J Mater Sci: Mater Med (2014) 25:1819–1824 DOI 10.1007/s10856-014-5203-7
Metallic ion content and damage to the DNA in oral mucosa cells patients treated dental implants Pı´a Lo´pez-Jornet • Francisco Parra Perrez Jose´ Luis Calvo-Guirado • Irene LLor-Ros Piedad Ramı´rez-Ferna´ndez
• •
Received: 24 December 2013 / Accepted: 19 March 2014 / Published online: 29 March 2014 Ó 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 assessed 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, Cr 52 , 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
P. Lo´pez-Jornet F. P. Perrez I. LLor-Ros Oral Medicine Department, Faculty of Medicine and Dentistry, Ageing Research Institute, University of Murcia, Murcia, Spain P. Lo´pez-Jornet (&) Clı´nica Odontolo´gica Universitaria, Hospital Morales Meseguer, Adv. Marques de los Velez s/n, 30008 Murcia, Spain e-mail: [email protected] J. L. Calvo-Guirado P. Ramı´rez-Ferna´ndez Department of Implant Dentistry, School of Medicine and Dentistry, University of Murcia, Murcia, Spain
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 surrounding implants must adapt to their presence and periodontal 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
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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 presence of metal particles in subjects with dental implants, comparing these with a control group.
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(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)
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. (b) 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 mouthwash users, taking antioxidant dietary supplements,patients receiving local or systemic therapy or suffering any illness
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(c)
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 sulcular 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 supernatant 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. DAPI Staining: Cell samples were stained with DAPI (40 ,6-diamidino-2-phenylindole dihydrochloride) (Sigma-Aldrich, D9542, USA) at a concentration 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. Scoring Method: Gingival buccal cell samples were collected and processed following recommendations
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1821 Table 1 Characteristics of study group subjects: age, and gender distribution, DNA damage-cytokinetic defects, (Student t test, and Pearson´s 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
Male
14 (46.66)
12 (40)
Female
16 (53.3)
18(60)
Yes
6 (20)
7(20.4)
No
24(80.3)
23 (76.6)
7 (20.4)
6 (20)
Sex: n (%)
NS
Smoking: n (%) Fig. 1 Photomicrography of gingival cell presenting micronuclei (stained with 1009 magnifications)
Alcohol consumption: n (%) Yes
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.
Ns
Ns
No 23 (76.6) DNA damage-cytokinetic defects
24 (80)
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
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 concentration 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 (SPSSÒ 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 independent samples was applied to quantitative variables, determining in each case whether variances were homogeneous. Bivariate analysis was also performed, considering the binary absence/presence of micronuclei as the outcome variable. Odds ratios and CIs were calculated with exact conditional logistic regression. Statistical significance was established as P B 0.05.
2.4 Microchemical analysis Metal concentrations were determined by using inductively coupled plasma mass spectrometry (Algilent 7500ce; Agilent Technologies, Santa Clara, California, USA). For this, the samples (10 mL) were acidified with 0.25 mL of 69 %
3 Results A total of 60 patients took part in the study, 30 with osteointegrated dental implants and 30 control patients
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Table 2 Concentration of metal ions (lg/l) detected in patients’ gingival cells: mean, standard deviation (SD) Elements
Implants (N = 30) Mean ± SD
Control (N = 30) Mean ± SD
P-value
Al27
1.40 ± 2.2
1.390 ± 2.50
P [ 0.05
Ag107
0 .00 ± 0.0
0 .00 ± 0.0
Co59 Cr52
0±0 0.340 ± 0.31
0±0 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
60
Ni
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
Pvalue
Age (\50 vs. [50) Sex (male vs. females)
1.02 1.32
1.00–1.22 0.61–1.90
0.048 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
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 significant 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 significantly associated with the presence of micronuclei in the gingival epithelial (P = 0.048).
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Fig. 2 Failed human dental implant showing tissue in contact with the metallic surface
4 Discussion Gingival epithelial cells are a useful target for biomonitoring 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 cytokinetics 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 different 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
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corrosion or from peroxide degradation, is fundamental to the production of adverse biological effects such as toxicity, allergy and mutagenicity. In an experimental study in rat tibia, Piozzi et al. [13] observed an absence of cytotoxicity 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 titanium 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 periimplantitis 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 compared 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
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patients with dental implants (without mucositis or peri-implantitis). 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 periimplantar 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 epithelial cells in patients with implants is scarce and so it is difficult to compare results. There seems to be a consensus that, immediately following the placement of metals in the mouth, ion release peaks as a result of corrosion, which is followed by stabilization and a reduction in ion release due to the formation of a protective biofilm over the metal surface [32]. For this reason, in order to assay accumulated DNA damage, 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 systematic 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 limitations 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, noninvasive 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
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Metallic ion content and damage to the DNA in oral mucosa cells patients treated dental implants Pı´a Lo´pez-Jornet • Francisco Parra Perrez Jose´ Luis Calvo-Guirado • Irene LLor-Ros Piedad Ramı´rez-Ferna´ndez
• •
Received: 24 December 2013 / Accepted: 19 March 2014 / Published online: 29 March 2014 Ó 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 assessed 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, Cr 52 , 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
P. Lo´pez-Jornet F. P. Perrez I. LLor-Ros Oral Medicine Department, Faculty of Medicine and Dentistry, Ageing Research Institute, University of Murcia, Murcia, Spain P. Lo´pez-Jornet (&) Clı´nica Odontolo´gica Universitaria, Hospital Morales Meseguer, Adv. Marques de los Velez s/n, 30008 Murcia, Spain e-mail: [email protected] J. L. Calvo-Guirado P. Ramı´rez-Ferna´ndez Department of Implant Dentistry, School of Medicine and Dentistry, University of Murcia, Murcia, Spain
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 surrounding implants must adapt to their presence and periodontal 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
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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 presence of metal particles in subjects with dental implants, comparing these with a control group.
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(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)
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. (b) 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 mouthwash users, taking antioxidant dietary supplements,patients receiving local or systemic therapy or suffering any illness
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(c)
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 sulcular 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 supernatant 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. DAPI Staining: Cell samples were stained with DAPI (40 ,6-diamidino-2-phenylindole dihydrochloride) (Sigma-Aldrich, D9542, USA) at a concentration 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. Scoring Method: Gingival buccal cell samples were collected and processed following recommendations
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1821 Table 1 Characteristics of study group subjects: age, and gender distribution, DNA damage-cytokinetic defects, (Student t test, and Pearson´s 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
Male
14 (46.66)
12 (40)
Female
16 (53.3)
18(60)
Yes
6 (20)
7(20.4)
No
24(80.3)
23 (76.6)
7 (20.4)
6 (20)
Sex: n (%)
NS
Smoking: n (%) Fig. 1 Photomicrography of gingival cell presenting micronuclei (stained with 1009 magnifications)
Alcohol consumption: n (%) Yes
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.
Ns
Ns
No 23 (76.6) DNA damage-cytokinetic defects
24 (80)
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
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 concentration 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 (SPSSÒ 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 independent samples was applied to quantitative variables, determining in each case whether variances were homogeneous. Bivariate analysis was also performed, considering the binary absence/presence of micronuclei as the outcome variable. Odds ratios and CIs were calculated with exact conditional logistic regression. Statistical significance was established as P B 0.05.
2.4 Microchemical analysis Metal concentrations were determined by using inductively coupled plasma mass spectrometry (Algilent 7500ce; Agilent Technologies, Santa Clara, California, USA). For this, the samples (10 mL) were acidified with 0.25 mL of 69 %
3 Results A total of 60 patients took part in the study, 30 with osteointegrated dental implants and 30 control patients
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Table 2 Concentration of metal ions (lg/l) detected in patients’ gingival cells: mean, standard deviation (SD) Elements
Implants (N = 30) Mean ± SD
Control (N = 30) Mean ± SD
P-value
Al27
1.40 ± 2.2
1.390 ± 2.50
P [ 0.05
Ag107
0 .00 ± 0.0
0 .00 ± 0.0
Co59 Cr52
0±0 0.340 ± 0.31
0±0 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
60
Ni
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
Pvalue
Age (\50 vs. [50) Sex (male vs. females)
1.02 1.32
1.00–1.22 0.61–1.90
0.048 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
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 significant 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 significantly associated with the presence of micronuclei in the gingival epithelial (P = 0.048).
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Fig. 2 Failed human dental implant showing tissue in contact with the metallic surface
4 Discussion Gingival epithelial cells are a useful target for biomonitoring 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 cytokinetics 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 different 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
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corrosion or from peroxide degradation, is fundamental to the production of adverse biological effects such as toxicity, allergy and mutagenicity. In an experimental study in rat tibia, Piozzi et al. [13] observed an absence of cytotoxicity 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 titanium 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 periimplantitis 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 compared 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
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patients with dental implants (without mucositis or peri-implantitis). 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 periimplantar 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 epithelial cells in patients with implants is scarce and so it is difficult to compare results. There seems to be a consensus that, immediately following the placement of metals in the mouth, ion release peaks as a result of corrosion, which is followed by stabilization and a reduction in ion release due to the formation of a protective biofilm over the metal surface [32]. For this reason, in order to assay accumulated DNA damage, 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 systematic 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 limitations 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, noninvasive 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
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