ORIGINAL ARTICLE

Cytotoxicity of esthetic, metallic, and nickel-free

orthodontic brackets: Cellular behavior and
viability
Luciana Borges Retamoso,a Tatiana Blaya Luz,b Daniel Rodrigo Marinowic,c Denise Cantarelli Machado,d
Luciane Macedo De Menezes,e Maria Perpe
tua Mota Freitas,f and Hugo Mitsuo Silva Oshimag
Porto Alegre and Canoas, Brazil

Introduction: In this study, we evaluated the cellular viability of various esthetic, metallic, and nickel-free
orthodontic brackets. Methods: The sample was divided into 11 groups (n 5 8): cellular control, negative control,
positive control, metallic, polycarbonate, 2 types of monocrystalline ceramic, 3 types of nickel free, and polycrys-
talline ceramic brackets. Cell culture (NIH/3T3-mice broblasts) was added to the plates of 96 wells containing the
specimens and incubated in 5% carbon dioxide at 37
C for 24 hours. Cytotoxicity was analyzed qualitatively and
quantitatively. Cell growth was analyzed with an inverted light microscope, photomicrographs were obtained, and
the results were recorded as response rates based on modications of the parameters of Stanford according to the
size of diffusion halo of toxic substances. Cell viability was analyzed (MTT assay); a microplate reader recorded the
cell viability through the mitochondrial activity in a length of 570 nm. The values were statistically analyzed.
Results: All tested brackets had higher cytotoxicity values than did the negative control (P\0.05), with the excep-
tion Rematitan and Equilibrium (both, Dentaurum, Ispringen, Germany) (P .0.05), suggesting low toxicity effects.
The values showed that only polycarbonate brackets were similar (P .0.05) to the positive control, suggesting high
toxicity. Conclusions: The brackets demonstrated different ranges of cytotoxicity; nickel-free brackets had better
biocompatibility than the others. On the other hand, polycarbonate brackets were made of a highly cytotoxic
material for the cells analyzed. (Am J Orthod Dentofacial Orthop 2012;142:70-4)

S
tainless steel is a metallic alloy widely used in or-
thodontics for brackets. The main advantages of
this material are its low cost and good mechanical
properties. However, these materials have a tendency to
corrode, with consequent release of metal ions.1-3
a
Postgraduate student, Department of Dental Materials, Pontical Catholic
University of Rio Grande do Sul, Porto Alegre, Brazil.
b
Postgraduate student, Department of Orthodontics, Lutheran University of
Brazil, Canoas, Brazil.
c
Postgraduate student, Department of Neuroscience, Pontical Catholic Univer-
sity of Rio Grande do Sul, Porto Alegre, Brazil.
d
Professor and chair, Department of Immunology, Pontical Catholic University
of Rio Grande Do Sul, Porto Alegre, Brazil.
e
Professor, Department of Orthodontics, Pontical Catholic University of Rio
Grande do Sul, Porto Alegre, Brazil.
f
Professor, Department of Orthodontics, Lutheran University of Brazil, Canoas,
Brazil.
g
Professor, Department of Dental Materials, Pontical Catholic University of Rio
Grande do Sul, Porto Alegre, Brazil.
The authors report no commercial, proprietary, or nancial interest in the prod-
ucts or companies described in this article.
Reprint requests to: Luciana Borges Retamoso, Department of Dental Materials,
PUCRS, Anita Garibaldi, 1940/301, 90480-200, Porto Alegre, RS, Brazil; e-mail,
retamosolb@gmail.com.
Submitted, November 2011; revised and accepted, February 2012.
0889-5406/$36.00
Copyright 2012 by the American Association of Orthodontists.
doi:10.1016/j.ajodo.2012.02.025
70
To decrease corrosion, nickel has been incorporated
into the alloys.4 Metal release and associated biologic
effects of nickel-containing orthodontic alloys have re-
ceived some attention in the literature.5 The biocompat-
ibility concerns from the use of nickel alloys in the
human oral cavity for extended periods of time have
prompted the study of alternative materials.6 Thus, non-
metallic or esthetic, polycarbonate and nickel-free, or
steels with reduced nickel content have been tried in
brackets for orthodontic use.
Previous studies have evaluated cellular viability with
esthetic orthodontic brackets and demonstrated that
ceramic brackets are chemically inert in oral uids,
with values similar to the control groups.7,8 Ceramic
brackets are made from alumina, which exists in nature
in monocrystalline and polycrystalline forms.
On the other hand, authors have advocated that poly-
carbonate brackets are more potentially harmful, because
their degradation causes the release of bisphenol-A.9
In this study, we analyzed the null hypothesis that
nickel-free and esthetic brackets used in orthodontics
are not cytotoxic for broblast cell cultures, considering
the possible toxic effects of orthodontic materials on the
tissues, as previously reported in the literature.
Retamoso et al 71

Table I. Group, material compositions, and manufacturers

Group Composition Manufacturer
Cellular control 3T3 cells -
Negative control Austenitic stainless steel Chromium (17%- Morelli, S~ao Paulo, Brazil
20%), nickel (8%-10.5%), iron (65%-69%)
Positive control Silver (40%), tin (31.3%), copper (28.7%), SDI Ultramat, Victoria, Australia
mercury (47.9%)
Metallic Chromium (24.97%), nickel (3.74%), iron American Orthodontics, Sheboygan, Wis
(70.83%), aluminum (0.46%)
Polycarbonate Bisphenol-A Composite, Morelli, S~ao Paulo, Brazil
Polycrystalline ceramic Aluminum Radiance, American Orthodontics,
Sheboygan, Wis
Polycrystalline ceramic Aluminum Inspire Ice, Nickel Free, Ormco, Glendora, Calif
Nickel free Titanium (100%) Equilibrium, Dentaurum, Ispringen, Germany
Nickel free Titanium (62.36%), chromium (32.50), Topic, Dentaurum, Ispringen, Germany
molybdenum (3.57%), iron (0.64%)
Nickel free Titanium (100%) Rematitan, Dentaurum, Ispringen, Germany
Polycrystalline ceramic Chromium (24.97%), nickel (3.74%), iron Clarity, 3M Unitek, Monrovia, Calif
(70.83%), aluminum (0.46%)

MATERIAL AND METHODS manipulated in the laboratory of cellular and molecular

This in-vitro cytotoxicity study was approved by the
ethical committee of the Pontical Catholic University
of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul,
Brazil. Tests were performed in cell cultures (lineage
NIH/3T3, mice broblasts) to evaluate the response
rates, determined by modications of the Stanford pa-
rameters and MTT assays.
The specimens were divided into 11 groups (n 5 8):
cellular control group, represented by the cell growth;
negative control group (stainless steel wire), whose ma-
terial does not produce a cytotoxic response10; positive
control group (amalgam disks), whose material is highly
cytotoxic10; metallic group; polycarbonate group; 2
monocrystalline ceramic groups (Radiance and Inspire
Ice), 3 nickel-free groups (Equilibrium, Topic, and Rema-
titan), and polycrystalline ceramic group with metallic
slot bracket (Clarity), as shown in Table I.
The specimens of the positive control group (amal-
gam disks) were prepared in the amalgamator (SDI,
Bayswater, Victoria, Australia) for 7 seconds. After, the
mixture was placed on a rectangular glass plate, a second
plate was pressed onto the mixture until a 2-mm space
was established between the 2 plates (nal thickness of
the amalgam disk). To determine the disk width, we
used an amalgam carrier with a 2-mm diameter opening.
Next, the specimens were polished with abrasive rubber
points to obtain a smooth surface, rinsed, and dried.
The specimens of all groups were sterilized for 60
minutes in an autoclave (Vitale 12 Plus; Crist
ofoli Equi-
pamentos de Biosseguranca, Paran
a, Brazil) before
immersion in the cell culture.
Mouse broblasts (lineage NIH/3T3), purchased from
American Type Culture Collection (Manassas, Va), were
biology at the Institute of Biomedical Research of S~ao
Lucas Hospital at the Pontical Catholic University of
Rio Grande do Sul. The cells were defrosted and cultured
in Dulbecco modied eagle medium (Invitrogen, Carlsbad,
Calif) supplemented with 10% bovine fetal serum, 100 U
per milliliter of penicillin, 100 mg per milliliter of strepto-
mycin, and 50 mg per milliliter of gentamicin (complete
Dulbecco modied eagle medium) in culture bottles
(Techno Plastics Products, Trasadingen, Switzerland).
The cells were incubated at a temperature of 37
C in
a humidied oven containing 5% carbon dioxide (Sanyo,
Electric Biomedical Co, Osaka, Japan) that was changed
twice a week until the cells reached 80% conuence.
After conuence was obtained, the cells were re-
moved by enzymatic action by using 0.1% trypsin-
ethylenediaminetetraacetic acid (Gibco, Grand Island,
NY) and counted in a Neubauer chamber (Optik Labor,
Friedrichsdorf, Germany). The suspension was added to
plates of 96 wells (n 5 8), in 500-mL increments, with
a density of 4.5 3 105 cells per well. Finally, the cultures
containing the specimens were again incubated for 24
hours.
After the 24-hour incubation period, the plates were an-
alyzed on an inverted light microscope (Axiovent 25; Carl
Zeiss SMT, Thornwood, NY) with a 10-times objective,
and photomicrographs were obtained. The results were re-
corded as response rates, according to the modied param-
eters of Stanford. The rates were calculated in relation to the
halos observed as 2 numbers separated by a bar: the rst
represented the size of the diffusion halo of the toxic sub-
stance, and the second indicated the quantity of cell lysis.
This qualitative analysis was based on the characteristics
of cell proliferation, growth, morphology, and adhesion.10

American Journal of Orthodontics and Dentofacial Orthopedics July 2012  Vol 142  Issue 1
72
The modied parameters of Stanford included (1) the
index of halo size: 0, no halo detected around or under
the specimen; 1, halo limited to the area under the spec-
imen; 2, halo not greater than 25% of the extent of the
specimen; 3; halo not greater than 50% of the extent of
the specimen; 4, halo greater than 50% of the extent of
the specimen but not involving the entire plate; 5, halo
involving the entire plate; and (2) the index of quantity
of cell lysis: 0, no lysis; 1, up to 20% of the halo with ly-
sis; 2, 20% to 40% of the halo with lysis; 3, 40% to 60%
of the halo with lysis; 4, 60% to 80% of the halo with
lysis; 5, more than 80% of the cells with lysis in the halo.
Cell viability was evaluated by the MTT assay (Gibco-
Invitrogen, Grand Island, NY), which is based on the abil-
ity of the mitochondrial enzyme succinate dehydrogenase
to convert the yellow water-soluble tetrazolium salt
(MTT) into formazan crystals in metabolically active cells.
This water-insoluble, dark-blue product is stored in the
cytoplasm of cells and is soluble afterward, generating
a blue color.11
After 24 hours, 200 mL of MTT was added to each well
of the plate, followed by 4 hours of incubation at 37
C and
5% carbon dioxide (Sanyo). Then the medium was re-
moved, and formazan crystals were dissolved with 120 mL
per well of dimethyl sulfoxide (Sigma-Aldrich Corporation,
St Louis, Mo), generating a blue color. Optical densities
were measured at 570 nm in an ELISA reader, and cell via-
bility was calculated according to the following formula:
Cell viability % 5 optical density of test groupO
optical density of cellular control
group3100
RESULTS
The response rates of the modied parameters of
Stanford are presented in Table II. The qualitative cellu-
lar analysis showed that the cellular and negative control
groups had greater numbers of fusiform cells, typical of
normal broblast development, and conuent growth
(Fig, A and B).
On the other hand, the positive control and polycar-
bonate bracket groups (Fig, C and D) demonstrated se-
vere inhibition of cell proliferation and growth, with
signicant alterations indicated by greater numbers of
round cells, mostly with darkened and granular aspects,
suggesting lysis with cell death.
The nickel-free brackets had fusiform cells with con-
uent growth, indicating normal broblast develop-
ment. But some areas of the photomicrographs showed
round and darkened cells, indicating cellular lysis in
a slight halo of diffusion (Fig, E-G).
July 2012  Vol 142  Issue 1 American Journal of Orthodontics and Dentofacial Orthopedics
Retamoso et al
Table II. Response rates observed in tested groups
Group n Response rate
Cellular control 8 0/0
Negative control 8 0/0
Positive control 8 5/5
Metallic 8 2/2
Equilibrium 8 0/0
Topic 8 1/0
Rematitan 8 0/0
Inspire Ice 8 1/2
Radiance 8 1/2
Polycarbonate 8 5/5
Clarity 8 2/2
Monocrystalline ceramic brackets showed equilib-
rium between fusiform and round cells, indicating
a greater spread of the toxic substance in the plates.
Consequently, we observed more dead cells in the mono-
crystalline ceramic brackets than in the nickel-free
brackets (Fig, H and I). Polycrystalline ceramic brackets
(Fig, J) had more dead cells in a halo of diffusion com-
pared with monocrystalline ceramic brackets, indicating
similar results to metallic brackets (Fig, K).
The MTT assay demonstrated that all tested brackets
showed cytotoxicity effects, but only polycarbonate
brackets were similar to the positive controls (P \0.05).
All nickel-free brackets demonstrated similar results
(P .0.05), but the Rematitan brackets had the highest
percentage of cell viability, with no statistically signicant
difference compared with the control group (P .0.05).
The metallic and ceramic brackets showed intermodal
values for toxicity, with no statistically signicant differ-
ence among them (P .0.05), as shown in Table III.
DISCUSSION
Nowadays, there are many studies on the biocompat-
ibility of orthodontic materials because this is a real con-
cern for clinicians, who do not want to place orthodontic
appliances with a risk of adverse toxic effects in their pa-
tients. We evaluated the null hypothesis that nickel-free
and esthetic brackets are not cytotoxic in cell cultures.
Various cell characteristics and functions are used to
investigate the cytotoxicity of metals. Some researchers
have evaluated the adhesion, proliferation, and metabo-
lism of cells such as 3T3, L929, and W138, and human -
broblasts and osteoblasts.12,13 In our study, mouse
broblast behavior was assessed by modications of the
parameters of Stanford.10 This was similar to the study
of Grill et al,12 who advocated the investigation of prolif-
eration by microscopic analysis of cell growth and divi-
sion. Thus, cell viability was assessed by the MTT assay.
In our study, the null hypothesis was partially
rejected. The Topic nickel-free brackets showd
Retamoso et al 73

Fig. Photomicrographs of various brackets evaluated by the modied parameters of Stanford: A, cel-
lular control; B, negative control (stainless steel wire); C, positive control (amalgam disks); D, polycar-
bonate brackets; E-G, nickel-free brackets (Rematitan, Equilibrium, and Topic, respectively); H and I,
monocrystalline ceramic brackets (Inspire Ice and Radiance, respectively); J, polycrystalline bracket
(Clarity); and K, metallic bracket.

some toxic effects compared with the Equilibrium
and Rematitan brackets. Under light microscopy,
the 3T3 cell culture showed some alterations in
cell morphology, and the cell monolayer was not
preserved.
It is commonly accepted that titanium alloying ele-
ments are biocompatible. Perhaps the molybdenum
(3.57%) added to the titanium increases the cytotoxicity.
Li et al14 hypothesized that cytotoxicity is linked to the
ions released from the metals and established that
a safe molybdenum ion concentration (below which
the ion concentration is nontoxic) is 8.5 mg per liter.
So, we suggest that the Topic bracket has an ion concen-
tration over this limit.

American Journal of Orthodontics and Dentofacial Orthopedics July 2012  Vol 142  Issue 1
74 Retamoso et al

1. Nickel-free brackets had better biocompatibility

Table III. Cell viability percentages and standard devi-
than did the other bracket types.
ations by MTT assay
2. Polycarbonate brackets showed the most cytotoxic
Group n Mean SD behavior, similar to the positive control group results.
Cellular control 8 100 A 0.06
Negative control 8 93.65 A, B 0.16
Positive control 8 45.76 C 0.11 REFERENCES
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