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Aust Endod J 2013; 39: 126–130
ORIGINAL RESEARCH
Analysis of six heavy metals in Ortho mineral trioxide aggregate and ProRoot mineral trioxide aggregate by inductively coupled plasma–optical emission spectrometry 1,
2,
2
aej_349
126..130
1
Kee-Yeon Kum, DDS, PhD *; Qiang Zhu, DDS, PhD *; Kamran Safavi, DMD, MEd ; Yu Gu, MD, MSD ; Kwang-Shik Bae, DDS, PhD1; and Seok Woo Chang, DDS, PhD3 1 Department of Conservative Dentistry, Dental Research Institute, Seoul National University Dental Hospital, Seoul National University School of Dentistry, Seoul, Korea 2 Department of Endodontology, School of Dental Medicine, University of Connecticut Health Center, Farmington, Connecticut, USA 3 Department of Conservative Dentistry, Institute of Oral Health Science, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
Keywords cadmium, heavy metal, inductively coupled plasma–optical emission spectrometry (ICP-OES), iron, Ortho MTA, ProRoot MTA. Correspondence Dr Seok Woo Chang, Department of Conservative Dentistry, Institute of Oral Health Science, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul 135-710, Korea. Email: [email protected] doi:10.1111/j.1747-4477.2012.00349.x *These authors contributed equally to this work.
Abstract Ortho mineral trioxide aggregate (MTA) is a mineral aggregate newly developed for perforation repair, root end filling and pulp capping. The aim of this study was to investigate the levels of cadmium (Cd), copper (Cu), iron (Fe), manganese (Mn), nickel (Ni) and zinc (Zn) in Ortho MTA and ProRoot MTA. A total of 0.2 g of each MTA was digested using a mixture of hydrochloric and nitric acids and filtered. Six heavy metals in the resulting filtrates were analyzed by inductively coupled plasma–optical emission spectrometry (n = 5). The results were statistically analyzed using the Mann–Whitney U-test. The concentrations of Cd, Cu, Fe, Mn, Ni and Zn in Ortho MTA were 0.10, 7.73, 49.51, 2.58, 0.82 and 10.09 p.p.m., respectively. The concentrations of Cd, Cu, Fe, Mn, Ni and Zn in ProRoot MTA were 0.16, 9.38, 1438.11, 74.51, 18.98 and 4.05 p.p.m., respectively. In conclusion, Ortho MTA had lower levels of Cd, Cu, Fe, Mn and Ni than ProRoot MTA.
Introduction Since its development in 1993 (1), mineral trioxide aggregate (MTA) has been successfully used for root perforation repair (2), root end filling (3), pulp capping (4) and one-visit apexification (5). Furthermore, recent studies showed MTA could be used as root canal sealer (6–11). The clinical success of MTA is due to its good sealing ability (12), biocompatibility (3) and potential to promote mineralised tissue formation (13). However, MTA is composed of mineral aggregate, which raises a concern regarding its heavy metal content. Recent studies (14–19) reported different results on the arsenic content of ProRoot MTA (from 0.0002 to 5.25 p.p.m.) (14,19). The difference could be attributed to the different leaching methods used in these experiments (14–19). According to the International Organization for 126
Standardization (ISO 9917-1) (20), the arsenic and lead contents in water-based cements should be less than 2 and 100 p.p.m., respectively. Our previous study showed that Ortho MTA and ProRoot MTA met the ISO specification 9917-1 regulation regarding the safety limits of the arsenic and lead contents (21). However, as of now, there is no regulation on heavy metals other than arsenic and lead. Considering that there are other heavy metals that could be toxic, it is necessary to investigate the heavy metals other than arsenic and lead contained in MTA. Up to now, there are few studies that investigated the various heavy metal content of ProRoot MTA (16,22), which showed conflicting results. Dammaschke et al. (22) reported that cadmium (Cd), manganese (Mn) and zinc (Zn) were not detected in ProRoot MTA and the concentrations of copper (Cu), iron (Fe) and nickel (Ni) in ProRoot MTA
© 2012 The Authors Australian Endodontic Journal © 2012 Australian Society of Endodontology
Six Heavy Metal Analysis of Ortho MTA
K.-Y. Kum et al.
Table 1 Components of the MTA materials tested in this study† Commercial brand of MTA White ProRoot MTA
Ortho MTA
Composition Tricalcium silicate (CaO)3•SiO2 Dicalcium silicate (CaO)2•SiO2 Tricalcium aluminate (CaO)3•Al2O3 Tetracalcium aluminoferrite (CaO)4•Al2O3•Fe2O3 Gypsum, CaSO4•2H2O Free calcium oxide, CaO Bismuth oxide, Bi2O3 Tricalcium silicate (CaO)3•SiO2 Dicalcium silicate (CaO)2•SiO2 Tricalcium aluminate (CaO)3•Al2O3 Tetracalcium aluminoferrite (CaO)4•Al2O3•Fe2O3 Free calcium oxide, CaO Bismuth oxide, Bi2O3
†The ProRoot MTA was from Dentsply (Tulsa Dental), and the Ortho MTA was from BioMTA. MTA, mineral trioxide aggregate.
were 43, 1489 and 17 p.p.m., respectively. However, Chang et al. (16) reported that the contents of Cd, Cu, Fe, Mn, Ni and Zn in ProRoot MTA were 2.09, 3.20, 23229.16, 31.75, 6.43 and 28.92 p.p.m., respectively. Considering that MTA comes into direct contact with pulpal and periradicular tissues, the contents of possibly toxic heavy metal in MTA need to be precisely investigated and confirmed. Ortho MTA is a newly developed MTA which was reported to contain less arsenic than ProRoot MTA (21). However, other heavy metal contents of Ortho MTA have not been studied yet. The aim of this study was to investigate the six heavy metal contents (Cd, Cu, Fe, Mn, Ni and Zn) of Ortho MTA and compare them with those of ProRoot MTA.
Materials and methods The materials used in this study were ProRoot MTA (tooth coloured formula, LOT number: 10003598A, Dentsply Tulsa Dental, Tulsa, OK, USA) and Ortho MTA (LOT number: O 1005 30 A 1, BioMTA, Seoul, Korea) (Table 1). For both ProRoot MTA and Ortho MTA, five samples were prepared and analyzed. The experimental procedures were described in detail in our previous report (21). A total of 0.2 g of each MTA was weighed to the nearest of 0.001 g and transferred to a 100 mL Teflon tube (Sanplatec, Osaka, Japan). A mixture of 7.0 mL of nitric acid (70% by volume) and 21 mL of hydrochloric acid (37% by volume) was added to the Teflon tube and left to stand for 2 h. The tube was capped and moved to a hood with exhaust vent, where it was heated to 105°C on a heating
© 2012 The Authors Australian Endodontic Journal © 2012 Australian Society of Endodontology
block (SCP Science, Baie D’Urfé, Québec, Canada) and then allowed to equilibrate for 2 h. The temperature of the mixture was increased gradually to avoid the loss of metal traces by abrupt boiling. After the reaction, the tube was cooled to room temperature; the mixture was filtered through a Whatman No. 40 filter paper (Whatman plc, Maidstone, UK) to get a filtrate. A blank test was performed in parallel by the same procedure with the use of the same quantities of all reagents but omitting the test sample.
Contamination control during the analytic procedure To avoid leaching of the trace metals from the walls of the sample bottles and pipette tips, these were soaked in an 8:1:1 (by volume) mixture of water, nitric acid and hydrochloric acid for more than 48 h. Then, they were rinsed with ultrapure water with a resistance of 18 MW, dried at room temperature on a clean bench and stored in a clean air environment before use. To avoid contamination, the analyst wore polyvinyl chloride gloves during cleaning and the analytic procedure.
Determination of heavy metal elements The resulting filtrates were analyzed by inductively coupled plasma–optical emission spectrometry (ICPOES; Varian Inc., Santa Clara, CA, USA). The concentrations of six heavy metals (Cd, Cu, Fe, Mn, Ni and Zn) were analyzed by ICP-OES in five replicates following the method suggested in ISO 11466 (23). The detection wavelengths of Cd, Cu, Fe, Mn, Ni and Zn were 226.502, 324.754, 259.940, 257.610, 231.604 and 213.857 nm, respectively; a 27.12 MHz and 1.2 kW radiofrequency generator was used in this experiment. Plasma gas flow was 15 L min-1, and the entrance/exit slit width was 20/30 mm, respectively.
Reagents and calibration Quantities of 1000 mg L-1 or 10 000 mg L-1 of stock standard solutions of six heavy metal species (Cd (Catalogue #140-001-485), Cu (Catalogue #140-001-295), Fe (Catalogue #140-001-265), Mn (Catalogue #140-001-255), Ni (Catalogue #140-001-285) and Zn (Catalogue #140-001305); SCP Science) were purchased and diluted into five different concentrations. Deionised water, having a resistance of 18 MW, was obtained with the use of a threestage ion exchange filter system. All reagents used in this experiment were of analytic grade or higher. The equipment was calibrated using a 5-point calibration method. Once the spectrometer was calibrated, the calibration was 127
Six Heavy Metal Analysis of Ortho MTA
(a)
K.-Y. Kum et al.
90000
Intensity (c s–1)
80000
R2 = 0.9998
70000 60000 50000 40000 30000 20000 10000 0 0
0.1
0.2
0.3
0.4
0.5
Concentration (mg L–1) (b) Intensity 110000
Intensity 80000
Cd 226.502
Intensity 300000
Cu 324.754
60000 50000
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0
8000
Intensity 600000
226.466
226.500 Mn 257.610
200000 100000 0 324.698
226.520 226.54 Intensity 14000
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Fe 259.940
324.750 Ni 231.604
324.807
259.897 259.920 Intensity 80000
259.940 Zn 213.857
259.960 259.981
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20000 0
600 257.567
257.600
257.620
257.652
231.566
231.600
231.620
231.640
213.821 213.840
213.860
213.891
Figure 1 (a) The standard curve for Cd determination using a 5-point calibration method. (b) The representative peaks (nm) of Cd, Cu, Fe, Mn, Ni and Zn.
verified with the use of standard solutions. A representative calibration curve for Cd is shown in Figure 1a.
Statistical analysis The results were analyzed statistically using the Mann– Whitney U-test. Differences with a P value < 0.05 were considered significant.
Results The concentrations of Cd, Cu, Fe, Mn, Ni and Zn in Ortho MTA and in ProRoot MTA were shown in Table 2. The concentrations of Cd, Cu, Fe, Mn and Ni in Ortho MTA were significantly lower than that in ProRoot MTA (P < 0.05). The concentration of Zn in Ortho MTA was significantly higher than that in ProRoot MTA (P < 0.05).
Discussion In this study, concentrations of six heavy metals (Cd, Cu, Fe, Mn, Ni and Zn) were investigated in Ortho MTA and compared with those in ProRoot MTA. 128
Table 2 Concentrations (p.p.m.) of Cd, Cu, Fe, Mn, Ni and Zn in ProRoot MTA and Ortho MTA Heavy metal
Ortho MTA
ProRoot MTA
Cd Cu Fe Mn Ni Zn
0.10† ⫾ 0.01 7.73† ⫾ 0.76 49.51† ⫾ 13.03 2.58† ⫾ 0.13 0.82† ⫾ 0.45 10.09† ⫾ 1.19
0.16 ⫾ 0.01 9.38 ⫾ 0.33 1438.11 ⫾ 35.49 74.51 ⫾ 1.40 18.98 ⫾ 0.37 4.05 ⫾ 1.44
ProRoot MTA was obtained from Dentsply (Tulsa Dental) and the Ortho MTA was obtained from BioMTA. †Statistically significant difference between Ortho MTA and ProRoot MTA. MTA, mineral trioxide aggregate.
Cadmium is currently known as one of the most important occupational and environmental pollutants and to be associated with genotoxicity and carcinogenicity (24). Lethal dose (LD) 50 value of cadmium was reported to be 2330 mg/kg in rat (25). Although Cd contents in ProRoot MTA were higher than those in Ortho MTA, the concentrations in both MTA were far less than the LD 50 value
© 2012 The Authors Australian Endodontic Journal © 2012 Australian Society of Endodontology
Six Heavy Metal Analysis of Ortho MTA
K.-Y. Kum et al.
(26). The Cd content of ProRoot MTA (0.16 p.p.m.) is similar to the result of a previous study (0.23 p.p.m.) (16). However, Dammaschke et al. (22) reported that Cd was not detected in ProRoot MTA. The difference could be attributed to the different leaching methods (16,22). Copper has been known to be one of the chromospheres of MTA (22). LD 50 value of copper is 370 mg L-1 for fish (26). The contents of Cu in ProRoot MTA and Ortho MTA were 9.38 and 7.73 p.p.m., respectively. The results are different from the study of Dammaschke et al. (22), which reported that copper was not detected in ProRoot MTA. Again, the difference could be the result of a different LOT number of the tested materials or the methods used in sample treatment (22). Tooth-coloured formula of ProRoot MTA (white ProRoot MTA) has been known to contain far less iron content than grey ProRoot MTA. Our previous study (16) reported that the iron contents of Gray ProRoot MTA and White ProRoot MTA were 23229.16 and 1108.33 p.p.m., respectively. The present study showed that the iron content of ProRoot MTA was 1438.11 p.p.m., which was consistent with the previous study (16). The finding that tooth-coloured MTA still contains considerable amount of Fe could be a possible reason for tooth discolouration by white ProRoot MTA (27). The cause and effect correlation between the Fe content and tooth discolouration effect of White ProRoot MTA needs further investigation. Another interesting and clinically important finding of the present study is that Ortho MTA contains far less Fe (49.51 p.p.m.) than white ProRoot MTA (1438.11 p.p.m.). This could be attributed to be the difference in starting raw materials or the manufacturing process between the two products. Manganese is an essential trace element for the adequate functioning of physiological processes (28). However, it has been also known that chronic Mn exposure can cause neurotoxicity (28). Oral LD 50 for Mn was reported to be 9000 mg/kg for a rat (29). Mn was also known to be one of the chromophores in ProRoot MTA (22). Our previous study has shown that Mn in grey ProRoot MTA and white ProRoot MTA were 31.75 and 61.66 p.p.m., respectively. It was interesting that white ProRoot MTA contained larger amount of Mn than grey ProRoot MTA. The present study found that Mn in white ProRoot MTA was 74.51 p.p.m., which is in agreement with our previous study (16). It was also a noteworthy finding that Ortho MTA (2.58 p.p.m.) contained far less manganese than white ProRoot MTA (74.51 p.p.m.). Nickel has been reported to be allergen and possible carcinogen (30). This study found that Ortho MTA contained far less nickel (0.82 p.p.m.) than white ProRoot MTA (18.98 p.p.m.). The result of Ni in ProRoot MTA (19.89 p.p.m.) is similar to the previous report (27.40 p.p.m.) (16). In the present study, Zn in ProRoot
© 2012 The Authors Australian Endodontic Journal © 2012 Australian Society of Endodontology
MTA was 4.05 p.p.m., which was much less than that in Ortho MTA (10.09 p.p.m.). This result is consistent with the previous study, which reported the Zn in ProRoot MTA to be 4.40 p.p.m. (16). Ortho MTA contained less heavy metal contents (Cd, Cu, Fe, Mn, Ni) than ProRoot MTA except for Zn. Considering the relatively small amount of MTA used clinically, the inclusion of these heavy metals in either MTA does not appear to impose a serious health problem. For example, the oral LD of cadmium is 350–3500 mg (31). Supposing that we use 1 g of MTA in a clinical procedure which contains 0.1–0.2 mg of cadmium; this amount is quite safe considering the above mentioned LD.
Conclusion In conclusion, the present study found that Ortho MTA had lower concentrations of heavy metals than ProRoot MTA except zinc. However, both MTAs might be useful as safe biomaterials when considering permitted concentration in human body. Further study is needed to evaluate the effect of the composition difference on the physical and mechanical properties of MTA cements.
Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) (2011-0014231). This study was supported by NRF of Korea funded by the MEST (2009-0086835). This study was also supported by Samsung Biomedical Research Institute grant (SBRI C-B1-310-1) and Samsung Medical Center Clinical Research Development Program grant (CRS-111-14-1).
References 1. Parirokh M, Torabinejad M. Mineral trioxide aggregate: a comprehensive literature review – Part I: chemical, physical, and antibacterial properties. J Endod 2010; 36: 16–27. 2. Mente J, Hage N, Pfefferle T et al. Treatment outcome of mineral trioxide aggregate: repair of root perforations. J Endod 2010; 36: 208–13. 3. Torabinejad M, Hong CU, Lee SJ, Monsef M, Pitt Ford TR. Investigation of mineral trioxide aggregate for rootend filling in dogs. J Endod 1995; 21: 603–8. 4. Parirokh M, Asgary S, Eghbal MJ, Kakoei S, Samiee M. A comparative study of using a combination of calcium chloride and mineral trioxide aggregate as the pulpcapping agent on dogs’ teeth. J Endod 2011; 37: 786–8.
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5. Torabinejad M, Turman M. Revitalization of tooth with necrotic pulp and open apex by using platelet-rich plasma: a case report. J Endod 2011; 37: 265–8. 6. Massi S, Tanomaru-Filho M, Silva GF et al. pH, calcium ion release, and setting time of an experimental mineral trioxide aggregate-based root canal sealer. J Endod 2011; 37: 844–6. 7. Oliveira AC, Tanomaru JM, Faria-Junior N, TanomaruFilho M. Bacterial leakage in root canals filled with conventional and MTA-based sealers. Int Endod J 2011; 44: 370–5. 8. Camilleri J, Gandolfi MG, Siboni F, Prati C. Dynamic sealing ability of MTA root canal sealer. Int Endod J 2011; 44: 9–20. 9. Scarparo RK, Haddad D, Acasigua GA, Fossati AC, Fachin EV, Grecca FS. Mineral trioxide aggregate-based sealer: analysis of tissue reactions to a new endodontic material. J Endod 2010; 36: 1174–8. 10. Bae WJ, Chang SW, Lee SI, Kum KY, Bae KS, Kim EC. Human periodontal ligament cell response to a newly developed calcium phosphate-based root canal sealer. J Endod 2010; 36: 1658–63. 11. Gandolfi MG, Prati C. MTA and F-doped MTA cements used as sealers with warm gutta-percha. Long-term study of sealing ability. Int Endod J 2010; 43: 889–901. 12. 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: 467–70. 13. Maeda H, Nakano T, Tomokiyo A et al. Mineral trioxide aggregate induces bone morphogenetic protein-2 expression and calcification in human periodontal ligament cells. J Endod 2010; 36: 647–52. 14. Monteiro Bramante C, Demarchi AC, de Moraes IG et al. Presence of arsenic in different types of MTA and white and gray Portland cement. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008; 106: 909–13. 15. Schembri M, Peplow G, Camilleri J. Analyses of heavy metals in mineral trioxide aggregate and Portland cement. J Endod 2010; 36: 1210–15. 16. 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 Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 109: 642–6. 17. 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: 887– 90.
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18. Matsunaga T, Tsujimoto M, Kawashima T et al. Analysis of arsenic in gray and white mineral trioxide aggregates by using atomic absorption spectrometry. J Endod 2010; 36: 1988–90. 19. Duarte MA, De Oliveira Demarchi AC, Yamashita JC, Kuga MC, De Campos Fraga S. Arsenic release provided by MTA and Portland cement. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005; 99: 648–50. 20. International Standardization Organization. International Standardization Organization 9917-1. Dentistry: waterbased cements – Part 1: powder/liquid acid-base cements. Geneva, Switzerland: International Standardization Organization; 2007. 21. Chang SW, Baek SH, Yang HC et al. Heavy metal analysis of Ortho MTA and ProRoot MTA. J Endod 2011; 37: 1673–6. 22. Dammaschke T, Gerth HU, Zuchner H, Schafer E. Chemical and physical surface and bulk material characterization of white ProRoot MTA and two Portland cements. Dent Mater 2005; 21: 731–8. 23. Organization IS. Extraction of trace elements soluble in aqua regia. Switzerland; 1995. 1–6. 24. Matovic V, Buha A, Bulat Z, Dukic-Cosic D. Cadmium toxicity revisited: focus on oxidative stress induction and interactions with zinc and magnesium. Arh Hig Rada Toksikol 2011; 62: 65–76. 25. Korea Occupational Safety and Health Agency. Material Safety Data Sheet: Cadmium. [Internet]. [Cited 25 Oct 2011.] Available from URL: http://www.kosha.or.kr 26. Korea Occupational Safety and Health Agency. Material Safety Date Sheet: Copper. [Internet]. [Cited 25 Oct 2011.] Available from URL: http://www.kosha.or.kr 27. Belobrov I, Parashos P. Treatment of tooth discoloration after the use of white mineral trioxide aggregate. J Endod 2011; 37: 1017–20. 28. Hernandez RB, Farina M, Esposito BP, Souza-Pinto NC, Barbosa F Jr, Sunol C. Mechanisms of manganeseinduced neurotoxicity in primary neuronal cultures: the role of manganese speciation and cell type. Toxicol Sci 2011; 124: 414–23. 29. Korea Occupational Safety and Health Agency. Material Safety Data Sheet: Mn. [Internet]. [Cited 25 Oct 2011.] Available from URL: http://www.kosha.or.kr 30. Cameron KS, Buchner V, Tchounwou PB. Exploring the molecular mechanisms of nickel-induced genotoxicity and carcinogenicity: a literature review. Rev Environ Health 2011; 26: 81–92. 31. Korea Soil Environment Center. Cadmium Toxicity. [Internet]. [Cited 15 Nov 2011.] Available from URL: http:// sec.keiti.re.kr
© 2012 The Authors Australian Endodontic Journal © 2012 Australian Society of Endodontology
Aust Endod J 2013; 39: 126–130
ORIGINAL RESEARCH
Analysis of six heavy metals in Ortho mineral trioxide aggregate and ProRoot mineral trioxide aggregate by inductively coupled plasma–optical emission spectrometry 1,
2,
2
aej_349
126..130
1
Kee-Yeon Kum, DDS, PhD *; Qiang Zhu, DDS, PhD *; Kamran Safavi, DMD, MEd ; Yu Gu, MD, MSD ; Kwang-Shik Bae, DDS, PhD1; and Seok Woo Chang, DDS, PhD3 1 Department of Conservative Dentistry, Dental Research Institute, Seoul National University Dental Hospital, Seoul National University School of Dentistry, Seoul, Korea 2 Department of Endodontology, School of Dental Medicine, University of Connecticut Health Center, Farmington, Connecticut, USA 3 Department of Conservative Dentistry, Institute of Oral Health Science, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
Keywords cadmium, heavy metal, inductively coupled plasma–optical emission spectrometry (ICP-OES), iron, Ortho MTA, ProRoot MTA. Correspondence Dr Seok Woo Chang, Department of Conservative Dentistry, Institute of Oral Health Science, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul 135-710, Korea. Email: [email protected] doi:10.1111/j.1747-4477.2012.00349.x *These authors contributed equally to this work.
Abstract Ortho mineral trioxide aggregate (MTA) is a mineral aggregate newly developed for perforation repair, root end filling and pulp capping. The aim of this study was to investigate the levels of cadmium (Cd), copper (Cu), iron (Fe), manganese (Mn), nickel (Ni) and zinc (Zn) in Ortho MTA and ProRoot MTA. A total of 0.2 g of each MTA was digested using a mixture of hydrochloric and nitric acids and filtered. Six heavy metals in the resulting filtrates were analyzed by inductively coupled plasma–optical emission spectrometry (n = 5). The results were statistically analyzed using the Mann–Whitney U-test. The concentrations of Cd, Cu, Fe, Mn, Ni and Zn in Ortho MTA were 0.10, 7.73, 49.51, 2.58, 0.82 and 10.09 p.p.m., respectively. The concentrations of Cd, Cu, Fe, Mn, Ni and Zn in ProRoot MTA were 0.16, 9.38, 1438.11, 74.51, 18.98 and 4.05 p.p.m., respectively. In conclusion, Ortho MTA had lower levels of Cd, Cu, Fe, Mn and Ni than ProRoot MTA.
Introduction Since its development in 1993 (1), mineral trioxide aggregate (MTA) has been successfully used for root perforation repair (2), root end filling (3), pulp capping (4) and one-visit apexification (5). Furthermore, recent studies showed MTA could be used as root canal sealer (6–11). The clinical success of MTA is due to its good sealing ability (12), biocompatibility (3) and potential to promote mineralised tissue formation (13). However, MTA is composed of mineral aggregate, which raises a concern regarding its heavy metal content. Recent studies (14–19) reported different results on the arsenic content of ProRoot MTA (from 0.0002 to 5.25 p.p.m.) (14,19). The difference could be attributed to the different leaching methods used in these experiments (14–19). According to the International Organization for 126
Standardization (ISO 9917-1) (20), the arsenic and lead contents in water-based cements should be less than 2 and 100 p.p.m., respectively. Our previous study showed that Ortho MTA and ProRoot MTA met the ISO specification 9917-1 regulation regarding the safety limits of the arsenic and lead contents (21). However, as of now, there is no regulation on heavy metals other than arsenic and lead. Considering that there are other heavy metals that could be toxic, it is necessary to investigate the heavy metals other than arsenic and lead contained in MTA. Up to now, there are few studies that investigated the various heavy metal content of ProRoot MTA (16,22), which showed conflicting results. Dammaschke et al. (22) reported that cadmium (Cd), manganese (Mn) and zinc (Zn) were not detected in ProRoot MTA and the concentrations of copper (Cu), iron (Fe) and nickel (Ni) in ProRoot MTA
© 2012 The Authors Australian Endodontic Journal © 2012 Australian Society of Endodontology
Six Heavy Metal Analysis of Ortho MTA
K.-Y. Kum et al.
Table 1 Components of the MTA materials tested in this study† Commercial brand of MTA White ProRoot MTA
Ortho MTA
Composition Tricalcium silicate (CaO)3•SiO2 Dicalcium silicate (CaO)2•SiO2 Tricalcium aluminate (CaO)3•Al2O3 Tetracalcium aluminoferrite (CaO)4•Al2O3•Fe2O3 Gypsum, CaSO4•2H2O Free calcium oxide, CaO Bismuth oxide, Bi2O3 Tricalcium silicate (CaO)3•SiO2 Dicalcium silicate (CaO)2•SiO2 Tricalcium aluminate (CaO)3•Al2O3 Tetracalcium aluminoferrite (CaO)4•Al2O3•Fe2O3 Free calcium oxide, CaO Bismuth oxide, Bi2O3
†The ProRoot MTA was from Dentsply (Tulsa Dental), and the Ortho MTA was from BioMTA. MTA, mineral trioxide aggregate.
were 43, 1489 and 17 p.p.m., respectively. However, Chang et al. (16) reported that the contents of Cd, Cu, Fe, Mn, Ni and Zn in ProRoot MTA were 2.09, 3.20, 23229.16, 31.75, 6.43 and 28.92 p.p.m., respectively. Considering that MTA comes into direct contact with pulpal and periradicular tissues, the contents of possibly toxic heavy metal in MTA need to be precisely investigated and confirmed. Ortho MTA is a newly developed MTA which was reported to contain less arsenic than ProRoot MTA (21). However, other heavy metal contents of Ortho MTA have not been studied yet. The aim of this study was to investigate the six heavy metal contents (Cd, Cu, Fe, Mn, Ni and Zn) of Ortho MTA and compare them with those of ProRoot MTA.
Materials and methods The materials used in this study were ProRoot MTA (tooth coloured formula, LOT number: 10003598A, Dentsply Tulsa Dental, Tulsa, OK, USA) and Ortho MTA (LOT number: O 1005 30 A 1, BioMTA, Seoul, Korea) (Table 1). For both ProRoot MTA and Ortho MTA, five samples were prepared and analyzed. The experimental procedures were described in detail in our previous report (21). A total of 0.2 g of each MTA was weighed to the nearest of 0.001 g and transferred to a 100 mL Teflon tube (Sanplatec, Osaka, Japan). A mixture of 7.0 mL of nitric acid (70% by volume) and 21 mL of hydrochloric acid (37% by volume) was added to the Teflon tube and left to stand for 2 h. The tube was capped and moved to a hood with exhaust vent, where it was heated to 105°C on a heating
© 2012 The Authors Australian Endodontic Journal © 2012 Australian Society of Endodontology
block (SCP Science, Baie D’Urfé, Québec, Canada) and then allowed to equilibrate for 2 h. The temperature of the mixture was increased gradually to avoid the loss of metal traces by abrupt boiling. After the reaction, the tube was cooled to room temperature; the mixture was filtered through a Whatman No. 40 filter paper (Whatman plc, Maidstone, UK) to get a filtrate. A blank test was performed in parallel by the same procedure with the use of the same quantities of all reagents but omitting the test sample.
Contamination control during the analytic procedure To avoid leaching of the trace metals from the walls of the sample bottles and pipette tips, these were soaked in an 8:1:1 (by volume) mixture of water, nitric acid and hydrochloric acid for more than 48 h. Then, they were rinsed with ultrapure water with a resistance of 18 MW, dried at room temperature on a clean bench and stored in a clean air environment before use. To avoid contamination, the analyst wore polyvinyl chloride gloves during cleaning and the analytic procedure.
Determination of heavy metal elements The resulting filtrates were analyzed by inductively coupled plasma–optical emission spectrometry (ICPOES; Varian Inc., Santa Clara, CA, USA). The concentrations of six heavy metals (Cd, Cu, Fe, Mn, Ni and Zn) were analyzed by ICP-OES in five replicates following the method suggested in ISO 11466 (23). The detection wavelengths of Cd, Cu, Fe, Mn, Ni and Zn were 226.502, 324.754, 259.940, 257.610, 231.604 and 213.857 nm, respectively; a 27.12 MHz and 1.2 kW radiofrequency generator was used in this experiment. Plasma gas flow was 15 L min-1, and the entrance/exit slit width was 20/30 mm, respectively.
Reagents and calibration Quantities of 1000 mg L-1 or 10 000 mg L-1 of stock standard solutions of six heavy metal species (Cd (Catalogue #140-001-485), Cu (Catalogue #140-001-295), Fe (Catalogue #140-001-265), Mn (Catalogue #140-001-255), Ni (Catalogue #140-001-285) and Zn (Catalogue #140-001305); SCP Science) were purchased and diluted into five different concentrations. Deionised water, having a resistance of 18 MW, was obtained with the use of a threestage ion exchange filter system. All reagents used in this experiment were of analytic grade or higher. The equipment was calibrated using a 5-point calibration method. Once the spectrometer was calibrated, the calibration was 127
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(a)
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90000
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80000
R2 = 0.9998
70000 60000 50000 40000 30000 20000 10000 0 0
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600 257.567
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Figure 1 (a) The standard curve for Cd determination using a 5-point calibration method. (b) The representative peaks (nm) of Cd, Cu, Fe, Mn, Ni and Zn.
verified with the use of standard solutions. A representative calibration curve for Cd is shown in Figure 1a.
Statistical analysis The results were analyzed statistically using the Mann– Whitney U-test. Differences with a P value < 0.05 were considered significant.
Results The concentrations of Cd, Cu, Fe, Mn, Ni and Zn in Ortho MTA and in ProRoot MTA were shown in Table 2. The concentrations of Cd, Cu, Fe, Mn and Ni in Ortho MTA were significantly lower than that in ProRoot MTA (P < 0.05). The concentration of Zn in Ortho MTA was significantly higher than that in ProRoot MTA (P < 0.05).
Discussion In this study, concentrations of six heavy metals (Cd, Cu, Fe, Mn, Ni and Zn) were investigated in Ortho MTA and compared with those in ProRoot MTA. 128
Table 2 Concentrations (p.p.m.) of Cd, Cu, Fe, Mn, Ni and Zn in ProRoot MTA and Ortho MTA Heavy metal
Ortho MTA
ProRoot MTA
Cd Cu Fe Mn Ni Zn
0.10† ⫾ 0.01 7.73† ⫾ 0.76 49.51† ⫾ 13.03 2.58† ⫾ 0.13 0.82† ⫾ 0.45 10.09† ⫾ 1.19
0.16 ⫾ 0.01 9.38 ⫾ 0.33 1438.11 ⫾ 35.49 74.51 ⫾ 1.40 18.98 ⫾ 0.37 4.05 ⫾ 1.44
ProRoot MTA was obtained from Dentsply (Tulsa Dental) and the Ortho MTA was obtained from BioMTA. †Statistically significant difference between Ortho MTA and ProRoot MTA. MTA, mineral trioxide aggregate.
Cadmium is currently known as one of the most important occupational and environmental pollutants and to be associated with genotoxicity and carcinogenicity (24). Lethal dose (LD) 50 value of cadmium was reported to be 2330 mg/kg in rat (25). Although Cd contents in ProRoot MTA were higher than those in Ortho MTA, the concentrations in both MTA were far less than the LD 50 value
© 2012 The Authors Australian Endodontic Journal © 2012 Australian Society of Endodontology
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(26). The Cd content of ProRoot MTA (0.16 p.p.m.) is similar to the result of a previous study (0.23 p.p.m.) (16). However, Dammaschke et al. (22) reported that Cd was not detected in ProRoot MTA. The difference could be attributed to the different leaching methods (16,22). Copper has been known to be one of the chromospheres of MTA (22). LD 50 value of copper is 370 mg L-1 for fish (26). The contents of Cu in ProRoot MTA and Ortho MTA were 9.38 and 7.73 p.p.m., respectively. The results are different from the study of Dammaschke et al. (22), which reported that copper was not detected in ProRoot MTA. Again, the difference could be the result of a different LOT number of the tested materials or the methods used in sample treatment (22). Tooth-coloured formula of ProRoot MTA (white ProRoot MTA) has been known to contain far less iron content than grey ProRoot MTA. Our previous study (16) reported that the iron contents of Gray ProRoot MTA and White ProRoot MTA were 23229.16 and 1108.33 p.p.m., respectively. The present study showed that the iron content of ProRoot MTA was 1438.11 p.p.m., which was consistent with the previous study (16). The finding that tooth-coloured MTA still contains considerable amount of Fe could be a possible reason for tooth discolouration by white ProRoot MTA (27). The cause and effect correlation between the Fe content and tooth discolouration effect of White ProRoot MTA needs further investigation. Another interesting and clinically important finding of the present study is that Ortho MTA contains far less Fe (49.51 p.p.m.) than white ProRoot MTA (1438.11 p.p.m.). This could be attributed to be the difference in starting raw materials or the manufacturing process between the two products. Manganese is an essential trace element for the adequate functioning of physiological processes (28). However, it has been also known that chronic Mn exposure can cause neurotoxicity (28). Oral LD 50 for Mn was reported to be 9000 mg/kg for a rat (29). Mn was also known to be one of the chromophores in ProRoot MTA (22). Our previous study has shown that Mn in grey ProRoot MTA and white ProRoot MTA were 31.75 and 61.66 p.p.m., respectively. It was interesting that white ProRoot MTA contained larger amount of Mn than grey ProRoot MTA. The present study found that Mn in white ProRoot MTA was 74.51 p.p.m., which is in agreement with our previous study (16). It was also a noteworthy finding that Ortho MTA (2.58 p.p.m.) contained far less manganese than white ProRoot MTA (74.51 p.p.m.). Nickel has been reported to be allergen and possible carcinogen (30). This study found that Ortho MTA contained far less nickel (0.82 p.p.m.) than white ProRoot MTA (18.98 p.p.m.). The result of Ni in ProRoot MTA (19.89 p.p.m.) is similar to the previous report (27.40 p.p.m.) (16). In the present study, Zn in ProRoot
© 2012 The Authors Australian Endodontic Journal © 2012 Australian Society of Endodontology
MTA was 4.05 p.p.m., which was much less than that in Ortho MTA (10.09 p.p.m.). This result is consistent with the previous study, which reported the Zn in ProRoot MTA to be 4.40 p.p.m. (16). Ortho MTA contained less heavy metal contents (Cd, Cu, Fe, Mn, Ni) than ProRoot MTA except for Zn. Considering the relatively small amount of MTA used clinically, the inclusion of these heavy metals in either MTA does not appear to impose a serious health problem. For example, the oral LD of cadmium is 350–3500 mg (31). Supposing that we use 1 g of MTA in a clinical procedure which contains 0.1–0.2 mg of cadmium; this amount is quite safe considering the above mentioned LD.
Conclusion In conclusion, the present study found that Ortho MTA had lower concentrations of heavy metals than ProRoot MTA except zinc. However, both MTAs might be useful as safe biomaterials when considering permitted concentration in human body. Further study is needed to evaluate the effect of the composition difference on the physical and mechanical properties of MTA cements.
Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) (2011-0014231). This study was supported by NRF of Korea funded by the MEST (2009-0086835). This study was also supported by Samsung Biomedical Research Institute grant (SBRI C-B1-310-1) and Samsung Medical Center Clinical Research Development Program grant (CRS-111-14-1).
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