Cytotoxicity of Two Bioactive Root Canal Sealers

Abstract

Objective

The aim of this study was to investigate the cytotoxicity of two different bioactive root canal sealers: one based on mineral trioxide aggregate, MTA Fillapex (Angelus, Solucoes Odontologicas, Londrina, PR, Brazil), and the other based on bioceramics, Endosequence BC Sealer (Brasseler, Savannah, Georgia, USA), in culture of mouse L929 fibroblasts.

Materials and methods

Mouse fibroblasts (L929), obtained from subcutaneous connective tissue of mouse line C3Hf, were cultivated in plastic culture flasks in an incubator at 37şC, with 5% CO2 and 90% humidity. Freshly mixed Endosequence BC Sealer and MTA Fillapex (0.1 g each) were placed on sterile teflon discs, 6 mm in diameter. Teflon discs with the materials as well as empty discs serving as control were placed in wells of 12-well plate. After incubation times of 1, 6, 20 and 24 hours, the teflon discs were removed from the wells and the number of viable cells was determined using trypan blue in Neubauer chamber.

Results

In comparison to the control group, MTA Fillapex had significantly less viable cells for all incubation periods (p≤0.05), while Endosequence BC sealer had significantly less viable cells after 6, 20, and 24 hours of incubation (p≤0.05). MTA Fillapex comprised significantly less viable cells in comparison to Endosequence BC sealer after the first hour and after 20 hours of incubation (p≤0.05), while for the other incubation periods there were no significant differences (p≥0.05).

Conclusion

MTA Fillapex and Endosequence BC sealer were both cytotoxic in cultures of mouse L929 fibroblasts.

Introduction

The primary goals of endodontic treatments are to clean and shape the root canal system to the greatest possible extent and to achieve a hermetic seal. Although different materials have been used as root canal sealers, new materials are constantly being developed in order to improve their physical-mechanical and biological properties. Recently, mineral trioxide aggregate (MTA) and bioceramic based root canal sealers have been introduced in endodontics.

MTA is an endodontic material currently used for pulp capping, apexification, perforation repair and root-end filling (1-4). The main advantages of this material are biocompatibility, good sealing ability and regeneration of periodontal ligament tissues with formation of bone and cementum (5, 6). Moreover, MTA forms a chemical bond to hard dental tissues through crystals of hydroxylapatite created at the interstitial layer (7). MTA Fillapex (Angelus, Solucoes Odontologicas, Londrina, PR, Brazil) is a bioactive root canal sealer consisting of two pastes. After mixing the material, a semipermeable structure is formed with MTA dispersed throughout. Therefore, MTA activity is possible due to permeability of the mixed material (8) while an alkaline pH explains its extended antibacterial action (9).

Other types of bioactive materials proposed for root canal obturation are based on bioceramics. One of the new bioceramic root canal sealers is Endosequence BC Sealer (Brassler, Savannah, GA, USA), consisting of premixed calcium silicate cement with particle size less than 2 microns. This material has advantages including: high pH (12.8), hydrophilic properties, no shrinkage or resorption, excellent sealing ability and fast setting (10). Endosequence BC sealer has the ability to form hydroxyapatite due to calcium silicates in powder, which in a hydration reaction produces a calcium silicate hydrate gel and calcium hydroxide (11). The calcium hydroxide reacts with the phosphate ions to precipitate hydroxyapatite and water (11).

Root canal sealers come in close contact with the periodontal tissues and it has been shown that they constantly dissolve when exposed to an aqueous environment for extended periods (12). The components of these materials can act as toxins causing cellular injury that can lead to tissue damage or delay and impede tissue repair (13). Biocompatibility tests for root canal sealer are important since tissue response after their placement may influence the success of endodontic treatment (14). For cytotoxicity testing, different animal cell cultures can provide information on basic biological behavior of the material (15). Cell cultures, such as mouse L929 fibroblasts, are useful models since they provide large amounts of consistent cells and because of the fact that most cellular characteristics are maintained, providing reliable experimental results (16).

The aim of this study was to investigate the cytotoxicity of two different bioactive root canal sealers, one based on mineral trioxide aggregate and the other based on bioceramics, in culture of mouse L929 fibroblasts.

Materials and methods

Cell lines

Mouse L929 fibroblasts, obtained from subcutaneous connective tissue of mouse line C3Hf, were cultivated in plastic culture flasks, 75 cm² in diameter (Sterile Tissue Culture Flask) in an incubator at a temperature of 37şC, with 5% CO₂ and 90% humidity. The cells were constantly enriched with a nourishing medium: 10% Dulbecco’s Modified Eagle medium (DMEM – Gibco BRL-Life Technologies) with 10% fetal bovine serum (FCS – Fetal Bovine Serum, Gibco BRL), antibiotics (100 IU/ml penicillin and 50 μl/ml streptomycin) and 200 mM L-glutamine (Gibco BRL). The cells grew in plastic cell culture flasks for 20 days until they covered the entire bottom of the flask. The cells were then trypsinized in sterile tubes and centrifuged (1200 rotary/min for 4 min). The cells were resuspended in a new medium and counted in Neubaer chamber using trypan blue and the number of cells was set at 3 x 10⁵ cells/ml.

Cytotoxicity study

Freshly mixed materials (0.1 g), Endosequence BC Sealer (Table 1) and MTA Fillapex (Table 1), were placed on sterile teflon discs, 6 mm in diameter. Teflon discs with tested materials and empty teflon discs, serving as a control group, were placed in wells of 12-well plate (Techno Plastic Products AG, Trasadingen, Switzerland). The teflon discs were covered with cell suspension (3000 μl) with number of cells 3 x 10⁵/ml. 12-well plate was placed in a CO₂ incubator at 37şC. After incubation time of one, six, 20 and 24 hours, the teflon discs were removed from the wells and the number of viable cells was determined using trypan blue in Neubaer chamber. All samples were done in triplicate and testing for each material and each incubation period was done three times.

Table 1. Composition of Endosequence BC Selaer and MTA Fillapex.

MATERIAL
Endosequence BC Sealer	Zirconium oxide, calcium silicates, calcium phosphate monobasic, calcium hydroxide, filler and thickening agents.
MTA Fillapex	Paste A: salicylate resin, bismut trioxide, silica Paste B: silica, titanium dioxide, MTA (40%), resin

Statistical analysis

The Mann-Whitney U test was used for statistical analysis of data. The level of significance was set at 5%.

Results

In the control group, there was no statistically significant difference in the number of viable cells during four incubation periods (p≥0.05). In comparison to the control group, MTA showed less viable cells for all incubation periods (p≤0.05), while BC sealer showed less viable cells from hour 6 to hour 24 of incubation (p≤0.05), (Figure 1). The MTA sealer showed less viable cells in comparison to the BC sealer after the first hour and after 20-hour incubation period (p≤0.05), while for other incubation periods there was no statistically significant difference (p≥0.05).

Figure 1.

Number of viable cells per ml (median) during four incubation periods

Discussion

This study was designed to determine the cytotoxicity of two bioactive root canal sealers, based on MTA and bioceramics, in a cell line of mouse L929 fibroblasts. Cytotoxicity tests are usually the initial screening tests assessing the biocompatibility of dental materials. The results of the present study showed that both root canal sealers had a cytotoxic effect.

MTA Fillapex was developed in an attempt to take advantage of excellent biological properties of MTA for root canal sealers. According to the present results, MTA Fillapex showed cytotoxicity for all tested incubation periods. The findings of this study agree with several previous studies which showed that the material strongly affected cell viability although different methodologies were used (17-19). The observed cytotoxicity can be explained by its chemical composition. MTA Fillapex is composed of two pastes, one containing MTA and the other containing salicylate resin. The exact chemical formula of salicylate resin is 1,2 butylene glycol disalicylate which should be biologically compatible (20). Salicylate resin showed less toxicity in comparison to epoxy resin based materials (20), which have shown well documented toxicity and mutagenicity (21). Different derivates of salicylates resins with differences in molecular structures and size of carbon chains may influence different physical properties of the material such as solubility (22). High solubility of root canal sealer is undesirable because dissolution may cause the release of the components that could irritate periapical tissues (23). Although the solubility of MTA Fillapex material is just 0.1% (20), this might be enough for release of salicylate resin from the material, which explains the toxic effect on L929 fibroblasts. Interestingly, Vitti et al. (24) showed that MTA Fillapex solubility increases over time, from -9.31% weight variation at day one to -25.55% weight variation after 28 days. Another possible explanation for the cytotoxicity of MTA Fillapex is a highly alkaline pH environment, which is associated with setting products (calcium hydroxide) that releases hydroxyl ions (24). The increase of pH value may kill bacteria, and host cells as well, by damaging the cytoplasmatic membrane and DNA and denaturing proteins (25). Over time, hydroxyl ions release from the material decreases creating a physiological pH, which is beneficial for cell activity (26). According to Vitti et al. (24), physiological pH is not obtained even after 28 days. Alkaline pH may also explain the results obtained for Endosequence BC sealer which was also found to be cytotoxic for all incubation time periods except in the first hour of incubation. Based on the composition of Endosequence, no toxicity is expected. However, Loushine et al. (27) have demonstrated that the final setting time of this material occurs between 160 and 240 hours in a moist medium. Long setting times may be responsible for some components of the material to leach for extended periods of time and influence adversely cell viability, which may also explain the cytotoxic effect of Endosequence BC sealer in this study. The same sealer was also found to be cytotoxic in a cell culture of mouse osteoblasts (27) while Zhang et al. (28) obtained similar results in a culture of mouse fibroblast which corresponds to the results of the present study even though different cell cultures were used. However, cytotoxicity is just one aspect of biocompatibility and the results should be interpreted with caution in order not to state that a material is not biocompatible just due to its cytotoxicity.

Conclusion

MTA Fillapex and Endosequence BC sealers were both cytotoxic in a culture of mouse L929 fibroblasts.