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ORIGINAL ARTICLE
In vivo study on metal release from xed orthodontic appliances and DNA damage in oral
mucosa cells
Fiorenzo Faccioni, DDS, MD,a Paola Franceschetti, PhD,b Marzia Cerpelloni, TA,c and
Maria E. Fracasso, MHPd Verona, Italy Interest in the amount of metal ion intake from dental
alloys has grown. Fixed orthodontic appliances usually include brackets, bands, and
archwires made of stainless steel, nickeltitanium, or nickelcobalt alloys, and these can
release metal ions. The purpose of this study was to investigate the biocompatibility in vivo of
xed orthodontic appliances, evaluating the presence of metal ions in oral mucosa cells, their
cytotoxicity, and their possible genotoxic effects. Mucosa samples were collected by gentle
brushing of the internal part of the right and left cheeks of orthodontic patients and control
subjects who were not receiving orthodontic treatment. The cells were immediately prepared
for cell viability and the comet assay. Nickel and cobalt cellular content was quantied by
inductively coupled plasma mass spectrometry ICPMS. The results indicate that nickel and
cobalt concentrations were .fold and .fold higher, respectively, in the patients than in the
controls cellular viability was signicantly lower in the patients than in the controls, and there
was a signicant negative correlation with metal levels. The biologic effects, evaluated by
alkaline comet assay, indicated that both metals induced DNA damage more cells with
comets and apoptotic cells. There were signicant positive correlations between cobalt levels
and the number of comets and apoptotic cells, nickel levels and number of comet cells, and
cobalt levels and comet tails. This study corroborates that nickel and cobalt released from
xed orthodontic appliances can induce DNA damage in oral mucosa cells. Am J Orthod
Dentofacial Orthop
everal studies have investigated whether orthodontic appliances release metal ions through
emission of electrogalvanic currents, with saliva as the medium or through continuous
erosion over time., The oral environment is particularly ideal for the biodegradation of metals
because of its thermal, microbiologic, and enzymatic properties. Intraoral xed orthodontic
appliances include brackets, bands, and archwires that are made of alloys containing nickel,
cobalt, and chromium in different percentages. At present, the different types of orthodontic
archwires
From the Faculty of Medicine, University of Verona. a Professor of orthodontics, Department
of Biomedical Morphological Sciences, Section of Dentistry. b Assistant research scientist,
Department of Medicine and Public Health, Section of Pharmacology. c Technical assistant,
Department of Medicine and Public Health, Section of Occupational Health. d Professor of
toxicology, Department of Medicine and Public Health, Section of Pharmacology. Reprint
requests to Prof Maria Enrica Fracasso, Department of Medicine and Public Health, Section
of Pharmacology, Faculty of Medicine, University of Verona. Policlinico Borgo Roma, P. le
Scuro, , Verona, Italy email, mariaenrica.fracassounivr.it. Submitted, January revised and
accepted, July . Copyright by the American Association of Orthodontists. //. doi./j.ajodo...
S
contain to nickel, to chromium, and to cobalt., Therefore, data on the biocompatibility of
alloys are great interest. Nickel is a strong immunologic sensitizer, although nickel sensitivity
has been reported to be lower in subjects who have received orthodontic treatment perhaps
they develop immunological tolerance over the long period of treatment., On the other hand,
chromium and cobalt ions can also cause hypersensivity, dermatitis, and asthma. These
metals can induce other adverse biologic effects, such as cytotoxicity, and they are
suspected genotoxic agents. Many metal compounds are carcinogenic to animals or humans
their mechanisms are not overall known, but pathway might be the involvement of the metals
with DNA interaction, either directly or indirectly. Several metals have been shown to have
cogenotoxicity mechanisms seem to be predominant the generation of oxidative DNA
damage and the interference with DNA repair and DNA replication processes., Recently, an
electrophoretic technique capable of detecting DNA single strand breaks and alkali labile
sites in individual cells has been developed by Singh et al. The importance of the singlecell
gel electrophoresis or comet assay comes from its ability to evaluate
Italy. .p. Previous studies have investigated. One milliliter of cell suspension cells from
buccal mucosa cells of each patient was treated with nitric acid mL. Nickel and cobalt
standard solutions of between . and aliquots of buccal mucosa cells of each patient were
immediately used for comet assay. V/cm and mA at room temperature. This is not sufcient to
assess the biocompatibility of orthodontic appliances that are in the mouth for several years.
nickel. . stainless steel TruChrome SS. but less information exists about adverse biologic
effects of these metals in vivo.. iron. . iron. One hundred randomly selected cells of each
subject were analyzed cells for each of replicate slides. The rst group comprised orthodontic
patients with xed appliances in both arches. and urine of patients with xed orthodontic
appliances. The brushes were stirred in ml of phosphatebuffer saline solution PBS. both in
vitro and in vivo. and possible DNA damage in the buccal cells by metal ion release.
Haverhill. The metal content in the buccal mucosa cells was measured according to Forrer et
al. Rocky Mountain Orthodontics. Glendora. nickel. measured from the center of the comet
head and by tail moment. Milan. Hewlett Packard S. and then diluted with deionized distilled
water. exposed to alkali pH for minutes. measurable but not toxic levels of metals in blood.
These studies have reported in vitro. their viability was determined by using the trypan blue
exclusion technique. molybdenum. or chromiumcobaltnickel alloy Elgiloy. chromium. such as
nickel and chromium. the material was American Iron and Steel Institute AISI type for the
bands and type for the brackets Ormco. Table I gives the general characteristics of patients
and controls. cell suspensions were centrifuged. Suffolk. The alkaline version of the comet
assay was performed as described previously. The slides were analyzed with a uorescent
microscope Axioplan Zeiss LSM Carl Zeiss. Florence.l. Lysis was performed overnight at pH
. The slides were neutralized. The results were presented as mean SD of medians of each
subject. The archwires used in this study were nickeltitanium alloy Memoria Anat Arches.
and the electrophoresis was performed for minutes at V . Cells were then placed in an
electrophoresis chamber.. Italy. Faccioni et al American Journal of Orthodontics and
Dentofacial Orthopedics December Table I. Denver. All steps were performed under yellow
light to prevent additional DNA damage. which was dened as the product of the comet length
and the amount of DNA in the tail. titanium. Milan. and added to a slide precoated with L of
agarose . Colo. Epithelial cells of buccal mucosa from each patient were collected. tail length
m. Rocky Mountain Orthodontics. the release of these metals from orthodontic appliances.
this is the rst study that examines directly in vivo on each patient the concentration of metal
ions in oral mucosa cells. chromium. Leone S. To measure the amount of nickel and cobalt
release. The purpose of this study was to measure the metal levels in the buccal mucosa
cells and to evaluate their possible adverse biological effects.a. according to the method of
Besarti Nia et al.. dried with ethanol. by gentle brushing of the internal part of the right and
left cheeks with an interdental brush. The second group comprised subjects who were not
undergoing orthodontic treatment and had no dental restorations. and ltered through
polyamide gauze m mesh opening. suspended in PBS. General characteristics of subjects
Controls / Patients / Parameters Number of subjects Fixed appliances y Male/female Age y
Smokers Drinkers intercellular differences in DNA damage and repair in very small samples
of proliferating or nonproliferating cells. Calif. with a Babington nebulizer. Sesto Fiorentino.
cobalt. the biocompatibility of orthodontic appliances cellular viability and apoptosis. United
Kingdom. nickel. evaluated with comet assay. Series .. . the cells were counted. . Perceptive
Instruments..r. The xed appliances consisted of an average of to bands and bonded
brackets. Briey. . pH . . Italy. after washing out the mouth many times with tepid water to
remove exfoliated dead cells. Informed consent was obtained after the objective of the study
was fully explained. saliva. Italy and image analysis Comet Assay II. the presence of metals..
MATERIAL AND METHODS Eighty ve subjects were included in this study. To our
knowledge. and. Milan. in vivo. L of cell suspension about cells were mixed with L of
lowmeltingpoint agarose . . we used inductively coupled plasmamass spectrometry IPCMS
Hewlett Packard .. and stained with ethidium bromide g/ml. The ltrates were pelleted by
centrifugation and resuspended in RPMI Life Technology. . DNA damage was evaluated by
percentage of DNA in the tail DNA. . . in simulated saliva medium.
Correlations were evaluated with Spearman r tests. GraphPad Software. than those in the
control subjects Table II. and the mean number of comets and apoptosis was assessed on
random cells cells of replicate slides for each subject. The statistical analysis by
nonparametric test MannWhitney Utest showed that the group of subjects with xed
orthodontic appliances had mean values of tail moment and tail length significantly higher
than the control subjects P . the mean levels of cobalt and nickel were . . and. which can
show DNA damage strand breaks. Representative images of cells with an undetectable
degree of DNA fragmentation A. ng/L were used. . for the measurements. in this case. .
Number Faccioni et al Table II. between groups. the product of amount of DNA in the tail.
Signicance determined by tailed MannWhitney U test. Patients . The potential genotoxic
effects of metals on buccal mucosa cells were evaluated by comet assay. respectively.
Nickel Table III.. incomplete excision repair sites. . . we observed a remarkable increase of
both metals in the subjects with xed orthodontic appliances the cobalt and nickel
concentrations were about . is equivalent to ng/ cells. San Diego.. and mean distance of the
tail migration. and the sample concentrations were calculated from the standard curve.
ng/mL . . The orthodontic patients had been wearing different types of metallic alloy xed
appliances in both arches for to years. . respectively. smoking. respectively. Simple linear
regression analysis was carried out among the comet parameters. The results showed a
marked increase of these parameters in the oral mucosa cells of the patients compared with
the controls P . The biologic compatibility of xed orthodontic appliances in buccal cells was
evaluated by cytogenetic variables. The nonparametric MannWhitney Utest from the Prism .
the general characteristics of the subjects in this study age. there was a signicant decrease
in cellular viability P . cells per sample Viability . respectively. The assay is named for the
characteristic shape when the DNA exits the nucleus and the cell body.fold higher. indicating
a low variability in each group. . and by cellular viability. whereas the same metals were .
Table III. ng/L and . a typical comet B. including the frequencies of cells with comets and
apoptotic cells.American Journal of Orthodontics and Dentofacial Orthopedics Volume . . .
When we examined the amount of nickel and cobalt ions in the buccal mucosa cells. the
percentage of DNA in the tail or tail intensity and tail moment. Cytotoxicity and frequency of
buccal cells with comet and apoptotic cells in controls n and patients n with xed orthodontic
appliances for years Controls . The control group consisted of subjects who were not
undergoing orthodontic treatment. P value . these differences were signicantly higher in the
statistical analysis with the Student t test P . All P values were tailed. ng/mL and . Comets . .
In this study. ng/mL. Calif statistics program was used to analyze the signicance of the
differences between controls and the subjects with appliances. The frequencies of cells with
a tail comet and of apoptotic cells were scored. ng/mL. and P .fold and . Values are mean SD
of median of each subject. . Parameters Statistical analysis with Student t test. . viability. P .
In the control group. parameters characterizing DNA strand breaks were evaluated tail
length.. we used the isotopes Ni and Co with an internal standard of Rh . and an apoptotic
cell C are shown in Figure . Table IV analyzes the data of comet parameters between groups
the mean and the median values of each parameter were very similar. while the percentage
of DNA in the tail was similar in both groups. . and alcohol drinking were similar in both
groups. and the presence of different metals in the cells. . There were signicant positive
correlations between cobalt levels in the buccal cells and tail . RESULTS As indicated in
Table I. g/L. alkalilabile sites. ng/mL and . sex. Nickel and cobalt release from the xed
orthodontic appliances was analyzed by ICPMS. The statistical difference between groups of
metal levels cobalt and nickel was calculated with the Student t test. in the patient group. . P .
the number of cells with comet or apoptotic cells. Results are given as ng/mL that. cells per
sample Apoptosis . Mean SD values of cobalt and nickel levels ng/mL evaluated by ICPMS
in buccal cells of controls n and patients n Cobalt Subjects Controls Patients .
. Our aim was to evaluate possible damage of cellular DNA induced by these metals by
analyzing the levels of nickel and cobalt. was signicantly below the average dietary intake
and did not reach toxic concentrations. . EF. including saliva. released from orthodontic
appliances in saliva or blood samples. they can be in the mouth for years or more. Several in
vitro and in vivo methods have been used to study the release of metals and their content in
biologic uids. . . median and range of comet assay in buccal cells of control subjects and
patients with xed orthodontic appliances Group n subjects Controls Statistical data mean SD
median range mean SD median range Tail moment DNA in tail Tail length m Patients . .
apoptotic cell. Signicance determined by tailed MannWhitney U test. we chose to evaluate
nickel and cobalt cellular levels as biomarkers of cytotoxicity and genotoxicity.. . . and a
signicant negative correlation between nickel levels with cellular viability Fig . . . A. . . B.
Fluorescence photomicrographs showing individual oral mucosa cells from comet assay. The
most common alloy constituents are cobalt. The studies have shown that these metals were
released during the rst or months of orthodontic therapy. Descriptive parameters mean SD.
Cell without DNA damage. and urine. The alloys used in dentistry are exposed to several
aggressive physicalchemical events. because these metals were the higher constituents with
harmful characteristics of the appliances used during orthodontic therapy. A signicant
positive correlation was found between nickel ions and number of cells with comet r . length.
Faccioni et al American Journal of Orthodontics and Dentofacial Orthopedics December Fig .
it cannot be excluded that even nontoxic concentrations might be sufcient to induce
important biologic effects in cells of oral mucosa.. brackets. blood.. NS P .... chromium..
followed by other metals in different amounts. . and number of comet and apoptotic cells. and
nickel. . . . DISCUSSION We investigated the presence of metal ions in oral mucosa cells in
orthodontic patients wearing xed appliances.. Corrosion events are very frequent in the oral
cavity. and both stainless steel and nickeltitanium archwires. . C. . . and the metals were
actually absorbed by patients with systemic distribution. Experimental and epidemiologic
studies . A standard orthodontic appliance consists of bands.. P . . and to assess the
cytogenetic damage by comet assay in the buccal mucosa cells.. . Not signicant. such as
high concentrations of oxygen and chloride mixtures in saliva. and a signicant negative
correlation with cell viability Fig . . and acid product deposit from microbiologic metabolism.
NS. and plaque. P . . Although the orthodontic appliances had no effect on the general levels
of metals. Table IV. which are the cells directly exposed. AD. . typical comet of damaged cell.
tartar. The main conclusions indicate that the measurable amount of metal.
and cellular viability F. cellular viability D. Correlations between cobalt levels in buccal cells
and tail length A. cellular nickel levels with number of comets E. . Number Faccioni et al Fig .
number of apoptosis C.American Journal of Orthodontics and Dentofacial Orthopedics
Volume . number of comets B.
Lyon. Biodegradation of orthodon This study corroborates the potential toxicologic effects of
nickel and cobalt. Biometals . Monographs on the evaluation of carcinogenic risk to humans
chromium. the parameters characterizing DNA breaks tail moment. this might be expected
for nickel. Scheper RJ. chlorination byproducts. emit metal ions in sufcient quantities to
induce evident cytotoxic effects. J Dent Res . Bencko V. . We noted signicant positive
correlations between metal concentration ie.. Am J Orthod Dentofacial Orthop . Franceschetti
P. Yarat A. Our results. Lee HJ. In general. Nickel a review of its occupational and
environmental toxicology. We thank Emma Orsolini. Exp Cell Res . Moreover. van
Hoogstraten IMW. such as the interaction of metals with DNA crosslinks. Burrows D. Even if
a genotoxic potential has been demonstrated in certain systems. Shin JS. nickel and
welding. . Fisher JR.. CONCLUSIONS might be an initiating event of adverse biologic
effects. Paik RS. Lee KH. Hartwing A. Agaoglu G. which are in the mouth for or years.
Rosenblum GA. IARC. To our knowledge. Hong YC. .. In any case. Monographs on the
evaluation of carcinogenic risk to humans chlorinated drinking water. Cha JY. Verbeeck R.
for her skillful technical assistance. are consistent with the evidence that both metals induce
these adverse biologic effects we found high frequencies of cells with comets and apoptosis
in the patient group.. Hwang CJ. Lyon. Bruynzeel DP. was signicantly lower in the patients
than in the control group. . Induction of immunological tolerance by oral administration of
nickel and chromium. Material Science Forum . von Blomberg BME. we found that cellular
viability. considering the low levels of extraneous sources eg. vol.. Because of possible
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correlated with the cobalt cellular content. but also are safe for patients. student in the
Department of Orthodontics. . We found that the oral mucosa cells from patients with xed
appliances had nickel and cobalt concentrations signicantly higher than those obtained from
the subjects without orthodontic appliances. Hypersensivity to mercury. Nitinol arch wire in a
stimulated oral environment changes in mechanical properties. Quinn JK. Arun T. Newman
SM. Talamini G. These data indicate that nickel and cobalt alloys of orthodontic appliances.
Nicholson JA. Thomson BD. normal cells can repair these lesions. Vreeburg KJJ. McCoy
LT.. Tice RR. . . Clin Exp Immunol . cobalt and cobalt compounds. although nickel and cobalt
content was different in the archwires studied. there were signicant negative correlations
between cobalt and nickel levels and cellular viability. Measurable metal values were also
found in the control group. Electrochemical research on the corrosion of orthodontic
nickeltitanium wires. Barrett RD. the generation of oxidative DNA damage. evaluated a few
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several possible pathways seem to be involved. Magnesium inhibits nickelinduced
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with the comet assay in relationship to genotoxicity and cytotoxicity. The results of these
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function were found for the cells exposed COMMENTARY What makes this study important.
. evidence of genotoxicity of ions derived from orthodontic alloys on human cells is
presented. The use of the Student t test is inappropriate in this case. Gruythuysen RJ. Part II.
Modulation by Co II of UVinduced DNA repair. Biodegradation of orthodontic appliances.
Barrett RD. brackets and wires. Bosch JA. The importance of this effect in a journal of mainly
clinical readership might be fold. Snyder RD. . Bishara SE. Van Zandwijk N. presents points
which need further clarication and revision. Speit G. .. and nickel. . the effect of metallic ions
might be exaggerated in cells with as high apoptotic rates as the oral mucosal epithelial cells.
Lutz H. Direct determination of selenium and other trace elements in serum samples by
ICPMS. Van Straaten HWM. Number Faccioni et al . Stroh A.. Factors inuencing DNA
migration from individual cells subjected to gel electrophoresis. mainstream techniques. .
HenstenPettersen A. Second. An additional body of literature pertains to the assessment of
cytotoxicity of ions released from orthodontic alloys by using various cell types and alloy
aging treatments. Contemporary orthodontics. the complexity of biological action of alloys
coupled with the multiplicity of modes of application and patterns of intraoral aging should
prevent the reader from extrapolating direct conclusions for the clinical performance of
materials. These . accepting the conclusions and endorsing the claims without critically
examining the integrity of the arguments. Am J Orthod Dentofacial Orthop . tic appliances. . .
Changes in the blood level of nickel. Am J Orthod Dentofacial Orthop . Olive PL. and the
plastic components of facemasks. Kocadereli L. . Merk O. Burgaz S.. differentiating it from
the pool of conventional cytotoxicity experiments. Assessment of cytogenetic damage in
lymphocytes and in exfoliated nasal cells of dental laboratory technicians exposed to
chromium.. The incidents reported were mostly hypersensitivity reactions. Proft WR. is the
assessment of the DNA damage incurred by the ions in oral mucosa cells. the statistical
analysis of cobalt and nickel cell contents is misleading. Ertas N. The state of knowledge on
the adverse effects of orthodontic materials is restricted to a series of case reports showing
an immune response to headgear bows and straps. . .. Salivary nickel and chromium in
patients with xed orthodontic appliances. . Fields HW Jr. Demircigil GC. However.. The
comet assay singlecell gel test a sensitive genotoxicity test for the detection of DNA damage
and repair. Kale PS. for the rst time in the orthodontic literature. Durand RE. Kemaloglu Y.
This study. Thus. The authors examined variables population control and patients and metal
cobalt and nickel. and it is possible that more than type of epithelial cells with varying
apoptotic potentials were grouped in the assays. . J Trace Elements Med Biol . Caries Res .
Environ Mol Mutagen . Yilmazer M. adhesives. impressive as it may be.. retrieved.. . St Louis
Mosby. Most importantly. Besarati Nia A.American Journal of Orthodontics and Dentofacial
Orthopedics Volume . Kleinjans JCS.. First. Wlodek D. Hartmann A. . Forrer R. Am J Orthod
Dentofacial Orthop . The study includes advanced analytical instrumentation and complex
biological assays. Eliades T. further studies are required to unequivocally establish the
critical importance of these ndings. mutagenesis and sisterchromatid exchanges in
mammalian cells. The cell population harvested was not characterized. Selim MI.