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ORIGINAL ARTICLE
Year : 2020  |  Volume : 12  |  Issue : 2  |  Page : 91-95

Evaluation of the Genotoxic Effects of Orthodontic NiTi Wires on Oral Mucosal Cells: An In Vivo Study


1 Department of Orthodontics and Dentofacial Orthopedics, Yenepoya Dental College and Hospital, Mangalore, Karnataka, India
2 NMC Royal Hospital Dubai, UAE

Date of Submission29-Jan-2020
Date of Decision08-May-2020
Date of Acceptance29-Sep-2020
Date of Web Publication16-Feb-2021

Correspondence Address:
Dr. Katheesa Parveen
Department of Orthodontics, Yenepoya Dental College, Mangalore, Karnataka, 575018
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jofs.jofs_18_20

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  Abstract 


Introduction: Orthodontic wires are one of the main components of fixed appliance treatment. Bio-degradation of the metals in these wires could be a source of genotoxicity in the oral cavity. Materials and Methods: After the ethical committee approval and obtaining patient consent, the oral buccal mucosal smears were collected from buccal mucosa using a metal spatula from 27 patients before bonding, during NiTi, and during SS wire stage. The MN assay test was performed using Papanicolaou staining procedure. The micronuclei were examined and counted using previously reported criteria under an electron microscope before bonding, during NiTi, and during SS wire stage. Result: The data collected were subjected to analysis of variance test with Greenhouse–Geisser correction for comparison. It determined that mean mucosa cells differed statistically significantly between time points. Post hoc tests using the Bonferroni correction showed that there was an increase in MN cells from before bonding brackets to the tooth (mean value 6.15 ± 2.769) to during NiTi wire stage (mean value 344.85 ± 64.73), and a reduced count from NiTi wire stage (mean value 344.85 ± 64.73) to SS wire stage (mean value 160.52 ± 47.52). Conclusion: The orthodontic wires were observed to be genotoxic. The NiTi wire is considered to have more potential to cause genotoxicity when compared to SS wires. This could be because of the Ni element present in a higher percentage in NiTi wire as compared to SS wire, hence, element Ni can be considered to be genotoxic. Whether the effect of these wires on oral buccal mucosal cells is reversible has to be evaluated further.

Keywords: Nickel, NiTi wires, micronuclei, genotoxicity, SS wire


How to cite this article:
Parveen K, Bhat M, Husain A, Kinchanakodi AM. Evaluation of the Genotoxic Effects of Orthodontic NiTi Wires on Oral Mucosal Cells: An In Vivo Study. J Orofac Sci 2020;12:91-5

How to cite this URL:
Parveen K, Bhat M, Husain A, Kinchanakodi AM. Evaluation of the Genotoxic Effects of Orthodontic NiTi Wires on Oral Mucosal Cells: An In Vivo Study. J Orofac Sci [serial online] 2020 [cited 2021 Apr 18];12:91-5. Available from: https://www.jofs.in/text.asp?2020/12/2/91/309583




  Introduction Top


Orthodontic patients mainly undergo fixed orthodontic treatment that encompasses orthodontic brackets, wires, bands, and so on, and these components of the fixed appliance are manufactured using metals like Nickel, Cobalt, Chromium, and so on. Orthodontic wires are one of the main components of fixed appliance treatment and among these wires, nickel-titanium (NiTi) wires and stainless steel (SS) wires are commonly used in routine orthodontic practice. The metals in orthodontic wires may undergo biodegradation in the oral cavity and may become a source of genotoxicity.[1]

Nickel Titanium Naval Ordnance Laboratory (NITINOL) wire is made up of an equiatomic alloy of Nickel and Titanium and was introduced into orthodontics by Andreasen along with a metallurgist, Dr. William J. Buehler, in 1978.[2] Nickel is a silvery-white lustrous metal with a slight golden tinge, has symbol Ni and atomic number 28, belongs to the transition metals, and is said to be biologically incompatible; its adverse reactions are hypersensitivity, chronic sinusitis, rhinitis, pneumonia, lung cancer, and cytotoxicity. Nickel is present in at a higher percentage in NITINOL wires. Titanium alloy is the most biocompatible alloy.[1] Stainless steel wires are made up of iron, chromium, and nickel, and nickel constitutes a minor percentage and is used in orthodontics since long and frequently. The genotoxic action of these wires through the dental route is questionable.

Genotoxicity is the property of chemical agents to damage the genetic information within a cell, thereby causing mutations, and leading to cancer.[3] A more hazardous effect of metal alloys is the possibility of causing DNA damage in human cells. Genotoxicity can be a mutagenic or carcinogenic process. Various studies are done for the evaluation of genotoxicity and these include comet assay (CA), micronuclei (MN) assay, metaphase analysis, and transgenetic assay; these tests have found evidence of DNA damage in oral mucosa cells.

The formation of MN is considered to be an active biomarker of diseases and is associated with the induction of DNA damage.[4] MN are damaged chromosomes in the form of acentric chromatids or chromosome fragments and are present as extranuclear cytoplasmic bodies. These damaged chromosomes lag in the anaphase phase of cell division in which centric elements move toward the spindle poles. After the last phase of cell division that is telophase, the undamaged chromosomes and the centric fragments give rise to regular daughter nuclei. The lagging elements are included in the daughter nuclei cells too, but a considerable portion is transformed into the nucleus or several secondary nuclei that appear in the cytoplasm of the daughter cells as a small nuclear particle, termed an MN.[5] Thus, the aim of the study was to evaluate the genotoxic potential of orthodontic wires on oral buccal mucosal cells using these MN.


  Materials and Methods Top


The investigation was conducted on the patients reporting to the Department of Orthodontics of the university and the study proceeded after the approval from the Yenepoya University Ethical Committee bearing protocol number 2016/265 on November 15, 2016. Twenty-seven subjects between the age of 14 and 30 years were enrolled in this study after obtaining consent. The power of the study is 80%. The inclusion criteria were (1) permanent dentition without amalgam fillings and metal restorations that could cause corrosion in the mouth, and (2) no previous history of orthodontic treatment. The exclusion criteria were (1) subjects with palatal or lingual appliances welded to the bands, (2) syndromic patient, (3) subjects with the debilitating disease and those under treatment with antibiotics or steroids during the study period, (4) subjects with prosthesis or tooth restorations with sharp edges or any lesions on the buccal mucosa, and (5) subjects with a nickel allergy. The alcohol-based mouthwashes were avoided during the study.

The details of the study, methodology, and the benefits of the study were explained in detail to the subjects, and consent was obtained. The scaling of the selected subject was done. After an hour subject was asked to rinse the mouth with the tepid distilled water to remove exfoliated dead cells. The oral mucosal cells were collected from each subject by gentle scraping of the inside part of the lips and buccal mucosa with a metal spatula in a sweeping motion. The sample obtained was immediately smeared onto the center of a clean glass slide. The smears were immediately fixed in absolute alcohol (isopropyl alcohol, 70%). Then the slides were hydrated with distilled water and stained with the Papanicolaou (PAP) method. Staining procedures include hydration, nuclear staining, developing, dehydration, cytoplasmic staining, washing, dehydration, and mounting.[6],[7]

In the PAP method, the nucleus appears blue, and the cytoplasm appears pink. Cells that are not smeared, clumped, or overlapped, and those that contained intact nuclei are included as MN cells. Cells undergoing degenerative processes such as karyorrhexis, karyolysis fragmentation of the nucleus, broken egg, and pyknosis are excluded.

The MN were identified according to the standard protocol,[8] described as follows: (1) a rounded smooth perimeter suggesting a membrane, (2) less than a third the diameter of the associated nucleus, but large enough to discern the shape and color, (3) Feulgen positive (i.e., pink in bright field illumination) (4) staining intensity similar to that of the nucleus, (5) texture similar to that of the nucleus, (6) the same focal plane as the nucleus, and (7) no overlap with, or bridge to, the nucleus.

We sampled oral mucosal cells with the same procedure at three time intervals in the study groups; T0, before the start of treatment; T1, after 3 months of the NiTi wire stage; and T2, after 3 months of SS wire stage. The oral smears were observed at 40× magnification under a light microscope by the same operator two times to avoid the interobserver error. One thousand cells from each subject were examined to determine the presence of MN.


  Result Top


In this study, the number of MN cells were counted on the microscope by the two observers to avoid errors at three time points; T0, before the start of treatment; T1, after 3 months of the NiTi wire stage; and T2, after 3 months of SS wire stage and tabulated. The data obtained were subjected to statistical analysis and the following results were drawn.

Descriptive status of the study explains [Table 1] that there is a difference in the mean value of MN cells at three time points; T0, T1, and T2. The highest mean value was seen in the T1 time interval, whereas the least at T0.
Table 1 Descriptive status of number of micronuclei at three time points.

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The mean values were subjected to analysis of variance test with Greenhouse–Geisser correction for comparison. A repeated-measures ANOVA with a Greenhouse–Geisser correction determined that mean mucosa cells differed statistically significantly between time points.

Post hoc tests using the Bonferroni correction revealed that there is an increase in MN cells from before bond to during NiTi (6.15 ± 2.769 vs 344.85 ± 64.73, respectively), which was statistically significant (P < 0.001), and there was a reduction in mean scores from during NiTi to during SS wire stage (344.85 ± 64.73 vs 160.52 ± 47.52), which was also statistically significant (P < 0.001) [Figure 1]. The MN cells score also increased before bonding to during SS (6.15 ± 2.769 vs 160.52 ± 47.52), which was statistically significant (P < 0.001) [Table 2].
Figure 1 Comparison of mean micronuclei at three time points.

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Table 2 Pairwise comparison of mean difference of micronuclei between threetime intervals.

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  Discussion Top


Orthodontic appliances are made up of various metal elements. Utmost care is taken that they are 100% biocompatible with oral tissues. But still, these materials are made of elements that have corrosive property. Because of the corrosive property, the metal ions are released into the oral cavity. These ions can be mutagenic, cytogenic, or allergic. Orthodontic NiTi wires are one of the materials with the highest quantity of nickel; nickel ions are considered to be a strong immunologic sensitizer.

Genotoxicity tests can be defined as in vitro and in vivo approaches designed to detect compounds that induce genetic damage, including DNA lesions, gene mutation, chromosomal breakage, altered DNA repair capacity, and cellular transformation.[9] Different biomarkers of exposure, such as saliva, blood, urine, hair, nails, and oral mucosa cells, are used, with each of them having their advantages and disadvantages. Oral mucosa cells are a non-invasive biomarker that is easy to collect. The advantage of this biomarker is that fixed orthodontic appliances are in direct contact with the cells, which can supply information about ion concentration. Thus, oral mucosa cells are the first tissue where a localized corrosion effect takes place resulting in hypersensitivity, contact dermatitis, asthma, cytotoxicity, and genotoxicity. [10]

In this study, there is an increase in mucosal cells from before bonding to NiTi wire stage. The highest number of MN was in NiTi stage as compared to the other two stages. Hence, NiTi wire is said to be more genotoxic. Further during the SS wire stage, there was a decrease in the number of MN as compared to NiTi stage but still increased number compared to before the bonding stage. This gives the impression that a reduced number of MN from NiTi wire to SS wire may be because of reduced Nickel content in SS wires.

Previously, many studies have been conducted on the genotoxicity of various components of the fixed appliance system. Westphalen et al.[11] did a comparative study of genotoxicity of the orthodontic appliances using CA and MN assay, and concluded that CA detects chromosomal damage at an early stage, whereas MN assay detects chromosomal damage at a later stage. This was in agreement with Van Goethem et al.,[12] Vrzoc, and Petras,[13] as cited by Westphalen et al. They also concluded that MN assay was more sensitive than CA.

Faccioni et al.[14] investigated the biocompatibility of fixed orthodontic appliances in orthodontic patients with non-orthodontic cases using CA and concluded that the orthodontic appliances induce DNA damage in oral buccal mucosal cells, but the study lacked time specificity and the smokers were not excluded. Gangadhara et al.[15] found a significant increase in MN in smokers; therefore, smoking was an exclusion criterion in our study. Hafez et al.[16] conducted a similar genotoxic study of various bracket archwire combinations using CA. They found that there was increased DNA damage in the titanium bracket and SS wire combination. There was no genotoxicity with the NiTi wire with SS bracket, and SS wire and SS bracket. So, their study affirmed that DNA damage was highest in the titanium brackets with NiTi wire combination; furthermore, they added that the biocompatibility of titanium has to be reconsidered. Heravi et al.[17] in his two-time point study using CA said that fixed orthodontic appliances did not expose healthy individuals to increased risk of DNA damage in oral mucosa cells; the mean MN frequency reduced before bonding to 9 months after bonding.

Our study outcomes harmonized with the results of Natarajan et al.[18] to a certain extent. Both the studies affirm that there is genotoxicity from the fixed orthodontic appliance system. The difference in the above two studies was at time points. The study by Natarajan et al. concluded that 30 days after debonding, oral mucosal cells reversed back to normal indicating genotoxicity to be transient. Similar studies were conducted by Quadras et al.[19] and their study findings agreed with our findings.

The major limitation of our study was the lack of specificity. We cannot highlight nickel element of NiTi wire is responsible for genetic damage; as there were other components in the oral cavity of fixed appliance like brackets and bands that may also release nickel element; but the presence of nickel element in brackets is in a small quantity compared to NiTi wires as they are made up of stainless steel. There are few previously conducted studies by Angelieri et al.[20] where they affirmed that brackets do not induce genotoxicity. Goncalves et al.[21] have also conducted a study on cytotoxicity and genotoxicity of orthodontic bands where they proved that there is increased genotoxicity with orthodontic bands. Therefore, patients with bands or any other additional appliance such as habit breaking appliance, transpalatal arch, and so on were avoided in our study.

The study was a simple, inexpensive, the comparison was done between NiTi and SS archwires; the comparison was within the same individual over a long period of time. Limitations of this study are the consumption of the food items containing Nickel such as black tea, nuts and seeds, chocolate, cocoa powder, canned and processed food, grains like oats, whole wheat, and wheat germ.


  Conclusion Top


Orthodontic NiTi wires are genotoxic; toxicity maybe because of the nickel content in NiTi wire and SS wire. Further, the reparability of DNA damage has to be checked after a long-term follow-up, following debonding. The proper evaluation of the patient for the high-risk group before starting with fixed appliance treatment is mandatory. As these patients are at a high chance of developing the cancerous disease.

Acknowledgement

The authors are thankful to Dr. Shahista Parveen, Dr. Vishnudas Prabhu and Dr. Riaz Abdulla for their guidance, sincere support and helpful suggestions.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Palve DH, Tupkari JV. Clinico-pathological correlation of micronuclei in oral squamous cell carcinoma by exfoliative cytology. J Oral Maxillofac Pathol 2008;12:2-7.  Back to cited text no. 5
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Van Goethem F, Lison D, Kirsch-Volders M. Comparative evaluation of the in vitro micronucleus test and the alkaline single cell gel electrophoresis assay for the detection of DNA damaging agents: genotoxic effects of cobalt powder, tungsten carbide and cobalt–tungsten carbide. Mutat Res Genet Toxicol Environ Mutagen 1997;392:31-43.  Back to cited text no. 12
    
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Vrzoc M, Petras ML. Comparison of alkaline single cell gel (Comet) and peripheral blood micronucleus assays in detecting DNA damage caused by direct and indirect acting mutagens. Mutat Res Fund Mol Mechan Mutagen 1997;381:31-40.  Back to cited text no. 13
    
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Faccioni F, Franceschetti P, Cerpelloni M, Fracasso ME. In vivo study on metal release from fixed orthodontic appliances and DNA damage in oral mucosa cells. Am J Orthod Dentofacial Orthop 2003;124:687-93.  Back to cited text no. 14
    
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20.
Angelieri F, Marcondes JP, de Almeida DC, Salvadori DM, Ribeiro DA. Genotoxicity of corrosion eluates obtained from orthodontic brackets in vitro. Am J Orthod Dentofacial Orthop 2011;139:504-9.  Back to cited text no. 20
    
21.
Gonçalves TS, de Menezes LM, Trindade C, da Silva Machado M, Thomas P, Fenech M et al. Cytotoxicity and genotoxicity of orthodontic bands with or without silver soldered joints. Mutat Res 2014;762:1-8.  Back to cited text no. 21
    


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    Tables

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