In the current study, all methods and experiments were carried out in accordance with relevant guidelines and regulations.
Synthesis of Zeo\ZnONPs as the photosensitizer
Zeo\ZnONPs was prepared as previously described by Alswata et al.  with a slight modification. Briefly, 5 gr of Zeo powder (Sigma-Aldrich, United Kingdom) was dissolved into 100 mL deionized water to form a solution. 3 g of Zn (NO3)2·4H2O (98.5%; obtained from Merck, Germany) was then added to the suspension. The mixture was stirred under reflux reaction at 80 °C for 3 h. 2 M of sodium hydroxide (NaOH) of 99% (Merck, Germany) was dropped into the solution for precipitation of ZnO onto the Zeo. The suspension was re-stirred until pH 11 was adjusted and the color changed to black. After that, the black product was filtered, washed three times with deionized water, and dried at 80 °C for 12 h. After drying, the final product was obtained after annealing at 450°C for 2 h.
Characterization of Zeo/ZnONPs
Field emission scanning electron microscopy (FESEM)
The surface morphology of Zeo/ZnONPs was studied by FESEM (ZEISS, German) under the voltage of 15 kV
Transmission electron microscope (TEM)
The transmission electron microscope (TEM; Zeiss EM10C) with an accelerating voltage of 80 Kv was used to assess the particle size and size distribution of Zeo\ZnONPs.
Dynamic light scattering (DLS) and Zeta potential analysis
The size distribution profiles of nanometer-sized particles in suspension and Zeta potential measurements were carried out using a MALVERN Zetasizer Ver. 6.01 (Malvern Instruments, UK) at approximately 25 °C.
The absorption spectrum of synthesized Zeo/ZnONPs was carried out by UV-visible spectrophotometer, scanning the absorbance spectra in the range of 350–500 nm wavelength.
Energy dispersive spectroscopy (EDS) mapping was employed to confirm the presence of chemical elements in the structure of Zeo/ZnONPs.
Hemolytic activity of Zeo/ZnONPs
The hemolysis experiment was performed to test the biocompatibility of the synthesized NET according to Pourhajibagher et al. study . In summary, after the collection and addition of sodium citrate to 2 mL of fresh human blood samples obtained from returned unused blood bag in blood bank (Iranian Blood Transfusion Organization), they were washed and centrifuged at 1000 rpm for 10 min to get the red blood cells (RBCs) as a pellet. The pellet was re-suspended with phosphate-buffered saline (PBS) and treated with an equal volume of synthesized Zeo/ZnONPs at different concentrations (0.5, 1, and 2×10–4 g/L). 2% Triton X and PBS buffer were used as the positive and negative controls, respectively. Following incubation of the samples at 25 °C for 30 min, RBCs were collected as a pellet by centrifugation at 2000 rpm for 3 min. The supernatant was utilized to evaluate the absorbance at 540 nm using a microplate reader. The percentage of hemolysis was then calculated using the following equation:
Cytotoxicity evaluation of Zeo/ZnONPs
Cell culture procedure
Human gingival fibroblast cells (HuGu; IBRC C10459) obtained from Iranian Biological Resource Center, Tehran, Iran were seeded in a 96-well plate at a plating density of 4×104 cells/well. The cells were maintained in 100 μL DMEM containing 10% fetal bovine serum (FBS) solution and antibiotics (200 μL/mL penicillin G, 200 μg/mL streptomycin, and 2 μg/mL fungizone) at 37 °C, 5% CO2 95% air, and 100% relative humidity. After 24 h, Zeo\ZnONPs at the concentrations of 0.5, 1, and 2×10–4 g/L were added into the wells and the well-plate was incubated for 24 h at 37 °C, 5% CO2, and 95% air with 100% relative humidity.
Tetrazolium (MTT)-based cytotoxicity assay
The cell viability was evaluated using a modified 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium (MTT) assay. The solutions were removed from each well and 50 μL of MTT reagent (5 mg/mL) was added and the well-plate was incubated for 4 h at 37 °C in the CO2 incubator. The MTT solution was then discarded, 100 μL of dimethyl sulfoxide (DMSO) was added to dissolved the formed purple crystal formazan, and the absorbance was measured at a wavelength of 570 nm using a microplate reader. DMEM prepared without the addition of samples were used as the control. Percentage cell viability was determined using the following equation:
Sulforhodamine B (SRB)-based cytotoxicity assay
The SRB assay was used to investigate cytotoxicity in cells based on the measurement of cellular protein content . After 24 h incubation, HuGu cells were fixed with 10% (wt/vol) trichloroacetic acid for 30 min. The cells were then washed repeatedly with 1% (vol/vol) acetic acid to remove the excess dye. 50 μL of SRB solution (0.04% [wt/vol]; Merck, Germany) was added to each well and the well-plate was incubated at room temperature. After 1 h, the wells were washed four times with 1% (vol/vol) acetic acid to remove unbound dye. Finally, 200 μL of 10 mM Tris base solution (pH 10.5) was used to dissolve the protein-bound dye and OD was determined at a wavelength of 510 nm using a microplate reader. Percentage cell viability was determined similar to the MTT assay.
Microorganisms and growth conditions
Standard strains of Streptococcus mutans ATCC 35668, Lactobacillus acidophilus ATCC 314, and Candida albicans ATCC 10231 obtained from Iranian Biological Resource Center, Tehran, Iran were cultured in Brain heart infusion (BHI) broth (Merck, Germany) and incubated at 37 °C in a shaker incubator at 150 rpm to achieve an optical density (OD) at a wavelength of 600 nm between 0.08–0.13, in the same range as equivalent to that of the 0.5 McFarland standard.
A blue laser (Laser Diode Stabilizer LDS201, Asha beam profile, Iran) in a continuous beam was used as a light source with an output intensity of 150 mW/cm2, 4.2 V, and 0.34 A at the wavelength of 405 ± 10 nm. A photodiode power meter (PMB- 104 power meter, Asha beam profile, Iran) was utilized to measure the output power at the optic tip. All the steps from the operatory protocol were set according to the manufacturer's recommendations.
Enamel slab preparation
Human premolars without visible cracks, enamel irregularities, and WSLs in the buccal and lingual enamel surfaces which extracted for orthodontic purpose were selected. All teeth experiments have been approved by the Ethics Committee of Tehran University of Medical Sciences (Application No. IR.TUMS.MEDICINE.REC.1400.52106), and the need for informed consent was waived by the ethical committee. All the teeth were disinfected and stored in thymol solution (0.1%) at 4 °C before use.
Enamel slabs (approximately 3×3×1 mm) were prepared from mid-labial tooth parts by excision using a water-cooled carborundum disc. For removal of about a 100 μm depth of enamel, the flat surface of slabs was then polished with diamond paste (water-based 0.25-μm diamond particles). Finally, the prepared enamel slabs were ultrasonically cleaned for 15 min.
Specimen preparation for microbial experiments
Prepared enamel slabs were etched with the use of 37% phosphoric acid gel for 30 s, rinsed, and dried for 10 s. A thin layer of a liquid resin primer (Transbond XT primer, 3M Unitek, Orthodontic Products, Monrovia, USA) was brushed on the etched enamel surface and light-cured with the visible light-curing for 20 s. The composite resin was placed onto the titanium orthodontic bracket (Parmis Teb, Isfahan, Iran) and the bracket was bonded on the enamel surface with a consonant force and light-cured for 30 s. The brackets were bonded on the metallic supports made with stainless steel orthodontic wire to suspended the samples for more accumulation of microbial bioﬁlms (Fig. 12 a-c). Before the experiments, enamel slabs with bonded brackets and the metallic supports were sterilized using 4.08 kGy of gamma radiation and stored in a humid atmosphere at 4 °C.
Formation of polymicrobial biofilms on the orthodontic brackets
The orthodontic brackets were placed in the 24-well plates (Ningbo Fuchun, China) with 1.5 mL BHI broth supplemented with 5% sucrose, and inoculated with 100 μL of each bacterial suspension at the concentration of 0.5 McFarland standard. The orthodontic brackets were incubated at 37 °C for 72 h under capnophilic conditions (5% CO2) for the formation of polymicrobial biofilms . After this time, the brackets were rinsed with PBS (pH 7.4) for removing non-adhered microorganisms. Eventually, the stainless-steel orthodontic wires were detached (Fig. 12 d) and the preformed polymicrobial biofilms on the orthodontic bracket were treated according to the study design (Fig. 12 e).
The orthodontic brackets were randomly divided into the experimental groups with 10 samples in each group by Random Allocation Software based on the selected type of blocking as follow:
B. Blue laser
D. Positive control: chlorhexidine (CHX)
E. Negative control: Normal saline
Group A: 100 μL of Zeo/ZnONPs at the concentrations of 0.5, 1, and 2×10–4 g/L was added separately to the orthodontic brackets containing polymicrobial biofilms and the samples were incubated in the dark at room temperature for 5 min.
Group B: Polymicrobial biofilms on the orthodontic brackets were exposed with blue laser irradiation at the wavelength of 405 ± 10 nm for 1 min and output intensity of 150 mW/cm2. The optical ﬁber reached up to 2 cm shorter than working length.
Group C: Polymicrobial biofilms on the orthodontic brackets were treated by Zeo/ZnONPs similar to group A and the samples were then exposed with a blue laser similar to group B.
Group D: 100 μL of 0.2% CHX was added to the orthodontic brackets and the samples were incubated at room temperature for 5 min.
Group E: 100 μL of normal saline was added to the orthodontic brackets and the samples were incubated at room temperature for 5 min.
Detection of intracellular ROS production
The production of ROS by microorganisms involved in polymicrobial biofilms after treatment with different groups was evaluated using 2′-7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma-Aldrich, United Kingdom) as described by Pourhajibagher et al 63. In summary, 100 μL of 5 μM DCFH-DA was added to the mixture (100 μL) of the microorganisms involved in polymicrobial biofilms and shaken at 37 °C for 10 min. The microbial cells were then centrifuged at 5000 rpm for 15 min and the pellets were rinsed with PBS. The cleaned microbial cells were treated based on the experimental design section and the fluorescence emission of DCFH-DA was measured at 535 nm using a microplate reader with an excitation wavelength of 488 nm.
Evaluation of anti-biofilm potency
After treatment of polymicrobial biofilms on the orthodontic brackets according to the experimental design described, each bracket was transferred to a sterile 5 mL eppendorf containing 2 mL of PBS and was sonicated at a frequency of 20 kHz and output power of 5 W for 20 s to remove the remaining polymicrobial biofilms from the surface of the orthodontic bracket. Then, the serial dilutions were prepared, and 10 μL of each dilution was cultured onto BHI agar plates. The plates were incubated at 37 °C for 24 h under capnophilic conditions and log10 CFUs/mL was counted based on the previous study . As well as, the anti-biofilm activities of study groups were assessed by SEM.
Evaluation of anti-metabolic activity
As Coraça-Hubér et al.  study, the metabolic activity was assessed using the XTT (2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay (Roche Applied Science, Indianapolis, IN, US). Following sonication (a frequency of 20 kHz and output power of 5 W for 20 s) of treated orthodontic brackets based on the experimental design section, the microbial suspensions were centrifuged at 2000 rpm for 10 min. The supernatants were removed and microbial cell sediments were dissolved in 150 μL of XTT-menadione-PBS solution in 96-well plates and incubated at 37°C for 3 h. 100 μL of the solution was then transferred to a new 96-well plate and the OD was measured at 492 nm using a microplate reader.
Polymicrobial biofilm induced enamel demineralization/ treatment induced remineralization assays
Prepared enamel slabs with a microhardness ranging from 2.52 to 3.09 gigapascals (GPa) were used for multi-species biofilm induced enamel demineralization and treatments induced remineralization assays. For these purposes, enamel slabs were separately transferred aseptically into a sterile 24-well tissue culture plate containing 500 μL of sterile artificial saliva and incubated 1 h at room temperature (22 ± 2 °C). After that, the saliva was discarded and 1.5 mL of BHI medium containing 1% sucrose as the growth medium were added. Plates were incubated in 5% CO2, for up to 10 days at 37 °C. The growth medium was replaced every 48 h. At the end of the experimental periods, enamel slabs were washed for 10 s in sterile PBS and transferred to microtubes for enamel demineralization using DIAGNOdent Pen reading and microhardness assays. In this study accepted range of microhardness for enamel demineralization was defined as the previous study (2-3 GPa) [56-58], the demineralized samples would be sorted into distill water for enamel remineralization assays using DIAGNOdent Pen reading and microhardness assays.
Evaluation of the treatment effects on enamel remineralization
Enamel demineralized samples distributed randomly into 7 groups (n=10) as follows:
A. Remineralization effect of Zeo/ZnONPs
B. Remineralization effect of blue laser
C. Remineralization effect of aPDT at three concentrations of Zeo/ZnONPs (0.5, 1, and 2×10–4 g/L)
D. Remineralization effect of sodium fluoride (NaF) varnish as treatment-control group
E. Remineralization effect of artificial saliva as the negative control group.
The treatment duration times were one month (t1) and three months (t2). Enamel remineralization was evaluated based on the following assays:
DIAGNOdent Pen reading
The surface change presented on each experimental enamel slabs after treatment was evaluated at baseline (t0) and at the end of treatment duration times (t1 and t2) using Type B probe of DIAGNOdent Pen 2190 (Kavo, Biberach, Germany) as recommended by the manufacturer guideline. In this assay, NaF varnish and artificial saliva also stood for the treatment-control group and the negative control group, respectively. The experiment was performed in triplicate and the mean value was calculated.
Surface microhardness measurement
After reading the treated enamel slabs using DIAGNOdent Pen, the slabs were used to measurement of surface microhardness. Enamel surface microhardness (ESM) was assessed using a digital hardness testing machine (FM-700, Future Tech, Tokyo, Japan) as described previously . The mean ESM measurement value of three indentations at intervals of 0.1 mm which conducted on the surface of each enamel slabs after treatment using a square-based diamond pyramid Vickers’s indenter (at a load of 50 g for 15 s) was calculated at baseline (t0) and the end of treatment duration time.
All assays were done in triplicate and the data were represented as mean values with standard deviation (± SD). The results were statistically evaluated by one-way analysis of variance (ANOVA) and Tukey test. A value of P<0.05 was considered statistically significant.