Preparation of Nanocomposites for Antibacterial Orthodontic Invisible Appliance Based on Piezoelectric Catalysis

Compared to fixed orthodontic appliances with brackets, thermoplastic invisible orthodontic aligners offer several advantages, such as high aesthetic performance, good comfort, and convenient oral health maintenance, and are widely used in orthodontic fields. However, prolonged use of thermoplastic invisible aligners may lead to demineralization and even caries in most patients’ teeth, as they enclose the tooth surface for an extended period. To address this issue, we have created PETG composites that contain piezoelectric barium titanate nanoparticles (BaTiO3NPs) to obtain antibacterial properties. First, we prepared piezoelectric composites by incorporating varying amounts of BaTiO3NPs into PETG matrix material. The composites were then characterized using techniques such as SEM, XRD, and Raman spectroscopy, which confirmed the successful synthesis of the composites. We cultivated biofilms of Streptococcus mutans (S. mutans) on the surface of the nanocomposites under both polarized and unpolarized conditions. We then activated piezoelectric charges by subjecting the nanocomposites to 10 Hz cyclic mechanical vibration. The interactions between the biofilms and materials were evaluated by measuring the biofilm biomass. The addition of piezoelectric nanoparticles had a noticeable antibacterial effect on both the unpolarized and polarized conditions. Under polarized conditions, nanocomposites demonstrated a greater antibacterial effect than under unpolarized conditions. Additionally, as the concentration of BaTiO3NPs increased, the antibacterial rate also increased, with the surface antibacterial rate reaching 67.39% (30 wt% BaTiO3NPs). These findings have the potential for application in wearable, invisible appliances to improve clinical services and reduce the need for cleaning methods.


Introduction
Malocclusion is a developmental disorder of the craniofacial complex that affects the jaws, tongue, and facial muscles and is known as one of the three major oral diseases that affect human oral function, aesthetics, social interactions, and health-related quality of life [1]. Malocclusion represents a high prevalence, ranging from 20% to 100%, as reported by different researchers [2][3][4][5]. Several studies have indicated that fixed orthodontic treatment can have adverse effects on the oral health of patients with malocclusion. These effects can range from mild to severe and include issues such as enamel demineralization, white lesions, caries, periodontics, and halitosis [6][7][8]. Consequently, traditionally fixed orthodontic treatment is no longer adequate for meeting the orthodontic needs of modern patients. Aesthetic considerations play a crucial role in the practice of dentistry. In addition, studies have shown that thermoplastic invisible orthodontic aligners pose fewer risks to oral hygiene compared to traditionally fixed orthodontic treatments [6], which has resulted in the thermoplastic invisible orthodontic appliances being widely used in the field of shown that BaTiO 3 nanocomposites possess favorable biocompatibility, making them a suitable platform for biomaterials [27].
In this study, we have developed, for the first time, a dental composite device with selfpowering and antibacterial properties, designed to be worn as an orthodontic aligner and retainer. These appliances can exhibit catalytic antibacterial effects by responding to minor mechanical stimulations caused by routine oral activities (e.g., speaking, drinking, and exercising) without the need for any auxiliary devices. The continuous production of active radicals by the appliances will effectively target bacteria and prevent biofilm formation on tooth surfaces. Furthermore, as the appliances do not require an external energy source, such as photostimulation or ultrasonic agitation, their antibacterial activity is a spontaneous process. Additionally, minor mechanical stimulations in the oral environment are ongoing, ensuring a long lifespan for the appliances.

Fabrication of Nanocomposites Diaphragm
As schematically illustrated in Figure 1, the nanocomposites diaphragm was synthesized through a solution blending method. Initially, BaTiO 3 NPs (Shanghai Lin En Chemical Technology Co., Ltd., Shanghai, China) with varying mass fractions (10 wt%, 20 wt%, and 30 wt%) were introduced into dichloromethane, an organic solvent. The resulting mixture was subjected to ultrasonic dispersion for 48 h. PETG matrix particles ( SK JN200, Seoul, Republic of Korea) with different mass fractions were then added to the solution system, followed by sealing the container. The mixed slurry was transferred to a mold for performing diaphragms after shaking in a constant temperature shaker at 200 rpm and 37 • C until completely dissolved, and 10 wt%, 20 wt%, and 30 wt% PETG/BaTiO 3 NPs composite diaphragms were prepared. The PETG diaphragm was also synthesized to serve as a control group: a certain amount of PETG matrix particles were dissolved in dichloromethane organic solvent and sealed the container at 200 rpm, 37 • C constant temperature oscillation, until completely dissolved. The slurry was transferred to a mold for performing diaphragm. Finally, some samples were cut into circular sheets with a diameter of 0.5 cm, covered with conductive adhesive, and then subjected to polarization treatment in a high-pressure polarization device at 10 KV/mm for 20 min.  [27].
In this study, we have developed, for the first time, a dental composite device with self-powering and antibacterial properties, designed to be worn as an orthodontic aligner and retainer. These appliances can exhibit catalytic antibacterial effects by responding to minor mechanical stimulations caused by routine oral activities (e.g., speaking, drinking, and exercising) without the need for any auxiliary devices. The continuous production of active radicals by the appliances will effectively target bacteria and prevent biofilm formation on tooth surfaces. Furthermore, as the appliances do not require an external energy source, such as photostimulation or ultrasonic agitation, their antibacterial activity is a spontaneous process. Additionally, minor mechanical stimulations in the oral environment are ongoing, ensuring a long lifespan for the appliances.

Fabrication of Nanocomposites Diaphragm
As schematically illustrated in Figure 1, the nanocomposites diaphragm was synthesized through a solution blending method. Initially, BaTiO3NPs (Shanghai Lin En Chemical Technology Co., Ltd., Shanghai, China) with varying mass fractions (10 wt%, 20 wt%, and 30 wt%) were introduced into dichloromethane, an organic solvent. The resulting mixture was subjected to ultrasonic dispersion for 48 h. PETG matrix particles ( SK JN200, Seoul, Republic of Korea) with different mass fractions were then added to the solution system, followed by sealing the container. The mixed slurry was transferred to a mold for performing diaphragms after shaking in a constant temperature shaker at 200 rpm and 37 °C until completely dissolved, and 10 wt%, 20 wt%, and 30 wt% PETG/BaTiO3NPs composite diaphragms were prepared. The PETG diaphragm was also synthesized to serve as a control group: a certain amount of PETG matrix particles were dissolved in dichloromethane organic solvent and sealed the container at 200 rpm, 37 °C constant temperature oscillation, until completely dissolved. The slurry was transferred to a mold for performing diaphragm. Finally, some samples were cut into circular sheets with a diameter of 0.5 cm, covered with conductive adhesive, and then subjected to polarization treatment in a high-pressure polarization device at 10 KV/mm for 20 min.

Characterization
The nanocomposites diaphragm microstructure was characterized by scanning electron microscopy (SEM; Hitachi S-4800, Tokyo, Japan), X-ray diffraction (XRD; Rigaku D/MAX-2400, Tokyo, Japan), and Raman spectra (HORIBA, XPloRAPLUS, Kyoto, Japan). First, the BaTiO 3 nanoparticles and fracture surface of PETG/BaTiO 3 (30 wt%) nanocomposites were analyzed and characterized using a scanning electron microscope. Images were captured at magnifications ranging from 100 to 50,000 and an acceleration voltage of 10 KV. To enhance the conductivity, all samples were coated with a thin layer (20-30 • ) of gold via sputtering. Then, the presence of phases in both PETG diaphragm and PETG/BaTiO 3 NPs (30 wt%) nanocomposite diaphragm was investigated using X-ray diffraction (XRD) technique. The diffraction angle range of 10-80 • was utilized for this purpose. The Raman spectra of both PETG diaphragms and PETG/BaTiO 3 NPs (30 wt%) nanocomposites diaphragms were obtained using a Raman microscope equipped with an Olympus microscope and a 532 nm laser. The Raman spectra were obtained by scanning 32 times in two seconds.

Contact Angle and Salivary Absorption
The contact angle of PETG diaphragm and PETG/BaTiO 3 NPs composite diaphragm was determined using a contact angle measurement setup with 1 mL of deionized water. The sample is polished with ultra-fine sandpaper prior to testing to achieve a consistent surface roughness. For measuring the salivary absorption rate, test samples were prepared by making PETG diaphragms and PETG/BaTiO 3 NPs composite diaphragms, drying them, and recording their weight. The samples were then placed into artificial saliva at 37 • C and left to soak for different time periods of 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 168 h. After soaking, the samples were quickly dried and weighed separately.

Mechanical Properties
Microhardness tester was used to determine the microhardness of samples. Four groups of samples to be tested were PETG diaphragm and PETG/BaTiO 3 NPs composite diaphragm, and 6 parallel samples were prepared in each group. The microhardness tester was loaded at 200 N for 10 s. After the diamond press head was pressed into the sample, a pit was created on the surface of the sample. The diagonal length of the pit was measured by the eyepiece micrometer. The microhardness values of the samples were calculated according to the load and the diagonal length of the pits. Five points were tested for each sample, and the average value was obtained at the end. In addition, according to the thickness of the test sample GB6672, the thickness of each sample is measured at three points within the range of marking, and the average value is taken. After marking the sample, the sample is clamped, and the longitudinal axis of the sample is required to coincide with the center line of the upper and lower fixtures of the tester, and the tightness is appropriate. The tensile test is conducted at a speed of 5 mm/min.

In Vitro Antibacterial Test
We established an in vitro model using a single-species biofilm to assess the antibacterial effectiveness of PETG/BaTiO 3 NPs composites. We selected Streptococcus mutans as our bacterial strain due to their strong correlation with dental caries and enamel demineralization. The bacterial strain was cultivated on a brain-heart infusion (BHI) agar plate and incubated at 37 • C for 24 h. A single colony was then collected and incubated in fresh BHI while being stirred at 200 rpm overnight. The resulting solution was diluted in fresh BHI to achieve OD 600 = 0.1 (~10 6 cfu/mL) [31].
For the biofilm inhibition experiments, both unpolarized and polarized PETG/BaTiO 3 NPs (0 wt%, 10 wt%, 20 wt%, and 30 wt%) were utilized. To ensure sterility, both unpolarized and polarized samples underwent sterilization by being immersed in 75% ethanol for one hour and subsequently air-dried under UV light exposure inside a biological safety cabinet. Each sample was then individually submerged in 3 mL of the diluted bacterial solution. To facilitate the adherence of bacteria to the surfaces of the material samples, they were statically incubated at 37 • C for a period of two hours. Subsequently, the samples were transferred to a centrifuge tube containing fresh media and placed in an ultrasonic vibrator. During the incubation period, the samples were subjected to cyclic loading at a frequency of 10 Hz. Take apart the samples after coculture and gently washing them with 1 mL of PBS to remove unattached cells. The biofilm present on the sample surface was then collected using a scraper and placed in fresh BHI medium. The resulting solution was subsequently diluted at three different concentrations, and the number of colonies (unit: CFU) was measured by incubating the samples on BHI agar plates at 37 • C for 24 h. To assess the biomass of the surface biofilms, crystal violet (CV) assays were utilized. To conduct biomass measurements, the biofilm on both unpolarized and polarized samples was treated with 1 mL of a 0.1% CV solution at room temperature for a duration of 20 min. The samples were then subjected to a destaining process involving the addition of 30% acetic acid and shaking at room temperature until the biofilm was completely dissolved. The resulting solution was subsequently transferred to a 96-well plate, and the absorbance at 620 nm was measured in order to quantify the amount of biomass present. The potential antibacterial mechanism of piezoelectric charges was investigated by assessing the accumulation of reactive oxygen species (ROS). To this end, the PETG diaphragm, unpolarized 30 wt% PETG/BaTiO 3 NPs composite diaphragm, and polarized 30 wt% PETG/BaTiO 3 NPs composite diaphragm were co-cultured with bacterial solution for a duration of 30 min at 37 • C. Subsequently, 1 mL of nitrotetrazolium blue chloride (NBT) solution, with a concentration of 1 mg/mL, was added to the bacterial solution. This was performed to evaluate the level of ROS accumulation. At the same time, ROS accumulation was evaluated to assess the potential antibacterial mechanism of piezoelectric charges. After the samples (PETG diaphragm, unpolarized 30 wt% PETG/BaTiO 3 NPs composite diaphragm and polarized 30 wt% PETG/BaTiO 3 NPs composite diaphragm) were cocultured with bacterial solution for 30 min at 37 • C, 1 mL of nitrotetrazolium blue chloride (NBT) solution at a concentration of 1 mg/mL was added to the bacterial solution. To halt the reaction, 0.1 mL of hydrochloric acid was added to the solution. The suspension was then centrifuged for 10 min to obtain bacterial cells after the supernatant was removed. Subsequently, dimethyl sulfoxide (DMSO) was utilized to extract the nitrotetrazolium blue chloride (NBT) present inside the cells, which had been reduced. The NBT was then diluted, and the absorbance at 540 nm under UV light was measured to assess intracellular oxidative pressure.

In Vitro Cytotoxicity
Firstly, we conducted a cell toxicity test using the CCK-8 assay. Unpolarized and polarized PETG/BaTiO 3 NPs (30 wt%) composite diaphragms were selected as the experimental group and PETG diaphragms as the control group. Prior to testing, all samples were immersed in 75% alcohol for 24 h. Following this, both sides of the diaphragm were exposed to UV disinfection for 1 h and subsequently washed with a sterile PBS solution. Extract-based cytotoxicity screening assays were conducted on L-929 cells (L-929 cells: Shanghai Cell Bank, Chinese Academy of Sciences, Shanghai, China) to determine the potential toxicity of the materials. Cells were incubated in DMEM (Shanghai, China) at 37 • C in a humidified atmosphere of 5% CO 2 until 80-90% confluence. To perform extract testing according to ISO guidelines for surface area/volume (3 cm 2 /mL), both the unpolarized and polarized PETG/BaTiO 3 NPs (30 wt%) composite diaphragm and the control PETG diaphragm were immersed in serum-free DMEM. For cytotoxicity screening, cells were seeded in 100 µL of culture media until adherent (5000 cells/ well in 96-well plates). The next day, the culture media was replaced with 90 µL of fresh media, and 10 µL of extracts from the materials were added for a treatment time of 24 h, 48 h, and 72 h. After treatment, 10 µL of CCK-8 reagent (purchased from Sigma-Aldrich, Yeasen Biotech Co., Ltd., Shanghai, China) was added to 90 µL of fresh serum-free media. The culture plates were then incubated in an incubator at 37 • C and 5% CO 2 for 2 h, and the absorbance at 450 nm was measured with an enzyme marker. The experiment was conducted independently three times in triplicate. To further evaluate the cytotoxicity of the composite material, we conducted live/dead staining cytotoxicity experiments. Cells were seeded in a 6-well plate (~5000 cells/well) and cultured in a 37 • C, 5% CO 2 incubator until they adhered to the wall. The old culture medium was then removed, and 90 µL of fresh DMEM high-glucose culture medium containing fetal bovine serum was added to each well, along with 10 µL of the extracted liquid culture medium. The cells were then cocultured with the materials for 24 h, 48 h, and 72 h. The culture medium was removed from the 6-well plate, and the cells were thoroughly washed three times with prepared PBS buffer to remove any residual esterase activity. Next, 100 µL of Calcein-AM/PI staining working solution was added to the wells, covering the bottom of the plate, and incubated at 37 • C for 15 min. After discarding the staining solution, the cells were washed three times with PBS to remove the fluorescence reagents. The number of live cells (yellow-green) was detected using an inverted fluorescence microscope with a blue excitation light filter, while the number of dead cells was observed using a green excitation light filter. The effect of the materials on the activity of L-929 cells was analyzed. Figure 2 presents the SEM images of BaTiO 3 NPs and PETG/BaTiO 3 NPs composites. In Figure 2a,b, a random distribution of BaTiO 3 NPs was observed, and they are agglomerated. Figure 2c,d present the SEM images of PETG/BaTiO 3 NPs (30 wt%), and uniformly distributed BaTiO 3 NPs in the PETG matrix was observed. Figure S1 shows a scanning electron microscopy (SEM) image of BaTiO 3 nanoparticles, and the size distribution data of the nanoparticles. It can be seen that the BaTiO 3 nanoparticles generally have an irregular sphere morphology with an average size of~100 nm. Moreover, the element mapping of C, O, Ba, and Ti presented by randomly selected positions in Figure 2e directly provides sufficient evidence for the uniform distribution and existence of BaTiO 3 NPs. In Figure 2f-i, the mappings of C, O, Ba, and Ti elements are shown in red, blue, pink, and yellow, respectively. 10 µL of CCK-8 reagent (purchased from Sigma-Aldrich, Yeasen Biotech Co., Ltd., S hai, China) was added to 90 µL of fresh serum-free media. The culture plates wer incubated in an incubator at 37 °C and 5% CO2 for 2 h, and the absorbance at 450 n measured with an enzyme marker. The experiment was conducted independently times in triplicate.

Characterization
To further evaluate the cytotoxicity of the composite material, we cond live/dead staining cytotoxicity experiments. Cells were seeded in a 6-well plate cells/well) and cultured in a 37 °C, 5% CO2 incubator until they adhered to the wa old culture medium was then removed, and 90 µL of fresh DMEM high-glucose c medium containing fetal bovine serum was added to each well, along with 10 µL extracted liquid culture medium. The cells were then cocultured with the materials h, 48 h, and 72 h. The culture medium was removed from the 6-well plate, and th were thoroughly washed three times with prepared PBS buffer to remove any re esterase activity. Next, 100 µL of Calcein-AM/PI staining working solution was ad the wells, covering the bottom of the plate, and incubated at 37 °C for 15 min. Aft carding the staining solution, the cells were washed three times with PBS to remo fluorescence reagents. The number of live cells (yellow-green) was detected using verted fluorescence microscope with a blue excitation light filter, while the num dead cells was observed using a green excitation light filter. The effect of the mater the activity of L-929 cells was analyzed. Figure 2 presents the SEM images of BaTiO3NPs and PETG/BaTiO3NPs comp In Figure 2a,b, a random distribution of BaTiO3NPs was observed, and they are ag erated. Figure 2c,d present the SEM images of PETG/BaTiO3NPs (30 wt%), and uni distributed BaTiO3NPs in the PETG matrix was observed. Figure S1 shows a sca electron microscopy (SEM) image of BaTiO3 nanoparticles, and the size distributio of the nanoparticles. It can be seen that the BaTiO3 nanoparticles generally have an ular sphere morphology with an average size of ~100 nm. Moreover, the element ma of C, O, Ba, and Ti presented by randomly selected positions in Figure 2e directly pr sufficient evidence for the uniform distribution and existence of BaTiO3NPs. In Fig  i, the mappings of C, O, Ba, and Ti elements are shown in red, blue, pink, and y respectively.     [25]. As the strong peak of BaTiO 3 NPs masks the peak of the polymer PETG, the XRD data of the composites do not show the peaks of PETG. The PETG/BaTiO 3 NPs composites and PETG were also characterized by Raman spectroscopy, and the Raman spectra are shown in Figure S2b, with the distinct bands of PETG observed at 477 cm −1 . No other element-related Raman peaks were observed. However, the distinct bands of PETG/BaTiO 3 NPs composites were observed at 477, 633, 703, and 859 cm −1 . The addition of BaTiO 3 NPs to the PETG resulted in other peaks. The peaks of PETG/BaTiO 3 NPs composites are usually associated with the asymmetric vibration of the octahedra [32].

Contact Angle and Salivary Absorption
As shown in Figure 3a-c, the contact angle of the composite surface increases with the increase in BaTiO 3 NPs content, and when the test lasted for 10 min, the contact angles of each group were 66.2 ± 4.7 • , 70.6 ± 5.5 • , 72.3 ± 3.9 • , and 80.5 ± 7.3 • . Wettability is one of the most important characteristics of solid surfaces, and the wetting phenomenon is the macroscopic result of microscopic properties of solid surface properties, liquid properties, and molecular interactions between solid-liquid interfaces [33]. Additionally, according to the existing studies, bacterial adhesion is firmly related to the surface wettability [34]. Prabhu Suraj Nanduru et al. [35] found that the hydrophobic surface can inhibit biofilm formation. The antibacterial results in this study suggest that a relatively hydrophobic surface is more effective at inhibiting the adhesion and reproduction of bacteria.
posites and PETG were also characterized by Raman spectroscopy, and the Raman spectra are shown in Figure S2b, with the distinct bands of PETG observed at 477 cm −1 . No other element-related Raman peaks were observed. However, the distinct bands of PETG/Ba-TiO3NPs composites were observed at 477, 633, 703, and 859 cm −1 . The addition of Ba-TiO3NPs to the PETG resulted in other peaks. The peaks of PETG/BaTiO3NPs composites are usually associated with the asymmetric vibration of the octahedra [32].

Contact Angle and Salivary Absorption
As shown in Figure 3a-c, the contact angle of the composite surface increases with the increase in BaTiO3NPs content, and when the test lasted for 10 min, the contact angles of each group were 66.2 ± 4.7°, 70.6 ± 5.5°, 72.3 ± 3.9°, and 80.5 ± 7.3°. Wettability is one of the most important characteristics of solid surfaces, and the wetting phenomenon is the macroscopic result of microscopic properties of solid surface properties, liquid properties, and molecular interactions between solid-liquid interfaces [33]. Additionally, according to the existing studies, bacterial adhesion is firmly related to the surface wettability [34]. Prabhu Suraj Nanduru et al. [35] found that the hydrophobic surface can inhibit biofilm formation. The antibacterial results in this study suggest that a relatively hydrophobic surface is more effective at inhibiting the adhesion and reproduction of bacteria. During the process of infiltrating the invisible appliance in the oral saliva environment, saliva absorption is likely to occur, affecting the performance of the invisible appliance. Figure 3d shows the curve of the saliva absorption rate of PETG/BaTiO3NPs (0 wt%, 10 wt%, 20 wt%, and 30 wt%) over time. The results showed that both PETG and the PETG/ BaTiO3NPs composites had a small amount of salivary absorption during the initial immersion in saliva. However, the saliva absorption rate of the composite was lower than that of the PETG, and all sample materials reached a stable state after approximately 3 days of immersion. The final salivary absorption rates of PETG/ BaTiO3NPs (0 wt%, 10 wt%, 20 wt%, and 30 wt%) at 7 days were 0.66%, 0.37%, 0.61%, and 0.52%, respectively. During the process of infiltrating the invisible appliance in the oral saliva environment, saliva absorption is likely to occur, affecting the performance of the invisible appliance. Figure 3d shows the curve of the saliva absorption rate of PETG/BaTiO 3 NPs (0 wt%, 10 wt%, 20 wt%, and 30 wt%) over time. The results showed that both PETG and the PETG/ BaTiO 3 NPs composites had a small amount of salivary absorption during the initial immersion in saliva. However, the saliva absorption rate of the composite was lower than that of the PETG, and all sample materials reached a stable state after approximately 3 days of immersion. The final salivary absorption rates of PETG/ BaTiO 3 NPs (0 wt%, 10 wt%, 20 wt%, and 30 wt%) at 7 days were 0.66%, 0.37%, 0.61%, and 0.52%, respectively.

Mechanical Properties
Statistical results in Figure S4a show that the microhardness of PETG/BaTiO 3 NPs (10 wt%, 20 wt%, and 30 wt%) composites was higher than PETG diaphragm, but the difference was not statistically significant, and the increase in microhardness was not concentration dependent. The results of Figure S4b show that the yield strength and tensile strength of PETG/BaTiO 3 NPs (10 wt%, 20 wt%, and 30 wt%) composites are higher than those of PETG diaphragm.  Figure 4d shows the antibacterial principle of a polarized composite diaphragm. The surface of nanocomposite materials generates an electric charge under micromechanical stimulation, and the electric charge interacts with bacteria to inhibit their growth and adhesion. Figure 4a shows the count results of the antibacterial colony plate of unpolarized and polarized PETG/BaTiO 3 NPs composites (0 wt%, 10 wt%, 20 wt%, and 30 wt%). The results show that the unpolarized composite diaphragm has a certain antibacterial effect, and with the increasing BaTiO 3 NPs content, the antibacterial effect was gradually enhanced, and the antibacterial rate of the composites containing 10 wt%, 20 wt%, and 30 wt% BaTiO 3 NPs could reach 19.35 ± 4.35%, 37.64 ± 5.75%, and 42.90 ± 3.72%, respectively. The results also show that the count results of the bacterial colony plates of polarized PETG/BaTiO 3 NPs (0 wt%, 10 wt%, 20 wt%, and 30 wt%) increased the BaTiO 3 NPs content, the antibacterial effect was gradually enhanced. The antibacterial rate of the polarizing composites containing 10 wt%, 20 wt%, and 30 wt% BaTiO 3 NPs reached 41.65 ± 2.34%, 63.15 ± 4.98%, and 67.39 ± 5.35%, respectively ( Figure S3). The polarized PETG/BaTiO 3 NPs composites showed a better antibacterial effect than the unpolarized ones. In order to further verify this antibacterial result, Figure 4b,c show the quantitative results of the surface biomass of unpolarized and polarized PETG/BaTiO 3 NPs diaphragm (0 wt%, 10 wt%, 20 wt%, and 30 wt%), which are consistent with the results of colony plate counting, indicating that unpolarized BaTiO 3 NPs has certain antibacterial properties. The polarized PETG/ BaTiO 3 NPs composites showed a better antibacterial effect than the unpolarized ones. Aadil Abass Shah et al. [26] also found that BaTiO 3 NPs had good antibacterial activity and antibacterial film performance. In a word, the results showed that the polarized PETG/BaTiO 3 NPs had good bacteriostatic performance under micromechanical stimulation. In order to further explore the mechanism of this antibacterial result, Figure 4e shows ROS concentrations of PETG (control), unpolarized PETG/BaTiO 3 NPs (30 wt%) (unpolarized), and polarized PETG/BaTiO 3 NPs (30 wt%) (polarized) composites after coculture with bacteria under mechanical stimulation. This result indicates that when the PETG/BaTiO 3 NPs complex is subjected to mechanical stimulation after polarization, ROS is produced in S. mutans cells, indicating that mechanical stimulation could activate the charge on the surface of the polarized PETG/BaTiO 3 NPs composites. Previous studies have found that piezoelectric materials have a unique piezoelectric effect. When subjected to external mechanical stress, piezoelectric materials can immediately generate an internal electric field and surface voltage potential [16]. Moreover, studies have found that piezoelectric materials are very sensitive to mechanical vibration, and water flow, muscle movement, and breathing can induce the generation of surface charge [28][29][30]. Therefore, when subjected to slight mechanical stimulation caused by daily oral activities (such as drinking, talking, exercising, etc.) in the mouth, the material surface can be induced to generate electric charges, and the surface molecules of bacterial cells are electrolyzed to produce reactive oxygen species (ROS). Meanwhile, these surface charges also contribute to the production of intracellular ROS. Such as superoxide anion (O 2− ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (•OH), lipid hydroperoxide, and singlet oxygen (O 2 1 ) [33]. The increase in reactive oxygen species exceeds the antioxidant capacity of cells, resulting in oxidative stress, destruction of lipids, proteins, and DNA, and ultimately the death of bacterial cells. The experimental results of this work also indicate that the polarized composites may activate surface charges and produce ROS under micromechanical stimulation, showing excellent antibacterial effects.

Cytotoxicity toward L-929
We studied the antibacterial activity of PETG/BaTiO3NPs composite discs against an oral pathogen (S. mutans), and cytotoxicity testing is essential. In this work, L-929 cells were selected for the cytotoxicity of PETG/BaTiO3NPs (30 wt%) composite discs. Here, we followed ISO guidelines to perform an extract cytotoxicity test on PETG/BaTiO3NPs (30 wt%) composite discs and control discs. The effect of the PETG/BaTiO3NPs composite discs on the viability of L-929 cells was evaluated using the CCK-8 assay. In Figure 5a, the data from CCK-8 assays showed that the viabilities of L-929 cells cocultured with the extract media (media in contact with samples for 24 h at 37 °C and 5% CO2) demonstrated no significant drop in cell viability for the cell line with treatment times of 24 h, 48 h, and 72 h. After 72 h of coculture, the cells in the control, unpolarized, and polarized groups maintained a high viability of 96.02 ± 3.38%, 96.74 ± 1.6%, and 95.67 ± 2.3%, respectively. In Figure 5b, the results of the live-dead staining cell toxicity experiment showed that after 24 h of exposure to each sample in the extraction solution culture medium (cultured at 37

Cytotoxicity toward L-929
We studied the antibacterial activity of PETG/BaTiO 3 NPs composite discs against an oral pathogen (S. mutans), and cytotoxicity testing is essential. In this work, L-929 cells were selected for the cytotoxicity of PETG/BaTiO 3 NPs (30 wt%) composite discs. Here, we followed ISO guidelines to perform an extract cytotoxicity test on PETG/BaTiO 3 NPs (30 wt%) composite discs and control discs. The effect of the PETG/BaTiO 3 NPs composite discs on the viability of L-929 cells was evaluated using the CCK-8 assay. In Figure 5a, the data from CCK-8 assays showed that the viabilities of L-929 cells cocultured with the extract media (media in contact with samples for 24 h at 37 • C and 5% CO 2 ) demonstrated no significant drop in cell viability for the cell line with treatment times of 24 h, 48 h, and 72 h. After 72 h of coculture, the cells in the control, unpolarized, and polarized groups maintained a high viability of 96.02 ± 3.38%, 96.74 ± 1.6%, and 95.67 ± 2.3%, respectively. In Figure 5b, the results of the live-dead staining cell toxicity experiment showed that after 24 h of exposure to each sample in the extraction solution culture medium (cultured at 37 • C and 5% CO 2 ), a large number of live cells (represented by green fluorescence) and a small number of dead cells (represented by red fluorescence) were visible in the fields of view of the PETG (control), polarized PETG/BaTiO 3 NPs (30 wt%, polarized), and unpolarized PETG/BaTiO 3 NPs (30 wt%, unpolarized) groups. However, there was no statistically significant difference in the number of live and dead cells among the three groups. After 48 h of treatment with the extraction solution culture medium, a large number of live cells and a small number of dead cells were visible in the fields of view of the PETG (control), polarized PETG/BaTiO 3 NPs (30 wt%, polarized), and unpolarized PETG/BaTiO 3 NPs (30 wt%, unpolarized) groups, and the number of live and dead cells increased significantly compared to 24 h, but there was still no statistically significant difference among the three groups. After 72 h of treatment with the extraction solution culture medium, a large number of live cells and a small number of dead cells were visible in the fields of view of the PETG (control), polarized PETG/BaTiO 3 NPs (30 wt%, polarized), and unpolarized PETG/BaTiO 3 NPs (30 wt%, unpolarized) groups, and there was no statistically significant difference in the number of live and dead cells among the three groups. Furthermore, previous research has also shown that BaTiO 3 NPs can serve as a platform for biomaterials [27,32]. Similarly, a new adhesive reported by Gang Ge et al. has high biocompatibility, good sealing ability, and excellent adhesion [36]. These materials with good biocompatibility have a good application prospect in bioengineering and medical implants.
view of the PETG (control), polarized PETG/BaTiO3NPs (30 wt%, polarized), and un larized PETG/BaTiO3NPs (30 wt%, unpolarized) groups. However, there was no sta cally significant difference in the number of live and dead cells among the three gro After 48 h of treatment with the extraction solution culture medium, a large numbe live cells and a small number of dead cells were visible in the fields of view of the P (control), polarized PETG/BaTiO3NPs (30 wt%, polarized), and unpolarized PETG TiO3NPs (30 wt%, unpolarized) groups, and the number of live and dead cells incre significantly compared to 24 h, but there was still no statistically significant differ among the three groups. After 72 h of treatment with the extraction solution culture dium, a large number of live cells and a small number of dead cells were visible in fields of view of the PETG (control), polarized PETG/BaTiO3NPs (30 wt%, polarized), unpolarized PETG/BaTiO3NPs (30 wt%, unpolarized) groups, and there was no sta cally significant difference in the number of live and dead cells among the three gro Furthermore, previous research has also shown that BaTiO3NPs can serve as a platf for biomaterials [27,32]. Similarly, a new adhesive reported by Gang Ge et al. has biocompatibility, good sealing ability, and excellent adhesion [36]. These materials w good biocompatibility have a good application prospect in bioengineering and med implants.

Conclusions
In this study, we have introduced piezoelectric nanoparticles (BaTiO 3 ) into PETG and evaluated their potential to inhibit pathogenic biofilm growth at the interface between teeth and orthodontic aligners. Our findings suggest that these composites have significant antibacterial properties, resulting in a marked decrease in biofilm biomass and viable bacteria when compared to the control. In the meantime, we also discovered that the antimicrobial activity of the composites can be affected by changes in filler content and the polarization of the piezoelectric composite. This work demonstrates the potential of piezoelectric BaTiO 3 NPs as a novel material for combating pathogenic bacteria in thermoplastic orthodontic appliances. The composite materials offer several advantages, such as long-term delivery of therapeutic effects, no concern over the increased bacterial resistance to drugs, no leaching of the compounds, and biocompatibility. This study provides new ideas for the development of invisible antibacterial appliances.
Author Contributions: X.A. and Q.S., project development; Y.S., N.Z. and J.L., data collection and manuscript writing; S.S. and J.W., literature review; J.Z., supervision. All authors have contributed to the preparation of the manuscript and agreed to its publication. All authors have read and agreed to the published version of the manuscript.