This in vivo study will include 42 participants undergoing fixed orthodontic treatment. All participants will be treated using fixed pre-adjusted appliances with metal brackets (slot size 0.022 x 0.028 inch, MBT prescription). The aim is to evaluate the antimicrobial properties of nickel titanium (NiTi) orthodontic archwires coated with titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles.

Surface Modification:

Surface modification of NiTi wires with photocatalytic titanium dioxide will be done using the sol-gel dip coating method.

1. Preparation of TiO2 precursor sol: 0.4 g titanium isopropoxide is mixed with 10 mL deionized water to form titanium hydroxide. The precipitate is washed to remove alcohol byproducts and then dissolved in 7.5 mL hydrogen peroxide solution to obtain a transparent orange peroxo-titanium sol, used as the coating precursor.
2. Coating with TiO2 thin film: NiTi wires are degreased, cleaned, dried, then dipped into the peroxo sol and pulled out at a steady rate to form a uniform wet film. The film is air dried and heated at 50 degrees Celsius in a furnace to form a stable TiO2 coating.

ZnO Coating:

NiTi archwires are ultrasonically cleaned with ethanol for 10 minutes at 30 degrees Celsius, then immersed in 4 M potassium hydroxide at 100 degrees Celsius for 30 minutes to remove oxides and increase surface roughness. For ZnO coating, 0.1 g zinc nitrate is dissolved in distilled water and ammonia is added dropwise with stirring until pH 12. The solution is heated to 50 degrees Celsius, and pretreated wires are immersed for nanoparticle deposition.

Allocation Method:

Participants will be selected as per inclusion and exclusion criteria and randomly assigned into three groups:

Group 1: TiO2 nanoparticle-coated 0.014 inch NiTi archwire

Group 2: ZnO nanoparticle-coated 0.014 inch NiTi archwire

Group 3: Uncoated 0.014 inch NiTi archwire (control)

This will be a single-blinded study. Participants will begin treatment with fixed appliances. The upper arch will be bonded with MBT bracket system (slot 0.022 x 0.028 inch). During initial leveling, a 0.014 inch NiTi archwire will be placed for four weeks. After four weeks, the wire will be retrieved and analyzed for antimicrobial properties.

Instructions to Patients: Patients will be instructed to brush after every meal and avoid using other oral hygiene aids such as mouthwash.

Sample Collection: After four weeks, plaque samples will be collected from a 2 cm segment cut from the posterior part of the maxillary archwire and placed in phosphate buffer saline for microbiology testing. Samples will be sent to Genexis Biotech Pvt. Ltd., Vadodara, Gujarat.

Microbiological Analysis: Samples will be inoculated in BHI broth for the isolation of Streptococcus mutans. Cultures will undergo serial dilution and be plated on chromogenic agar for bacterial identification. Isolated colonies will be further incubated in BHI broth at 37 degrees Celsius overnight. Bacterial DNA will be extracted using a genomic DNA kit. 16S rDNA amplification will be done using universal primers. PCR samples will be confirmed on 0.8 percent agarose gel and purified using a PCR cleanup kit and PCR thermocycle Sequencing will be done.

For Results  Colony Forming Units (CFU) will be counted.