Research Article
ISSN: 2471 657X

Cytotoxicity and metal content of tricalcium silicate-based endodontic cements

Athanasia Karypidou*1, Evangelos Trikas2, Garyfalia Dimosiari3, Elisabeth Koulaouzidou4, Nikolaos Economides5, Panagiotis Beltes6
1DDS, MSc Candidate, Division of Dental Tissues Pathology and Therapeutics, Department of Endodontology, Dental School, Aristotle University of Thessaloniki, Greece
2PhD Candidate, Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, Greece
3DDS, MSc, Division of Dental Tissues Pathology and Therapeutics, Department of Operative Dentistry, Dental School, Aristotle University of Thessaloniki, Greece
4DDS, MSc, PhD, Division of Dental Tissues Pathology and Therapeutics, Department of Basic Dental Sciences and Operative Dentistry, Dental School, Aristotle University of Thessaloniki, Greece
5DDS, MSc, PhD, Division of Dental Tissues Pathology and Therapeutics, Department of Endodontology and Basic Dental Sciences, Dental School, Aristotle University of Thessaloniki, Greece
6DDS, PhD, Division of Dental Tissues Pathology and Therapeutics, Department of Endodontology and Basic Dental Sciences, Dental School, Aristotle University of Thessaloniki, Greece
Corresponding author: Athanasia Karypidou
DDS, MSc Candidate, Division of Dental Tissues Pathology and Therapeutics, Department of Endodontology, Dental School, Aristotle University of Thessaloniki, Greece. E mail: operdent@gmail.com
Received Date: February 10, 2018 Accepted Date: February 23, 2018 Publlised Date: March 13, 2018
Citation:Athanasia Karypidou et al. (2018), Cytotoxicity and metal content of tricalcium silicate-based endodontic cements. Int J Dent & Oral Heal. 4:2, 20-26. DOI:
Copyright: ©2018 Athanasia Karypidou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited


Aim: To compare the cytotoxicity of six silicate endodontic cements and evaluate the concentrations of eleven metals (Ni, Cr, Fe, Pb, Cd, Zn, Cu, Al, As, Mn, Bi) in the materials.
Methods: Extracts derived from MTA CAPS, MTA Angelus White, Cerkamed MTA+, Masterdent MTA, Biodentine and ProRoot MTA White were incubated with MRC5 cells for 24 and 72 hours. The cytotoxicity was assessed by sulforhodamine-B assay. Samples of the same materials were also prepared and analyzed by inductively coupled plasma atomic emission spectrometry (ICP-AES). The statistical analysis was performed using one-way analysis of variance (ANOVA) and Tukey multiple comparison tests.
Results: MTA Angelus White displayed its higher antiproliferative effect after 24 hours (p<0.05). At 72 hours all the cements had similar effects on cell viability (p>0.05). Ni, Cr, Cd, Cu and Mn were not detected in any of the materials tested. Pd, As and Zn were found only in MTA-CAPS.
Conclusions: All the materials tested were proved highly biocompatible, except for MTA Angelus White, which showed mild cytotoxicity. Regarding the metal content the cements showed presence of some metals, although the detected concentrations were below the safety limits.

Keywords: Silicate Cements, Cytotoxicity, ICP-AES, Mineral Trioxide Aggregate, Biodentine



Introduction:


Mineral trioxide aggregate (MTA) has been used in numerous clinical procedures including root perforation repair(1, 2), one-visit apexification(3, 4), retrogate filling(5), pulp capping(6) and pulpotomy(7).
Several studies have demonstrated that MTA has good sealing ability(8-10) and marginal adaptation(11), excellent biological properties(12, 13) and the potential to promote hard-tissue formation and odontoblastic differentiation(14, 15). However, there are some drawbacks accompanying the use of MTA including long setting time and difficult manipulation(16). The first mineral trioxide aggregate formulation was a mixture of Portland cement and bismuth oxide in a ratio of 4:1. Several new calcium-silicate materials have been subsequently introduced aiming to alleviate the drawbacks of MTA, with minor variations in composition such as the inclusion of calcium chloride, calcium carbonate, silicon dioxide, or the omission of calcium sulfate(17-19).

Given the clinical applications of calcium-silicate cements and the fact that they are placed in direct contact with pulpal or periradicular tissues, it is essential for them to be non-toxic and biocompatible. There have been several in vitro and in vivo studies evaluating the biocompatibility of MTAs and alternative tricalcium silicates. Nevertheless, studies comparing the biocompatibility of different MTA brands and new calcium-silicate cements remain sparse.

The fact that mineral trioxide aggregate is refined from Portland cement leads to suspicion of possible contaminants or trace metals constituents in the commercial products. To ensure lack of contamination, MTA manufacturers claim to use materials obtained from refined laboratory-grade materials(20). However, previous researchers have detected traces of arsenic, lead and chromium in MTA cements(21-26).
The aim of this study was to assess the cytotoxicity of six calcium-silicate endodontic cements currently available on the market and to evaluate the concentrations of eleven heavy metals in the same materials using inductively coupled plasma-atomic emission spectrometry (ICP-AES). The null hypotheses were that the tested materials had no differences in terms of cytotoxicity and in the heavy metal content.

Materials and methods:


Cytotoxicity
Cell culture:

MRC5 cells (human lung fibroblasts) supplied by the American Type Culture Collection (ATCC) were used to evaluate the biocompatibility of the calcium-silicate cements. The cells were grown in T-75 culture flasks (Costar/Corning, NY, USA) and sub-cultured twice a week at 37 ⁰C in an atmosphere containing 5% CO2in air and 100% relative humidity. The culture medium was Dulbecco’s Modified Eagle Medium (DMEM, Life, NY, USA) supplemented with 10% FBS (Fetal Bovine Serum, Gibco, Glaskow, UK) and antibiotics (100IU/mL penicillin and 100mg/mL streptomycin).

Cell Inoculation:

Adherent cells at a logarithmic growth phase (eight to ninth passage) were detached by the addition of 2-3 ml of a 0.05% trypsin (Gibco, 1:250) and 0.02% EDTA mixture and incubated for 2-5 minutes at 37⁰C. The cells were then plated in 96-well flat-bottom microtiter plates (Costar/Corning, NY, USA) at a density of 3,000 cells per well (growth area 0.32 cm2) in 100 μL culture medium, and were left for 24 h in the incubator to resume exponential growth.

Preparation of Cement Extracts:

The composition and the manufacturers of the MTA-cements examined are shown in Table 1.

IJDOH
Table 1: Composition and manufactures of cements tested.

The materials were prepared according to the manufacturers’ instructions under aseptic conditions and placed in cylindrical teflon tubes (15 mm in diameter and 3 mm in height). The samples were allowed to set at 37⁰C in 100% relative humidity for 24 hours. After setting, the pellets were weighed to the nearest of 0.001 g and exposed to ultraviolet light for 20 minutes to ensure sterility and transferred into vials containing 5 mL DMEM. The weight to volume ratio used for extract preparation was about 0.2 gr/ml. Five milliliters of extract were drawn from each well after incubation at 37 ⁰C in 100% relative humidity for 24 hours and filtered using a 0.22-μm syringe filter. A volume of 100 μL of extract was added to the cells (thus the final volume in each well was 200 μL) and incubated for either 24 or 72 hours. Cells treated with 100 μL of DMEM were used as negative controls. Three replicate wells were prepared for each cement and the experiments were carried out at least twice. At the end of the incubation period, cell numbers were estimated by means of the sulforhodamine-B (SRB) assay.

Sulforhodamine-B (SRB) colorimetric assay

The SRB assay was performed as described by Skenan et al.(27) and modified by Papazisis et al.(28). Briefly, the culture medium was aspirated prior to fixation and 75 μL of 10% cold trichloroacetic acid were gently added to the wells. The plates were left for 30 minutes at 4 °C, washed five times with deionized water and left to dry at room temperature for at least 24 hours. Subsequently, 70 μL 0.4% (w/v) sulforhodamine B (Sigma Aldrich Corp., St. Louis, MO, USA) in 1% acetic acid solution were added to each well and left at room temperature for 20 minutes. The SRB was removed and the plates were washed five times with 1% acetic acid before air-drying. Bound SRB was solubilized with 70 μL 10 Mm unbuffered Tris-base solution (E.Merck, Darmstadt, Germany) and the plates were left on a plate shaker for at least 10 minutes. Absorbance was read at 492 nm by subtracting the background measurement of 620 nm. The test optical density (OD) was defined as the mean absorbance of each individual well minus the blank value (‘blank’ is the mean OD of the background control wells). Mean values and coefficient of variation (CV) were calculated. Each experiment was carried out at least twice. The results were expressed as a ‘survival fraction’, which was calculated as the percentage of test OD to control OD (plain medium was added to the control wells).

Heavy metal analysis:
Sample preparation

Four samples were prepared for each material (n=4). Each sample (0.2 g of MTA in unhydrated powder form weighed to the nearest of 0.001 g)was transferred to a 100 mL Teflon tube (Sanplatec, Osaka, Japan). A mixture of 2 mL HNO3(ΗΝΟ3 65%, Chem-Lab, Zedelgem, Belgium), 3 mL HCL(HCl 37%, Carlo Erba Reagents, Italy), and 2 mL HF(HF 48%, Merck Millipore, Massachusetts, USA) was added to the Teflon tube and left to stand for 1hour. The tube was capped and heated to 120°C on a heating block and then allowed to equilibrate for 2hours. Special precautions were taken when handling the hydrofluoric acid and the digestions were made in a ventilated hood. The temperature of the mixture was increased gradually to avoid the loss of metal traces by abrupt boiling. After the reaction, the mixture was cooled to room temperature, filtered through Whatman No. 40 filter paper (WhatmanPlc, Maidstone, UK) and the clear digests were quantitatively transferred to 25 mL volumetric flasks and diluted to final volumes with de-ionized water. A blank test was performed in parallel using the same procedure and the same quantities of all reagents except for the test sample.

Instrumentation

The resulting filtrates were analyzed by inductively coupled plasma atomic emission spectrometry (ICP-AES). All ICP-AES measurements were carried out using a Perkin Elmer (MA, USA) Optima 3100XL axial viewing spectrometer according to the operating condition described in Table 2. The injector was made from alumina, which is sufficientlyresistant to acidified and hydrofluoric solutions. Otherwise suchsolutions may erode the injector tube and impact the finalresults.A peristaltic pump was used to introduce sample solutionsinto the ICP-AES at a flow rate of 2 mLmin−1. A cyclonic spraychamber with a cross-flownebulizerwas employed to allowhigherrates of sample introduction into the plasma. The detection wavelengths of Ni, Cr, Fe,Pb, Cd, Zn, Cu, Al, As, Mn, and Bi were 341.476, 357.869, 259.939, 217.000, 226.502, 202.548, 324.752, 308.215, 193.696, 257.610 and 306.766 nmrespectively.

Reagents and calibration

The single-element standards used for preparation of multi-element standards were Merck traceable to NIST standards. An intermediate standard solution containing 10 mg L-1of all the above mentioned analytes was prepared by mixing suitable aliquots of single- element stock solutions (Merck) containing Ni, Cr, Fe, Pb, Cd, Zn, Cu, Al, As, Mn, and Bi at 1000 mg L-1each, and appropriate dilution. This solution was further diluted in 0.5 mol L-1HNO3 to obtain a series of low concentration working standards (0-50mg L-1). Five-point calibration curves were prepared for elements and their slope was used to estimate the sensitivity in presence of yttrium as IS.

Statistical analysis

At the first part of the experiment we compared the survival fraction resulting from SRB-assay and data were statistically analysed using paired-t test, one-way analysis of variance (ANOVA) and Bonferonni post-hoc tests. To test the hypothesis that there were differences in heavy metal content we performed one-way analysis of variance (ANOVA) and Tukey multiple comparison tests. Significance level for hypotheses’ rejection was set to p=0.05.

Results:


The results of SRB assay are presented in Figure 1.

IJDOH
Figure 1: Survival fraction after incubation of cells with the cements for 24 and 72 hours and post-hoc p-values for 24 hours. No statistical significant difference was observed for 72 hours.

One-way analysis of variance determined that after 24 hours exposure, there was a significant difference between the groups (p=0.02), though there was no difference at 72 hours (p=0.503). Post-hoc analysis revealed that the cell survival fraction after exposure to MTA Angelus White was significantly reduced in comparison with all other groups (p<0.05). Within subjects’ analysis was also performed between the 24 and 72 hours and showed that the 72 hours effect was significantly higher (p<0.05), except for Masterdent MTA (p>0.05).The concentrations of Ni, Cr, Fe, Pb, Cd, Zn, Cu, Al, As, Mn, and Bi in the tested materials are shown in Table 2.

IJDOH
Table 2: Parameters of ICP-AES and metal content analysis in the tested meterials.

For those elements where was available we hypothesized that the concentration levels would be equal for all the materials. The null hypothesis was retained for Fe (p=0.111), but was rejected for Al (p=0.002) and Bi (p=0.005). Since the null hypotheses were rejected, post-hoc analyses were performed. For the concentration of Al, MTA Angelus White differs from WhiteProRoot MTA (p=0.002) and Masterdent MTA (p=0.04). As for the Bi concentration MTA-CAPS differed from ProRoot White (p=0.041) and MTA Angelus White (p=0.028).

Discussion:


The present study showed that the cytotoxicity of the tested materials depends on the time of exposure and the material. All materials were significantly more cytotoxic at 72h exposure than at 24h, except for Masterdent. MTA Angelus White had a significantly higher cytotoxic effect compared to the other cements tested at 24hours exposure whereas at 72hours no statistical significant difference was observed between materials. No previous information regarding the cytotoxicity of MTA CAPS, Cerkamed MTA+, and Masterdent MTA is available in the literature.

Moreover, the present study demonstrated that the cytotoxities of CerkamedMTA+, Masterdent, Biodentine and White ProRoot MTA were comparable. Previous studies have indicated that grey ProRoot MTA and MTA Angelus behave similarly in terms of cytotoxicity(29, 30). Biodentine has also shown equivalent cytotoxic effect to MTA in previous studies(31, 32).

Cell culture models evaluate cytotoxicity by cell growth measurements, changes in membrane permeability, metabolic alteration and cell viability(13). In the present study, we used MRC5 fibroblasts derived from an established cell-line, because of their well-defined cultural characteristics; this eliminated any variability due to different donors and gave the possibility to achieve reproducibility. The SRB assay is a fluorescent colorimetric assay, measuring the cellular protein content of cells, as it is bonded to the basic amino acids of cell proteins. As cements are in direct contact with pulpal and periradicular tissues, their trace metal content may leach out into the body. Standard ISO 9917-1 recommends that the levels of leachable arsenic and lead in dental water-based cements should be < 2 and 100 ppm, respectively. Arsenic (As) is an element, which has been used historically and fictionally. The toxicity of As is compound dependent(33).

Previous studies have examined either the As released from MTAs or their total arsenic content. The results are controversial, with Bramante et al.(19) and Schembri et al.(26) contending that the levels of As in some commercial forms of MTA are considerably in excess of those recommended by the ISO 9917-1 standard, but Duarte et al.(21) and De-Deus et al.(23) reporting negligible levels. In our experiment, As was detected only in MTA-Caps at the level of 1,675 ppm. Other studies using similar methodologies have also detected As in White ProRoot MTA(24, 34) and in White MTA-Angelus(35). Lead (Pb) is an omnipresent toxic heavy metal, the mutagenic and genotoxic effects of which are still the subject of controversy(36). This study found Pb only in MTA-Caps (7600ppm). The other tested materials were found to be free of Pb, which is in agreement with other studies(24, 37). However, Camillieri et al.(35) detected Pb in Biodentine and White MTA-Angelus and Schembri et al.(26) detected Pb in White ProRoot MTA.

Aluminum (Al) was detected in all the tested materials in this study, except Biodentine, at different concentrations. White MTA Angelus differs significantly from White ProRoot and Masterdent MTA regarding the concentration of aluminum. The U.S. EPA secondary maximum contaminant level (MCL) for total Al is 0.05-0.2 mg/L(38). Considering that the amount of MTA used clinically is about 0.5 g, both the total amount of Al and the leaching of the metal into tissue fluids, is unlikely to exceed these values. Thus, the Al content could be considered safe from a clinical point-of-view.
The iron (Fe) content in MTA-Caps, Masterdent MTA and White ProRoot MTA was 200 ppm, 200 ppm and 250 ppm respectively, whereas no iron was detected in MTA Angelus White, Cerkamed MTA+ and Biodentine. Chang et al.(24) and Kum et al.(34) also detected Fe in White ProRoot MTA, but at significantly higher levels. The finding that some calcium silicate cements contain Fe could raise a concern that their application could cause tooth discoloration(39). However, the correlation between the iron content in MTAs and tooth discoloration needs further investigation.

Zinc (Zn) was found only in MTA at the amount of 1725 ppm. Other researchers have also detected Zn in White ProRoot MTA, but at a lower level(24, 34). Bismuth (Bi)is less toxic compared to other metals and showed no carcinogenic effect in long-term in vivo tests(40). Bismuth oxide was added to Portland cement to enhance its radiopacity. ProRoot MTA has a 20% loading of bismuth oxide as reported by the manufacturer. The results of the present study confirm this claim, as the percentage of Bidetected in White ProRoot MTA was 20%. Biodentine contains zirconium oxide instead of bismuth oxide as radiopacifier.

In the present study, the tested cements were also scanned for nickel, chromium, cadmium, copper and manganese and none of these were detected. Interestingly, in previous studies many of these metals were detected in various silicate cements. The disagreement could be attributed to the different sample digestion methods, the method applied for the measurements and the detection limit of the equipment used. The digestion in the present study was performed using a mixture of strong acids (HNO3, HCl, HF), because it has been proved that this mixture is suitable for the recovery of inorganic elements from the samples(41). The detection limit of the apparatus can also be affected by a number of factors such as additional noise resulting from line overlap and ICP operating parameters(42).

The results of this study cannot be directly compared with the maximum allowable levels for As and Pb in water-based cements proposed by ISO 9917-1 due to the difference in the methods for extraction of trace elements used. ISO 9917-1 measures metal ions leached from dental cements using 2.4 mol L-1 of hydrochloric acid. The method applied in our study is slightly modified from the one used in ISO 11466 for extraction of the total metal amount from specimens. However, under in vivo conditions, trace metals in MTAs may be slowly released into body fluids, indicating that the selected method is probably less clinically relevant. Since the aim of this study was to compare very similar products, we chose this method due to its sensitivity and its ability to differentiate the minute differences of trace metals in each material tested.

Conclusions:


This study indicated that the silicate-based cements tested demonstrated similar cytotoxic profiles, except for White MTA Angelus which showed a significantly higher effect. In parallel, all cements except for Biodentine, contain traces of metals, although in concentrations below the safety limits.

Acknowledgements:


The authors deny any conflicts of interest related to this study.




References:

  1. Ford TR, Torabinejad M, McKendry DJ, Hong CU, Kariyawasam SP. Use of mineral trioxide aggregate for repair of furcal perforations. Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics 1995;79(6):756-763.
  2. Mente J, Leo M, Panagidis D, Saure D, Pfefferle T. Treatment outcome of mineral trioxide aggregate: repair of root perforations-long-term results. J Endod 2014;40(6):790-796.
  3. Hayashi M, Shimizu A, Ebisu S. MTA for obturation of mandibular central incisors with open apices: case report. J Endod 2004;30(2):120-122.
  4. Simon S, Rilliard F, Berdal A, Machtou P. The use of mineral trioxide aggregate in one-visit apexification treatment: a prospective study. International endodontic journal 2007;40(3):186-197.
  5. Torabinejad M, Hong CU, Lee SJ, Monsef M, Pitt Ford TR. Investigation of mineral trioxide aggregate for root-end filling in dogs. J Endod 1995;21(12):603-608.
  6. Ford TR, Torabinejad M, Abedi HR, Bakland LK, Kariyawasam SP. Using mineral trioxide aggregate as a pulp-capping material. Journal of the American Dental Association (1939) 1996;127(10):1491-1494.
  7. Asgary S, Shirvani A, Fazlyab M. MTA and ferric sulfate in pulpotomy outcomes of primary molars: a systematic review and meta-analysis. The Journal of clinical pediatric dentistry 2014;39(1):1-8.
  8. Wu MK, Kontakiotis EG, Wesselink PR. Long-term seal provided by some root-end filling materials. J Endod 1998;24(8):557-560.
  9. Weldon JK, Jr., Pashley DH, Loushine RJ, Weller RN, Kimbrough WF. Sealing ability of mineral trioxide aggregate and super-EBA when used as furcation repair materials: a longitudinal study. J Endod 2002;28(6):467-470.
  10. Gandolfi MG, Sauro S, Mannocci F, Watson TF, Zanna S, Capoferri M, et al. New tetrasilicate cements as retrograde filling material: an in vitro study on fluid penetration. J Endod 2007;33(6):742-745.
  11. Torabinejad M, Smith PW, Kettering JD, Pitt Ford TR. Comparative investigation of marginal adaptation of mineral trioxide aggregate and other commonly used root-end filling materials. J Endod 1995;21(6):295-299.
  12. Koh ET, McDonald F, Pitt Ford TR, Torabinejad M. Cellular response to Mineral Trioxide Aggregate. J Endod 1998;24(8):543-547.
  13. Torabinejad M, Parirokh M. Mineral trioxide aggregate: a comprehensive literature review--part II: leakage and biocompatibility investigations. J Endod 2010;36(2):190-202.
  14. Min KS, Park HJ, Lee SK, Park SH, Hong CU, Kim HW, et al. Effect of mineral trioxide aggregate on dentin bridge formation and expression of dentin sialoprotein and heme oxygenase-1 in human dental pulp. J Endod 2008;34(6):666-670.
  15. Maeda H, Nakano T, Tomokiyo A, Fujii S, Wada N, Monnouchi S, et al. Mineral trioxide aggregate induces bone morphogenetic protein-2 expression and calcification in human periodontal ligament cells. J Endod 2010;36(4):647-652.
  16. Parirokh M, Torabinejad M. Mineral trioxide aggregate: a comprehensive literature review--Part III: Clinical applications, drawbacks, and mechanism of action. J Endod 2010;36(3):400-413.
  17. Wiltbank KB, Schwartz SA, Schindler WG. Effect of selected accelerants on the physical properties of mineral trioxide aggregate and Portland cement. J Endod 2007;33(10):1235-1238.
  18. Akbari M, Zebarjad SM, Nategh B, Rouhani A. Effect of nano silica on setting time and physical properties of mineral trioxide aggregate. J Endod 2013;39(11):1448-1451.
  19. Bramante CM, Kato MM, Assis GF, Duarte MA, Bernardineli N, Moraes IG, et al. Biocompatibility and setting time of CPM-MTA and white Portland cement clinker with or without calcium sulfate. Journal of applied oral science : revista FOB 2013;21(1):32-36.
  20. Chen CC, Ho CC, David Chen CH, Ding SJ. Physicochemical properties of calcium silicate cements for endodontic treatment. J Endod 2009;35(9):1288-1291.
  21. Duarte MA, De Oliveira Demarchi AC, Yamashita JC, Kuga MC, De Campos Fraga S. Arsenic release provided by MTA and Portland cement. Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics 2005;99(5):648-650.
  22. Monteiro Bramante C, Demarchi AC, de Moraes IG, Bernadineli N, Garcia RB, Spangberg LS, et al. Presence of arsenic in different types of MTA and white and gray Portland cement. Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics 2008;106(6):909-913.
  23. De-Deus G, de Souza MC, Sergio Fidel RA, Fidel SR, de Campos RC, Luna AS. Negligible expression of arsenic in some commercially available brands of Portland cement and mineral trioxide aggregate. J Endod 2009;35(6):887-890.
  24. Chang SW, Shon WJ, Lee W, Kum KY, Baek SH, Bae KS. Analysis of heavy metal contents in gray and white MTA and 2 kinds of Portland cement: a preliminary study. Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics 2010;109(4):642-646.
  25. Matsunaga T, Tsujimoto M, Kawashima T, Tsujimoto Y, Fujiwara M, Ookubo A, et al. Analysis of arsenic in gray and white mineral trioxide aggregates by using atomic absorption spectrometry. J Endod 2010;36(12):1988-1990.
  26. Schembri M, Peplow G, Camilleri J. Analyses of heavy metals in mineral trioxide aggregate and Portland cement. J Endod 2010;36(7):1210-1215.
  27. Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, et al. New colorimetric cytotoxicity assay for anticancer-drug screening. Journal of the National Cancer Institute 1990;82(13):1107-1112.
  28. Papazisis KT, Geromichalos GD, Dimitriadis KA, Kortsaris AH. Optimization of the sulforhodamine B colorimetric assay. Journal of immunological methods 1997;208(2):151-158.
  29. De Deus G, Ximenes R, Gurgel-Filho ED, Plotkowski MC, Coutinho-Filho T. Cytotoxicity of MTA and Portland cement on human ECV 304 endothelial cells. International endodontic journal 2005;38(9):604-609.
  30. Rezende TM, Vargas DL, Cardoso FP, Sobrinho AP, Vieira LQ. Effect of mineral trioxide aggregate on cytokine production by peritoneal macrophages. International endodontic journal 2005;38(12):896-903.
  31. Silva EJ, Senna PM, De-Deus G, Zaia AA. Cytocompatibility of Biodentine using a three-dimensional cell culture model. International endodontic journal 2016;49(6):574-580.
  32. Attik GN, Villat C, Hallay F, Pradelle-Plasse N, Bonnet H, Moreau K, et al. In vitro biocompatibility of a dentine substitute cement on human MG63 osteoblasts cells: Biodentine versus MTA((R)). International endodontic journal 2014;47(12):1133-1141.
  33. Mandal BK, Suzuki KT. Arsenic round the world: a review. Talanta 2002;58(1):201-235.
  34. Kum K-Y, Zhu Q, Safavi K, Gu Y, Bae K-S, Chang SW. Analysis of six heavy metals in Ortho mineral trioxide aggregate and ProRoot mineral trioxide aggregate by inductively coupled plasma–optical emission spectrometry. Australian Endodontic Journal 2013;39(3):126-130.
  35. Camilleri J, Kralj P, Veber M, Sinagra E. Characterization and analyses of acid-extractable and leached trace elements in dental cements. International endodontic journal 2012;45(8):737-743.
  36. Garcia-Leston J, Mendez J, Pasaro E, Laffon B. Genotoxic effects of lead: an updated review. Environment international 2010;36(6):623-636.
  37. Chang SW, Baek SH, Yang HC, Seo DG, Hong ST, Han SH, et al. Heavy metal analysis of ortho MTA and ProRoot MTA. J Endod 2011;37(12):1673-1676.
  38. Willhite CC, Ball GL, McLellan CJ. Total allowable concentrations of monomeric inorganic aluminum and hydrated aluminum silicates in drinking water. Critical reviews in toxicology 2012;42(5):358-442.
  39. Belobrov I, Parashos P. Treatment of tooth discoloration after the use of white mineral trioxide aggregate. J Endod 2011;37(7):1017-1020.
  40. Babula P, Adam V, Opatrilova R, Zehnalek J, Havel L, Kizek R. Uncommon heavy metals, metalloids and their plant toxicity: a review. Environ Chem Lett 2008;6(4):189-213.
  41. Zachariadis GA, Sahanidou E. Multi-element method for determination of trace elements in sunscreens by ICP-AES. Journal of pharmaceutical and biomedical analysis 2009;50(3):342-348.
  42. Boumans PWJM, Vrakking JJAM. Detection limit including selectivity as a criterion for line selection in trace analysis using inductively coupled plasma-atomic emission spectrometry (ICP-AES)—a tutorial treatment of a fundamental problem of AES. Spectrochimica Acta Part B: Atomic Spectroscopy 1987;42(6):819-840.



...

Information Menu

Upcoming Conferences

...