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J Dent Res 86(5):441-445, 2007
© 2007 International and American Associations for Dental Research

RESEARCH REPORT
Biomaterials & Bioengineering

In vitro Evaluation of Corrosion and Cytotoxicity of Orthodontic Brackets

M.T. Costa1, M.A. Lenza1,*, C.S. Gosch2, I. Costa3, and F. Ribeiro-Dias2

1 Graduate Program in Orthodontics, School of Dentistry,
2 Immunology Section, Institute of Tropical Pathology and Public Health, Federal University of Goiás–Goiânia-Goiás, Brazil; and
3 Nuclear and Energy Research Institute (IPEN/CNEN-SP), São Paulo–SP, Brazil;

* corresponding author, Av. Americano do Brasil, 904, Setor Marista, Goiânia-Goiás-Brazil CEP 74180-010, marcoslenza{at}lenza.com.br


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The corrosion resistance of AISI 304 stainless steel (AISI 304 SS) and manganese stainless steel (low-nickel SS) brackets in artificial saliva was investigated. The cytotoxic effects of their corrosion products on L929 cell culture were compared by two assays, crystal violet, to evaluate cell viability, and MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide), for cell metabolism and proliferation. The atomic absorption spectroscopic analysis of the corrosion products demonstrated that nickel and manganese ion concentrations were higher for the AISI 304 SS-bracket immersion solution as compared with the low-nickel SS brackets. Scanning electron microscopy and energy-dispersive spectroscopy demonstrated less corrosion resistance for the AISI 304 SS brackets. Although none of the bracket extracts altered L929 cell viability or morphology, the AISI 304 SS-bracket extracts decreased cellular metabolism slightly. The results indicated that the low-nickel SS presents better in vitro biocompatibility than AISI 304 SS brackets. Abbreviations used: AISI, American Iron and Steel Institute; EDS, energy-dispersive spectroscopy; OD, optical density; ISO, International Organization for Standardization; MTT, (3-{4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NiSO4, nickel sulfate; SEM, standard error of the mean; WHO, World Health Organization; and TNF, tumor necrosis factor.

KEY WORDS: corrosion • cytotoxicity • nickel • manganese • orthodontic


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The increasing use of metallic biomaterials in the medical field and in the population in general has led to a large number of studies on the detrimental effects of corrosion products in relation to health. The oral cavity is a potentially corrosive environment, and the release of metallic ions from orthodontic devices is a genuine concern.

Among the metallic corrosion products that may enter the body, nickel ion has received considerable attention, due to its carcinogenic (Oller et al., 1997), allergenic (Blanco-Dalmau et al., 1984), mutagenic (Lee et al., 1998), cytotoxic (Wataha, 2000), and genotoxic (Faccioni et al., 2003) effects. Alternative low-nickel and nickel-free alloys have come to supersede the traditional stainless steel alloy in the orthodontic bracket manufacturing process, especially for persons with nickel hypersensitivity. However, the biocompatibility of low-nickel alloy has not been widely evaluated (Rose et al., 1998; Mockers et al., 2002; Montanaro et al., 2006). The corrosion resistance of orthodontic alloys depends on the oral environment, which is influenced by several variables, such as quantity and quality of saliva, and pH of food and beverages, among others (Eliades and Bourauel, 2005). Although in vitro studies do not reproduce this complex environment, standard assays (ISO 1999) are useful to evaluate the cytotoxicity and biocompatibility of metallic medical devices.

In this study, the corrosion resistance of two types of orthodontic brackets, the AISI 304 stainless steel and low-nickel manganese brackets, was evaluated in artificial saliva. The in vitro cytotoxicity of corrosion extracts was also investigated.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sample Preparation
The two types of orthodontic brackets tested were: (i) the 0.022 slot AISI 304 SS (Roth Light®, Morelli-Brazil), and (ii) the 0.022 slot manganese SS (Nickel-free Monobloc®, Morelli-Brazil). Their compositional elements were analyzed by energy-dispersive spectroscopy (EDS, XL-30 model; EDAX, Philips) (Appendix Table). Three sets of each bracket system were weighed, autoclaved, and immersed in 6.75 ± 0.15 pH artificial saliva in sterile airtight glass tubes (Becton Dickinson Ind., Cirúrgicas Ltda, Juiz de Fora, MG, Brazil). The artificial saliva was composed of 0.40 mg/L of NaCl, 0.40 mg/L of KCl, 0.80 mg/L of CaCl2.H2O, and 1.0 mg/L of CO(NH2)2 in distilled water, and the pH was adjusted and controlled with 10 N NaOH solution (Hwang et al., 2001). The amount of saliva used was calculated by 1 mL of artificial saliva to 0.2 g of bracket ratio (ISO 1999). The brackets were maintained in immersion and stored at 37°C under stationary conditions for 21, 42, and 63 days. Tubes containing only artificial saliva were stored under the same conditions as controls. After the immersion periods, the brackets were removed from the tubes, washed in de-ionized water, dried, and stored in airtight tubes, and the artificial saliva with the corrosive product extracts was stored at 4°C until analysis.

Bracket Corrosion and Extract Characterization
We used atomic absorption spectroscopy (model CG AA 7000 BC), with air-acetylene gas flame at the 232.0- and 279.5-nm wavelength, to analyze the extracts for nickel and manganese ion concentrations, respectively. This was determined in relation to each standard curve, with a detection limit of 0.04 µg/mL for nickel and 0.02 µg/mL for manganese.

Scanning electron microscopy (EDAX Philips, XL-30 model with EDS detector) was used to analyze the bracket surfaces prior to and after artificial saliva exposure.

L929 Cell Culture
The L929 murine fibroblast cells were maintained in RPMI 1640 culture media (Sigma Chemical Co., St. Louis, MO, USA), supplemented with 10 mM HEPES and 10% fetal bovine serum (Gibson BRL, Grand Island, NY, USA), 2 mM L-glutamine, 11 mM sodium bicarbonate, 100 units/mL of penicillin, and 100 µg/mL of streptomycin. Cell preparation for cytotoxicity assays was based on Flick and Gifford (1984). The L929 cells were cultured in 96-well flat-bottomed plates (Costar, Cambridge, MA, USA) at 3.5 x 105 cells/mL concentrations (100 µL). After 24-hour culture in a humidified atmosphere containing 5% CO2, a cell monolayer was obtained.

MTT and Crystal Violet Assays
We evaluated the cytotoxicity of the corrosion products by adding 20 µL of each extract to the L929-cell monolayer. Nickel sulfate (Sigma) at increasing concentrations was used (0.01 mM, 0.1 mM, 1 mM, 10 mM) as a positive control, with artificial saliva as negative. Cytotoxicity was estimated by crystal violet (Flick and Gifford, 1984) and MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide) assays (Ribeiro-Dias et al., 2000).

Cell viability was measured by the crystal violet assay. Dead cells were detached from the plates and the cell monolayer stained by the crystal violet assay, indicating the residual cell number after exposure to toxic substances. After a 48-hour incubation, 10 µL of crystal violet (0.5% in 30% acetic acid) were added to the plate wells, which were rinsed with water after 10 min, and dried at 37°C; methanol (100 µL) was added. After wells were shaken, the optical density (OD) was measured at 620 nm on a microplate reader (Thermo Labsystems, Shanghai, China).

MTT assay is suitable for the detection of alterations in cellular metabolism, proliferation, or activation. It evaluates the mitochondrial dehydrogenase activity and depends on the degree of cell activation (Mosmann, 1983; Gerlier and Thomasset, 1986). MTT is converted to formazan by living cells, and the color intensity is directly proportional to the mitochondrial activity. Ten µL of MTT solution (Sigma) in 5 mg/mL of phosphate-buffered saline were added to each well, and the plates were incubated for an additional 4 hrs. The dissolution of MTT-formazan precipitates was achieved by the use of 10% sodium dodecyl sulfate-0.01 N HCl solution (100 µL). The plates were maintained at 35°C for 18 hrs, and the OD was measured at 550 nm on a microplate reader.

Cells cultured in the medium plus saliva without the bracket extract were used as controls for 100% of cell viability or metabolic activity, and the OD was used as reference for determination of the cytotoxicity (%) in the assays. All results were expressed in OD.

Since cell metabolism and growth vary daily in cell line cultures, we included standard murine recombinant tumor necrosis factor (TNF, Sigma) in each assay, to check for possible cell susceptibility variation. The TNF cytokine has been reported to induce L929 cell death, and the crystal violet assay is appropriate for the measurement of TNF effects in biological fluids (Flick and Gifford, 1984; Ribeiro-Dias et al., 1999). In crystal violet assays, the TNF levels detected varied from 64 to 128 lytic units/mL, and, in MTT assays, from 512 to 1024 lytic units/mL. No significant variations were found between the experiments.

Statistical Analysis
Results were expressed as a mean ± SEM (standard error of the mean). One-way ANOVA/Bonferroni’s post-test was performed with GraphPad PRISM (GraphPad Software, Inc., San Diego, CA, USA). Statistical significance was defined at the level of p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The highest concentration of nickel was detected from the AISI 304 SS-bracket extracts after a 42-day immersion period (4.46 ± 0.68 µg/mL), and from the low-nickel SS-bracket extracts after 63 days (0.07 ± 0.01 µg/mL). Maximum manganese release was also detected from the AISI 304 SS-bracket extracts after 42 days (0.90 ± 0.05 µg/mL), and from the low-nickel SS-bracket extracts after 21 days (0.11 ± 0.02 µg/mL, TableGo).


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  		Table. Nickel and Manganese Ion Concentrations in Bracket Extracts Exposed to Artificial Salivaa
 
Under scanning electron microscopy, the SS bracket surfaces presented small irregularities, which increased after saliva exposure. Low-nickel SS brackets did not present these irregularities after the same observation period (Fig. 1Go). Both bracket-surface analyses by EDS, prior to and after 63 days of artificial saliva exposure, did not demonstrate any alteration in surface components (data not shown).


Figure 1
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  		Figure 1. Scanning electron micrographs of brackets, before and after artificial saliva exposure. (A) AISI 304 stainless steel brackets before and (B) after 63-day saliva exposure. In (C), low-nickel steel brackets prior to and (D) after 63-day exposure. AISI 304 stainless steel bracket surface (B), with irregular areas suggestive of corrosive attack, and low-nickel steel bracket surface (D), presenting regular and less-altered surfaces after artificial saliva exposure.

 
Different degrees of cytotoxicity of SS and low-nickel SS bracket extracts were observed in L929 cell cultures. By observing the loss of cell monolayer integrity/cell viability by crystal violet assay, we noted that neither the SS bracket (3.0%, Fig. 2AGo) nor the low-nickel SS extracts significantly altered cell viability (2.7%, Fig. 2AGo). None of the 21-day exposure extracts affected cell viability, and a cell monolayer was preserved under light microscopy (data not shown).


Figure 2
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  		Figure 2. AISI 304 stainless steel brackets’ cytotoxic-corrosion extracts. L929 cells cultured (3.5 x 104 cells/100 µL of medium) to obtain a cell monolayer. After 24 hrs (37°C, 5% CO2), 20 µL of control saliva, low-nickel steel or AISI 304 stainless steel-bracket extracts, obtained after 42 days of incubation, were added to the culture separately. After a 48-hour exposure period (37°C, 5% CO2), crystal violet (A) and MTT (B) assays were performed as described in MATERIALS & METHODS. Data represent mean ± SEM (n = 6). *p < 0.05.

 
When the mitochondrial metabolic activity was observed by MTT assay, a slight decrease in metabolic activity (37%, p < 0.05) was detected in the 42-day SS-bracket extract cells (Fig. 2BGo), similar to the results from the 63-day extracts (data not shown). Low-nickel SS-bracket extracts did not lead to significant alterations in cell metabolism (10.7%, Fig. 2BGo). Cell metabolic activity was not altered by any of the 21-day extracts (data not shown).

We used increasing amounts of nickel sulfate as a positive control, to evaluate the nickel cytotoxic concentration in cell culture. Nickel adverse effects were clearly demonstrated, with an IC50 (50% inhibition concentration) value of 10 mM (588 µg/mL) in the crystal violet assay, and an IC50 value of 0.57 mM (33.51 µg/mL) in the MTT assay (Fig. 3Go). The increased nickel concentration induced cell death, leading to loss of cell monolayer integrity (Figs. 3A–3DGo).


Figure 3
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  		Figure 3. L929 cell culture nickel sulfate cytotoxicity. L929 cells were cultured (3.5 x 104 cells/100 µL of medium) for 24 hrs (37°C, 5% CO2) to obtain a monolayer, after which different nickel sulfate concentrations were added. After a 48-hour incubation period (37°C, 5% CO2), the cytotoxicity was assessed by crystal violet and MTT assays, as described in MATERIALS & METHODS. The data represent optical density (OD), mean ± SEM (n = 6), obtained at 620 nm (crystal violet assay) and 550 nm (MTT assay). In parentheses are percentages of extract cytotoxicity. Light-microscopic images of the L929-cell monolayer after exposure to different nickel sulfate concentrations are depicted (A,B,C,D) (scale bar: 100 µm) (magnification x 400). *p < 0.05 (medium vs. NiSO4).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In vitro corrosion of AISI 304 SS and low-nickel orthodontic brackets was evaluated. Nickel and manganese ion concentrations, detected from 63-day bracket extracts, indicated that SS brackets presented lower corrosion resistance than did low-nickel brackets when exposed to artificial saliva. The amount of nickel ions released in this study was similar to those previously reported, in spite of methodological differences (Grimsdóttir et al., 1992; Hwang et al., 2001; Mockers et al., 2002; Shin et al., 2003).

Stainless steel brackets demonstrated surface roughness suggestive of corrosion attack, which increased with increasing saliva exposure. In contrast, low-nickel brackets did not present surface morphology alteration with increasing saliva exposure, indicating a higher corrosion resistance. This is in agreement with results from other studies on stainless steel wires (Shin et al., 2003) and manganese steel alloys (Fitjer et al., 2002) facing a corrosion attack.

However, SS and low-nickel SS bracket corrosion extracts did not alter cellular viability. Under light microscopy, after extract exposure, the L929 cell culture did not present any alteration in cell morphology, and the cell monolayer was preserved. However, a slight decrease in cellular metabolism was detected when cells were exposed to SS bracket extracts. These results indicate mitochondrial metabolism as a target for corrosion products. Nickel ions undergo mitochondrial redox metabolism when in trivalent form, leading to intermediate reactive oxygen radical formation, which is toxic for the cell. Manganese ions may also lead to mitochondrial dysfunction, which could result in cellular oxidative stress (WHO, 2000a).

The nickel concentration that could lead to a 50% decrease in cellular viability and 50% inhibition of mitochondrial metabolism in L929 cell culture was estimated with the use of different nickel sulfate concentrations (Fig. 3Go). In the crystal violet assay, the IC50 mean value corresponded to 10 mM of nickel sulfate, whereas in the MTT assay it corresponded to 0.57 mM. These results demonstrate that cellular mitochondrial metabolism may be affected by a low nickel ion concentration, although higher concentrations are required for cell death. The AISI 304 SS-corrosion extracts led to a slight inhibition of cell metabolism, but not to cell death. Eliades et al.(2004) obtained an IC50 value of 1.98 mM for nickel in gingival fibroblast cultures, and 2.05 mM in periodontal ligament cell cultures through the MTT cytotoxicity assay. By comparison, the IC50 obtained in this study with L929 cells (MTT assay) highlighted cell culture differences in nickel sensitivity. Primary cells’ and cell lines’ susceptibility to nickel ions may vary according to cell line characteristics and laboratory conditions, which strengthens the importance of evaluating different cell cultures and experimental conditions to obtain an accurate estimate of nickel biocompatibility.

Although MTT assay is recommended by ISO (1999), other methods have been performed for the evaluation of nickel cytotoxicity (Taira et al., 2001; Montanaro et al., 2006). The results from the MTT assay were similar to those from Taira et al.(2001), although an assay dependent on active lysosome engulfment instead of cell metabolism was used for cell viability. Crystal violet has not been reported to evaluate cytotoxic effects of orthodontic devices. The results indicate that a higher nickel ion concentration is required to lead to in vitro cell death and loss of the integrity of a fibroblast monolayer, as opposed to a relatively low concentration, which affects cellular metabolism.

The extrapolation of the data obtained in vitro, when applied to in vivo conditions, deserves caution, because the amount of nickel ingested or absorbed via the oral mucosa during orthodontic treatment has not been well-established. In vitro corrosion assays generate corrosion products, which are added to cell cultures under static conditions for a longer period and do not mimic dynamic in vivo conditions. Considering the daily intake of nickel and manganese, respectively, established by the WHO (2000a,b) through food (300 µg, 8 µg) or water consumption (20 µg, 24 µg), the amounts of nickel and manganese ions obtained in the extracts are not of toxicological relevance. Nevertheless, the results demonstrated that brackets had undergone corrosion, and that nickel and manganese ions were detected mainly in the AISI 304 SS-bracket extracts. Thus, for persons with nickel hypersensitivity, to whom the minimal dosage for inducing an allergic reaction is unknown, corrosion of orthodontic brackets may have clinical relevance.

The results from this study suggest that low-nickel SS brackets present higher biocompatibility than do AISI 304 SS brackets. Further studies on the need for accurate determination of the in vivo biocompatibility of these biomaterials are of paramount importance.


   ACKNOWLEDGMENTS
 
This study was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)-Brazil. The authors thank Drs. Miriam Dorta, Milton A.P. Oliveira, Lourival Faria, and Hélio Galdino, Jr. for their technical contributions, and Drs. Birte Melsen, Dorthe Bindslev, and Raman Aulakh for carefully reviewing this manuscript. This paper is based on a thesis submitted to the Graduate Program in Orthodontics at the Federal University of Goiás School of Dentistry, in partial fulfillment of the requirements for the Master’s degree. A preliminary report was presented at the 6th International Orthodontic Congress, Paris, France, September 10–14, 2005.


   FOOTNOTES
 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received December 19, 2005; Last revision December 8, 2006; Accepted January 1, 2007


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Blanco-Dalmau L, Carrasquillo-Alberty H, Silva-Parra J (1984). A study of nickel allergy. J Prosthet Dent 52:116–119.[ISI][Medline]

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Eliades T, Pratsinis H, Kletsas D, Eliades G, Makou M (2004). Characterization and cytotoxicity of ions released from stainless steel and nickel-titanium orthodontic alloys. Am J Orthod Dentofacial Orthop 125:24–29.[ISI][Medline]

Faccioni F, Franceschetti P, Cerpelloni M, Fracasso ME (2003). In vivo study on metal release from fixed orthodontic appliances and DNA damage in oral mucosa cells. Am J Orthod Dentofacial Orthop 124:687–693.[ISI][Medline]

Fitjer LC, Jonas IE, Kappert HF (2002). Corrosion susceptibility of lingual wire extensions in removable appliances. J Orofac Orthop 63:212–226.[Medline]

Flick DA, Gifford GE (1984). Comparison of in vitro cell cytotoxicity assays for tumor necrosis factor. J Immunol Methods 68:167–175.[ISI][Medline]

Gerlier D, Thomasset N (1986). Use of MTT colorimetric assay to measure cell activation. J Immunol Methods 94:57–63.[ISI][Medline]

Grímsdóttir MR, Gjerdet NR, Hensten-Pettersen A (1992). Composition and in vitro corrosion of orthodontic appliances. Am J Orthod Dentofacial Orthop 101:525–532.[ISI][Medline]

Hwang CJ, Shin JS, Cha JY (2001). Metal release from simulated fixed orthodontic appliances. Am J Orthod Dentofacial Orthop 120:383–391.[ISI][Medline]

ISO (1999). Biological evaluation of medical devices—Part 5: Tests for in vitro cytotoxicity. International Organization for Standardization: ISO document 10993–5, pp. 1–7.

Lee YW, Broday L, Costa M (1998). Effects of nickel on DNA methyltransferase activity and genomic DNA methylation levels. Mutat Res 415:213–218.[ISI][Medline]

Mockers O, Deroze D, Camps J (2002). Cytotoxicity of orthodontic bands, brackets and archwires in vitro. Dent Mater 18:311–317.[ISI][Medline]

Montanaro L, Cervellati M, Campoccia D, Arciola CR (2006). Promising in vitro performances of a new nickel-free stainless steel. J Mater Sci Mater Med 17:267–275.[ISI][Medline]

Mosmann T (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63.[ISI][Medline]

Oller AR, Costa M, Oberdörster G (1997). Carcinogenicity assessment of nickel compounds. Toxicol Appl Pharmacol 143:152–166.[ISI][Medline]

Ribeiro-Dias F, Russo M, Marzagão Barbuto JA, Fernandes do Nascimento FR, Timenetsky J, Jancar S (1999). Mycoplasma arginini enhances cytotoxicity of thioglycollate-elicited murine macrophages towards YAC-1 tumor cells through production of NO. J Leukoc Biol 65:808–814.[Abstract]

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Wataha JC (2000). Biocompatibility of dental casting alloys: a review. J Prosthet Dent 83:223–234.[ISI][Medline]

World Health Organization (2000a). Manganese. In: Air quality guidelines. Chapter 6.8. Geneva: WHO.

World Health Organization (2000b). Nickel. In: Air quality guidelines. Chapter 6.10. Geneva: WHO.





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