Metallic ion content and damage to the DNA in oral mucosa cellspatients treated dental implants

Pıa Lopez-Jornet • Francisco Parra Perrez •

Jose Luis Calvo-Guirado • Irene LLor-Ros •

Piedad Ramırez-Fernandez

Received: 24 December 2013 / Accepted: 19 March 2014 / Published online: 29 March 2014

� Springer Science+Business Media New York 2014

Abstract The aim of this study was to assess the potential

genotoxicity of dental implants, evaluating biomarkers of

DNA damage (micronuclei and/or nuclear buds), cytokinetic

defects (binucleated cells) and the presence of trace metals in

gingival cells of patients with implants, comparing these with

a control group. A total of 60 healthy adults (30 patients with

dental implants and 30 control patients without) were included

in the study. Medical and dental histories were made for each

including life-style factors. Genotoxicity effects were asses-

sed by micronucleus assays in the gingival epithelial cells of

each patient; 1,000 epithelial cells were analyzed, evaluating

the frequency of micronucleated cells and other nuclear

anomalies. The concentration of metals (Al27, Ag107, Co 59, Cr52, Cu63, Fe56, Sn118, Mn55, Mo92, Ni60, Pb208, Ti47) were

assayed by means of coupled plasma-mass spectrophotometry

(ICP-MS). The frequency of micronuclei in the patient group

with implants was higher than in the control group but without

statistically significant differences (P [ 0.05). Similar results

were found for binucleated cells and nuclear buds (P [ 0.05).

For metals assayed by ICP-MS, significant differences were

found for Ti47 (P B 0.045). Univariate analysis identified a

significant association between the presence of micronuclei

and age. Dental implants do not induce DNA damage in

gingival cells, the slight effects observed cannot be indicated

as biologically relevant.

1 Introduction

Dental implants restore patients’ oral health as well as

function, esthetics and comfort. The soft tissues sur-

rounding implants must adapt to their presence and peri-

odontal and peri-implant tissues are important for

establishing a protective barrier [1–3]. It has already been

documented in both in vitro and in vivo research that dental

metals can produce alterations in surrounding cells arising

mainly from corrosion [4–7]. The oral mucosa is covered

by a stratified epithelium composed of multiple layers of

cells that show various patterns of differentiation between

the deepest cell layer and the surface [8].

Biocompatibility is a fundamental requirement for the

success of dental implant treatments; the release of elements

from a biomaterial, whether this is through metal ions

released by alloy corrosion or peroxide degradation, is a

fundamental factor in the production of adverse biological

effects such as toxicity, allergy and mutagenicity [9–14].

Titanium is widely used in implant dentistry due to its

good physical, chemical and mechanical properties as well

as its resistance to corrosion [14–17]. Nevertheless, the oral

environment is an ideal medium for the biodegradation of

metals (pH variation, temperature, salivary conditions,

microbiological and enzymatic activity) and the release of

metal ions can cause a wide variety of biological responses.

However, understanding of the mechanisms of biological

interactions between metallic dental and oral or systemic

tissues remains limited [3, 7].

The Micronuclei test (MN) on human oral gingival cells

is a minimally-invasive and simple technique to detect

P. Lopez-Jornet � F. P. Perrez � I. LLor-Ros

Oral Medicine Department, Faculty of Medicine and Dentistry,

Ageing Research Institute, University of Murcia, Murcia, Spain

P. Lopez-Jornet (&)

Clınica Odontologica Universitaria, Hospital Morales Meseguer,

Adv. Marques de los Velez s/n, 30008 Murcia, Spain

e-mail: majornet@um.es

J. L. Calvo-Guirado � P. Ramırez-Fernandez

Department of Implant Dentistry, School of Medicine and

Dentistry, University of Murcia, Murcia, Spain

123

J Mater Sci: Mater Med (2014) 25:1819–1824

DOI 10.1007/s10856-014-5203-7

genotoxic damage. The MN in oral epithelial cells is being

used as a biomarker of exposure to genotoxic agents [18].

Some reports in the literature have shown that metal can

affect the close contact between implant and tissue. The

hypothesis of this study was that the titanium alloy used for

fabricating dental implants releases fewer metallic ions but

might nevertheless produce changes to the DNA of the oral

gingival mucosa. Olmedo et al. [10] measured the presence

of metal particles in cells exfoliated from peri-implant oral

mucosa around titanium dental implants; the concentration

of titanium was higher in patients with peri-implantitis.

The detection of metal ions in relation to genotoxic and

mutagenic effects has been studied in depth in vitro and in

animal studies [4, 16, 18], but in vivo research into metal

release in patients with dental implants and possible DNA

damage to gingival mucosa cells is scarce.

In this way, the aim of the present study was to assess

the potential genotoxicity of dental implants, evaluating

biomarkers of DNA damage (micronuclei and/or nuclear

buds), cytokinetic defects (binucleated cells) and the pre-

sence of metal particles in subjects with dental implants,

comparing these with a control group.

2 Materials and methods

The study protocol was designed and performed following

Spanish Ethical Guidelines and the Declaration of Helsinki

for research involving humans; it was approved by the

Bioethical Committee of the University of Murcia. All

subjects were fully informed of the purpose of the study

and gave their consent to take part.

This cohort study included 60 patients attending the

University of Murcia dental clinic. Thirty subjects were

selected consecutively who had received dental implants

more than one year previously without any associated

pathologies. A further 30 control subjects with the same

age and gender characteristics as the study group were

selected from the same local area. Medical and dental

histories were prepared including information about dietary

habits, alcohol consumption, smoking and exposure to

chemical carcinogens or radiation.

2.1 Inclusion criteria

Healthy subjects older than 18 years, in good general

health and without oral disease.

2.2 Exclusion criteria

The study excluded edentulous subjects, regular mouth-

wash users, taking antioxidant dietary supplements,patients

receiving local or systemic therapy or suffering any illness

(sensitivity to metals, oral lesions or ulcers, dental

pathologies, diabetics or subjects with a previous history of

cancer) or pregnant women. Subjects wearing complete

fixed or removable dental prostheses were also excluded, as

well as subjects with piercings, dental amalgams, previous

orthodontic treatment or with any clinical signs of mucosal

metallosis in the mucosa. None of the participants had

received radiological diagnosis during the six months prior

to sample collection.

2.3 Micronucleus assay technique [18–20]

(a) Cell Sampling and Preparation: Exfoliated oral

mucosal cells were collected from each subject by a

single practitioner (FP). According to the technique

proposed by Thomas et al. [18] in 2009 , the mouth

was rinsed with water to remove saliva, food particles

and any other debris prior to cell collection. Samples

were collected from the gingival margin of the sul-

cular epithelium with a conventional toothbrush,

applying a circular motion 20 times, covering a wide

area without damaging the gingival mucosa. Sample

sites for cell collection were uniform for all subjects.

Two 30-ml yellow-capped containers were prepared

containing 20 ml of gingival mucosal cell buffer

(EDTA 0.1 M, Tris–HCl 0.01 M, NaCl 0.02 M,

pH 7) (Sigma-Aldrich E6758, USA). The brushes

were placed in their respective buffer containers and

rotated repeatedly to dislodge the cells and release

them into the buffer medium. The cells were then

transferred to centrifuge tubes and centrifuged for

10 min. at 1,500 rpm. After centrifuging, the super-

natant was aspirated and the cells were resuspended in

another 5 ml of oral mucosal cell buffer, followed by

repeat centrifugation. The process was repeated once

again to eliminate bacteria and inactivate enzymes.

The cells were transferred using a pipette, placing

120–150 ll of cell suspension onto two clean and

labeled microscope slides. After drying, the slides

were placed in an oven at 55 �C for 15 min. and were

then fixed with 50 % methanol (Panreac SAU,

E-08211, Barcelona, Spain) at 0 �C for 15 min.

(b) DAPI Staining: Cell samples were stained with

DAPI (40,6-diamidino-2-phenylindole dihydrochlo-

ride) (Sigma-Aldrich, D9542, USA) at a concentra-

tion of 200 lg/ml, for 15 min. The slides were then

washed in Milli-Q water. Slides were scored using a

Leica DRMB fluorescence microscope equipped

with a DAPI band filter (excitation wavelength filter

set [BP340-380], dichroic filters RKP 400 and

emission filters LP 425) under 1009 magnifications.

(c) Scoring Method: Gingival buccal cell samples were

collected and processed following recommendations

1820 J Mater Sci: Mater Med (2014) 25:1819–1824

123

made by Thomas et al. [18] by a single practitioner

blinded to study group assignation.

Micronuclei, nuclear buds and other nuclear anomalies

were scored over 1,000 cells; they were identified by the

following characteristics:

Normal differentiated cells have a uniformly stained

nucleus and are distinguished from basal cells by their

larger size and by a smaller nucleus-to-cytoplasm ratio.

Cells with micronuclei are identified by the presence of

both a main nucleus and one or more smaller nuclear

structures called micronuclei (MN). The MN must be

located within the cytoplasm of the cells, with round or

oval shapes and their diameter should range between 1/3

and 1/16 of the main nucleus. MN have the same staining

intensity and texture as the main nucleus. Most cells with

MN will contain only one MN but it is possible to find cells

with two or more MN.(Fig. 1).

Cells with nuclear buds contain nuclei with an apparent

sharp constriction at one end of the nucleus suggestive of a

budding process and elimination of nuclear material by

budding. The nuclear bud and the nucleus are usually in

very close proximity and appear to be attached to each

other. The nuclear bud has the same morphology and

staining properties as the nucleus; however, its diameter

may range from a half to a quarter of that of the main

nucleus.

Binucleated cells are cells containing two main nuclei

instead of one. The nuclei are usually very close and may

touch each other and usually have the same morphology as

that observed in normal cells.

2.4 Microchemical analysis

Metal concentrations were determined by using inductively

coupled plasma mass spectrometry (Algilent 7500ce; Ag-

ilent Technologies, Santa Clara, California, USA). For this,

the samples (10 mL) were acidified with 0.25 mL of 69 %

nitric acid (Hiperpur, Panreac, Barcelona, Spain). The

metals measured in all samples were those included in the

metal composition Al27, Ag107, Co59, Cr52, Cu63, Fe56,

Sn118, Mn55, Mo92, Ni60, Pb208, Ti47. Three determinations

were obtained per sample, so the result for each sample

was the mean of these three values. The metal concentra-

tion is in the range of parts per billion (lg/l to ppb).

2.5 Statistical analysis

Data were analyzed using SPSS version 19.0 statistical

software (SPSS� Inc., Chicago, IL, USA). A descriptive

study was made of each variable. Associations between the

different qualitative variables were studied using the

Pearson Chi squared test. The Student t test for two inde-

pendent samples was applied to quantitative variables,

determining in each case whether variances were homo-

geneous. Bivariate analysis was also performed, consider-

ing the binary absence/presence of micronuclei as the

outcome variable. Odds ratios and CIs were calculated with

exact conditional logistic regression. Statistical signifi-

cance was established as P B 0.05.

3 Results

A total of 60 patients took part in the study, 30 with os-

teointegrated dental implants and 30 control patients

Fig. 1 Photomicrography of gingival cell presenting micronuclei

(stained with 1009 magnifications)

Table 1 Characteristics of study group subjects: age, and gender

distribution, DNA damage-cytokinetic defects, (Student t test, and

Pearsons Chi squared test)

Characteristics Implant

dental

(n = 30)

Control

group

(n = 30)

P-value

Age: mean ± SD** 50.3 ± 10.41 51.4 ± 9.4 NS

Sex: n (%) NS

Male 14 (46.66) 12 (40)

Female 16 (53.3) 18(60)

Smoking: n (%) Ns

Yes 6 (20) 7(20.4)

No 24(80.3) 23 (76.6)

Alcohol consumption: n (%) Ns

Yes 7 (20.4) 6 (20)

No 23 (76.6) 24 (80)

DNA damage-cytokinetic defects

Micronuclei

mean ± SD

1.05 ± 0.0 0.91 ± 0.8 NS

Binucleated cells

mean ± SD

7.17 ± 5.4 6.8 ± 3.0 NS

Nuclear buds

mean ± SD

2.71 ± 1.8 2.21 ± 1.98 NS

J Mater Sci: Mater Med (2014) 25:1819–1824 1821

123

without implants Their average age was 50.61 ±

6.15 years. Table 1 shows the characteristics of the two

groups.

In patients wearing implants, the average time since

implant placement was 2.6 ± 1.13 years. The average

number of implants per patient was three with a range of

one to ten. Micronuclei frequency (mean ± standard

deviation) per 1,000 gingival epithelial cells quantified for

each subject in the dental implant group was higher than

for control group subjects but without statistically signifi-

cant differences (P [ 0.05). Similar results were found for

binucleated cells and nuclear buds. (Table 1) (Fig. 1).

When the presence of metals was assayed by means of

ICP-MS, a significant difference was found between the

groups for titanium alloy, 2.42 ± 5.049 versus 0.461 ±

1.13 (P B 0.045) (Table 2).

Univariate analysis (Table 3) showed that age was sig-

nificantly associated with the presence of micronuclei in

the gingival epithelial (P = 0.048).

4 Discussion

Gingival epithelial cells are a useful target for biomoni-

toring due to their accessibility [19]; the micronucleus

(MN) assay is one of the most widely applied used in

genotoxicity studies and has become one of the most

important tests used for the evaluation of mutagenicity and

carcinogenicity [18, 19]. The results of the present study

were negative in that no statistically significant effect

relating to exposure to dental implants was observed on the

induction of MN and/or BN in the gingival epithelium. In

this way, the present study did not produce any evidence to

suggest that titanium alloy dental interventions increase

mutagenic and carcinogenic risks in humans.

The significance of binucleated cells remains uncertain,

though the phenomenon appears to indicate failed cytoki-

netics following the last step in nuclear division. The

binucleated/mononucleated cell ratio could be an important

biomarker indicating failed cytokinetics due to an increase

in aneuploid DNA rates [20, 21].

When considering DNA damage, factors such as sex,

age, smoking and alcohol consumption must be taken into

account [22–28]. According to Barnett and King [22], the

influence of age on genotoxic and cytotoxic endpoints may

reflect the increase in spontaneous chromosome instability

associated with an accumulation of DNA damage due to

progressive impairment of overall DNA-repair capacity.

Tissue repair capacity decreases with age and so the oral

mucosa becomes more permeable to nocive substances and

more vulnerable to damage produced by mechanical agents

[20]. In this way, the present results showed a significant

association between age and MN frequency.

Biomaterials used in dental medicine may release dif-

ferent cytotoxic elements that can cause toxic reactions,

allergy, mutagenic or inflammatory effects on cells [9, 29,

30](Fig. 2). The release of elements from a biomaterial,

whether from metal ions released through from alloy

Table 2 Concentration of metal ions (lg/l) detected in patients’

gingival cells: mean, standard deviation (SD)

Elements Implants (N = 30) Control (N = 30) P-value

Mean ± SD Mean ± SD

Al27 1.40 ± 2.2 1.390 ± 2.50 P [ 0.05

Ag107 0 .00 ± 0.0 0 .00 ± 0.0

Co59 0 ± 0 0 ± 0

Cr52 0.340 ± 0.31 0.010 ± 0.02 P [ 0.05

Cu63 0.160 ± 0.18 0.141 ± 0.14 P [ 0.05

Fe56 4.007 ± 3.96 3.570 ± 2.97 P [ 0.05

Sn118 0.001 ± 0.001 0.001 ± 0.001 P [ 0.05

Mn55 0.193 ± 0.16 0.187 ± 0.10 P [ 0.05

Mo92 0.004 ± 0.015 0 .001 ± 0.01 P [ 0.05

Ni60 0.106 ± 0.171 0.100 ± 0.03 P [ 0.05

Pb208 0.080 ± 0.062 0.019 ± 0.02 P [ 0.05

Ti47 2.42 0 ± 5.049 0.461 ± 1.13 0.0450

Table 3 Association with study subjects’ characteristics:Logistic

regression model for ‘‘presence of micronucleus/1,000 cells

gingival.’’

Variables Odds

ratio

95 % Confidence

interval

P-

value

Age (\50 vs. [50) 1.02 1.00–1.22 0.048

Sex (male vs. females) 1.32 0.61–1.90 0.150

Smoking (Yes/No) 1.74 0.29–3.69 0.670

Alcohol (Yes/NO) 0.09 0.01–1.85 0.303

Metal (Yes/No) 0.40 0.83–2.01 0.272

Year implant (\3

vs. [3)

2.6 0.61–2.10 0.122

Fig. 2 Failed human dental implant showing tissue in contact with

the metallic surface

1822 J Mater Sci: Mater Med (2014) 25:1819–1824

123

corrosion or from peroxide degradation, is fundamental to

the production of adverse biological effects such as toxic-

ity, allergy and mutagenicity. In an experimental study in

rat tibia, Piozzi et al. [13] observed an absence of cyto-

toxicity and genotoxicity from titanium. Thirty, 90, and

180 days after implantation, no statistically significant

differences in DNA damage were found in any study

groups, for any of the organs evaluated when compared

with the negative control group.

Flatebø, et al. [12] made a histological evaluation of

non-perforated mucosas covering maxillary submerged

titanium implants, finding no tissue sensitivity to the tita-

nium implants in spite of the fact that all the biopsies taken

at six months contained dense metal particles.

Olmedo et al. [10] measured the presence of metal

particles in cells exfoliated from peri-implant oral mucosas

around titanium dental implants. The concentration of

titanium was higher in the group of patients with peri-

implantitis compared to the group without peri-implantitis;

no traces of titanium were observed in control subjects.

In metal detection it should be remembered that in some

cases metal levels can be below the lower detection limit of

the instruments used for analysis; in other words, they are

present but undetectable. This may be the case even when the

ICP-MS technique is used, which can quantify parts per

billion. Furthermore, some titanium particles present in the

epithelium might not have anything to do with the implant

but originate from quite different sources. For example, TiO2

is widely used in food products, in toothpaste, prophylactic

pastes, etc. In vitro research has reported that various metal

elements such as Ni, Co and Cr can modulate immune

responses. Furthermore, human fibroblast and epithelial cell

cultures show that Cu, Co and Zn significantly increase

prostaglandin synthesis, a proinflammatory mediator

derived from arachidonic acid [14]. Nevertheless, while

in vitro tests are quick and simple and ensure controlled

laboratory conditions, they do not exactly reflect phenomena

occurring in the oral environment. In this way, actual oral

tissues might show the cumulative effects of metal release.

The results obtained in the present study correspond to

the data analyzed by Di Pietro et al. [31] who observed the

effects of dental restorative materials on peripheral blood

lymphocytes in patients with composite restorations com-

pared to those without restorations, whereby comet assay

results showed that DNA damage was two times higher in

the exposed group than in the control group. Furthermore,

DNA damage increased with the time of exposure and

number of restorations.

Soft tissues around teeth and implants present anatomic

similarities represented by the presence of an oral epithelium,

continuous with a junctional epithelium. The present study

assayed gingival cells in patients with natural teeth in good

periodontal health (control subjects) and gingival cells in

patients with dental implants (without mucositis or peri-im-

plantitis). Differences were observed in the positioning of the

most apical portion of the junctional epithelium, which in

tooth sections was close to the level of the cementoenamel

junction but in implant sections was at a variable distance from

the gingival margin. Another difference concerns the peri-

implantar absence of cementum layer or Sharpey’s fibers.

Consequently, collagen fiber bundles in teeth (dentogingival

fibers, dento-periodontal fibers and circular fibers) are inserted

perpendicular to the surface, while at implant sites, a dense

network of collagen fibers is observed extending from the

alveolar bone crest to the gingival margin, arranged parallel to

the implant surface [32]. Such anatomical variations should be

considered when contemplating the results of an assay such as

the present one, given that research involving gingival epi-

thelial cells in patients with implants is scarce and so it is

difficult to compare results.

There seems to be a consensus that, immediately fol-

lowing the placement of metals in the mouth, ion release

peaks as a result of corrosion, which is followed by sta-

bilization and a reduction in ion release due to the for-

mation of a protective biofilm over the metal surface [32].

For this reason, in order to assay accumulated DNA dam-

age, cytogenetic defects and trace metals, the present study

avoided the initial ion release peak period and assayed

responses during the stabilized phase.

Most studies have evaluated the metal ions in saliva [33,

34]. However, Mikulewicz and Chojnacka [35], in a system-

atic literature review, suggest that this procedure may have

limitations since the saliva is continuously washed and

swallowed and so will give information at the moment of

sampling only. For the purposes of the present study, it was

decided to use gingival mucosa cells since they are in direct

contact with implants and it has been reported that oral tissues

take up the metal ions released by an adjacent implant [29].

Given its design, the present study had certain limita-

tions and prospective studies with larger case numbers that

assay metal concentrations are needed to confirm findings.

It should also be remembered that metal degradation not

only alters the implants integral structure but brings about

systemic metal release. The microorganisms of the oral

flora must play an important role, which has not been

assayed in this study.

The method used for sample collection was simple, non-

invasive and well tolerated by the participants and so useful

for evaluating biomarkers of DNA damage and detecting

trace metals in gingival cells. Dental implants do not

induce DNA damage in gingival cells, the slight effects

observed cannot be indicated as biologically relevant.

Conflict of interest The authors declare no conflicts of interest

J Mater Sci: Mater Med (2014) 25:1819–1824 1823

123

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