphany points were heated to 80°C before condensation into the molds. The materials were packed into Teflon molds (1 mm thick ⫻ 10 mm diameter), covered on both sides with Mylar sheets and allowed to set overnight at room temperature (25°C) before testing. The specimens were tested for cytotoxicity in cell-culture using Balb/c 3T3 fibroblasts (CCL 163, American Type Culture Collection, Rockville, MD). For testing the liquid materials, the cells were plated at a density of 15,000 cells/cm2 and incubated for 24 hours at 37°C in 5% CO2 24 hours before the addition of the materials, to allow fibroblast attachment. Cell response was evaluated 24 and 48 hours after addition of the liquids. For the solid materials, cells were plated at 15,000 cells/ cm2 in 24-well format containing 1 ml of medium (surface area-tovolume ratio of the material to the medium was 1.76 cm2/ml), which conforms to the requirements of the International Standards Organization (18). The cells and materials were incubated for 24 hours and cell response was measured (24-hours reading). After this interval, the specimens were removed from the cell-culture, rinsed twice with sterile phosphate-buffered saline (PBS) then immediately added to a second cell-culture. This second culture was incubated for 48 hours before assessing cell response (72-hours reading). Cell response was estimated by measuring succinic dehydrogenase (SDH) activity. Procedural details for this method have been published elsewhere (19). The method in the current study used 1 mg/ml of MTT with a 2.0 molal sodium succinate substrate exposed to the cells for 1 h, dissolving the formazan reaction product into a dimethylsulfoxide/ NaOH solution and reading formazan concentration at 562 nm. The amount of formazan (indicative of SDH activity) was expressed as a percentage of the Teflon negative controls. Groups were compared statistically using one-way ANOVA and Tukey multiple comparison intervals (␣ ⫽ 0.05).
For acetone and ethanol, SDH activities of the cells were depressed by approximately 10%, whereas unpolymerized HEMA elicited a strong cytotoxic effect after 24 and 72 hour exposures (95% reduction in SDH activity, Fig. 1). At 24 h, the GuttaFlow primer was only slightly toxic but SDH activity significantly decreased to approximately 50% at 72 hours. Epiphany primer was strongly cytotoxic at both 24 and 72 hours (95% reduction in SDH activity). Results for the cytotoxicity of the mixed sealers are presented in Fig. 2. Overall, our results suggested significant differences between materials, and that cytoxicity worsens with time regardless of the material. At 24 h, GuttaFlow was indistinguishable from the Teflon control and significantly less cytotoxic than AH Plus, Epiphany sealer, and Resilon. SDH activities of the cells exposed for 24 hours to AH Plus, Epiphany sealer and Resilon were significantly depressed to 58, 38, and 35% respectively. At 72 hours, metabolic activities significantly decreased for all materials although, GuttaFlow remained significantly less toxic than AH Plus, Epiphany sealer, and Resilon. These latter materials were statistically equivalent at 72 hours.
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Figure 1. Cellular response to liquid primers (Epiphany and GuttaFlow) and resin solvents (water, acetone, HEMA, ethanol) placed into contact with cultured cells for 24 and 72 h. Succinic dehydrogenase (SDH) activity was measured and expressed as a percentage of Teflon (Tf) negative controls. Error bars indicate standard deviation of the mean (n ⫽ 4). Within each set of columns, different letters indicate statistical differences (ANOVA, Tukey, ␣ ⫽ 0.05).
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Basic Research—Technology
Figure 2. Cellular response to endodontic sealers placed into contact with cultured cells for 24 and 72 h. Succinic dehydrogenase (SDH) activity was measured and expressed as a percentage of Teflon (Tf) negative controls. Error bars indicate standard deviation of the mean (n ⫽ 4). Within each set of columns, different letters indicate statistical differences (ANOVA, Tukey, ␣ ⫽ 0.05). Uppercase letters compare 72 hour times and lowercase letters compare 24 hour times.
Discussion Although a variety of methods have been used to assess the cytotoxicity of biomaterials, the use of cell-culture assays is a well accepted, biochemically based, and commonly used initial method (20). We report here for the first time the cytotoxicity of Epiphany and GuttaFlow compared with AH Plus. AH Plus was moderately toxic initially (24 hours exposure), but the toxicity increased with time (72 hours exposure). This result is in agreement with previous reports (21, 22) that used a direct-cell contact test. Other study that measured the expression of IL-6 and IL-8 in human osteoblastic cells has confirmed that AH Plus induces significant cellular biochemical changes (23). The antibacterial activity that has been demonstrated for AH Plus suggests that components released from the material also may elicit a toxic response (11). Furthermore, the presence of porosities in hand-mixed cements, may favor the release of un-reacted components responsible for toxicity (24). The Epiphany primer, sealer, and the Resilon points elicited severe cytotoxic responses at all intervals. For the Epiphany primer, toxicity may be explained by the presence of hydrophilic monomers (such as HEMA) that can easily diffuse into the cell-culture medium (25). As shown in Fig. 1, unpolymerized HEMA can cause toxicity. It must be noted both the concentration used in this study (e.g. a 0.5 % concentration, commonly used in primers) and the absence of polymerization are relevant to clinical conditions because the primer cannot be cured at the apex of the root. Although the specimens of the Epiphany sealer were polymerized before testing, the inherently high resin content of the sealer may explain the poor biological properties of this material. There is evidence that resin components that are released into the cell-culture medium elicit significant toxicity (26). Other reports indicate that the complete setting of the Epiphany sealer vary from 30 minute to 7 days depending on the environment (27). Therefore, the risk that the methacrylate-based sealer was not completely polymerized cannot be ruled out. This assumption is supported by particles that were observed in the cell-cultures, meaning that dissolution occurred. Resilon points also were cytotoxic after 24 and 72 hour exposure. This result could be explained by the biodegradable nature of this material. Further, Hirashi
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et al. (2005) have reported that phase separation may occur because of the immiscible characteristics of polycaprolactone and urethane dimethacrylate that are the major constituents of the material (28). Another report indicates that Resilon is susceptible to enzymatic biodegradation via ester bond cleavage (29). The results of the study also indicate that GuttaFlow was significantly less cytotoxic than the other materials tested. This result is in agreement with a previous report that evaluated the biological properties of the previous formulation of this material called RoeKo Seal (21). Further, Al-Awadhi et al. (2004) have demonstrated that RoeKo Seal induced significantly less apoptotic activity in cultured osteoblasts than a conventional zinc-oxide eugenol-based sealer (30). However, in the current study, the toxic response to GuttaFlow increased with time. The increasing toxicity of GuttaFlow could be attributed to the release of silver particles that have been added as a preservative. A recent study has indicated that silver nanoparticles are cytotoxic to Escherichia coli at very low concentrations (31). Further, a recent research has indicated that small voids are frequently observed within the core of GuttaFlow thereby promoting the release of unreacted components that accumulate in such porosities (32). Although the relevance of in vitro toxicity tests to clinical conditions has been frequently questioned, it appears that the biological risks of these new resin-based sealers is relatively high compared with resin composites or ceramics tested under the same conditions (26, 33). This is unfortunate knowing that these materials might be placed in close contact with living tissues for long periods of time. Therefore, the current results support the need to evaluate longer periods of time to assess theresponse to cements over the long term and to develop new materials that combine the excellent sealing and bonding properties of resins with acceptable biological properties.
References 1. Schlider H. Filling root canal in three dimensions. Dent Clin North Am 1967;11:723– 44. 2. Orstavik D, Nordahl I, Tibballs JE. Dimensional change following setting of root canal sealer materials. Dent Mater 2001;17:512–9. 3. Hovland EJ, Dumsha TC. Leakage evaluation in vitro of the root canal sealer cement Sealapex. Int Endod J 1985;18:179 – 82. 4. Torabinejad M, Pitt Ford TR. Root end filling materials: a review. Endod Dent Traumatol 1996;12:161–78. 5. Pommel L, About I, Pashley D, Camps J. Apical leakage of four endodontic sealers. J Endod 2003;29:208 –10. 6. Saunders WP, Saunders EM. Coronal leakage as a cause of failure in root-canal therapy: a review. Endod Dent Traumatol 1994;10:105– 8. 7. Ray HA, Trope M. Periapical status of endodontically treated teeth in relation to the technical quality of the root filling and the coronal restoration. Int Endod J 1995;28:12– 8. 8. Teixeira FB, Teixeira ECN, Thompson JY, Trope M. Fracture resistance of endodontically treated roots using a new type of resin filling material. J Am Dent Assoc 2004;135: 646 –52. 9. Schafer E, Zandbiglari T. Solubility of root-canal sealers in water and artificial saliva. Int Endod J 2003;36:660 –9. 10. McMichen FR, Pearson G, Rahbaran S, Gulabivala K. A comparative study of selected physical properties of five root-canal sealers. Int Endod J 2003;36:629 –35. 11. Kayaoglu G, Erten H, Alacam T, Orstavik D. Short-term antibacterial activity of root canal sealers towards Enterococcus faecalis. Int Endod J 2005;38:483– 8. 12. Shipper G, Ørstavik G, Teixeira FB, Trope M. An evaluation of microbial leakage in roots Filled with a thermoplastic synthetic polymer-based root canal filling material (Resilon). J Endod 2004;30:342–7. 13. Teixeira FB, Teixeira EC, Thompson JY, Trope M. Fracture resistance of roots endodontically treated with a new resin filling material. J Am Dent Assoc 2004;135:646 –52. 14. Ricucci D, Langeland K. Apical limit of root canal instrumentation and obturation: part 2. A histological study. Int Endod J 1998;31:394 – 409. 15. Bratel J, Jontell M, Dahlgren U, Bergenholtz G. Effects of root canal sealers on immunocompetent cells in vitro and in vivo. Int Endod J 1998;31:178 – 88.
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25. Bouillaguet S, Wataha JC, Hanks CT, Ciucchi B, Holz J. In vitro cytotoxicity and dentin permeability of HEMA. J Endod 1996;22:244 – 8. 26. Wataha JC, Lockwood PE, Bouillaguet S, Noda M. In vitro biological response to core and flowable dental restorative materials. Dent Mater 2003;19:25–31. 27. Nielsen BA, Beeler WJ, Vy C, Baumgartner JC. Setting times of resilon and other sealers in aerobic and anaerobic environments. J Endod 2006;32:130 –2. 28. Hiraishi N, Papacchini F, Loushine RJ, Weller RN, Ferrari M, Pashley DH, Tay FR. Shear bond strength of Resilon to a methacrylate-based root canal sealer. Int Endod J 2005;38:753– 63. 29. Tay FR, Pashley DH, Yiu CK, et al. Susceptibility of a polycaprolactone-based root canal filling material to degradation. II. Gravimetric evaluation of enzymatic hydrolysis. J Endod 2005;31:737– 41. 30. Al-Awadhi S, Spears R, Gutmann JL, Opperman LA. Cultured primary osteoblast viability and apoptosis in the presence of root canal sealers. J Endod 2004;30:527–33. 31. Baker C, Pradhan A, Pakstis L, Pochan DJ, Shah SI. Synthesis and antibacterial properties of silver nanoparticles. J Nanosci Nanotechnol 2005;5:244 –9. 32. Elayouti A, Achleithner C, Lost C, Weiger R. Homogeneity and adaptation of a new gutta-percha paste to root canal walls. J Endod 2005;31:687–90. 33. Messer RL, Lockwood PE, Wataha JC, Lewis JB, Norris S, Bouillaguet S. In vitro cytotoxicity of traditional versus contemporary dental ceramics. J Prosthet Dent 2003;90:452– 8.
JOE — Volume 32, Number 10, October 2006
Initial In Vitro Biological Response to Contemporary Endodontic Sealers Serge Bouillaguet DMD, PD,* John C. Wataha, DMD, PhD,† Franklin R. Tay, DMD, PhD,‡ Martha G. Brackett, DDS, MSD,§ and Petra E. Lockwood† Abstract The objective of this study was to evaluate the cytotoxicity of three endodontic sealers (AH Plus/MailleferDentsply, Epiphany/Pentron, GuttaFlow, ColteneWhaledent). Materials were mixed according to the manufacturer instructions and packed into Teflon molds (10 ⫻ 1 mm). For cytotoxicity testing (MTT method), the specimens were placed in contact with cultured cells, then evaluated at two subsequent time points (24 or 72 h). In addition to testing the mixed materials, 5 l of primer liquid (GuttaFlow and Epiphany) and resin solvents (HEMA, ethanol, sterile water, or acetone) were added directly in culture for 24 and 72 h. The results showed that most materials pose significant cytotoxic risks and that cytotoxicity generally increased with time. At 72 h, GuttaFlow became significantly less toxic than AH Plus, Epiphany sealer, and Resilon. The current results support the need to continue to develop better endodontic sealers that combine the excellent sealing and bonding properties of resins with acceptable biological properties for endodontic applications. (J Endod 2006;32:989 –992)
Key Words Biocompatibility, cell-culture, endodontics, MTT, resilon
From the *Department of Cariology and Endodontology, University of Geneva, Geneva, Switzerland; †Department of Oral Biology and Maxillofacial Pathology, ‡Department of Endodontics, §Department of Oral Rehabilitation, Medical College of Georgia, Augusta, Georgia. Address requests for reprints to Dr. Serge Bouillaguet, Department of Cariology and Endodontology, School of Dental Medicine, University of Geneva, 19 rue B. Menn 1205, Geneva, Switzerland. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright © 2006 by the American Association of Endodontists. doi:10.1016/j.joen.2006.05.006
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or many years, gutta-percha has been used as a core material with zinc oxide eugenolbased or calcium hydroxide-based sealers to obturate the root canal system during endodontic therapy (1). However, these sealers shrink upon setting and dissolve over time, which compromise the quality and life expectancy of the apical seal (2, 3). None of these combinations of materials bond to root dentin and prevent apical leakage in root-end fills (4, 5). Thus, with current treatments, the sealing of the dentin provided by the coronal restoration is more important than the apical seal of the root-end fill (6, 7). Recently, endodontic sealers have been developed to improve the sealing and bonding to root dentin. These improvements rely on the incorporation of resin monomers into the sealer, or application of resins during a distinct conditioning step (primer). Other strategies have focused on substitutes for gutta-percha that bond to the root dentin, thereby establishing a so called “monoblock obturation” (8). AH plus is an epoxy-based endodontic sealer which can be used with threedimensional (3D) gutta-percha compaction techniques. AH plus exhibits very low shrinkage during setting and long-term dimensional stability (9, 10). Although interesting anti-bacterial properties have been reported for AH plus, its sealing ability remains controversial (11). More recently, a thermoplastic synthetic material based on polyester (copolymer of polycaprolactone and urethane dimethacrylate) has been developed for use as an endodontic root filling material (Resilon). Resilon replaces gutta-percha points while providing the same handling properties and improved bonding characteristics to the sealer. The Epiphany obturating system (Pentron) uses Resilon points and is bonded to root dentin via a dual curing resin-based sealer (12). Recent research indicates that endodontically treated teeth filled with Resilon exhibit higher resistance to fracture and better sealing than endodontically treated teeth filled with gutta-percha and conventional sealers (13). Based on a different approach, a new material (GuttaFlow) that contains a mixture of gutta-percha powder and polydimethylsiloxane has been introduced on the market. The gutta-percha powder has nanometer-sized silver particles added to act as a preservative. The manufacturer claims that GuttaFlow slightly expands (0.2%) during setting that results in an improved apical seal of the root end fill. The major advantages of this material are that it flows at room temperature and that a primer can be used to increase its wettability to radicular dentin. Although endodontic sealers are designed to be used only within the root canal during endodontic therapy, they are frequently extruded through the apical constriction (14). Thus, they are often placed in intimate contact with the periapical tissues for extended periods of time. It is generally accepted that the biocompatibility of endodontic sealers is critical to clinical success of endodontic therapy (15). Whereas the cytotoxicity of conventional endodontic sealers has been well documented (16, 17), little is known about the toxicity of newer endodontic sealers such as AHPlus, Epiphany, or GuttaFlow. Therefore, the purpose of the current study was to evaluate the early biological response of these new endodontic sealers. Cytotoxic responses were assessed in cell-culture. The hypothesis of this study was that the composition, formulation, and chemistry of these sealers affect their biological properties.
Biological Contemporary Endodontic Sealers
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Basic Research—Technology TABLE 1. Composition of the materials and their manufactures Materials
Components
AH Plus Dentsply/Maillefer, Konstanz, Germany
Component A: Epoxy resin, calcium tungstate, zirconium oxide, aerosil, iron oxide
GuttaFlow Coltene-whaledent Langenau, Germany
GuttaFlow Primer: Heptane
Epiphany Pentron Clinical Technologies, LLC, Wallingford, CT
Epiphany sealer: BisGMA, ethoxylated BisGMA UDMA, Hydrophilic monomers Fillers: calcium hydroxide, barium sulfate, barium glass, silica
Component B: Adamantane amine, N,N-Dibenzyl-5-oxanonane, TCD-Diamine, Calcium tungstate, Zirconium oxide, Aerosil GuttaFlow: Polydimethylsiloxane, silicone, paraffin oil, platinum catalyst, zirconium dioxide, nano-silver gutta-percha powder Epiphany points: polymers of polyester bioactive glass, bismuth oxychloride, barium sulfate
Materials and Methods
Results
Three endodontic sealers and their primer-adhesives were evaluated (Table 1). Materials were prepared under aseptic conditions to limit the risks of biological contamination. In addition to testing the mixed-materials, 5 l of primer liquid of GuttaFlow and Epiphany were added directly to cultures (see below). HEMA, ethanol, sterile water and acetone (all USP grade) were used as controls for these experiments. Solid materials that required mixing were prepared according to the instructions given by the manufacturers. AH Plus pastes A and B were hand-mixed for 20 seconds to obtain a homogeneous consistency; GuttaFlow caps were activated and vibrated for 15 seconds using a dental amalgamator, and the two components of the Epiphany sealer were mixed using the automix tip of the dual chamber syringe and polymerized. The Epiphany points were heated to 80°C before condensation into the molds. The materials were packed into Teflon molds (1 mm thick ⫻ 10 mm diameter), covered on both sides with Mylar sheets and allowed to set overnight at room temperature (25°C) before testing. The specimens were tested for cytotoxicity in cell-culture using Balb/c 3T3 fibroblasts (CCL 163, American Type Culture Collection, Rockville, MD). For testing the liquid materials, the cells were plated at a density of 15,000 cells/cm2 and incubated for 24 hours at 37°C in 5% CO2 24 hours before the addition of the materials, to allow fibroblast attachment. Cell response was evaluated 24 and 48 hours after addition of the liquids. For the solid materials, cells were plated at 15,000 cells/ cm2 in 24-well format containing 1 ml of medium (surface area-tovolume ratio of the material to the medium was 1.76 cm2/ml), which conforms to the requirements of the International Standards Organization (18). The cells and materials were incubated for 24 hours and cell response was measured (24-hours reading). After this interval, the specimens were removed from the cell-culture, rinsed twice with sterile phosphate-buffered saline (PBS) then immediately added to a second cell-culture. This second culture was incubated for 48 hours before assessing cell response (72-hours reading). Cell response was estimated by measuring succinic dehydrogenase (SDH) activity. Procedural details for this method have been published elsewhere (19). The method in the current study used 1 mg/ml of MTT with a 2.0 molal sodium succinate substrate exposed to the cells for 1 h, dissolving the formazan reaction product into a dimethylsulfoxide/ NaOH solution and reading formazan concentration at 562 nm. The amount of formazan (indicative of SDH activity) was expressed as a percentage of the Teflon negative controls. Groups were compared statistically using one-way ANOVA and Tukey multiple comparison intervals (␣ ⫽ 0.05).
For acetone and ethanol, SDH activities of the cells were depressed by approximately 10%, whereas unpolymerized HEMA elicited a strong cytotoxic effect after 24 and 72 hour exposures (95% reduction in SDH activity, Fig. 1). At 24 h, the GuttaFlow primer was only slightly toxic but SDH activity significantly decreased to approximately 50% at 72 hours. Epiphany primer was strongly cytotoxic at both 24 and 72 hours (95% reduction in SDH activity). Results for the cytotoxicity of the mixed sealers are presented in Fig. 2. Overall, our results suggested significant differences between materials, and that cytoxicity worsens with time regardless of the material. At 24 h, GuttaFlow was indistinguishable from the Teflon control and significantly less cytotoxic than AH Plus, Epiphany sealer, and Resilon. SDH activities of the cells exposed for 24 hours to AH Plus, Epiphany sealer and Resilon were significantly depressed to 58, 38, and 35% respectively. At 72 hours, metabolic activities significantly decreased for all materials although, GuttaFlow remained significantly less toxic than AH Plus, Epiphany sealer, and Resilon. These latter materials were statistically equivalent at 72 hours.
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Figure 1. Cellular response to liquid primers (Epiphany and GuttaFlow) and resin solvents (water, acetone, HEMA, ethanol) placed into contact with cultured cells for 24 and 72 h. Succinic dehydrogenase (SDH) activity was measured and expressed as a percentage of Teflon (Tf) negative controls. Error bars indicate standard deviation of the mean (n ⫽ 4). Within each set of columns, different letters indicate statistical differences (ANOVA, Tukey, ␣ ⫽ 0.05).
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Figure 2. Cellular response to endodontic sealers placed into contact with cultured cells for 24 and 72 h. Succinic dehydrogenase (SDH) activity was measured and expressed as a percentage of Teflon (Tf) negative controls. Error bars indicate standard deviation of the mean (n ⫽ 4). Within each set of columns, different letters indicate statistical differences (ANOVA, Tukey, ␣ ⫽ 0.05). Uppercase letters compare 72 hour times and lowercase letters compare 24 hour times.
Discussion Although a variety of methods have been used to assess the cytotoxicity of biomaterials, the use of cell-culture assays is a well accepted, biochemically based, and commonly used initial method (20). We report here for the first time the cytotoxicity of Epiphany and GuttaFlow compared with AH Plus. AH Plus was moderately toxic initially (24 hours exposure), but the toxicity increased with time (72 hours exposure). This result is in agreement with previous reports (21, 22) that used a direct-cell contact test. Other study that measured the expression of IL-6 and IL-8 in human osteoblastic cells has confirmed that AH Plus induces significant cellular biochemical changes (23). The antibacterial activity that has been demonstrated for AH Plus suggests that components released from the material also may elicit a toxic response (11). Furthermore, the presence of porosities in hand-mixed cements, may favor the release of un-reacted components responsible for toxicity (24). The Epiphany primer, sealer, and the Resilon points elicited severe cytotoxic responses at all intervals. For the Epiphany primer, toxicity may be explained by the presence of hydrophilic monomers (such as HEMA) that can easily diffuse into the cell-culture medium (25). As shown in Fig. 1, unpolymerized HEMA can cause toxicity. It must be noted both the concentration used in this study (e.g. a 0.5 % concentration, commonly used in primers) and the absence of polymerization are relevant to clinical conditions because the primer cannot be cured at the apex of the root. Although the specimens of the Epiphany sealer were polymerized before testing, the inherently high resin content of the sealer may explain the poor biological properties of this material. There is evidence that resin components that are released into the cell-culture medium elicit significant toxicity (26). Other reports indicate that the complete setting of the Epiphany sealer vary from 30 minute to 7 days depending on the environment (27). Therefore, the risk that the methacrylate-based sealer was not completely polymerized cannot be ruled out. This assumption is supported by particles that were observed in the cell-cultures, meaning that dissolution occurred. Resilon points also were cytotoxic after 24 and 72 hour exposure. This result could be explained by the biodegradable nature of this material. Further, Hirashi
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et al. (2005) have reported that phase separation may occur because of the immiscible characteristics of polycaprolactone and urethane dimethacrylate that are the major constituents of the material (28). Another report indicates that Resilon is susceptible to enzymatic biodegradation via ester bond cleavage (29). The results of the study also indicate that GuttaFlow was significantly less cytotoxic than the other materials tested. This result is in agreement with a previous report that evaluated the biological properties of the previous formulation of this material called RoeKo Seal (21). Further, Al-Awadhi et al. (2004) have demonstrated that RoeKo Seal induced significantly less apoptotic activity in cultured osteoblasts than a conventional zinc-oxide eugenol-based sealer (30). However, in the current study, the toxic response to GuttaFlow increased with time. The increasing toxicity of GuttaFlow could be attributed to the release of silver particles that have been added as a preservative. A recent study has indicated that silver nanoparticles are cytotoxic to Escherichia coli at very low concentrations (31). Further, a recent research has indicated that small voids are frequently observed within the core of GuttaFlow thereby promoting the release of unreacted components that accumulate in such porosities (32). Although the relevance of in vitro toxicity tests to clinical conditions has been frequently questioned, it appears that the biological risks of these new resin-based sealers is relatively high compared with resin composites or ceramics tested under the same conditions (26, 33). This is unfortunate knowing that these materials might be placed in close contact with living tissues for long periods of time. Therefore, the current results support the need to evaluate longer periods of time to assess theresponse to cements over the long term and to develop new materials that combine the excellent sealing and bonding properties of resins with acceptable biological properties.
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