The aim of this study was to develop a new approach to testing the impact of nickel antigen on in vitro
cell-proliferation assay, to identify adverse reactions to casting alloys among orthodontic patients. Cell-
proliferation assay in vitro was used as the basic methodology to assess the influence of such variables as
source of nickel antigen, type of serum used to supplement the culture medium, and number of cells in the
culture. We selected 35 orthodontic patients who were classified as nickel sensitive and non–nickel sensitive,
based on their clinical records. Our results showed that hexahydrated nickel sulfate at 10 g/mL, 10% of
autologous sera, and 2 ⫻ 105 cells was the best condition for inducing the most marked nickel proliferation
response in vitro. This optimized method was able to distinguish nickel-sensitive from non–nickel-sensitive
dental patients and also to discriminate those with positive skin tests. Our data suggest that continuous
exposure to nickel casting alloys might lead to oral tolerance mechanisms that modulate nickel sensitivity,
as evidenced by the lower cell proliferation index in patients undergoing orthodontic treatment over 24
months. Finally, our findings demonstrated a known nickel-induced type 2 immune response and a marked
lack of type 1 immunity (interferon ␥) as the hallmarks of nickel-sensitive patients. Further studies are needed
to clarify the major cell phenotype associated with this type 2 immune response and the lack of type 1
immunity observed in nickel-sensitive people. (Am J Orthod Dentofacial Orthop 2003;123:000-00) (Am J
Orthod Dentofacial Orthop 2003;124:46-52)
M
any objects contain nickel and can cause the immunologic standpoint, is considered type IV
nickel sensitivity, and many people are in hypersensitivity. In this context, nickel binding to
continuous contact with items that contain endogenous macromolecules can stimulate macro-
nickel.1 Nickel-containing objects are common in den- phages and cytotoxic cells, up-regulating the expres-
tistry, especially in dental implants and compounds sion of adhesion molecules.5-7 It has also being re-
used in crowns, bridgework, and orthodontic appli- ported that low-dose exposure can alter the metabolism
ances. Nickel-titanium alloys are widely used in orth- of human monocytes.8 Additionally, nickel induces T
odontic treatment, and all these devices can induce skin lymphocytes to produce several cytokines, including
and mucosal reactions, leading to tissue inflamma- interferon IFN-␥, interleukin IL-2, IL-5, and IL-10, and
tion.2-4 An immune response induced by nickel appli- stimulates cellular proliferation.9 The pattern and level
ances is generally called contact dermatitis and, from of cytokines secreted are critical to triggering differen-
a
Núcleo de Pesquisa em Imunologia, Faculdade de Ciências da Saúde, tial immune responses, which can lead to different
Universidade Vale do Rio Doce, Governador Valadares, Minas Gerais, Brazil. degrees of tissue damage. The temperature, microor-
b
Faculdade de Odontologia de Piracicaba, UNICAMP, São Paulo, Brazil.
c
Faculdade de Ciências Agrárias, Universidade Vale do Rio Doce, Governador
ganisms, enzymatic compounds, and ions normally
Valadares, Minas Gerais, Brazil. present in the oral cavity particularly favor the break-
d
Centro de Pesquisas René Rachou, Fundaçáo Oswaldo Cruz, Belo Horizonte, down and release of metallic elements from dental
Minas Gerais, Brazil.
Supported in part by Fundação de Amparo à Pesquisas do Estado de Minas casting alloys.10
Gerais, Brazil. Contact dermatitis generally appears clinically as
Reprint requests to: Dr Marcelo Marigo, Rua João Pinheiro, 610 Centro,
Governador Valadares, MG CEP 35020-270, Brazil; e-mail,
eczema. Reactions of the mucous membranes, such as
iomarigo@goval.com.br. stomatitis, also occur, and gum hyperplasias, cheilitis,
Submitted, July 2002; revised and accepted, October 2002. labial desquamation, and multiform erythemas are fre-
Copyright © 2003 by the American Association of Orthodontists.
0889-5406/2003/$30.00 ⫹ 0 quently noted. Nickel hypersensitivity has increased in
doi:10.1016/S0889-5406(03)00239-7 the last 10 years, and it is estimated that 15% to 30% of
46
American Journal of Orthodontics and Dentofacial Orthopedics Marigo et al 47
Volume 124, Number 1
Europeans and Americans, in a proportion of 1 to 8, owing to the limited quantity of PBMC we were able to
men to women, show symptoms.11 A prevalence of 7% extract from their blood samples. Informed consent was
to 28% of the general population has been also report- obtained from all subjects and their parents. The studies
ed.12,13 were approved by the Ethical Committee of Univer-
Nickel sensitivity has been diagnosed through bio- sidade Estadual de Campinas and Universidade Vale do
compatibility testing,14,15 including cutaneous sensitiv- Rio Doce.
ity tests,13 and also through clinical observations and The cutaneous sensitivity test was performed as
family history. However, major problems regarding described by Carvalho.19 Briefly, a 5% nickel sulfate
specificity of the cutaneous tests are frequently re- and petroleum jelly substrate (nickel sulfate 5%, Aler-
ported. Therefore, a great challenges in the diagnosis of gofar, Rio de Janeiro, Brazil) was kept in direct contact
nickel sensitivity has been to discover and develop with the skin of the forearm for 48 to 72 hours. The
alternative in vitro assays to improve specificity and results were classified as positive or negative, based on
minimize false-positive results. Lymphocyte stimula- the reaction.
tion assays with peripheral blood mononuclear cells Nickel sources used as stimulating agents for the
(PBMC) and different concentrations of nickel salts cellular proliferation assays in vitro were obtained from
have contributed to advances and show promising solutions containing nickel from in vitro corrosion of
results.16 In one study, lymphocytes from all suspected orthodontic appliances (nickel extract) and from hexa-
nickel-sensitive patients showed a significantly greater hydrated nickel sulfate solutions (NiSO4.6H2O, Sigma
response than did those of healthy controls.17 Lympho- Chemical, St. Louis, Mo). The nickel extracts were
cyte transformation, as measured by increased deoxyri- obtained from 4 different brands of orthodontic appli-
bonucleic acid (DNA) synthesis, seems to be an impor- ances. Alloys were kept in a sterile 0.85% saline
tant tool for investigating the problems arising from solution at 37°C for 7, 30, and 60 days. After centrif-
false-positive or false-negative patch tests.18 ugation to remove the particles produced during the
The purpose of this research was to develop a new corrosion process, the solutions were sterilized by
approach to testing the effect of nickel antigen on filtration (0.2 m, Filter Millex-HA Millipore Products
lymphoproliferative response in vitro, by using PBMC Division, Bedford, Mass). Then the dosage of nickel in
from patients with orthodontic appliances. Using this this solution was measured with an atomic absorption
strategy, we evaluated whether lymphoproliferative spectrophotometer (Hitachi Z-8200, Tokyo, Japan).
response is a useful method to discriminate nickel- The extracts thus obtained were diluted in culture
sensitive from non–nickel-sensitive dental patients. We media (RPMI-1640, Gibco BRL, Grand Island, NY)
also studied patients wearing orthodontic appliances containing a 3% solution of antibiotic-antimycotic
(either with or without clinical symptoms) to determine (stock solution: 10,000 IU penicillin, 10,000 IU strep-
the influence of certain variables on in vitro lympho- tomycin/mL, and 25 g amphotericin B/mL, Sigma
proliferation assay, including source of antigen, type of Chemical) and 1.6% L-glutamine (stock solution: 200
serum used to supplement the medium, and the number mmol/L, Gibco), labeled incomplete RPMI. Extracts
of cells used. containing nickel at concentrations of 1.25, 2.5, and 5
g/mL were used in the cellular proliferation assays.
MATERIAL AND METHODS The spectrophotometer readings of saline extract from
Thirty-five patients, aged 10 to 21 years (mean the 4 appliances (A, B, C, and D) showed similar levels
14.3), all undergoing orthodontic treatment with fixed of nickel after 60 days of spontaneous corrosion. No
appliances, were selected based on their clinical health significant differences on the yield of nickel between
records and classified into 2 groups. The first group the 4 different brands were observed (A, 40.23 g/mL;
comprised 26 patients (8 males, 18 females, aged 10 to B, 39.58 g/mL; C, 40.03g/mL, and D, 40.23 g/
21 years) with clinical manifestations of contact der- mL). Solution A was used in cell culture. The nickel
matitis, including angular cheilitis, labial desquama- sulfate (NiSO4.6H2O, Sigma) was also diluted in the
tion, or gum hyperplasia resulting from metallic com- above-described incomplete RPMI medium and used at
pounds, especially earrings, bracelets, and other concentrations of 2.5, 5, and 10 g/mL in the cellular
jewelry. The second group comprised 9 patients (3 proliferation assays.
males, 6 females, aged 11 to 21 years) who did not have In vitro cellular proliferation assays were performed
clinical signs or histories of nickel sensitivity. All with the procedure described by Gazzinelli et al.20 In
patients underwent cutaneous nickel sensitivity tests, 96-well, flat-bottom, tissue-culture plates, 2 different
and those selected for the control group had negative concentrations of PBMC (2.0 ⫻ 105 and 3.0 ⫻ 105 cells
results. We could not use all 26 patients for all tests per well) were incubated with 100 L of 2 different
48 Marigo et al American Journal of Orthodontics and Dentofacial Orthopedics
July 2003
Fig 2. Influence of serum-type supplement and number Fig 4. Down-regulation of nickel sulfate–induced lym-
of cells on in vitro lymphoproliferation assay. Results phoproliferation in vitro associated with exposure time
are expressed as mean cell proliferation index (E/C) ⫾ to orthodontic appliances. Results are expressed as
standard error. E/C ⬎ 2.0 was adopted as arbitrary mean cell proliferation index (E/C) ⫾ standard error. E/C
cutoff to signify significant cell proliferation index. ⬎ 2.0 was adopted as arbitrary cutoff to signify signif-
icant cell proliferation index.
Fig 6. Effect of nickel stimuli on cytokine profile. Results are expressed as cytokine concentration
in the cell culture supernatant (ng/mL) for control cultures (white bars) and nickel-stimulated cultures
(black bars) ⫾ standard error. Statistical analysis was performed by analysis of variance followed by
Student t test. Significance was considered when P ⬍ .05. Differences are marked by distinct letter.
treatment with nickel-containing appliances before sen- ● This optimized methodology for assessing cellular
sitization to nickel (ear piercing) can lead to reduced immune status could enable diagnostic testing of
frequency of nickel hypersensitivity.30 Tolerance in- nickel sensitivity without exposing patients to the
duction might be a possible benefit of using intraorally hazards of patch testing.
placed alloys.3 ● The exposure to nickel alloy castings for more than
We have also begun to investigate the role of type 24 months resulted in lower cell proliferation in-
1 and type 2 immune responses in the pathogenesis of dexes, suggesting that the development of oral toler-
nickel sensitivity. It seems that nickel stimuli can elicit ance mechanisms might play a role in modulating the
IL-5 production in nickel-sensitive patients. There was cellular response to nickel.
a significant difference between their cytokine levels ● A known nickel-induced type 2 immune response
and those observed in the control cultures. Moreover, and a marked lack of type 1 immunity (IFN-␥) were
we showed that our optimized method for lymphocyte the hallmarks of nickel-sensitive patients.
transformation in vitro is a useful tool for identifying
We thank Marlucy Rodrigues Lima, Maria de
the lack of type 1 (IFN-␥) immune response in nickel-
Fátima da Silva, and Lilia Cardoso Moreira for techni-
sensitive compared with non–nickel-sensitive patients
cal assistance.
(Fig 6). Further studies are needed to elucidate the
major cell phenotype associated with the type 2 im-
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