Evaluation of the Coating with TiO 2 Nanoparticles as an Option for the Improvement of the Characteristics of NiTi Archwires : Histopathological , Cytotoxic , and Genotoxic Evidence

1Departamento de Biologı́a Celular y Ultraestructura, Centro de Investigación Bioimédica, Facultad de Medicina, Universidad Autónoma de Coahuila, Torreón, COAH, Mexico 2Cĺınica de Ortodoncia, Facultad de Odontologı́a, Universidad Autónoma de Coahuila, Torreón, COAH, Mexico 3Departamento de Histologı́a, Facultad de Medicina, Universidad Autónoma de Coahuila, Torreón, COAH, Mexico 4Escuela de Medicina, Universidad Autónoma de Coahuila, Unidad Norte, Piedras Negras, COAH, Mexico 5Instituto Mexicano del Seguro Social, Centro de Investigación Biomédica del Noreste, Departamento de Biologı́a Celular y Molecular, Monterrey, NL, Mexico


Introduction
In the recent practice of orthodontics, the use of Nickel-Titanium (NiTi), Nickel-Titanium-Copper (NiTiCu), or Titanium-Molybdenum (TiMo) alloys predominates due to its elastic characteristics that facilitate the alignment and leveling of the arches, improving the elasticity and flexibility [1].The use of NiTi arches is widely accepted by the orthodontic community and offers biomechanical benefits difficult to match by some other materials on the market; however, a negative aspect of these arches is the roughness which has the ability to retain a greater amount of dental plaque because it favors its adhesion [2]; principally constituted by aerobic bacteria such as Streptococcus sanguinis and Streptococcus mutans, this plaque propitiates the corrosion of metals and alloys through the formation of organic acids during the glycolysis of sugars, reducing the pH [3].Currently, the search for improvement of existing materials in the field of orthodontics has been a subject of study; for example, the organically modified antibacterial silicates (ORMOSIL) such as quaternary ammonium methacryloxy silicate (QAMS) is added to the orthodontic acrylic resins in order to improve antimicrobial activity and toughness [4], resin-based adhesion materials contain Portland-type cement to provide adequate shear bond strength (SBS) and a caries-preventive effect [5], the incorporation of bioactive glass (BAG) into composite resins (BAG-Bonds) showed the capacity for buffering acidic oral conditions through the liberation of calcium in the environment [6], and also the modification of the arches with the application of nanomaterials, for example, nanoparticles (NPs), is an interesting topic due to popularity carried in recent times, and the advantages above the other modification techniques are low cost through the use of simple devices and easy handling.Between the diverse types of nanomaterials used in order to improve the arches' characteristics, the nanoparticles of TiO 2 have benn shown to be cost-effective [7] and they possess a unique photocatalytic property that results in enhanced microbicide activity, principally against bacterial strains of the plaque [8][9][10] besides their apparently low toxicity and excellent biocompatibility [11,12]; regardless, there is still controversy about it being harmless [13,14], since TiO 2 NPs have been related to the induction of cytotoxicity and genotoxicity due to the production of reactive oxygen species (ROS) in different cell types [15][16][17]; in addition, in vivo studies have shown apparently nanoscale and microscale toxicological effects associated with the size of the nanoparticles of TiO 2 [18,19].
The efficacy of electrochemical methods such as electrophoretic deposition (EPD) is an alternative, versatile, and inexpensive procedure for depositing of these nanomaterials [20] and is a very useful tool for the manufacture of films of nanostructures, where thickness can be controlled varying parameters such as voltage and time, principally with the use of TiO 2 NPs which have been already proven in the literature; moreover, compared with other wet, dry, or plasma deposition methods, attachment could be oriented more effectively due its nondestructive depositing method that does not affect the particle [21].The efficiency of EPD in the production of homogeneous and reliable films depends to a great extent on the surface chemistry of the particles, the behavior of surface-liquid interfaces under an electric field, and the development of the particle-particle network and particle-substrate network [22]; because of this, the good quality of TiO 2 NPs deposition on stainless steel bars [23] found in the literature does not necessarily correspond to a good deposition quality on other materials including NiTi arches.In our research, we propose an adequate technique for the correct deposition of TiO 2 NPs on the surface of NiTi arches, since the current bibliographic data in this type of material is virtually nonexistent and the methodology is poor described; for this reason, the objective of this study was to evaluate the electrophoretic deposition (EPD) of TiO 2 NPs in arches as well as their histopathological, genotoxic, and cytotoxic effects in Long-Evans rats.

Preparation of the NiTi Archwires.
Conventional NiTi archwires (0.017 × 0.025 inches) (Ah-Kim-Pech5, México) were sandblasted with aluminum oxide (Zogear Blaster, CHN) and subsequently etched with 10% oxalic acid at 80 ∘ C for 60 min followed by immersion in an ultrasonic bath in acetone for 3 min and rinsed with distilled water, according to Paoli et al. [22].To optimize the adhesion of NPs, later the archwires were cut into pieces of 16 mm.

Suspension Design.
A suspension was made for the nanoparticle dispersion with a mixture of H 2 O and C 2 H 5 OH (1 : 4) with a concentration of TiO 2 NPs (CAS: 13463-67-7; purity ≥ 99.5%; molecular weight: 79.87; particle size: 21 nm), 1% mass, and poly(diallyl dimethyl ammonium chloride) (PDADMAC) 2% mass [24] (all chemicals were obtained from Sigma-Aldrich Co., Ltd., St. Luis, MO, USA).The stability of the suspension was obtained by magnetic stirring (hotplate stirrer LMS-1003 Daihan Labtech Co., Ltd., Korea) during 12 h before the EPD process.This was carried out with two electrodes in a 500 mL glass vessel; the NiTi archwires were used as deposition substrates and a stainless steel sheet was used as a counter electrode, separated by a distance of 20 mm; both the working electrode and the counter electrode were connected to direct current power supply (Enduro 300V, Labnet International Inc., Woodbridge, NJ, USA).The NiTi archwire samples were weighed on an analytical balance with a sensitivity in g (Denver Instruments apx-200) and then divided into 6 groups which used a constant voltage () of 5, 10, 15, 20, 25, and 30 V, respectively, at different times (15,30,45,60, 75, and 90 s); this was carried out in triplicate.After the EPD the samples were allowed to dry for 24 h at room temperature; the samples were observed under a 10x optical microscope (Labomed S1100, Germany) with an AmScope MD700 digital microscope to determine the degree of TiO 2 NPs deposition.A new weighing of the samples was performed to measure the amount (g) of TiO 2 deposited.The amount of TiO 2 NPs deposited per area in the NiTi arcs was calculated (g/mm 2 ); the samples were left in a sterile container with 2 mL of PBS solution for 30 days to administer them later to the experimental group (G3).Twelve adult male Long-Evans rats ranging in age from 10 to 12 weeks, with an average weight between 240 and 280 grams, were provided by the Bioterium of the Faculty of Medicine of the Autonomous University of Coahuila.All animals were maintained under controlled conditions of temperature at 25-26 ∘ C in a 12 : 12 h light/dark cycle housed in individual cages with water and food ad libitum.

Experimental Groups and
Treatments.The rats were divided into 4 groups; ( = 3) the control group (CG) were given 1 mL of xylocaine5: 2% injectable solution of lidocaine without epinephrine intraperitoneally for 3 days, 1 dose daily; the sacrifice was 24 h after last dose.Group 1 (G1) is given 5 mg/kg body weight of TiO 2 dissolved in 1 mL xylocaine: 2% injectable solution of lidocaine without epinephrine intraperitoneally for 3 days, 1 dose daily.The sacrifice was at 48 h after last dose.Group 2 (G2) is given 5 mg/kg body weight of TiO 2 dissolved in 1 mL xylocaine: 2% injectable solution of lidocaine without epinephrine intraperitoneally for 3 days, 1 dose daily.The sacrifice was at 72 h after last dose.Group 3 (G3) received a solution composed of PBS (2 mL) NPs of TiO 2 plus the arcs NiTi (25 V 90 s).This solution remained at rest for 30 days prior to its administration in the rats; this solution was administered by nasogastric tube for 3 days, and the sacrifice was 72 h after last dose.After the exposure time, the rats were sacrificed by the veterinarian in charge through cervical dislocation; blood samples were extracted by cardiac puncture; the organs (liver and kidney) were removed and fixed in 10% neutral formalin for subsequent histopathological analysis.

Comet Assay in Lymphocytes.
DNA fragmentation analysis of individual cells for peripheral blood lymphocytes was performed based on the methodology of Singh et al. [25] with some minor modifications.This allows the differentiation and analysis of cells with fragmented DNA to determine the percentage of fragmentation using specialized software.From each treatment, 10 L of peripheral blood suspended in 0.5% low melting point agarose was used on slides pretreated with 0.5% normal melting point agarose and then covered with coverslips and the agarose was allowed to solidify at 4 ∘ C per 5 min; the slides were placed in a Köplin with lysis solution (2.5 M NaCl, 0.1 M EDTA, 10% DMSO, and 1% Triton X-100).At the end of the lysis, the samples were taken to horizontal electrophoresis chamber and incubated in electrophoresis buffer (NaOH 0.3 M, 200 mM EDTA) at pH = 13.0 to 20 min at 4 ∘ C for the unwinding of DNA in a dark room.The electrophoresis was completed with the following specifications: 25 V (1 V/cm), 300 mA for 20 min.After switching off the electrophoresis power source, the electrophoresis chamber slides were carefully removed and rinsed with a neutralization buffer (0.4 mol/L Tris-HCl, pH 7.5) for 5 min.The excess of neutralization buffer was drained off and placed in ethanol and then allowed to dry; once dried, the slides were stored for later reading.

DNA Fragmentation Analysis.
The DNA was stained in phosphate-buffered saline (PBS) with fluorescent solution of GelGreen6 (Nucleic Acid Gel Stains, Biotium5, Fremont, CA, USA).Comets lymphocytes were evaluated under fluorescence microscope, 40x and 100x (Labomed LX 400, Germany).The images were taken with a fluorescence, 16megapixel digital camera (AmScope, Digital Camera Microscope # MD700) and were converted into Bitmaps format ( * .bmp)and analyzed in TriTek's CometScore Freeware v1.5 software.ImageJ software V.1.8.0 was first used to remove background noise from the DNA images obtained.Automatic image processing software was used for analysis of the comet assay.The software was able to calculate the amount of DNA at specified location based on pixel intensity of images.DNA in the tail was computed as follows: DNA = total comet tail intensity total comet intensity × 100. (1)

Histopathological Analysis.
At the end of the experimental period, the rats were sacrificed by cervical dislocation; blood samples were collected by cardiac puncture and dissection of the abdominal organs was performed and fixed in 10% neutral formalin for subsequent histological analysis.
Representative samples of hepatic and renal tissue previously fixed to be included in paraffin blocks were taken by conventional histological technique, which were cut in a microtome (Leitz 1512, Austria) at a thickness of 5 m and mounted on slides stained with hematoxylin and eosin (H&E).The stained sections were examined under light microscopy to make the respective observations and to evaluate the morphological changes comparing them with the control tissues.

Statistical Analysis.
We performed Kruskal-Wallis test as a nonparametric test and Dunn test as a post hoc test and unidirectional ANOVA as a parametric test using Tukey as a post hoc test.Measures of central tendency and standard deviation were done.Statistical analysis was performed using the Minitab 17 software for Windows.In the microphotographs, we can see the different groups of arcs with their voltages and their effects (Figure 4).In the group of 5 volts (Figure 4(a)), it was observed that the deposition is minimal; increasing the voltage shows a larger deposition, considering the uniformity of the EPD at 10 V 75 s (Figure 4(c)), the greater amount of deposition is obtained at 25 V to 90 s (Figure 4(f)).The general behavior is that from 15 V there is a sustained increase of NPs deposited up to 30 V where there is a decrease because it exceeds the value of the critical cracking thickness (EAC).

Analysis of DNA Fragmentation by Exposure to
NPs of TiO 2

DNA Fragmentation in Lymphocytes.
When the alkaline comet test was performed, a stability was observed in the DNA chain, since there was no significant migration on this (Figure 5); treatment with TiO 2 NPs did not cause cytotoxicity at the concentration of 5 mg/kg for 3 days, 1 daily dose, with the sacrifice being conducted after the last dose at 48 h (G1).There was also no cytotoxicity at the concentration of 5 mg/kg for 3 days, 1 daily dose, with the sacrifice being conducted after the last dose at 72 h (G2) or in the PBS solution (2 mL) in which previously treated arches were submerged (25 V 90 s) and left to rest for 30 days and administered by nasogastric tube for 3 days and with a sacrifice conducted after the last dose of 72 h (G3), suggesting that there were no DNA breaks (see Figure 5(c)).Another parameter delivered by the comet test is the percentage of DNA contained in the comet tail.This parameter is the subtraction of 100% of DNA minus the percentage of DNA contained in the head of the comet.This parameter is the percentage of fragmentation that the evaluated cell has; the group with greater fragmentation was group 1 (G1) which had an average of 6.06 ± 1.49 percent.

Histopathological Findings.
Figure 6 shows the microphotographs with the observations found in liver tissue samples.In the analysis of the samples corresponding to the control group, which was administered only with 2% lidocaine for 3 days, no pathological data were found (Figure 6(a)).In those corresponding to group 1, which received 5 mg/kg TiO 2 for 3 days, being sacrificed 48 h after the last dose, the presence of vacuoles included within the hepatocyte cytoplasm was observed (Figure 6(b)).In the case of group 2 samples, they received 5 mg/kg of TiO 2 for three days and were sacrificed 72 h after the last dose; it is observed that the hepatocytes present a foamy cytoplasm and nucleus with granular chromatin (Figure 6(c)).Group 3, which was given PBS solution for 3 days in which the TiO 2 treated arches rested and were sacrificed 72 h after the last dose, extensive areas of cell necrosis were observed with destruction of hepatocytes (Figure 6(d)).Figure 7 shows the representative microphotographs, corresponding to the findings in renal tissue samples.In the analysis of the samples from the control group, no pathological data were found.
In the samples corresponding to groups 1, 2, and 3, a slight glomerular retraction and moderate vascular congestion were observed.greater deposition but with fractures, which increases the time of deposition/voltage.After having a constant increase, the samples treated at 30 V have a decrease in the deposited TiO 2 NPs, as can be seen in the deposition's interval graphs; the possible explanation for this is that it exceeds the value of the critical cracking thickness (CCT) as explained by Sadeghi et al. [30].When the layer becomes thicker during the deposition process, there is an increase in the resistance because the previously deposited layer is nonconductive, weakening the electric force field and causing weak connections between the particles along the final layer.Therefore, the accumulated NPs on that layer tend to have fractures that cause the detachment of the NPs due to the accumulated excess.It was decided not to perform any sintering technique after EPD because it has been proven that the exposure of the NiTi archwires at temperatures above 500 ∘ C alters their mechanical properties [34][35][36].

Comet Assay.
Although administration of TiO 2 NPs to the experiment animals was intraperitoneally (G1 and G2) and through nasogastric probe (G3), there was no significant induction of DNA breaks in peripheral blood lymphocytes in the results delivered by the comet assay; our negative results were similar to those found in keratinocytes, irradiated with TiO 2 at 20, 40, and 60 min, where there was no evidence of type IV comet damage and the number of comets II and III was approximately 30% [37] also in human diploid fibroblasts and human bronchial epithelial cells that were exposed at concentrations up to 50 g/cm 2 where the tail moment does not exceed [15].But, as mentioned above, there are studies where positive results were found in the comet assays, for example, in lymphocytes, which were treated with TiO 2 at concentrations up to 59.7 g/mL having a mean of olive moment of 7.30 ± 0.81 [38], human bronchial cell cultures that were treated with 1.77 g/cm 2 TiO 2 NPs causing olive moments up to 90 [17], Rtg cells exposed for 4 h to TiO 2 NPs (50 g/mL) in MEM cultures PBS, and H 2 O solution where there was a percentage of DNA in tail of 32, 45, and 32, respectively [39].These inconsistent results may be due to the different sizes and structures of the TiO 2 NPs used in the studies as suggested [40], making it difficult to compare the results between the studies.

Pathophysiological Findings.
The results of the present study show the existence of cytotoxic potential of TiO 2 NPs after acute exposure by intraperitoneal injection (G2 and G3) at a high dose as presented in our methodology and coinciding with Singh et al. [25] with progressive damage even after cessation of exposure, supporting the distribution and accumulation data in liver and kidney [41].In the case of G3, the liver damage was a lot greater, represented by the extensive areas of cellular necrosis.There was not enough time to find fibrous septa (cirrhosis) in the liver parenchyma as in the findings reported by Umbreit et al. [42] where central fibrous septa were found after 7 days of exposure.In the case of renal damage analysis, the findings were more discrete and there was no significant difference in the cases exposed to a controlled concentration compared to those that received the PBS solution in which the deposited archwires rested.
In this case, contrary to the findings of Chen et al. [43], what was observed in our study was a glomerular contraction and not a glomerular inflammation.Our observations seem to indicate that, after deposition of TiO 2 NP, NiTi arcs that exceed the CCT, for example, samples of 25 V/90 s, are susceptible to degradation of the coating in an aqueous medium (PBS solution), when ingests are absorbed and TiO 2 NPs enter the bloodstream producing a toxic effect on the organs, with the first step being the liver parenchyma, which results in extensive lesions such as those found in our study.
Other studies describe the ability of TiO 2 NPs to produce an inflammatory response and induce the production of ROS (reactive oxygen species) inducing apoptosis, as observed in our results [44], in addition to mentioning the relationship between the size of the NPs and their toxicity, suggesting that the smaller the size, the greater the metabolic activity and toxicity.Regarding the intracellular mechanisms of damage [45], when describing the importance of the internalization of NPs of TiO 2 , when they accumulate in lysosomes, this leads to their rupture and releases their content, such as cathepsin B with the subsequent activation of caspases to apoptosis.

Conclusion
The aim in this study was to improve the physical and biocompatibility characteristics of the NiTi arches; therefore the EPD technique was conducted to deposit TiO 2 NPs, thus proving the success of the method as a low-cost and versatile way of performing homogenous depositions even on complex surfaces such as NiTi alloys; the quality of the deposition was controlled with an adequate voltage, a precise time, and an ideal charging agent.With regard to genotoxicity and cytotoxicity, controversial results were still found, in agreement with other authors of the related literature; for example, our results in the comet assays suggest that there is no genotoxicity in any of the experimental groups under the conditions conducted.Nevertheless, in the cytotoxic findings, cytotoxic potential in the TiO 2 NPs was found, representing progressive damage even after the cessation of exposition and in some cases it was the G3 cellular necrosis.At the end, the use of NiTi arches coated with TiO 2 is not recommended to be placed in the mouth until more experimental research is done about friction resistance and degradation in the oral environment, as well as testing of TiO 2 NPs with long term exposition in vivo.

2
NPs 2.3.1.Animals.Our work was developed under the Official Mexican Standard for use and handling of animals in experimentation (SAGARPA in Mexico, NOM-062-ZOO, 1999).The study was approved by the Bioethics Committee and by the Internal Committee for the Care and Use of Laboratory Animals (CICUAL) of the Faculty of Medicine of the Autonomous University of Coahuila (number CONBIOET-ICA07CEI00320131015).All procedures with experimental animals were supervised by a veterinarian certified by the Secretary of Agriculture, Cattle Raising, Rural Development, Fishing and Food (SAGARPA, key code: MR-0716-33-001-1).

5 V 15 sFigure 4 :
Figure 4: Deposited arches.It is observed that, at higher voltage/deposition time, there are more deposited TiO 2 NPs, but there is also an increase in surface fractures ((f) and (g)) (10x).