Toxicity of bioceramic and resinous endodontic sealers using an alternative animal model: Artemia salina.

Aims: The present study assessed the toxicity of a novel calcium silicate-based root canal sealer (Bio-C Sealer) in comparison to Endosequence BC Sealer and AH Plus through a lethality assay involving brine shrimp (Artemia salina).
Methods: Brine shrimp cysts were incubated for 24 h for the hatching of the larvae, which were then exposed to different concentrations (2.5, 5, 10, 20, 40, 80, and 100 μg/mL) of the test endodontic sealers for 24 h, followed by the determination of the survival rate. Statistical Analysis Used: One-way repeated-measures ANOVA and the Newman-Keuls post hoc test were used to compare the different materials as well as different concentrations of the same material. Dunnett's test was used to compare the different concentrations and different sealers to the control. The lethal concentration of each endodontic sealer necessary to kill 50% of the brine shrimp larvae (LC50) was also determined.
Results: The toxicity of Bio-C (10, 20, 40, 80, and 100 μg/mL) and Endosequence BC Sealer (20, 80, and 100 μg/mL) was lower than that of AH Plus. No significant difference was found between Bio-C and Endosequence BC Sealer or among the different intragroup concentrations of these sealers. In the AH Plus group, concentrations ≥5.0 μg/mL exhibited greater toxicity compared to the concentration of 2.5 μg/mL and the control. AH Plus had the lowest LC50 (59.95 μg/mL), whereas Bio-C and Endosequence BC Sealer had LC50 values >200 μg/mL. Conclusions: Bio-C Sealer proved to be less toxic than AH Plus and exhibited similar toxicity to that of Endosequence BC Sealer. Copyright:

INTRODUCTION

Obturation is the final operative step of root canal treatment and the endodontic sealer plays the important function of filling in areas of difficult access in the root canal system.[1] Several sealers are currently available on the market and novel materials are constantly being developed in an attempt to provide the ideal physicochemical and biological properties in a single product.[2]

AH Plus (Dentsply DeTrey Gmbh, Konstanz, Germany) is an epoxy resin-based root canal sealer widely used in clinical practice and employed as the “gold standard” in scientific studies due to its excellent physicochemical properties.[3] However, there is a constant search for more biocompatible sealers,[2] and calcium silicate-based bioceramic materials have received considerable attention from the endodontic community in the last decade.[3]

The literature reports that Endosequence BC Sealer (Brasseler, Savannah, GA, USA) is a calcium silicate-based root canal sealer with favorable physicochemical[34] and biological[456] properties, despite concerns of its high solubility.[7] A novel calcium silicate-based material denominated Bio-C Sealer (Angelus, Londrina, Paraná, Brazil) has recently become commercially available and studies report its promising physicochemical[89] and biological[101112] characteristics. However, knowledge regarding this material is limited and further studies are needed to support these claims.

As endodontic materials come into direct contact with dental tissues, their chemical composition could affect the inflammatory response and compromise systemic health.[13] Therefore, biocompatibility is one of the most important requirements for root canal sealers.[14] Lethality assays involving brine shrimp (Artemia salina) larvae – a miniscule halophilic marine crustacean – have been used in scientific studies as a method for estimating the toxicity of drugs and nanomaterials.[1516] This lethality test is economical, simple, fast, reliable, and comprehensive.[15] Current research suggests that applications of A. salina will continue to be used in the most diverse fields,[171819] including dentistry.[20]

Therefore, the aim of the present study was to assess the toxicity of a novel calcium silicate-based root canal sealer (Bio-C Sealer) in comparison to Endosequence BC Sealer and AH Plus through a brine shrimp lethality assay. The null hypothesis is that there is no difference in the toxic effects of the materials evaluated.

METHODS

The lethality assay with A. salina was performed following a protocol adapted from Vanhaecke et al.,[21] Brine shrimp cysts (Maramar®, Arraial do Cabo, RJ, Brazil) were acquired locally (Santa Maria, RS, Brazil) and incubated for 24 h for the hatching of the larvae (nauplii) in a glass cube with an aqueous solution containing sea salt (3.5%) at a controlled temperature (25 to 30°C), pH between 7 and 8, constant artificial light (2000 lux) and constant aeration. After this period, the test organisms (brine shrimp larvae) were exposed to the root canal sealers (Bio-C Sealer, Endosequence BC Sealer, and AH Plus) for 24 h using test tubes (containing 10 mL) with 10 larvae in each tube and seven different concentrations of the test materials in quadruplicate sets.

The test materials [Table 1] were manipulated following the manufacturers’ instructions. As the root canal sealers had a hydrophobic characteristic, 0.3 g of polysorbate 80 was added during the preparation of the materials. In the first assay, the toxicity of each root canal sealer was tested at the following concentrations: 2.5, 5, 10, 20, 40, 80, and 100 μg/mL. A control experiment was also performed to ensure that the mortality of the test organisms was due to the toxicity of the root canal sealers and not polysorbate 80. After exposure, the survival rates (%) of the larvae were calculated, with counts based on normal locomotion, such as visible movement. The lethal concentration of each endodontic sealer necessary to kill 50% of the brine shrimp larvae (LC50) was also determined. The ratio between survival/lethality and toxicity and the ratio between LC50 and toxicity are inversely proportional – meaning that lower larvae survival or a lower LC50 indicates greater toxicity.

Table 1

Materials tested and respective compositions

Material	Presentation	Composition	Industry
BC®	Premixed	Tricalcium silicate, dicalcium silicate, tricalcium aluminate, calcium oxide, zirconium oxide, silicon oxide, polyethylene glycol, iron oxide	Angelus, Londrina, PR, Brazil
ES®	Premixed	Calcium silicates, zirconium oxide, calcium phosphate monobasic, calcium hydroxide, radiopacifiers, and thickeners	Brasseler, Savannah, GA, USA
AH®	Paste-paste	Paste A: Bisphenol A epoxy resin, bisphenol F epoxy resin, calcium tungstate, zirconium oxide, aerosol, and iron oxide Paste B: Dibenzyldiamine, adamantane amine, tricyclodecane diamine, calcium tungstate, zirconium oxide, aerosol, and silicon oil	Dentsply DeTrey GmbH, Konstanz, Germany

AH: AH Plus, BC: Bio-C Sealer, ES: Endosequence BC Sealer

Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS, version 20.0, IBM Corp, New York, NY, USA) and the data were expressed as mean ± standard deviation percentage values. One-way repeated-measures ANOVA and the Newman–Keuls post hoc test were used to compare the different materials as well as different concentrations of the same material. Dunnett's test was used to compare the different concentrations and different sealers to the control. The level of significance was set at 5% (P < 0.05). GraphPad Prism version 4.0 (GraphPad, San Diego, CA, USA) was used to calculate the LC50 of the test materials.

RESULTS

The data from the toxicity test of the root canal sealers (Bio-C Sealer, Endosequence BC Sealer, and AH Plus) are displayed in Table 2. The toxicity of Bio-C (10, 20, 40, 80, and 100 μg/mL) and Endosequence BC Sealer (20, 80, and 100 μg/mL) was lower compared to that of AH Plus (P < 0.05). No significant difference was found between Bio-C and Endosequence BC Sealer or among the different intragroup concentrations of the Bio-C and Endosequence BC sealers (P > 0.05). In the AH Plus group, concentrations ≥5.0 μg/mL exhibited greater toxicity compared to the concentration of 2.5 μg/mL and the control (P < 0.05). Moreover, a higher concentration of AH Plus was associated with greater toxicity and AH Plus had the lowest LC50 (59.95 μg/mL) (greatest toxicity), whereas Bio-C and Endosequence BC Sealer were less toxic (LC50>200 μg/mL).

Table 2

Survival of artemia salina exposed to test root canal sealers

Concentration (µg/mL)	Control	AH	BC	ES
0.00	95.3±0.19	-	-	-
2.5	-	95.0±2.88a	97.5±2.50	85.0±5.00
5.0	-	70.0±7.08 b	90±4.08	95.0±5.00
10.0	-	67.5±2.5 b	97.5±2.5*	87.5±6.29
20.0	-	52.5±4.78 c	95.0±5.00*	77.5±2.5*
40.0	-	60.0±0.00 b	90.0±4.08*	80.0±4.08
80.0	-	52.5±4.78 c	92.5±2.50*	75.0±5.00*
100.0	-	50.0±4.08 c	80.0±4.08*	85.0±8.66*

*Significant difference from AH Plus, a,b,cDifferent letters denote significantly different intragroup values. Data in bold differ significantly from control. Statistical significance: P<0.05. Data expressed as mean±SD%. AH: AH Plus, BC: Bio-C Sealer, ES: Endosequence BC Sealer, SD: Standard deviation

DISCUSSION

Although diverse root canal sealers are available, there is a constant search for the ideal material that meets all the requirements of biocompatibility, bioactivity, antimicrobial activity, and adequate physicochemical properties.[2] The present study evaluated the biological activity of a novel root canal sealer (Bio-C Sealer) in comparison to Endosequence BC Sealer and AH Plus using the toxicity test with brine shrimp (A. salina). The null hypothesis was rejected, as Bio-C Sealer was less toxic than AH Plus and had comparable toxicity to that of Endosequence BC Sealer.

Due to the current trend to limit the use of vertebrate animals in toxicological tests, alternative organisms are better accepted by the scientific community for the assessment of toxicity.[1517181920] A. salina is an advantageous way to test the biological activity of diverse drugs.[1516] The lethality test with this organism is used to detect cytotoxicity, the results of which can be extrapolated to cell lines.[1516] Therefore, the use of A. salina facilitates toxicity experiments, diminishes costs, and is also an alternative to in vitro cell culture assays.[16] The brine shrimp toxicity test has been widely used for decades in the most diverse fields of research.[151617181920] In dentistry, few studies have employed A. salina as an animal model[202223] and none of the studies cited investigated the toxicity of root canal sealers. Therefore, the present work presents an innovative and simplified method to be used in the field of endodontics.

In root canal therapy, it is of the utmost importance to know the toxicity of materials before applying them clinically, as constituent compounds can cause harm to the surrounding tissues.[13] In the present study, Bio-C Sealer proved to be less cytotoxic than AH Plus. Recent studies have also found that Bio-C Sealer had better cytocompatibility in terms of viability, adhesion, migration, and cellular morphology compared to AH Plus.[1012] The present findings also show that Endosequence BC Sealer achieved better results than AH Plus. This is consistent with data reported in other studies that compared the cytotoxicity of Endosequence BC Sealer and AH Plus using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay.[456]

The toxicity of Bio-C Sealer and Endosequence BC Sealer was similar, which may be explained by the fact that these materials have a similar chemical composition (calcium silicate base). However, no previous studies have directly compared the cytotoxicity of these two materials. Thus, more studies are needed with these root canal sealers to confirm the comparability of Bio-C Sealer and Endosequence BC Sealer. Nonetheless, the present results and data from other investigations[4561012] suggest that calcium silicate-based bioceramic endodontic sealers show less cytotoxicity compared to epoxy resin-based sealers.

It is also noteworthy that the LC50 of the AH Plus was lower than that found for Bio-C Sealer and Endosequence BC Sealer, which confirms the greater toxicity of AH Plus in comparison to the bioceramic materials. In a previous study, Giacomino et al.[6] also found that calcium silicate-based sealers were significantly more biocompatible with osteoblast precursor cells than AH Plus, requiring a concentration 100-fold greater to result in the death of 50% of the cells. Moreover, AH Plus exhibited greater toxicity at higher concentrations in the present study, whereas no significant intragroup differences were found among the different concentrations of Bio-C Sealer and Endosequence BC Sealer, as the survival of the A. salina larvae was maintained even at lower dilutions of these materials, which clearly demonstrates the superior biocompatibility of these sealers in comparison to the resinous product. In agreement with these findings, Alsubait et al.[5] and López-García et al.[10] reported that the bioceramic endodontic materials Endosequence BC Sealer and Bio-C Sealer are biocompatible in different dilutions and AH Plus is cytotoxic at different concentrations.

The chemical composition of endodontic materials plays an important role in biocompatibility, which explains the results found in the present study. Bio-C Sealer and Endosequence BC Sealer contain calcium silicate and promote the slow release of Ca+ for a long period of time, even after the curing reaction. This elution of calcium ions may be responsible for the biocompatibility of bioceramic materials.[5624] On the other hand, the toxicity of AH Plus may be related to the resin-epoxy component and the release of formaldehyde from amines added to accelerate the polymerization.[25]

The results of the present study furnish information that can help dentists in the selection of an adequate, safe root canal filling material for use in clinical practice. However, despite the fact that the in vitro toxicity assessment method of endodontic sealers is reliable and enables the rigorous control of the experimental variable, the failure to consider the host immune response is a limitation of this study, which could affect the clinical results. Thus, the toxicity test is only preliminary, and more in-depth in vivo studies should be conducted to investigate and confirm the biocompatibility of these root canal sealers.

CONCLUSION

Bio-C Sealer proved to be less toxic than AH Plus and exhibited similar toxicity to that of Endosequence BC Sealer, demonstrating potential as a root canal sealer of choice in clinical practice. Further studies are needed to analyze the behavior of this novel calcium silicate-based material and confirm the findings of the present investigation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.