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Abstract process of remineralization [13]. This demineralization is manifested as white spot lesions (WSLs) and can lead to further cavitation. Therefore, the occurrence of decalcification and their appearance in the form of WSLs at the time of debonding is one of the main concerns for both the patient and the orthodontist [14]. Therefore, efforts were made to control or even prevent the development of WSLs. Starting from maintaining good oral hygiene measures that include teeth brushing using fluoridated toothpaste and rinsing with fluoride mouthwash [15, 16]. Moving to the application of varnishes [17, 18], resin materials containing antibacterial agent [19, 20] and using modified orthodontic elastomerics [21]. Recently, attempts included the addition of nanoparticles to orthodontic adhesives [22, 23], resin modified glass ionomer cements [24, 25], elastomerics [26] and coating of orthodontic brackets, wires and bands [27, 28]. Physical vapor deposition (PVD) is one of the coating techniques that is characterized by its sustainability, as coatings can be reproduced with more efficiency and higher purity than when using other techniques [29]. Thermal evaporation, which is a type of PVD is considered advantageous over magnetron sputtering in its better control over the produced film thickness and higher purity [30]. Nanoparticles are defined as insoluble materials of size smaller than 100 nm [31]. Because of its small size, it has a higher surface-to-volume ratio and a closer interaction with microbial membranes, resulting in a larger surface area of antimicrobial activity [32]. Several metals as silver, copper, gold, titanium and zinc have been used since ages to act as antimicrobial materials, where each of them has different properties and range of activity [33, 34]. Using silver, silver ions and silver compounds have been considered as antibacterial agents in biomedical applications [35]. In dental applications, nanoparticles of silver were proven to be an effective antimicrobial component when added to dental resin composites, and also when coated on orthodontic brackets and wires [22, 36, 37]. Zinc oxide nanoparticles (ZnO) was proven to be a good antibacterial agent [38]. Also, on coating orthodontic wires with ZnO, it was found to have a good antibacterial activity [39]. Although, the nanoparticles of silver (Ag) have displayed higher antimicrobial activity than ZnO nanoparticles [40], several studies have shown that Ag nanoparticles are cytotoxic and genotoxic to human cells [41, 42]. However, a composite of Ag and ZnO nanoparticles exhibited an improved antibacterial activity against S. mutans [25]. Therefore, with the aim of benefiting from both ZnO nanoparticles and Ag nanoparticles, while reducing their individual cons which are the cytotoxicity of Ag nanoparticles [41, 42], its higher cost than ZnO nanoparticles [33], along with the reduced antibacterial effectiveness of ZnO nanoparticles when compared to Ag nanoparticles [40], a combination of Ag/ ZnO nanoparticles was used in this study for coating of orthodontic brackets and its antibacterial activity was compared to the antimicrobial effect of Ag and ZnO nanoparticles coatings individually. The null hypothesis was that the antibacterial effects of the three types of coatings on the orthodontic stainlesssteel brackets; Ag, ZnO and the combination of Ag/ ZnO nanoparticles were not to be significantly different. Methods This study aimed at assessing the antibacterial effect of three types of nanoparticles; Ag, ZnO and a combination of both Ag and ZnO (Ag/ZnO) when applied as coatings on orthodontic stainless-steel brackets through physical vapor deposition, on two different strains of bacteria; Streptococcus mutans and Lactobacillus acidophilus. This evaluation was to be carried out immediately after coating (T1) and after 3 months (T2) to see if the antibacterial effect, if present, persisted. The study was carried out at Faculty of Dentistry, Alexandria University, the Egyptian Nanotechnology center, Cairo University, El-Sheikh Zayed Campus and Faculty of Science, Cairo University. Sample grouping and preparation The sample size was estimated based on assumptions of alpha error to be equal 5% and study power 80% [37, 43, 44], a total of 48 brackets were to be included. The brackets were divided into four groups, each constituting 12 brackets: control group (brackets as received without modifications), Ag nanoparticles coated group, ZnO nanoparticles coated group and Ag/ ZnO nanoparticles coated group. The brackets used were stainless steel “American orthodontics” 0.018’’ slot size brackets of lower premolars. Before coating the brackets, ultrasonication was done at Faculty of Dentistry, Alexandria University to remove any adventitious macroscopic contamination [37]. Prior to storage in an airtight container, the brackets were thoroughly cleaned and sterilized using an autoclave. Coating procedure Physical vapor deposition was carried out using PROTOFLEX 1400 machine (USA; Figs. 1 and 2) at the Egyptian Nanotechnology center, Cairo University, El-Sheikh Zayed Campus. Thermal Evaporation was used in which Ag and/ or ZnO were vaporized followed by their deposition and coating of the surface of the orthodontic brackets. First vacuum environment was achieved through |