Synthesis and Antibacterial Activity of ZnO Nanoparticle Coatings for Titanium Orthodontic Fixed Retainers

INTRODUCTION

Oral hygiene becomes notably intricate after the placement of fixed orthodontic appliances. In a considerable number of patients undergoing fixed orthodontic treatment, these fixed appliances remain in use throughout the treatment duration, typically spanning 1.5 to 2 years [1,2]. Both the brackets and the adhesive materials employed for bonding may retain plaque, introducing a susceptibility to dental caries at this newly established site. This susceptibility arises due to a significant elevation in the levels of Streptococcus mutans detected in both saliva and plaque among patients undergoing fixed orthodontic appliance treatment [3]. Consequently, this situation escalates the risk of dental caries. To address these issues and to enhance the performance of orthopedic and dental implants, coatings comprising organic and inorganic materials are applied to dental appliances. These coatings serve the dual purposes of ensuring biocompatibility, resistance against inflammation, and demonstrating antibacterial properties. Among the inorganic coatings considered for this purpose are metal oxides such as ZnO, TiO₂, ZrO₂, Nb₂O₅, and Ta₂O₅ [4,5].

To further enhance the antibacterial, bone regeneration, and cohesion properties of bone implants, employing zinc oxide (ZnO) nanoparticle films as coatings is an exemplary choice [6]. The United States Federal Drug Administration has categorized ZnO as generally recognized as safe (GRAS), as per 21CFR182.8991 [7]. This can be achieved with both naturally occurring substances and those purposefully developed for their nanoscale properties [8].  Nonetheless, there is a scarcity of comprehensive research studies that have systematically characterized ZnO nanoparticles coated on Ti6Al4V alloy samples, particularly concerning their suitability for orthodontic fixed retainers. Existing literature reveals that ZnO coatings can be fabricated using various methods, including chemical vapor deposition, radio frequency magnetron sputtering, molecular beam epitaxy, sol-gel processes, hydrothermal synthesis, and electrochemical or electrophoretic deposition [9-15]. The technique of electrophoretic deposition (EPD) represents a cost-effective method for the production of highly uniform films, characterized by thickness ranging from the nanometer to micrometer scale. This uniformity is achieved by manipulating the applied voltage and deposition time. The key advantages associated with the EPD method encompass the following aspects [16,17]: its applicability to any solid material available in a finely powdered form, its capacity for rapid film deposition, cost-effectiveness, straightforward instrumentation, minimal restrictions concerning substrate shapes, and resource conservation.

EPD has gained prominence as a valuable technique for the creation of nanostructured ZnO films[17-26]. It is noteworthy that both aqueous and non-aqueous ZnO suspensions have been effectively employed to coat a wide array of substrates, including transparent conductive oxide glass, anodic alumina membranes, as well as steel, nickel, and titanium. In the context of depositing ZnO coatings on Ti6Al4V substrates, the electrophoretic coating technique has been chosen for its capacity to afford control over parameters such as coating thickness, chemical composition, and particle sizes [27,28]. The primary objective of this scholarly investigation is to rigorously examine the effects and implications of zinc oxide (ZnO) coatings, applied onto Ti6Al4V alloy substrates using the cathodic electrophoretic deposition (EPD) technique. This study aims to meticulously characterize and analyze the resultant surface properties utilizing advanced methodologies such as X-ray Diffraction (XRD) and Atomic Force Microscopy (AFM). Moreover, a significant aspect of this research is dedicated to evaluating the antimicrobial efficacy of the ZnO nanoparticle-coated Ti6Al4V alloy surfaces, specifically in the context of their application in orthodontic devices. This comprehensive analysis is poised to contribute valuable insights into the potential of ZnO coatings in enhancing the functional and hygienic properties of orthodontic materials.

MATERIALS AND METHODS

The ASTM Grade 5 titanium alloy, also known as Ti₆Al₄V, was procured from BAOJI JINSHENG METAL MATERIAL CO., LTD. The alloy composition consists of titanium (Ti) at 90%, aluminum (Al) at 6%, and vanadium (V) at 4%. It falls under the category of alpha-beta titanium alloys, with aluminum serving to stabilize the alpha phase and vanadium contributing to the stabilization of the beta phase. Ti6Al4V is widely favored due to its exceptional combination of material properties. It can undergo further processing to tailor its attributes for specific applications. Upon receipt of the alloy in sheet form for electrophoretic deposition, the samples were prepared by employing a cutting machine with a slow cutting speed. A cutting fluid was also employed to prevent the generation of excessive heat during the cutting process. The sample dimensions were determined in accordance with the required tests: samples measuring 2x10x20 mm for characterization and disc-shaped samples with a diameter of 6 mm for the antimicrobial diffusion test. The Ti6Al4V alloy samples were smoothed using silicon carbide-based smoothing paper with a granular size of 500 microns. Subsequently, they were subjected to chemical cleaning with a solution comprising oxygen (O₂), hydrofluoric acid (HF), and nitric acid (H₃HNO) in volume ratios of 3:1:6, respectively. The samples underwent a thorough washing process using 90% ethanol in an ultrasound bath for 10 minutes, repeated twice, followed by a final wash using distilled water in an ultrasound bath for an additional 10 minutes.

For the electrophoretic deposition process, a solution was prepared containing nano-sized ZnO particles (ranging from 2 to 5 g), 100 ml of 90% ethanol, and 2 g of Polyvinylpyrrolidone (PVP) binder. In this process, the Ti6Al4V samples served as the cathode, while a graphite electrode was employed as the anode. These electrodes were connected to a power supply, with varying electrode distances (1, 3, and 5 cm) and voltage settings (10, 30, and 50 Volts) applied for a duration of 5 minutes. Following deposition, the samples were air-dried for 1 hour. X-ray diffraction (XRD) measurements were carried out using a Shimadzu X-ray diffractometer with a wavelength of 1.54056 Å. Surface morphology analysis was conducted using an Atomic Force Microscope (NaioAFM, Switzerland). In accordance with Standard ISO 20645:2004 (BS EN ISO 20645:2004 or GB/T 20944.1), antimicrobial testing was performed utilizing Mueller Hinton agar for diffusion tests. Disc-shaped coated specimens measuring 6 mm in diameter and 2 mm in thickness were employed for this examination, following the guidelines of the Clinical and Laboratory Standards Institute (CLSI; 2018).

The density of test cultures was adjusted to an optimal value of 0.5 McFarland standards (equivalent to 1.5×10⁸ CFU/mL) for the antibacterial assays. Coated and uncoated specimens were systematically placed in the culture at specific millimeter intervals. Each test plate accommodated three samples, with a total of 10 samples for each group (control and optimized EPD). The plates were incubated at 37°C for 24-48 hours. The assessment of antibacterial activity was based on the measurement of the width of an inhibition zone (H) surrounding the samples, calculated using the formula H = (D − d)/2, where H represents the inhibition zone in millimeters, D is the combined diameter of the sample and inhibition zone in millimeters, and d is the diameter of the sample in millimeters. Following the measurement of the inhibition zone, the specimens were carefully removed from the agar using a pair of tweezers.

RESULTS AND DISCUSSION

Structure of the ZnO nanoparticles

X-ray diffraction (XRD) analysis was employed to elucidate the composition and structural characteristics of ZnO films that had been deposited onto Ti6Al4V substrates. In essence, the X-ray diffraction patterns exhibited discernible diffraction lines, which unequivocally corresponded to the ZnO phase (as per the ICDD file 36-1451). Additionally, traces of the Ti phase (according to the ICDD file 44-1494) were discerned (Fig. 1, 2, 3). The XRD patterns of the deposits, generated at varying concentrations of ZnO nanoparticles (2, 3, 4, and 5 g), voltage settings (10, 30, 50 Volts), and inter-electrode distances (1, 3, 5cm), consistently demonstrated the presence of polycrystalline phases and prominent diffraction lines associated with ZnO. Notably, there were no discernible alterations in the crystal structure in comparison to the uncoated alloy. Only the diffraction peaks attributable to Ti were observed, specifically at angles θ = 38.42º and 40.17º, corresponding to the (002) and (101) planes of titanium. Other alloy constituents remained inconspicuous in the diffraction pattern, primarily due to their lower proportion within the alloy, rendering them less amenable to X-ray refraction. The X-ray diffraction analysis effectively unveiled the characteristic diffraction peaks that can be ascribed to the well-defined hexagonal wurtzite structure of ZnO. Utilizing Scherrer’s equation [29], the average size of the ZnO crystallites was calculated and is presented in Table 1. Notably, when the XRD patterns were examined for the deposit produced at 50 V, a ZnO concentration of 5 mg, and an inter-electrode distance of 1 cm, all for a duration of 5 minutes, robust diffraction lines corresponding to ZnO were evident. This observation aligns with the expectation that the quantity of deposited ZnO exhibits an almost linear increase with escalating concentrations of nano-sized ZnO, and furthermore, the rate of ZnO deposit formation is positively correlated with augmented applied voltages. These findings are corroborated by existing literature [30].

AFM Image Analysis

In Fig. 4, we present Atomic Force Microscope (AFM) images depicting both uncoated Ti6Al4V alloy samples and ZnO-coated samples, where variations in suspension concentration, applied voltage, and inter-electrode spacing are observed. Complementary data, including grain sizes of the ZnO thin films (ranging from 42.20 to 102.7 nm) and root mean square (RMS) roughness values (ranging from 14.82 to 44.10 nm), are tabulated in Table 2. These findings collectively underscore the marked heterogeneity in size and shape exhibited by the deposited films. As discerned from the visual representations, elevating the concentration of ZnO nanoparticles (at 5mg), applying a voltage of 50 Volts, and reducing the inter-electrode spacing to 1 cm led to an enlargement in grain size. This phenomenon is likely attributed to an accelerated deposition rate and the plausible agglomeration of smaller grains into larger ones. In instances where the lowest applied deposition voltage is employed, the ZnO film’s morphology reveals a uniform layer with a random orientation, stemming from the presence of only a few grains characterized by an oriented texture (Fig. 4A). Notably, the surface exhibits an evident increase in roughness, with grain boundaries becoming more pronounced. The increase in grain size becomes more evident, accompanied by an augmentation in grain protrusions. Remarkably, surface porosity diminishes substantially, rendering the structure relatively discrete, with discernible deep valleys. The grain boundaries become distinctly delineated, and individual grains manifest as distinct microregions, each characterized by unique dimensions.

Conversely, the electrophoretic current exhibits an upward trend with increasing applied voltage, corresponding to greater deposition thickness. The associated increase in surface roughness is attributed to the migration of charged particles under the influence of the applied voltage. As the applied field strength amplifies, so does the force acting upon the particle, thereby augmenting the surface roughness of the film. Furthermore, as the suspension concentration of nano-sized ZnO escalates, the surface morphologies reveal the emergence of grain features on the film surface, manifesting with greater intensity. This phenomenon culminates in the formation of considerably larger grains within the ZnO films (Fig. 4B-H). This development is a consequence of enhanced surface mobility of adatoms, which are vital for the formation of continuous films. The intensified surface diffusion of these adatoms is facilitated by the elevated applied voltage, thus imparting momentum to the growing surface. Conversely, a higher suspension concentration of ZnO nanoparticles, coupled with elevated deposition voltage and reduced inter-electrode spacing, yields an increase in RMS roughness due to the prominence of larger grains. This observed trend in RMS roughness corresponds directly with the crystallite size data (Table 2).

Antimicrobial Activity of ZnO Nanoparticles

As depicted in Fig. 5 and 6, the influence of ZnO nanoparticles on the growth of microorganisms, such as Lactobacillus, Streptococcus mutans, and Candida albicans, exhibits variability in its effects. The findings reveal that smaller ZnO nanoparticles exert a more pronounced impact on microbial cells, with Lactobacillus and Streptococcus mutans displaying greater susceptibility compared to Candidan albicans. ZnO nanoparticles achieve this by disrupting the structural integrity of the cell membrane and impeding the normal budding process, thereby impeding the growth of Candida albicans. Nonetheless, the efficacy of this mechanism varies depending on the specific microorganism, chiefly due to disparities in cell wall thickness [31]. Tables 3 and 4 compile the results of experiments where ZnO nanoparticle-coated and uncoated specimens were introduced into microbial cultures at specified intervals. After 24 hours of exposure, the observable effect was quantified and recorded as an inhibition zone. Notably, smaller grain sizes are correlated with increased active or specific surface area, facilitating enhanced interaction and penetration of microbial cell membranes, consequently heightening their antimicrobial activity. The results indicate that ZnO nanoparticles exhibit relatively greater effectiveness against Lactobacillus and Streptococcus mutans in contrast to Candida albicans, an outcome attributed to variations in cell wall thickness. ZnO nanoparticles within the film potentially manifest antifungal activity by perturbing the structural integrity of the cell membrane and impeding the typical budding process. Differing degrees of sensitivity among microorganisms are likely due to disparities in the structure and/or composition of their cell walls and extracellular matrices. This study posits the possibility that nanoparticles may adhere to bacterial cell walls and subsequently infiltrate them, ultimately leading to cell demise or the generation of free silver nanoparticle radicals capable of inflicting damage to the cell membrane. While the precise mechanism underlying antimicrobial activity remains incompletely understood, these findings are consistent with earlier research endeavors, aligning with prior investigations [31-37].

In an academic context, ZnO nanoparticle-coated Ti6Al4V samples exhibited noteworthy antimicrobial effectiveness against Lactobacillus, Streptococcus mutans, and Candida albicans, as determined through the Shapiro Wilk test at a significance level of p>0.05. The results presented in Table 5 indicate that the diameter of the inhibition zones for all microorganisms, in both the control group (uncoated Ti6Al4V) and the group with electrophoretic deposited (EPD) ZnO on Ti6Al4V samples follows a normal distribution. Data description, analysis, and presentation were conducted using the Statistical Package for Social Science (SPSS version 22, Chicago, Illinois, USA), as detailed in Table 6. Fig. 7 presents a cluster chart displaying key parameters, including minimum, maximum, mean, standard deviation, Shapiro Wilk test for normality, and Student T test. Significance was established when the p-value was less than 0.05. The application of ZnO-coated Ti6Al4V demonstrates considerable potential as a method to enhance antimicrobial efficacy against lactobacillus, Streptococcus mutans, and Candida albicans.

CONCLUSION

The current research reveals that the application of cathodic Electrophoretic Deposition (EPD) in an aqueous environment using a zinc anode is an effective and efficient technique for the formation of uniform, densely packed nanostructures exhibiting enhanced sintering properties, even at lower direct current (dc) voltage levels. The EPD process successfully facilitated the coating of the Ti6Al4V alloy surface with a zinc oxide (ZnO) phase, the characteristics of which were significantly influenced by the chosen deposition parameters. Optimal conditions for creating a homogenous and continuous ZnO layer were established at 50 volts, with a 5 mg concentration of ZnO nanoparticles and a 1 cm electrode gap. Additionally, the methodology employed for the synthesis of ZnO nanoparticles (ZnONPs) in this study was notably straightforward and economical. This approach not only simplifies the production process but also makes it more cost-effective. The synthesized ZnONPs solution demonstrated in this research exhibits promising potential as an effective agent for antibacterial and antifungal applications. This suggests its viability for use in a range of applications, particularly in enhancing the antimicrobial properties of materials used in medical and dental fields.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interests regarding the publication of this manuscript.