Genotoxicity and cytotoxicity of lingual bonded retainers coated with silver and titanium dioxide nanoparticles – A randomized controlled trial

INTRODUCTION

With retention posing an ongoing challenge, fixed retainers have become extremely popular especially since Zachrisson in 1977 proposed the use of multi-stranded wires bonded canine-to-canine retainers.^[1] Besides being esthetic and not requiring patient cooperation, there is evidence that using a fixed bonded retainer reduces the chances of lower labial segment relapse.^[2]

The downside is an increased chance of plaque and calculus accumulation which can lead to the formation of carious lesions, gingival inflammation, and periodontal disease.^[3] Mechanical methods to remove plaque and antimicrobials to control the microflora can be used; however, these methods depend on patient compliance. In recent times, nanoparticles have been explored as coatings on orthodontic appliances or incorporated into bonding materials such as cement and composites. They have proved effective against various microorganisms thereby controlling the formation and composition of the oral biofilm.^[4]

TiO₂ is a popular nanomaterial due to its high photocatalytic activity resulting in the organic degradation processes and also due to its low cost, chemical stability, and resistance to photo corrosion.^[5] Silver (Ag) is a strong disinfectant, having a broad bactericidal spectrum with the ability to cause changes in the bacterial cell membrane leading to cell death.^[6]

Although these nanomaterials have antibacterial action on the oral biofilm, the potential cytotoxicity of these materials on normal cells must be considered. Potential hazards are inflammation, necrosis, reactive oxygen species, and apoptosis. Nanoparticles used in the oral cavity can be absorbed and their small size allows them to be transported to other sites in the body.^[7] The increased cytotoxicity of smaller particles is attributed to the “Trojan horse effect.”^[7]

A study on human periodontal fibroblasts has proved that Ag nanoparticles of size <20 nm are more cytotoxic than the size of 80–100 nm.^[8] Similarly, it has been demonstrated that the cytotoxicity of TiO₂ nanoparticles also differs with size, with 5 nm proving more cytotoxic than 32 nm.^[9]

The study of cytotoxicity of TiO₂, Ag, and other nanoparticles has been restricted to in vitro and cell line studies.^[10-13]

Genotoxicity is the ability of an agent to exert adverse effects on the cell’s genetic material whereas cytotoxicity is the ability of an agent to be virulent to living cells.^[14] The micronuclei

(MN) index is considered one of the standard cytogenetic endpoints and biomarkers in genetic toxicology. ^[15,16]

MN are extranuclear cytoplasmic bodies that have their origin from the acentric chromosome fragments, acentric chromatid fragments, or whole chromosomes that fail to be included in the daughter nuclei at the completion of telophase during the process of mitosis because they failed to attach properly with the spindle during segregation process in anaphase.^[17]

MATERIAL AND METHODS

Enrollment

The sample size was calculated using the alpha significance level of 0.05 with a power of 80 as per the study of Natarajan M et al^.[15] A total number of 60 subjects were enrolled in the study. Among them, 45 patients who had completed fixed orthodontic treatment and required retainers were recruited into three groups that received intervention. A fourth group of 15 patients (within the same age group) who were not orthodontically treated made up the baseline control [Figure 1].

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Figure 1:

Consort flow chart. Ag: Silver, TiO₂: Titanium dioxide, S.S: Stainless steel

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Figure 2:

Comparison of micronuclei count between groups at different time intervals.

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Slide preparation

The sample obtained was smeared onto the center of a clean glass slide and the smears were immediately fixed in absolute alcohol (Isopropyl alcohol - 100%) for a period of 20–30 min. Then, the slides were hydrated with distilled water and stained using the Papanicolaou (PAP) method according to the standard protocol (PAP, 1942).^[18]

The staining technique results in the blue-black appearance of nuclei and the blue-green appearance of cytoplasm. The keratinizing cells have a pinkish-orange hue.

The slides were observed at 40× magnification under the light microscope (Lawrence and Mayo XSZ-N107T). Cells were observed using high-power magnification in 10 fields in a zigzag fashion to determine the presence of MN [Figure 3].

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Figure 3:

Micronuclei stained with papanicolaou (PAP) highlighted in high-power magnified field.

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RESULTS

DISCUSSION

Fixed retention brings associated challenges of maintaining oral hygiene and increased plaque and microbial accumulation on the tooth surface.^[4]

The application of nanocoatings on orthodontic appliances has been widely explored and found effective but most studies have been restricted to in vitro situations without evaluating the toxicity. This study sought to evaluate the genotoxicity and cytotoxicity of multistranded lingual bonded retainers coated with Ag and TiO₂ nanoparticles in an in vivo environment.

The photocatalytic activity of TiO₂ when exposed to ultraviolet (UV) light has been employed since TiO₂ has a wide bandgap of 3.2 eV, where its absorption edge occurs below 400 nm (UV region) and only a small fraction of solar spectrum is absorbed.^[20] However, since exposure to UV light has its downsides, doping using non-metal ion elements has been favored to reduce the optical gap of TiO₂ to visible light. Nitrogen has gained popularity and it has been suggested by Asahi et al.^[21] that visible light of <500 nm would be sufficient to activate N-doped TiO₂. Since its efficacy has been proven in clinical settings, we chose N-doping to activate TiO₂ under visible light.^[20] Since the anatase phase has more photocatalytic activity and minimal cytotoxic effects as compared to the rutile phase, we chose to coat our wires with the anatase phase of TiO₂.^[10]

Ag nanoparticles have a broad bactericidal spectrum and have been used to coat orthodontic appliances including fixed and removable retainers demonstrating strong antibacterial effects.^[22,23]

In our study, the coating of the retainer wires was done using a magnetron sputtering machine since this technique has certain advantages such as a strong and uniform coating and high hydrophilicity.^[24] While previous studies have used buccal mucosal cells to evaluate genotoxicity, in this study, samples were collected from the tip of the tongue due to its direct contact with the lingual surface of the bonded retainer. MN assay is considered a simple, sensitive, and noninvasive method for evaluating DNA damage, proliferation of basal cells, and cell death. The presence of MN is indicative of chromosomal abnormalities that include breakage of chromosomes and subsequent damage to the DNA. This test is generally performed as a reliable screening test for the presence of genotoxic compounds.^[25]

A recent study concluded that PAP stain is the preferred method for detecting MN and this method was used. It encompasses a fixative that has the potential to demarcate the cell boundaries clearly so that the MN is visible in the transparent cytoplasm.^[26]

Since orthodontic appliances can also contribute to genotoxicity, we used an untreated control to mitigate the residual influence of fixed appliances for baseline comparison.^[14] At T_0, all three experimental groups and the untreated control group showed some presence of MN. Although the latter displayed the least MN count compared to the experimental group, the difference was not statistically significant.

At T₁, all three experimental groups showed an increase in the MN count, with group 1 (Ag) coated retainers showing the highest count and this was significant when compared to the uncoated retainers.

At T₂, there was a further increase in all the groups except group 1, which showed a small and insignificant decrease. There was no significant difference between the three groups.

It appears that Ag nanoparticles were more cytotoxic in the initial periods which reduced over the 3–6-month period. Although TiO₂-coated retainers showed a significant increase in the MN over 6 months, this was not significant when compared to the other groups since the uncoated samples group also showed an increase in the number of MN. This could probably be attributed to the inherent cytotoxicity of the metal and mechanical irritation caused by the retainer in contact with the tongue.^[14] Although both Ag and TiO₂ nanoparticles showed a significant increase over a period, the result was no different when compared to the uncoated stainless steel retainer group.

Despite statistical significance reflected at some time intervals, the increase in MN count from T₀ to T₂ was not high and leveled off by 6 months. The lack of clinically significant genotoxicity and cytotoxicity when compared with the uncoated retainer group advocates the usage of Ag and TiO₂ nanoparticles in orthodontic practice.

CONCLUSION

This study was done to evaluate and compare the genotoxicity and cytotoxicity of lingual bonded retainers coated with Ag and TiO₂ nanoparticles. The following conclusions were drawn from the study.

• Ag and TiO₂ nanoparticles coated retainers showed a significant increase in MN count over a period of 6 months although this increase was no different when compared to uncoated stainless-steel retainers.

Thus, orthodontic lingual bonded retainers coated with Ag and TiO₂ nanoparticles are biocompatible and can be used clinically.