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Cytotoxic outcomes of orthodontic bands with and without silver solder in different cell lineages
ORIGINAL ARTICLE
Cytotoxic outcomes of orthodontic bands with and without silver solder in different cell lineages ^s da Silva Rodrigues Junior,b Maria Martha Campos,b and Luciane Macedo de Letıcia Spinelli Jacoby,a Valne a Menezes Porto Alegre, Rio Grande do Sul, Brazil
Introduction: The safety of orthodontic materials is a matter of high interest. In this study, we aimed to assess the in-vitro cytotoxicity of orthodontic band extracts, with and without silver solder, by comparing the viability outcomes of the HaCat keratinocytes, the fibroblastic cell lineages HGF and MRC-5, and the kidney epithelial Vero cells. Methods: Sterilized orthodontic bands with and without silver solder joints were added to culture media (6 cm2/mL) and incubated for 24 hours at 37 C under continuous agitation. Subsequently, the cell cultures were exposed to the obtained extracts for 24 hours, and an assay was performed to evaluate the cell viability. Copper strip extracts were used as positive control devices. Results: The extracts from orthodontic bands with silver solder joints significantly reduced the viability of the HaCat, MRC-5, and Vero cell lines, whereas the viability of HGF was not altered by this material. Conversely, the extracts of orthodontic bands without silver solder did not significantly modify the viability index of all evaluated cell lines. Conclusions: Except for HGF fibroblasts, all tested cell lines showed decreased viability percentages after exposure to extracts of orthodontic bands containing silver solder joints. These data show the relevance of testing the toxicity of orthodontic devices in different cell lines. (Am J Orthod Dentofacial Orthop 2017;151:957-63)
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ytotoxicity is secondary to cellular function alterations, which are mainly related to changes in metabolic pathways or intracellular processes.1 When choosing a dental material, it is important to have a substantial knowledge of its composition, its allergenic properties, and its toxic effects.2 Nevertheless, there are great concerns regarding the biocompatibility of orthodontic materials and the ions related to their potential toxicity. The biocompatibility of orthodontic materials is a critical issue because of the long-term contact with the oral mucosa and the potential corrosion of different materials. From Pontifıcia Universidade Cat olica do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil. a Department of Orthodontics. b Institute of Toxicology and Pharmacology. All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest, and none were reported. Supported by Coordenac¸~ao de Aperfeic¸oamento de Pessoal de Nıvel Superior, Brazil, and Conselho Nacional de Desenvolvimento Cientıfico e Tecnol ogico, Brazil, Edital Universal 14/2014—Process Number 446056/2014-6. Address correspondence to: Luciane Macedo de Menezes, Pontifıcia Universidade Cat olica do Rio Grande do Sul, 6681/P6 Av Ipiranga, Porto Alegre 90619-900, RS, Brazil; e-mail, [email protected]. Submitted, July 2016; revised and accepted, October 2016. 0889-5406/$36.00 Ó 2017 by the American Association of Orthodontists. All rights reserved. http://dx.doi.org/10.1016/j.ajodo.2016.10.030
The use of orthodontic appliances has increased over the past years. Bishara3 published a case report that showed an association between an oral lesion and an orthodontic retainer containing silver solder. This led to much research and discussion over orthodontic material biocompatibility. A regular orthodontic treatment lasts for approximately 24 months, but appliances used in the mixed dentition can remain in place for many years. Orthodontic bands are largely used, especially when auxiliary appliances are required. Silver solder is frequently necessary to connect the orthodontic bands to wires in both fixed and removable appliances. Maxillary expanders are often used for as long as 6 months and may be used for longer periods when associated with facemasks. Lingual arches, used as space maintainers, can remain in the oral cavity for several years, especially during the development of the permanent dentition. Ideally, when considering the potential toxic effects, the biocompatibility of orthodontic materials should be evaluated before any clinical use.4 Host response can be influenced by corrosion parameters, ion release profiles, and metal ion toxicity.5 Saliva can act as a corrosive agent of orthodontic appliances.6 When compared with stainless steel, silver solder shows greater corrosion, which is the possible cause of cytotoxic effects.2 Silver solder coupled with a 957
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Table. Cell lines data Cell line Organism Tissue Cell type Properties Disease Age Sex Ethnicity
HaCaT Homo sapiens, human Skin Keratinocyte Adherent Normal 62 years Male White
HGF Homo sapiens, human Mouth Fibroblast Adherent Normal Not specified Female Not specified
high-resistance nickel-chromium alloy shows corrosion when exposed to a saline solution of 0.9% sodium chloride.7 In a cell culture model, Pianigiani et al8 evaluated the biocompatibility of metallic dental materials. Oxidation was observed on the surface of the soldering material, suggesting the release of metal ions. In-vitro assays provide relevant data when assessing the biocompatibility of dental materials, and it is crucial to understand the variables affecting their outcomes. The chosen cell line, the cell density, and the passage number are critical variables for in-vitro studies.9 Even under identical culture conditions, different cell lines can show different levels of cytotoxicity. In this study, we aimed to investigate the in-vitro cytotoxicity of orthodontic bands with and without silver solder and to compare the viability outcomes of HaCat, HGF, MRC-5, and Vero cell lines exposed to orthodontic band extracts. Our data showed different profiles of cytotoxicity after exposure to orthodontic materials, depending on the tested cell line. MATERIAL AND METHODS
This in-vitro cytotoxicity study was approved by the ethics committee of the Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil. The experiments were performed following the standards of the International Organization for Standardization (10993-5 and 10993-12).10,11 The chosen cell lines are shown in the Table. All cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Grand Island, NY), supplemented with 1% penicillin/streptomycin (Gibco), 0.1% fungizone (Gibco), and 10% fetal bovine serum (Gibco). The cells were incubated in 75-cm2 flasks at 37 C, with a minimum relative humidity of 95% and an atmosphere of 5% carbon dioxide. The culture medium was replaced every 48 hours, and trypsin (Sigma-Aldrich, St Louis, Mo) was used to detach the cells when 80% confluence was reached. Tests were performed with extracts of stainless steel orthodontic bands (36 mm; Morelli, Sorocaba, S~ao Paulo, Brazil) with and without silver solder joints.
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MRC-5 Homo sapiens, human Lung Fibroblast Adherent Normal 14 weeks Male White
Vero Cercopithecus aethiops, African green monkey Kidney Epithelial Adherent Normal Adult Not specified Not specified
Pure copper strips were used as the positive control. The stainless steel orthodontic bands were composed, according to the manufacturer, of 8% to 12% nickel and 18% to 20% chromium and iron (balance). For the orthodontic bands with silver solder joints, a segment of a stainless steel 1.0-mm diameter wire composed of 8% to 10% nickel and 17% to 19% chromium and iron (Morelli) was used; this was soldered onto an orthodontic band using a 10-cm silver solder alloy composed of 55% to 57% silver, 21% to 23% copper, 15% to 19% zinc, and 4% to 6% tin (Morelli), together with 20 mg of silver solder flux composed of boric acid, potassium bifluoride, potassium hydroxide, and water (Morelli). Carborundum discs were used to remove the excess wire, and polishing was accomplished with polishing discs (Dhpro, Paranagua, Parana, Brazil). The orthodontic bands and copper strips were autoclaved before the sample's preparation. DMEM supplemented with 10% fetal bovine serum was used as the extraction vehicle because of its ability to support cellular growth and to extract polar and nonpolar substances. A ratio (surface area of the bands 3 volume of culture medium) of 6 cm2 per milliliter was used. Extractions were carried out in 4 microtubes (each, 1.5 mL) filled with DMEM supplemented with 10% fetal bovine serum containing the stainless steel orthodontic bands, the stainless steel orthodontic bands with silver solder joints, or the copper strips. The microtubes were kept at 37 C under agitation in a shaker (180 rpm) for 24 hours. To determine cell viability, 20 mL of cell suspension was mixed with 20 mL of Trypan Blue, and the cells were counted with a Neubauer Chamber on an inverted microscope (Olympus Model CH30RF100; Olympus Optical, Shinjuku-ku, Tokyo, Japan). Approximately 7000 cells per well were seeded in 96-well plates, and 100 mL of DMEM was added. After a 24-hour incubation period, the medium was removed, and the cells were exposed to the culture medium containing extracts of the orthodontic bands with and without silver solder or to the extracts of the pure copper strips for additional 24 hours. The negative control was carried out by
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Fig. Cell viability evaluated by MTT assay after 24 hours of exposure to the culture medium alone (negative control, C–), the copper strip extracts (positive control, C1), the stainless steel orthodontic band extracts (group 1), and the stainless steel orthodontic bands with silver solder joints (group 2). Data are means 6 standard error of the mean of 3 independent experiments carried out in quadruplicate. A, HaCat; B, HGF; C, MRC-5; D, Vero. *P \0.05; **P \0.01; 1-way analysis of variance followed by the Dunnett multiple comparison test.
renewing the culture medium. Four replicates were used for the test samples and the controls. Three independent experiments were performed. The study groups were as follows. 1. 2.
3.
4.
Negative control: the cells were exposed to the culture medium alone (100 mL per well). Positive control: the cells were exposed to the culture medium containing extracts of pure copper strips (100 mL per well). Stainless steel orthodontic bands (group 1): the cells were exposed to the culture medium containing extracts of the stainless steel orthodontic bands as received from the manufacturer (100 mL per well). Stainless steel orthodontic bands with silver solder joints (group 2): the cells were exposed to the culture medium containing extracts of the stainless steel orthodontic bands with silver solder joints (100 mL per well).
Cytotoxicity was assessed by the measurement of the succinate dehydrogenase activity with the methodology used in previous studies.10,14,15 Briefly, the culture medium containing the extracts was removed, and
100 mL of 10% (v/v) MTT solution (Invitrogen, Eugene, Ore) was added. A 3-hour incubation period in a 37 C, humidified, 5% carbon dioxide/air incubator was used. Subsequently, the MTT solution was removed, and the formazan crystals were dried at room temperature for 24 hour. One hundred mL of dimethyl sulfoxide was used in each well to dissolve the formed crystals, and the absorbance was measured by a spectrophotometer (SpectraMax M2e; Molecular Devices, Sunnyvale, Calif). The cell survival was expressed in relation to the negative control. To calculate the percentage of cell viability, the following equation was used. Cell viability ð%Þ 5 AbsT=AbsCN 3 100 (AbsT, absorbance of the treated cells; AbsCN, absorbance of the negative control). Statistical analysis
The collected data were analyzed by 1-way analysis of variance, followed by the Dunnett multiple comparison test (Prism 5.0 software; GraphPad, San Diego, Calif). A P value less than 0.05 was considered statistically significant.
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RESULTS
Data presented in the Figure show that the extracts obtained from pure copper strips, used as the positive control, caused a marked reduction of the viability of all tested cell lines, compared with the negative control group. Furthermore, the exposure to the extracts from orthodontic bands with silver solder (group 2) resulted in a significant reduction of the viability of HaCat, MRC-5, and Vero cell lines, whereas the viability of HGF cells remained unaltered. The extracts from orthodontic bands without silver solder (group 1) did not significantly modify the viability index of any tested cell lineages. A further analysis of the inhibition percentages for each experimental group showed distinct profiles of susceptibility for the different tested cell lines, with HaCat keratinocytes the most sensitive cell lineage for either positive control or orthodontic bands with silver solder joints (data not shown). DISCUSSION
Orthodontic appliances and their corrosive products remain in contact with the oral mucosa for long periods of time. The damage to human health resulting from the release of metallic ions from orthodontic appliances is a cause of great concern.4 Therefore, evaluation of the biocompatibility of the orthodontic material is necessary. Previous studies have shown that ion release occurs during orthodontic treatment, especially at the initial stages of therapy, although further studies are needed regarding the long-term changes.16,17 Research has shown a great concern about nickel and chromium released from orthodontic materials. The release of iron, cobalt, silver, palladium, titanium, and zinc from orthodontic materials has also been investigated.16,18 Orthodontic patients showed higher concentrations of cobalt and nickel in their buccal mucosa cells than did subjects who were not undergoing orthodontic treatment.19 An in-vitro evaluation compared the concentrations of nickel, chromium, manganese, and iron released from stainless steel orthodontic appliances in artificial saliva to estimate the daily intake of these metals.20 The results indicated that the only possible risk of exposure for orthodontic patients with stainless steel appliances would be nickel, since stainless steel seems to release a significant amount of this ion.17,18,21 Previous cytotoxicity studies have demonstrated that iron and nickel are released from stainless steel orthodontic bands in concentrations that induce both cytotoxicity and genotoxicity.22 However, some authors have shown that stainless steel did not affect cellular proliferation and that it could be
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considered a noncytotoxic material.23-25 The resistance of stainless steel to corrosion is directly related to the chromium content because of oxide formation.26 Data obtained in this study suggest that stainless steel was unable to induce cytotoxic effects in the HaCat, HGF, MRC-5, and Vero cell lines when compared with the negative control. When orthodontic bands are associated with silver solder, higher concentrations of iron, nickel, chromium, copper, silver, zinc, and cadmium were detected.22 Silver is considered to be cytotoxic on the basis of the results of several research studies.8,22-24,27-30 Silver and copper can be considered the primary causes of cytotoxicity.28 In our study, extracts of the orthodontic bands with silver solder joints significantly reduced the viability of the HaCat, MRC-5, and Vero cell lines when compared with the negative control, although HGF fibroblast cells were not significantly affected. As previously reported, decreased alkaline phosphatase activity induced by silver solder might lead to a reduction in the differentiation process and in cell proliferation that can explain our data.30 Because silver solder appears to inhibit a keratinocyte migration, it has been suggested that it could interfere with the healing process of mucosal wounds.30 The HaCat keratinocyte cell line was greatly susceptible to the exposure of the orthodontic bands with silver solder, in relation to the other tested cell lines. The sensitivity of a cell line can affect the results of a cytotoxicity study.27 Traditionally, established cell lines—human and nonhuman—are the classic experimental paradigms for the evaluation of the cytotoxicity of orthodontic materials. According to a recent review, cell types from the contact area of orthodontic appliances—immortalized human gingival keratinocytes, fibroblast cells from the human gingiva, and the human osteosarcoma cell line—and cells with no relation to orthodontic treatments—HepG2 from a kidney and PK84 skin fibroblasts—are used as models for this purpose.4 Continuous and transformed cell lines such as the HaCaT, MRC-5, and Vero have increased proliferation rates, but they still preserve characteristics of the original tissue. Continuous cell lines are recommended for standardized tests and are widely used in scientific research.9 Human gingival fibroblasts have been used to assess cytotoxicity.31-34 Previous data have shown that compared with other cell lines, the HGF cell line was less sensitive to the toxic effects of metals.31 This agrees with our results, since the HGF cell line appeared to be less sensitive to the stainless steel orthodontic bands with and without silver solder joints compared with HaCaT, MRC-5, and Vero cell lines. The HGF cell line exhibits good in-vivo reproducibility and an accurate response when specific sensitivity is required.35
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It is important that experiments are carried out under the same conditions—immersion media, incubation conditions, static/dynamic conditions, and experiment duration—to allow comparisons among the results of various studies.18 In our study, although under the same experimental conditions, the HaCaT, HGF, MRC5, and Vero cell lines had different values of cell viability percentages when exposed to the tested materials. According to our results, fibroblasts (HGF and MRC-5) could be considered less sensitive to stainless steel, silver solder, and copper exposure. Keratinocytes and epithelial cells (HaCaT and Vero) exhibited greater reductions of cell viability (Fig). This agrees with a study that evaluated the effect of cell lines on in-vitro metal ion cytotoxicity. It was demonstrated that under identical culture conditions, different cell lines had different levels of cytotoxicity.9 Therefore, it could be suggested that more than 1 type of cell line should be used when evaluating cytotoxicity. The time interval used to evaluate in-vitro cytotoxicity should be selected with caution, since it can also strongly influence the results.28 Controlled laboratory conditions validate findings from in-vitro studies. The 37 C temperature used in this study met International Organization for Standardization recommendations, and, as reported by a previous systematic literature review, it is the most used temperature in studies of metal ions released from orthodontic appliances.18 Since copper has been used as a positive control in cytotoxicity assays, it was the material of choice for this research.11,23 Previous studies that have evaluated the biocompatibility of the components of metallic dental alloys have indicated that, after 24 hours in a culture situation, the cells exposed to copper showed a significant reduction in the number of viable cells.23,27 In our research, as expected, the positive control showed a statistically significant cell viability reduction for all cell lines, validating copper as a positive control material. An MTT (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to evaluate cell viability. This is a simple, accurate, and reproducible assay that measures mitochondrial dehydrogenase activity and is the most commonly used cytotoxicity assay in orthodontics.4 In this assay, yellow watersoluble MTT is metabolically reduced in viable cells to a blue-violet insoluble formazan.10 The color intensity is measured as a wavelength with a spectrophotometer and is directly correlated to the number of viable cells. A material can be considered cytotoxic when there is a reduction of cell viability by more than 30%.10 According to this information, it is possible to infer that the stainless steel orthodontic bands did not have cytotoxic
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effects in the HaCaT, HGF, MRC-5, and Vero cell lines. Orthodontic bands with silver solder joints reduced more than 30% of the cell viability in the HaCaT and the Vero cell lines because of a decrease in succinate dehydrogenase activity after 24 hours of incubation. As previously reported, the succinate dehydrogenase behavior may be correlated to the released silver and copper.28 Both silver and copper showed low biocompatibility values in previous investigations.27,28 Some studies have shown that silver solder induces severe cytotoxicity because of the inhibition of the proliferation, growth, and development of the cells.22,24,30,36 Limberger et al24 showed that silver solder is highly cytotoxic in a dose-dependent manner and suggested that it might be harmful in the oral cavity, not only to the mucosa cells, but also to normal microbial flora involved in maintaining health. Previous studies have demonstrated significant differences between the cytotoxicity of silver solder and laser welding.29,30,37,38 The latter showed results similar to the negative control group. Therefore, laser welding could be a more biocompatible alternative to silver soldering. To minimize the cytotoxic effects of orthodontic appliances, higher standards for corrosion resistance are required from manufacturers. Moreover, research must be undertaken to reduce the duration of orthodontic treatment.39 There is great concern about the release of trace elements during orthodontic treatment and their potential toxicity. Metals released from orthodontic appliances can be considered as chronic exposure to toxicants, and further studies should investigate the sites of metal accumulation in the human body: eg, in the liver, kidneys, and hair.16 However, most data have shown that the doses of metals released by orthodontic appliances are far below the toxic level of harm.16 It is important to isolate each orthodontic material to evaluate its toxicity, since different orthodontic appliance combinations can lead to variable responses.39 Patients with allergic responses to nickel should be identified before any orthodontic treatment.21,40 Previous studies have shown that orthodontic appliances have genotoxic effects,19,22,39,40 although some results have indicated that orthodontic therapy cannot induce DNA damage in oral mucosa cells.41,42 We suggest that genotoxicity and mutagenicity of orthodontic materials should be investigated to further our knowledge on these subjects. The negative effects of sample extracts on cells in culture do not lead to the conclusion that a material is biocompatible. Both material and host characteristics influence the biocompatibility phenomena and therefore should be carefully analyzed.43 Standardization of the
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methodology for in-vitro assessment of orthodontic material cytotoxicity would allow for a better understanding of this subject. We suggest that the role of different cytotoxicity assays and their results must be considered in future orthodontic research. CONCLUSIONS
HaCat, MRC-5, and Vero cell lines showed decreased viability percentages after exposure for 24 hours to the extracts of orthodontic bands containing silver solder joints. According to our results, it can be concluded that more than 1 type of cell line must be used to evaluate the cytotoxicity of orthodontic materials because the outcomes may vary from 1 cell line to another. REFERENCES 1. Freshney RI. Culture of animal cells: a manual of basic technique and specialized applications. 6th ed. Hoboken, NJ: John Wiley & Sons; 2010. 2. Locci P, Marinucci L, Lilli C, Belcastro S, Staffolani N, Bellocchio S, et al. Biocompatibility of alloys used in orthodontics evaluated by cell culture tests. J Biomed Mater Res 2000;51:561-8. 3. Bishara SE. Oral lesions caused by an orthodontic retainer: a case report. Am J Orthod Dentofacial Orthop 1995;108:115-7. 4. Martin-Camean A, Jos A, Mellado-Garcia P, Iglesias-Linares A, Solano E, Camean AM. In vitro and in vivo evidence of the cytotoxic and genotoxic effects of metal ions released by orthodontic appliances: a review. Environ Toxicol Pharmacol 2015;40:86-113. 5. Williams DF. On the mechanisms of biocompatibility. Biomaterials 2008;29:2941-53. 6. Mikulewicz M, Wolowiec P, Loster BW, Chojnacka K. Do soft drinks affect metal ions release from orthodontic appliances? J Trace Elem Med Biol 2015;31:74-7. 7. Shigeto N, Yanagihara T, Hamada T, Budtz-Jorgensen E. Corrosion properties of soldered joints. Part I: electrochemical action of dental solder and dental nickel-chromium alloy. J Prosthet Dent 1989;62:512-5. 8. Pianigiani E, Andreassi A, Lorenzini G, Alessandrini C, Fimiani M, Atrei A, et al. Evaluation of biocompatibility of metallic dental materials in cell culture model. Bull Group Int Rech Sci Stomatol Odontol 2004;46:63-71. 9. Wataha JC, Hanks CT, Sun Z. Effect of cell line on in vitro metal ion cytotoxicity. Dent Mater 1994;10:156-61. 10. International Organization for Standardization. ISO 10993-5: 2009. Biological evaluation of medical devices: part 5. Tests for in vitro cytotoxicity. Available at: https://www.iso.org/standard/ 36406.html. Accessed March 24, 2017. 11. Internation Organization for Standardization. ISO 10993–12: 2012. Biological evaluation of medical devices: part 12. Sample preparation and reference materials. Available at: https://www. iso.org/standard/53468.html. Accessed March 24, 2017. 12. ATCC: The Global Bioresource Center 2015. American Type Culture Collection. Available at: http://www.atcc.org/. Accessed March 8, 2015. 13. Banco de Celulas do Rio de Janeiro 2015. BCRJ. Available at: http://bcrj.org.br/. Accessed March 8, 2015. 14. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63.
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15. Renck D, Machado P, Souto AA, Rosado LA, Erig T, Campos MM, et al. Design of novel potent inhibitors of human uridine phosphorylase-1: synthesis, inhibition studies, thermodynamics, and in vitro influence on 5-fluorouracil cytotoxicity. J Med Chem 2013;56:8892-902. 16. Mikulewicz M, Chojnacka K. Trace metal release from orthodontic appliances by in vivo studies: a systematic literature review. Biol Trace Elem Res 2010;137:127-38. 17. Bhaskar V, Subba Reddy VV. Biodegradation of nickel and chromium from space maintainers: an in vitro study. J Indian Soc Pedod Prev Dent 2010;28:6-12. 18. Mikulewicz M, Chojnacka K. Release of metal ions from orthodontic appliances by in vitro studies: a systematic literature review. Biol Trace Elem Res 2011;139:241-56. 19. Faccioni F, Franceschetti P, Cerpelloni M, Fracasso ME. In vivo study on metal release from fixed orthodontic appliances and DNA damage in oral mucosa cells. Am J Orthod Dentofacial Orthop 2003;124:687-94. 20. Mikulewicz M, Chojnacka K, Wozniak B, Downarowicz P. Release of metal ions from orthodontic appliances: an in vitro study. Biol Trace Elem Res 2012;146:272-80. 21. Amini F, Jafari A, Amini P, Sepasi S. Metal ion release from fixed orthodontic appliances—an in vivo study. Eur J Orthod 2012;34: 126-30. 22. Gonc¸alves TS, Menezes LM, Trindade C, Machado Mda S, Thomas P, Fenech M, et al. Cytotoxicity and genotoxicity of orthodontic bands with or without silver soldered joints. Mutat Res Genet Toxicol Environ Mutagen 2014;762:1-8. 23. Mockers O, Deroze D, Camps J. Cytotoxicity of orthodontic bands, brackets and archwires in vitro. Dent Mater 2002;18:311-7. 24. Limberger KM, Westphalen GH, Menezes LM, Medina-Silva R. Cytotoxicity of orthodontic materials assessed by survival tests in Saccharomyces cerevisiae. Dent Mater 2011;27:e81-6. 25. Spalj S, Mlacovic Zrinski M, Tudor Spalj V, Ivankovic Buljan Z. Invitro assessment of oxidative stress generated by orthodontic archwires. Am J Orthod Dentofacial Orthop 2012;141:583-9. 26. Mikulewicz M, Wolowiec P, Michalak I, Chojnacka K, Czopor W, Berniczei-Royko A, et al. Mapping chemical elements on the surface of orthodontic appliance by SEM-EDX. Med Sci Monit 2014;20:860-5. 27. Cortizo MC, De Mele MF, Cortizo AM. Metallic dental material biocompatibility in osteoblastlike cells: correlation with metal ion release. Biol Trace Elem Res 2004;100:151-68. 28. Wataha JC, Malcolm CT, Hanks CT. Correlation between cytotoxicity and the elements released by dental casting alloys. Int J Prosthodont 1995;8:9-14. 29. Vande Vannet B, Hanssens JL, Wehrbein H. The use of threedimensional oral mucosa cell cultures to assess the toxicity of soldered and welded wires. Eur J Orthod 2007;29:60-6. 30. Sestini S, Notarantonio L, Cerboni B, Alessandrini C, Fimiani M, Nannelli P, et al. In vitro toxicity evaluation of silver soldering, electrical resistance, and laser welding of orthodontic wires. Eur J Orthod 2006;28:567-72. 31. Issa Y, Brunton P, Waters CM, Watts DC. Cytotoxicity of metal ions to human oligodendroglial cells and human gingival fibroblasts assessed by mitochondrial dehydrogenase activity. Dent Mater 2008;24:281-7. 32. Messer RL, Lucas LC. Evaluations of metabolic activities as biocompatibility tools: a study of individual ions' effects on fibroblasts. Dent Mater 1999;15:1-6. 33. Messer RL, Bishop S, Lucas LC. Effects of metallic ion toxicity on human gingival fibroblasts morphology. Biomaterials 1999;20: 1647-57.
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34. Messer RL, Lucas LC. Cytotoxicity of nickel-chromium alloys: bulk alloys compared to multiple ion salt solutions. Dent Mater 2000; 16:207-12. 35. Kasten FH, Felder SM, Gettleman L, Alchediak T. A model culture system with human gingival fibroblasts for evaluating the cytotoxicity of dental materials. In Vitro 1982;18:650-60. 36. Freitas MP, Oshima HM, Menezes LM, Machado DC, Viezzer C. Cytotoxicity of silver solder employed in orthodontics. Angle Orthod 2009;79:939-44. 37. Gonc¸alves TS, de Menezes LM, Ribeiro LG, Lindholz CG, MedinaSilva R. Differences of cytotoxicity of orthodontic bands assessed by survival tests in Saccharomyces cerevisiae. Biomed Res Int 2014;2014:143283. 38. Solmi R, Martini D, Zanarini M, Isaza Penco S, Rimondini L, Carinci P, et al. Interactions of fibroblasts with soldered and laser-welded joints. Biomaterials 2004;25:735-40. 39. Hafez HS, Selim EM, Kamel Eid FH, Tawfik WA, Al-Ashkar EA, Mostafa YA. Cytotoxicity, genotoxicity, and metal release in
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patients with fixed orthodontic appliances: a longitudinal invivo study. Am J Orthod Dentofacial Orthop 2011;140: 298-308. Natarajan M, Padmanabhan S, Chitharanjan A, Narasimhan M. Evaluation of the genotoxic effects of fixed appliances on oral mucosal cells and the relationship to nickel and chromium concentrations: an in-vivo study. Am J Orthod Dentofacial Orthop 2011; 140:383-8. Angelieri F, Carlin V, Martins RA, Ribeiro DA. Biomonitoring of mutagenicity and cytotoxicity in patients undergoing fixed orthodontic therapy. Am J Orthod Dentofacial Orthop 2011;139(4 Suppl):e399-404. Angelieri F, Marcondes JP, de Almeida DC, Salvadori DM, Ribeiro DA. Genotoxicity of corrosion eluates obtained from orthodontic brackets in vitro. Am J Orthod Dentofacial Orthop 2011; 139:504-9. Williams DF. There is no such thing as a biocompatible material. Biomaterials 2014;35:10009-14.
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Cytotoxic outcomes of orthodontic bands with and without silver solder in different cell lineages ^s da Silva Rodrigues Junior,b Maria Martha Campos,b and Luciane Macedo de Letıcia Spinelli Jacoby,a Valne a Menezes Porto Alegre, Rio Grande do Sul, Brazil
Introduction: The safety of orthodontic materials is a matter of high interest. In this study, we aimed to assess the in-vitro cytotoxicity of orthodontic band extracts, with and without silver solder, by comparing the viability outcomes of the HaCat keratinocytes, the fibroblastic cell lineages HGF and MRC-5, and the kidney epithelial Vero cells. Methods: Sterilized orthodontic bands with and without silver solder joints were added to culture media (6 cm2/mL) and incubated for 24 hours at 37 C under continuous agitation. Subsequently, the cell cultures were exposed to the obtained extracts for 24 hours, and an assay was performed to evaluate the cell viability. Copper strip extracts were used as positive control devices. Results: The extracts from orthodontic bands with silver solder joints significantly reduced the viability of the HaCat, MRC-5, and Vero cell lines, whereas the viability of HGF was not altered by this material. Conversely, the extracts of orthodontic bands without silver solder did not significantly modify the viability index of all evaluated cell lines. Conclusions: Except for HGF fibroblasts, all tested cell lines showed decreased viability percentages after exposure to extracts of orthodontic bands containing silver solder joints. These data show the relevance of testing the toxicity of orthodontic devices in different cell lines. (Am J Orthod Dentofacial Orthop 2017;151:957-63)
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ytotoxicity is secondary to cellular function alterations, which are mainly related to changes in metabolic pathways or intracellular processes.1 When choosing a dental material, it is important to have a substantial knowledge of its composition, its allergenic properties, and its toxic effects.2 Nevertheless, there are great concerns regarding the biocompatibility of orthodontic materials and the ions related to their potential toxicity. The biocompatibility of orthodontic materials is a critical issue because of the long-term contact with the oral mucosa and the potential corrosion of different materials. From Pontifıcia Universidade Cat olica do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil. a Department of Orthodontics. b Institute of Toxicology and Pharmacology. All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest, and none were reported. Supported by Coordenac¸~ao de Aperfeic¸oamento de Pessoal de Nıvel Superior, Brazil, and Conselho Nacional de Desenvolvimento Cientıfico e Tecnol ogico, Brazil, Edital Universal 14/2014—Process Number 446056/2014-6. Address correspondence to: Luciane Macedo de Menezes, Pontifıcia Universidade Cat olica do Rio Grande do Sul, 6681/P6 Av Ipiranga, Porto Alegre 90619-900, RS, Brazil; e-mail, [email protected]. Submitted, July 2016; revised and accepted, October 2016. 0889-5406/$36.00 Ó 2017 by the American Association of Orthodontists. All rights reserved. http://dx.doi.org/10.1016/j.ajodo.2016.10.030
The use of orthodontic appliances has increased over the past years. Bishara3 published a case report that showed an association between an oral lesion and an orthodontic retainer containing silver solder. This led to much research and discussion over orthodontic material biocompatibility. A regular orthodontic treatment lasts for approximately 24 months, but appliances used in the mixed dentition can remain in place for many years. Orthodontic bands are largely used, especially when auxiliary appliances are required. Silver solder is frequently necessary to connect the orthodontic bands to wires in both fixed and removable appliances. Maxillary expanders are often used for as long as 6 months and may be used for longer periods when associated with facemasks. Lingual arches, used as space maintainers, can remain in the oral cavity for several years, especially during the development of the permanent dentition. Ideally, when considering the potential toxic effects, the biocompatibility of orthodontic materials should be evaluated before any clinical use.4 Host response can be influenced by corrosion parameters, ion release profiles, and metal ion toxicity.5 Saliva can act as a corrosive agent of orthodontic appliances.6 When compared with stainless steel, silver solder shows greater corrosion, which is the possible cause of cytotoxic effects.2 Silver solder coupled with a 957
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Table. Cell lines data Cell line Organism Tissue Cell type Properties Disease Age Sex Ethnicity
HaCaT Homo sapiens, human Skin Keratinocyte Adherent Normal 62 years Male White
HGF Homo sapiens, human Mouth Fibroblast Adherent Normal Not specified Female Not specified
high-resistance nickel-chromium alloy shows corrosion when exposed to a saline solution of 0.9% sodium chloride.7 In a cell culture model, Pianigiani et al8 evaluated the biocompatibility of metallic dental materials. Oxidation was observed on the surface of the soldering material, suggesting the release of metal ions. In-vitro assays provide relevant data when assessing the biocompatibility of dental materials, and it is crucial to understand the variables affecting their outcomes. The chosen cell line, the cell density, and the passage number are critical variables for in-vitro studies.9 Even under identical culture conditions, different cell lines can show different levels of cytotoxicity. In this study, we aimed to investigate the in-vitro cytotoxicity of orthodontic bands with and without silver solder and to compare the viability outcomes of HaCat, HGF, MRC-5, and Vero cell lines exposed to orthodontic band extracts. Our data showed different profiles of cytotoxicity after exposure to orthodontic materials, depending on the tested cell line. MATERIAL AND METHODS
This in-vitro cytotoxicity study was approved by the ethics committee of the Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil. The experiments were performed following the standards of the International Organization for Standardization (10993-5 and 10993-12).10,11 The chosen cell lines are shown in the Table. All cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Grand Island, NY), supplemented with 1% penicillin/streptomycin (Gibco), 0.1% fungizone (Gibco), and 10% fetal bovine serum (Gibco). The cells were incubated in 75-cm2 flasks at 37 C, with a minimum relative humidity of 95% and an atmosphere of 5% carbon dioxide. The culture medium was replaced every 48 hours, and trypsin (Sigma-Aldrich, St Louis, Mo) was used to detach the cells when 80% confluence was reached. Tests were performed with extracts of stainless steel orthodontic bands (36 mm; Morelli, Sorocaba, S~ao Paulo, Brazil) with and without silver solder joints.
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MRC-5 Homo sapiens, human Lung Fibroblast Adherent Normal 14 weeks Male White
Vero Cercopithecus aethiops, African green monkey Kidney Epithelial Adherent Normal Adult Not specified Not specified
Pure copper strips were used as the positive control. The stainless steel orthodontic bands were composed, according to the manufacturer, of 8% to 12% nickel and 18% to 20% chromium and iron (balance). For the orthodontic bands with silver solder joints, a segment of a stainless steel 1.0-mm diameter wire composed of 8% to 10% nickel and 17% to 19% chromium and iron (Morelli) was used; this was soldered onto an orthodontic band using a 10-cm silver solder alloy composed of 55% to 57% silver, 21% to 23% copper, 15% to 19% zinc, and 4% to 6% tin (Morelli), together with 20 mg of silver solder flux composed of boric acid, potassium bifluoride, potassium hydroxide, and water (Morelli). Carborundum discs were used to remove the excess wire, and polishing was accomplished with polishing discs (Dhpro, Paranagua, Parana, Brazil). The orthodontic bands and copper strips were autoclaved before the sample's preparation. DMEM supplemented with 10% fetal bovine serum was used as the extraction vehicle because of its ability to support cellular growth and to extract polar and nonpolar substances. A ratio (surface area of the bands 3 volume of culture medium) of 6 cm2 per milliliter was used. Extractions were carried out in 4 microtubes (each, 1.5 mL) filled with DMEM supplemented with 10% fetal bovine serum containing the stainless steel orthodontic bands, the stainless steel orthodontic bands with silver solder joints, or the copper strips. The microtubes were kept at 37 C under agitation in a shaker (180 rpm) for 24 hours. To determine cell viability, 20 mL of cell suspension was mixed with 20 mL of Trypan Blue, and the cells were counted with a Neubauer Chamber on an inverted microscope (Olympus Model CH30RF100; Olympus Optical, Shinjuku-ku, Tokyo, Japan). Approximately 7000 cells per well were seeded in 96-well plates, and 100 mL of DMEM was added. After a 24-hour incubation period, the medium was removed, and the cells were exposed to the culture medium containing extracts of the orthodontic bands with and without silver solder or to the extracts of the pure copper strips for additional 24 hours. The negative control was carried out by
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Fig. Cell viability evaluated by MTT assay after 24 hours of exposure to the culture medium alone (negative control, C–), the copper strip extracts (positive control, C1), the stainless steel orthodontic band extracts (group 1), and the stainless steel orthodontic bands with silver solder joints (group 2). Data are means 6 standard error of the mean of 3 independent experiments carried out in quadruplicate. A, HaCat; B, HGF; C, MRC-5; D, Vero. *P \0.05; **P \0.01; 1-way analysis of variance followed by the Dunnett multiple comparison test.
renewing the culture medium. Four replicates were used for the test samples and the controls. Three independent experiments were performed. The study groups were as follows. 1. 2.
3.
4.
Negative control: the cells were exposed to the culture medium alone (100 mL per well). Positive control: the cells were exposed to the culture medium containing extracts of pure copper strips (100 mL per well). Stainless steel orthodontic bands (group 1): the cells were exposed to the culture medium containing extracts of the stainless steel orthodontic bands as received from the manufacturer (100 mL per well). Stainless steel orthodontic bands with silver solder joints (group 2): the cells were exposed to the culture medium containing extracts of the stainless steel orthodontic bands with silver solder joints (100 mL per well).
Cytotoxicity was assessed by the measurement of the succinate dehydrogenase activity with the methodology used in previous studies.10,14,15 Briefly, the culture medium containing the extracts was removed, and
100 mL of 10% (v/v) MTT solution (Invitrogen, Eugene, Ore) was added. A 3-hour incubation period in a 37 C, humidified, 5% carbon dioxide/air incubator was used. Subsequently, the MTT solution was removed, and the formazan crystals were dried at room temperature for 24 hour. One hundred mL of dimethyl sulfoxide was used in each well to dissolve the formed crystals, and the absorbance was measured by a spectrophotometer (SpectraMax M2e; Molecular Devices, Sunnyvale, Calif). The cell survival was expressed in relation to the negative control. To calculate the percentage of cell viability, the following equation was used. Cell viability ð%Þ 5 AbsT=AbsCN 3 100 (AbsT, absorbance of the treated cells; AbsCN, absorbance of the negative control). Statistical analysis
The collected data were analyzed by 1-way analysis of variance, followed by the Dunnett multiple comparison test (Prism 5.0 software; GraphPad, San Diego, Calif). A P value less than 0.05 was considered statistically significant.
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RESULTS
Data presented in the Figure show that the extracts obtained from pure copper strips, used as the positive control, caused a marked reduction of the viability of all tested cell lines, compared with the negative control group. Furthermore, the exposure to the extracts from orthodontic bands with silver solder (group 2) resulted in a significant reduction of the viability of HaCat, MRC-5, and Vero cell lines, whereas the viability of HGF cells remained unaltered. The extracts from orthodontic bands without silver solder (group 1) did not significantly modify the viability index of any tested cell lineages. A further analysis of the inhibition percentages for each experimental group showed distinct profiles of susceptibility for the different tested cell lines, with HaCat keratinocytes the most sensitive cell lineage for either positive control or orthodontic bands with silver solder joints (data not shown). DISCUSSION
Orthodontic appliances and their corrosive products remain in contact with the oral mucosa for long periods of time. The damage to human health resulting from the release of metallic ions from orthodontic appliances is a cause of great concern.4 Therefore, evaluation of the biocompatibility of the orthodontic material is necessary. Previous studies have shown that ion release occurs during orthodontic treatment, especially at the initial stages of therapy, although further studies are needed regarding the long-term changes.16,17 Research has shown a great concern about nickel and chromium released from orthodontic materials. The release of iron, cobalt, silver, palladium, titanium, and zinc from orthodontic materials has also been investigated.16,18 Orthodontic patients showed higher concentrations of cobalt and nickel in their buccal mucosa cells than did subjects who were not undergoing orthodontic treatment.19 An in-vitro evaluation compared the concentrations of nickel, chromium, manganese, and iron released from stainless steel orthodontic appliances in artificial saliva to estimate the daily intake of these metals.20 The results indicated that the only possible risk of exposure for orthodontic patients with stainless steel appliances would be nickel, since stainless steel seems to release a significant amount of this ion.17,18,21 Previous cytotoxicity studies have demonstrated that iron and nickel are released from stainless steel orthodontic bands in concentrations that induce both cytotoxicity and genotoxicity.22 However, some authors have shown that stainless steel did not affect cellular proliferation and that it could be
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considered a noncytotoxic material.23-25 The resistance of stainless steel to corrosion is directly related to the chromium content because of oxide formation.26 Data obtained in this study suggest that stainless steel was unable to induce cytotoxic effects in the HaCat, HGF, MRC-5, and Vero cell lines when compared with the negative control. When orthodontic bands are associated with silver solder, higher concentrations of iron, nickel, chromium, copper, silver, zinc, and cadmium were detected.22 Silver is considered to be cytotoxic on the basis of the results of several research studies.8,22-24,27-30 Silver and copper can be considered the primary causes of cytotoxicity.28 In our study, extracts of the orthodontic bands with silver solder joints significantly reduced the viability of the HaCat, MRC-5, and Vero cell lines when compared with the negative control, although HGF fibroblast cells were not significantly affected. As previously reported, decreased alkaline phosphatase activity induced by silver solder might lead to a reduction in the differentiation process and in cell proliferation that can explain our data.30 Because silver solder appears to inhibit a keratinocyte migration, it has been suggested that it could interfere with the healing process of mucosal wounds.30 The HaCat keratinocyte cell line was greatly susceptible to the exposure of the orthodontic bands with silver solder, in relation to the other tested cell lines. The sensitivity of a cell line can affect the results of a cytotoxicity study.27 Traditionally, established cell lines—human and nonhuman—are the classic experimental paradigms for the evaluation of the cytotoxicity of orthodontic materials. According to a recent review, cell types from the contact area of orthodontic appliances—immortalized human gingival keratinocytes, fibroblast cells from the human gingiva, and the human osteosarcoma cell line—and cells with no relation to orthodontic treatments—HepG2 from a kidney and PK84 skin fibroblasts—are used as models for this purpose.4 Continuous and transformed cell lines such as the HaCaT, MRC-5, and Vero have increased proliferation rates, but they still preserve characteristics of the original tissue. Continuous cell lines are recommended for standardized tests and are widely used in scientific research.9 Human gingival fibroblasts have been used to assess cytotoxicity.31-34 Previous data have shown that compared with other cell lines, the HGF cell line was less sensitive to the toxic effects of metals.31 This agrees with our results, since the HGF cell line appeared to be less sensitive to the stainless steel orthodontic bands with and without silver solder joints compared with HaCaT, MRC-5, and Vero cell lines. The HGF cell line exhibits good in-vivo reproducibility and an accurate response when specific sensitivity is required.35
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It is important that experiments are carried out under the same conditions—immersion media, incubation conditions, static/dynamic conditions, and experiment duration—to allow comparisons among the results of various studies.18 In our study, although under the same experimental conditions, the HaCaT, HGF, MRC5, and Vero cell lines had different values of cell viability percentages when exposed to the tested materials. According to our results, fibroblasts (HGF and MRC-5) could be considered less sensitive to stainless steel, silver solder, and copper exposure. Keratinocytes and epithelial cells (HaCaT and Vero) exhibited greater reductions of cell viability (Fig). This agrees with a study that evaluated the effect of cell lines on in-vitro metal ion cytotoxicity. It was demonstrated that under identical culture conditions, different cell lines had different levels of cytotoxicity.9 Therefore, it could be suggested that more than 1 type of cell line should be used when evaluating cytotoxicity. The time interval used to evaluate in-vitro cytotoxicity should be selected with caution, since it can also strongly influence the results.28 Controlled laboratory conditions validate findings from in-vitro studies. The 37 C temperature used in this study met International Organization for Standardization recommendations, and, as reported by a previous systematic literature review, it is the most used temperature in studies of metal ions released from orthodontic appliances.18 Since copper has been used as a positive control in cytotoxicity assays, it was the material of choice for this research.11,23 Previous studies that have evaluated the biocompatibility of the components of metallic dental alloys have indicated that, after 24 hours in a culture situation, the cells exposed to copper showed a significant reduction in the number of viable cells.23,27 In our research, as expected, the positive control showed a statistically significant cell viability reduction for all cell lines, validating copper as a positive control material. An MTT (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to evaluate cell viability. This is a simple, accurate, and reproducible assay that measures mitochondrial dehydrogenase activity and is the most commonly used cytotoxicity assay in orthodontics.4 In this assay, yellow watersoluble MTT is metabolically reduced in viable cells to a blue-violet insoluble formazan.10 The color intensity is measured as a wavelength with a spectrophotometer and is directly correlated to the number of viable cells. A material can be considered cytotoxic when there is a reduction of cell viability by more than 30%.10 According to this information, it is possible to infer that the stainless steel orthodontic bands did not have cytotoxic
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effects in the HaCaT, HGF, MRC-5, and Vero cell lines. Orthodontic bands with silver solder joints reduced more than 30% of the cell viability in the HaCaT and the Vero cell lines because of a decrease in succinate dehydrogenase activity after 24 hours of incubation. As previously reported, the succinate dehydrogenase behavior may be correlated to the released silver and copper.28 Both silver and copper showed low biocompatibility values in previous investigations.27,28 Some studies have shown that silver solder induces severe cytotoxicity because of the inhibition of the proliferation, growth, and development of the cells.22,24,30,36 Limberger et al24 showed that silver solder is highly cytotoxic in a dose-dependent manner and suggested that it might be harmful in the oral cavity, not only to the mucosa cells, but also to normal microbial flora involved in maintaining health. Previous studies have demonstrated significant differences between the cytotoxicity of silver solder and laser welding.29,30,37,38 The latter showed results similar to the negative control group. Therefore, laser welding could be a more biocompatible alternative to silver soldering. To minimize the cytotoxic effects of orthodontic appliances, higher standards for corrosion resistance are required from manufacturers. Moreover, research must be undertaken to reduce the duration of orthodontic treatment.39 There is great concern about the release of trace elements during orthodontic treatment and their potential toxicity. Metals released from orthodontic appliances can be considered as chronic exposure to toxicants, and further studies should investigate the sites of metal accumulation in the human body: eg, in the liver, kidneys, and hair.16 However, most data have shown that the doses of metals released by orthodontic appliances are far below the toxic level of harm.16 It is important to isolate each orthodontic material to evaluate its toxicity, since different orthodontic appliance combinations can lead to variable responses.39 Patients with allergic responses to nickel should be identified before any orthodontic treatment.21,40 Previous studies have shown that orthodontic appliances have genotoxic effects,19,22,39,40 although some results have indicated that orthodontic therapy cannot induce DNA damage in oral mucosa cells.41,42 We suggest that genotoxicity and mutagenicity of orthodontic materials should be investigated to further our knowledge on these subjects. The negative effects of sample extracts on cells in culture do not lead to the conclusion that a material is biocompatible. Both material and host characteristics influence the biocompatibility phenomena and therefore should be carefully analyzed.43 Standardization of the
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methodology for in-vitro assessment of orthodontic material cytotoxicity would allow for a better understanding of this subject. We suggest that the role of different cytotoxicity assays and their results must be considered in future orthodontic research. CONCLUSIONS
HaCat, MRC-5, and Vero cell lines showed decreased viability percentages after exposure for 24 hours to the extracts of orthodontic bands containing silver solder joints. According to our results, it can be concluded that more than 1 type of cell line must be used to evaluate the cytotoxicity of orthodontic materials because the outcomes may vary from 1 cell line to another. REFERENCES 1. Freshney RI. Culture of animal cells: a manual of basic technique and specialized applications. 6th ed. Hoboken, NJ: John Wiley & Sons; 2010. 2. Locci P, Marinucci L, Lilli C, Belcastro S, Staffolani N, Bellocchio S, et al. Biocompatibility of alloys used in orthodontics evaluated by cell culture tests. J Biomed Mater Res 2000;51:561-8. 3. Bishara SE. Oral lesions caused by an orthodontic retainer: a case report. Am J Orthod Dentofacial Orthop 1995;108:115-7. 4. Martin-Camean A, Jos A, Mellado-Garcia P, Iglesias-Linares A, Solano E, Camean AM. In vitro and in vivo evidence of the cytotoxic and genotoxic effects of metal ions released by orthodontic appliances: a review. Environ Toxicol Pharmacol 2015;40:86-113. 5. Williams DF. On the mechanisms of biocompatibility. Biomaterials 2008;29:2941-53. 6. Mikulewicz M, Wolowiec P, Loster BW, Chojnacka K. Do soft drinks affect metal ions release from orthodontic appliances? J Trace Elem Med Biol 2015;31:74-7. 7. Shigeto N, Yanagihara T, Hamada T, Budtz-Jorgensen E. Corrosion properties of soldered joints. Part I: electrochemical action of dental solder and dental nickel-chromium alloy. J Prosthet Dent 1989;62:512-5. 8. Pianigiani E, Andreassi A, Lorenzini G, Alessandrini C, Fimiani M, Atrei A, et al. Evaluation of biocompatibility of metallic dental materials in cell culture model. Bull Group Int Rech Sci Stomatol Odontol 2004;46:63-71. 9. Wataha JC, Hanks CT, Sun Z. Effect of cell line on in vitro metal ion cytotoxicity. Dent Mater 1994;10:156-61. 10. International Organization for Standardization. ISO 10993-5: 2009. Biological evaluation of medical devices: part 5. Tests for in vitro cytotoxicity. Available at: https://www.iso.org/standard/ 36406.html. Accessed March 24, 2017. 11. Internation Organization for Standardization. ISO 10993–12: 2012. Biological evaluation of medical devices: part 12. Sample preparation and reference materials. Available at: https://www. iso.org/standard/53468.html. Accessed March 24, 2017. 12. ATCC: The Global Bioresource Center 2015. American Type Culture Collection. Available at: http://www.atcc.org/. Accessed March 8, 2015. 13. Banco de Celulas do Rio de Janeiro 2015. BCRJ. Available at: http://bcrj.org.br/. Accessed March 8, 2015. 14. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63.
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15. Renck D, Machado P, Souto AA, Rosado LA, Erig T, Campos MM, et al. Design of novel potent inhibitors of human uridine phosphorylase-1: synthesis, inhibition studies, thermodynamics, and in vitro influence on 5-fluorouracil cytotoxicity. J Med Chem 2013;56:8892-902. 16. Mikulewicz M, Chojnacka K. Trace metal release from orthodontic appliances by in vivo studies: a systematic literature review. Biol Trace Elem Res 2010;137:127-38. 17. Bhaskar V, Subba Reddy VV. Biodegradation of nickel and chromium from space maintainers: an in vitro study. J Indian Soc Pedod Prev Dent 2010;28:6-12. 18. Mikulewicz M, Chojnacka K. Release of metal ions from orthodontic appliances by in vitro studies: a systematic literature review. Biol Trace Elem Res 2011;139:241-56. 19. Faccioni F, Franceschetti P, Cerpelloni M, Fracasso ME. In vivo study on metal release from fixed orthodontic appliances and DNA damage in oral mucosa cells. Am J Orthod Dentofacial Orthop 2003;124:687-94. 20. Mikulewicz M, Chojnacka K, Wozniak B, Downarowicz P. Release of metal ions from orthodontic appliances: an in vitro study. Biol Trace Elem Res 2012;146:272-80. 21. Amini F, Jafari A, Amini P, Sepasi S. Metal ion release from fixed orthodontic appliances—an in vivo study. Eur J Orthod 2012;34: 126-30. 22. Gonc¸alves TS, Menezes LM, Trindade C, Machado Mda S, Thomas P, Fenech M, et al. Cytotoxicity and genotoxicity of orthodontic bands with or without silver soldered joints. Mutat Res Genet Toxicol Environ Mutagen 2014;762:1-8. 23. Mockers O, Deroze D, Camps J. Cytotoxicity of orthodontic bands, brackets and archwires in vitro. Dent Mater 2002;18:311-7. 24. Limberger KM, Westphalen GH, Menezes LM, Medina-Silva R. Cytotoxicity of orthodontic materials assessed by survival tests in Saccharomyces cerevisiae. Dent Mater 2011;27:e81-6. 25. Spalj S, Mlacovic Zrinski M, Tudor Spalj V, Ivankovic Buljan Z. Invitro assessment of oxidative stress generated by orthodontic archwires. Am J Orthod Dentofacial Orthop 2012;141:583-9. 26. Mikulewicz M, Wolowiec P, Michalak I, Chojnacka K, Czopor W, Berniczei-Royko A, et al. Mapping chemical elements on the surface of orthodontic appliance by SEM-EDX. Med Sci Monit 2014;20:860-5. 27. Cortizo MC, De Mele MF, Cortizo AM. Metallic dental material biocompatibility in osteoblastlike cells: correlation with metal ion release. Biol Trace Elem Res 2004;100:151-68. 28. Wataha JC, Malcolm CT, Hanks CT. Correlation between cytotoxicity and the elements released by dental casting alloys. Int J Prosthodont 1995;8:9-14. 29. Vande Vannet B, Hanssens JL, Wehrbein H. The use of threedimensional oral mucosa cell cultures to assess the toxicity of soldered and welded wires. Eur J Orthod 2007;29:60-6. 30. Sestini S, Notarantonio L, Cerboni B, Alessandrini C, Fimiani M, Nannelli P, et al. In vitro toxicity evaluation of silver soldering, electrical resistance, and laser welding of orthodontic wires. Eur J Orthod 2006;28:567-72. 31. Issa Y, Brunton P, Waters CM, Watts DC. Cytotoxicity of metal ions to human oligodendroglial cells and human gingival fibroblasts assessed by mitochondrial dehydrogenase activity. Dent Mater 2008;24:281-7. 32. Messer RL, Lucas LC. Evaluations of metabolic activities as biocompatibility tools: a study of individual ions' effects on fibroblasts. Dent Mater 1999;15:1-6. 33. Messer RL, Bishop S, Lucas LC. Effects of metallic ion toxicity on human gingival fibroblasts morphology. Biomaterials 1999;20: 1647-57.
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34. Messer RL, Lucas LC. Cytotoxicity of nickel-chromium alloys: bulk alloys compared to multiple ion salt solutions. Dent Mater 2000; 16:207-12. 35. Kasten FH, Felder SM, Gettleman L, Alchediak T. A model culture system with human gingival fibroblasts for evaluating the cytotoxicity of dental materials. In Vitro 1982;18:650-60. 36. Freitas MP, Oshima HM, Menezes LM, Machado DC, Viezzer C. Cytotoxicity of silver solder employed in orthodontics. Angle Orthod 2009;79:939-44. 37. Gonc¸alves TS, de Menezes LM, Ribeiro LG, Lindholz CG, MedinaSilva R. Differences of cytotoxicity of orthodontic bands assessed by survival tests in Saccharomyces cerevisiae. Biomed Res Int 2014;2014:143283. 38. Solmi R, Martini D, Zanarini M, Isaza Penco S, Rimondini L, Carinci P, et al. Interactions of fibroblasts with soldered and laser-welded joints. Biomaterials 2004;25:735-40. 39. Hafez HS, Selim EM, Kamel Eid FH, Tawfik WA, Al-Ashkar EA, Mostafa YA. Cytotoxicity, genotoxicity, and metal release in
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patients with fixed orthodontic appliances: a longitudinal invivo study. Am J Orthod Dentofacial Orthop 2011;140: 298-308. Natarajan M, Padmanabhan S, Chitharanjan A, Narasimhan M. Evaluation of the genotoxic effects of fixed appliances on oral mucosal cells and the relationship to nickel and chromium concentrations: an in-vivo study. Am J Orthod Dentofacial Orthop 2011; 140:383-8. Angelieri F, Carlin V, Martins RA, Ribeiro DA. Biomonitoring of mutagenicity and cytotoxicity in patients undergoing fixed orthodontic therapy. Am J Orthod Dentofacial Orthop 2011;139(4 Suppl):e399-404. Angelieri F, Marcondes JP, de Almeida DC, Salvadori DM, Ribeiro DA. Genotoxicity of corrosion eluates obtained from orthodontic brackets in vitro. Am J Orthod Dentofacial Orthop 2011; 139:504-9. Williams DF. There is no such thing as a biocompatible material. Biomaterials 2014;35:10009-14.
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