This research aimed to evaluate the effect of titanium dioxide (TiO 2 ) coating on surface roughness (R a ) of nickel-titanium (NiTi) archwires and its influence on Streptococcus mutans ( S mutans ) adhesion and enamel mineralization at the end of 1 month in orthodontic patients and to evaluate the integrity of the TiO 2 coating.
Twelve patients undergoing orthodontic treatment with preadjusted edgewise appliance formed the sample for this prospective clinical study. Uncoated NiTi archwires and TiO 2 nanoparticle coated NiTi archwires in as-received condition and after 1 month of intraoral use were subjected to R a analysis using surface profilometry, and surface topography using scanning electron microscopy. S mutans adhesion was evaluated on the retrieved archwires using real-time polymerase chain reaction (PCR). Enamel mineral content in the arches related to the uncoated and coated archwires was evaluated using DIAGNOdent.
After 1 month of intraoral use, both coated and uncoated archwires exhibited a rougher surface with coated archwires demonstrating greater quantum of increase (control, P = 0.002; experimental, P = 0.002). S mutans adhesion was more in uncoated archwires ( P = 0.0005). The TiO 2 nanoparticle coating on the NiTi archwires showed delamination, deterioration and was lost by 60% at the end of 1 month. Laser fluorescence values did not show any significant difference (control, P = 0.182; experimental, P = 0.105).
TiO 2 nanoparticle coating on NiTi archwires causes an initial reduction in roughness; however, at the end of 1 month, the benefit was lost. S mutans adhesion was lesser on the coated wires, which could be attributed to reduced initial R a and antibacterial property of TiO 2 . Orthodontic archwire appears to have a limited role in enamel demineralization.
Coated and uncoated nickel-titanium archwires showed increased roughness after 1 month.
Titanium dioxide nanoparticle coating showed delamination and deterioration after 1 month.
Titanium dioxide nanoparticle coated nickel-titanium archwires had less Streptococcus mutans adhesion than uncoated wire.
Bacterial adhesion to archwires and other biomaterials is mainly influenced by the surface characteristics which plays a critical role in being a habitat for caries causing microorganisms like Streptococcus mutans (S mutans) . This increase in S mutans after orthodontic appliance placement has been well documented and even within one month of fixed appliance therapy. ,
Prevention of white spot lesions during fixed appliance therapy is a major challenge faced by clinical orthodontist. Methods to prevent white spot lesions, especially those with minimal or nil patient cooperation, have been extensively studied. Reducing the plaque around the fixed orthodontic attachments with semiconductor particles such as TiO 2 , silver oxide, zinc oxide, copper oxide, and ferric oxide, that have been coated on bracket and archwires have been recently studied. Previous studies in the literature have assessed TiO 2 as a photocatalytic agent, whereas the antibacterial effectiveness of TiO 2 coated brackets and archwires , , , has been proven in several in-vitro studies.
The surface roughness (R a ) of the archwires is an important parameter, and it has been shown to contribute to the S mutans aggregation on the archwire. , Abraham et al found that the NiTi archwire becomes rougher after 1 month of intraoral usage, and they suggested that this increase in R a contributed to an increase in S mutans count.
Ozylidiz et al and Ghasemi et al showed that TiO 2 coated brackets significantly reduced R a and thereby decrease in S mutans colonization. Although coated brackets render the above benefits, to the best of our knowledge, no studies have assessed the effect of coated archwires, specifically TiO 2 coating, and its effects on the R a of NiTi wires and also on S mutans adhesion.
Therefore, this clinical study was designed to evaluate and compare the R a of TiO 2 nanoparticle coated with uncoated 0.016-in NiTi archwires and S mutans adhesion in orthodontic patients at the end of one month. The integrity of the TiO 2 coating and the mineral content of enamel were also evaluated over 1 month against the background of the envisaged benefits.
Material and methods
Ethical approval was granted by the Institutional Ethics Committee, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India (approval no. CSP/18/JUN/71/199; approval date: February 7, 2018). This clinical study was conducted on patients who were to undergo orthodontic treatment with fixed appliance. The inclusion criteria were, patients with all permanent teeth mesial to first molars and who were to be treated without extractions as part of their treatment plan, aged 14-25 years, minimal crowding and/or spacing of 0 to 3 mm, and good or fair oral hygiene with a simplified debris index (DI-S) of 0.0 to 1.5 The exclusion criteria were smokers, any history of oral diseases, systemic diseases, occupational exposure to metals, patients receiving any medications or supplements, and allergies to jewelry and/or watches.
Based on data from the study by Abraham et al (power, 0.80; α = 0.05), the sample size was calculated to be 24 arches (ie, 12 per group). Thus, 12 maxillary and 12 mandibular arches comprised the study sample. Because a split-mouth design would eliminate the confounding factors, alternate allocation of patients was done to ensure that a patient receiving an uncoated wire (control) in 1 arch would receive a coated (experimental) wire in the opposing arch. Thus, 2 groups, namely group 1 (control; uncoated NiTi wires) and group 2 (experimental; TiO 2 nanoparticle coated NiTi wires), were generated from the 24 arches.
The procedure was explained to the patients who met the inclusion criteria, and they were invited to take part in the study. Informed consent was obtained from the willing participants, and informed assent was obtained from patients aged <18 years. Twelve patients were recruited for the study. Molars were banded, and preadjusted edgewise brackets were bonded (Ormco Mini 2000; Ormco Corp, Glendora, Calif), using a standard etchant, primer, and orthodontic adhesive. Patients were instructed on oral hygiene measures and use of identical dentifrices and advised not to use any chewing gum, fluoridated mouthwash, or antibiotics during the period of the study.
The archwires used in this study were, 0.016-in NiTi wires (Ormco Corp); for the experimental group, these wires were coated with TiO 2 nanoparticles.
Evaluation of R a of TiO 2 coated and uncoated NiTi wires at baseline (T 0 ) was done before allocation using 3-dimensional surface profilometry and was repeated on the retrieved wires after 1 month (T 1 ). Surface topography of coated archwires using scanning electron microscopy (SEM) was done at T 0 and T 1 . S mutans adhesion was also evaluated on the retrieved archwires using real-time PCR. Evaluation of the enamel mineral content in the arches related to the control and experimental group was done using DIAGNOdent.
Surface coating of 0.016-in NiTi wires with TiO 2 was done by the Radio Frequency magnetron sputtering method (Anelva Sputtering Unit Model SPF-332H; Canon Anelva Corp, Kawasaki, Japan).
TiO 2 coating was done on 18 archwires, 6 wires were taken for the assessment using Zeiss Ultra 55 “Gemini” with ultra-high-resolution SEM coupled with material spectroscopy tools, (Zeiss, Oberkochen, Germany). and the remaining 12 were used on the patients. For the SEM analysis, the coated wires were analyzed at the middle laser-etched portion of the archwire. Surface analysis was followed by cross-section analysis. The mean and standard deviation of coating thickness were taken at 4 random points for each wire. At the end of 1 month, all 12 samples that were used on patients were analyzed using SEM on the surface and cross-section view.
The 24 archwires, 12 coated and 12 uncoated intended for intraoral use, were subjected to 3-dimensional surface profilometry analysis to assess the mean R a . For the retrieved samples before the estimation, the samples were cleaned with isopropanol to remove the debris from the surface. The R a of in as-received condition and after 1 month of intraoral exposure of each specimen was measured using a 3-dimensional surface profilometer (Wyko NT1100; Veeco, Tucson, Ariz) at a magnification of ×30 and vertical scanning interferometry mode. Four profilometric scans were taken at different positions on the wire surface, and the mean value and standard deviation were calculated. Subsequently, the retrieved archwires were subjected to SEM analysis.
The patients were recalled 1-month postinsertion of the archwires, and the archwires were removed cautiously to avoid any contact with oral mucosa. These retrieved archwires were stored in phosphate-buffered saline (PBS) in a 50 mL sterile falcon tube.
The estimation of microbial adhesion was done after the procedure by Yang et al. Briefly, the retrieved archwires were washed with PBS. The adherent bacteria were then detached by vortexing and then by a sonication method using a sonicator in 15 mL PBS with four 30-second pulses and three 30-second intermittent cooling periods. After the detachment of the microbes, S mutans adhesion was evaluated using real-time PCR. To extract the bacterial DNA, we prepared a lysozyme enzyme solution containing Tris HCl, ethylenediaminetetraacetic acid, tritonex, lysozyme (20 mg per sample). To each sample, 1 mL of the above-prepared solution was added and incubated at 37°C for 30 min in a thermos mixer water bath. Then 20 μL of 10% sodium dodecyl sulfate was added to the sample, mixed in av and again incubated at 37°C for 30 min in a dry bath. After incubation, an equal volume of phenol-chloroform solution was added and centrifuged at 10,000 rpm for 10 min. The supernatant was collected and transferred to new Eppendorf tubes. Chloro-iso-amine alcohol of equal volume was added, and the samples were centrifuged at 10,000 rpm for 10 min. The supernatant was then removed and transferred into new Eppendorf tubes to which one-tenth the volume of sodium acetate and 300 μL of 100% ethanol (absolute) were added and stored at −50°C overnight. The following day, the samples were centrifuged at 10,000 rpm for 10 min. The supernatant was removed and discarded, and the pellets were air-dried. A total of 30 to 50 μL of distilled water was added (RNA/DNAase-free water).
The real-time PCR assay was performed to estimate the amount of bacterial adhesion on the archwires. The assay was performed using the 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, Calif) to study the bacterial quantification by SYBR green for relative quantification. The sequence for the forward primer S mutans F5 was 5′-AGC CAT GCG CAA TCA ACA GGT T-3′ with 22 bases, and the sequence for the reverse primer S mutans R4 was 5′-CGC AAC GCG AAC ATC TTG ATC AG-3′ with 23 bases.
A real-time PCR reaction mixture of 20 μL was prepared (SYBR green mix [×2], 10 μL; forward primer, 1 μL; reverse primer, 1 μL; template [bacterial genome], 1 μL; sterile water, 7 μL). The reactions were performed on 96 well trays with nontemplate control (negative control) that contained no template DNA. The PCR conditions were as follows: denaturation for 2 min at 50°C; annealing for 10 mins at 95°C; extension for 1 min at 60°C; and repeated for 40 cycles. The real-time PCR values were obtained in the form of a graph which was interpreted using the relative quantification from Manager Software (Applied Biosystems).
Enamel mineralization was assessed using the DIAGNOdent pen (KAVO Dental Corp, Lake, Zurich, Ill). The laser device was calibrated for each patient by placing the laser beam on the calibration tool (as recommended by the manufacturer), and the display was reset to zero. All teeth from the second premolar to the second premolar in each arch were evaluated for each patient. The buccal surfaces of each tooth were divided into 4 quadrants: gingival, mesial, occlusal, and distal, as recommended by Banks and Richmond. The peak fluorescence measurement shown on display was recorded. This measurement was done at T 0 and T 1 for the control and experimental groups. The average values for the 4 quadrants on the buccal surfaces for each tooth were taken. This average value from the second premolar to the second premolar was summed up for each arch to derive the mean and standard deviation for the control and experimental group.
The collected data were analyzed with SPSS statistical software (version 23.0; IBM Corp, Armonk, NY). To analyze data, we used descriptive statistics, mean, and standard deviation. To find the significant difference between the bivariate samples in paired groups, we used the paired sample t test was used, and for independent groups, unpaired sample t test was used. To assess the relationship between the variables, Pearson correlation test was used. In all the above statistical tools, the probability value of 0.05 was considered significant.
The R a of 0.016-in NiTi wires (group 1) at T 0 was greater than the R a of 0.016-in TiO 2 nanoparticle coated NiTi wires (group 2), and this difference was statistically significant ( P = 0.0005). The group 1 wires showed increased roughness in comparison with group 2 wires at T 1 . The intergroup comparison was not statistically significant ( P = 0.309). The R a of group 1 and group 2 wires increased from T 0 to T 1 , and the intragroup comparison showed it was statistically significant for both the groups (control, P = 0.002; experimental, P = 0.002) ( Table I ).
|Wires||T 0 (nm)||P value||T 1 (nm)||P value||Comparison between T 0 and T 1|
|0.016-in NiTi||1591.08 ± 260.41||0.0005||2296.18 ± 730.93||0.309||0.002|
|0.016-in TiO 2 nanoparticle coated NiTi wire||746.14 ± 150.45||1922.04 ± 1007.81||0.002|