2.1. Materials
Calcium hydroxide, cerium nitrate and hydrogen peroxide were purchased from Sigma (Germany). Mouse Fetal Fibroblast was commercially purchased from the national cell bank of Iran (Tehran, Iran). The extracted teeth were obtained from the Meimanat Clinic, (Tehran, Iran). Other chemicals were selected from the analytical grade/best grade.
2.2. Synthesis of cerium oxide nanozyme
Similar to HAp nanoparticles synthesis, CeO2 nanozymes were created through precipitation approach. In this regard, the diluted ammonia (1M) was added dropwise into cerium nitrate hexahydrate (1 M), until the pH of the mixture reaches to 10. After filtration, the produced precipitates were washed with distilled water and the dried for 2 hours in an oven, 110 ◦C. In the final stage, the resulted powders of CeO2 heat treated about 700 ◦C for 2 hours in a conventional furnace.
2.3. Synthesis of hydroxyapatite nanoparticles
Synthesis of the hydroxyapatite (HAp) nanoparticles conducted through wet precipitation approach, as was described in our previous report, with a calcium/phosphate molar ratio of 1.67 11. In brief, orthophosphoric acid solution (100 ml, 0.6 M) was added dropwise into calcium hydroxide solution (100 ml, 2 M) to create a milky solution under vigorous stirring at ambient condition within 24 hours; pH was adjusted on 11. After that, the free ions were removed through washing of the collected precipitation with distilled water. In the next stage, the obtained cake, first was dried under 100 ◦C in an oven, overnight. In the end, the generated HAp was sintered for 2 hours in the conventional furnace, at 1000 ◦C.
2.4. Characterization of the synthesized nanoparticles
2.4.1. Fourier transform infrared spectroscopy (FTIR)
The recognition of functional groups in CeO2 and HAp NPs were conducted using an FT-IR spectrophotometer (Shimadzu 43000, Shimadzu) viathe KBr disk method. The recorded spectra with resolution of 2 cm-1 were in the range of 4000 to 400 cm-1.
2.4.2. Morphology analysis with SEM
The surface morphology evaluation of the synthesized nanoparticles was executed through a scanning electron microscopy (SEM, MIRA3, TESCAN, USA), at 15 kV. Prior to examination, the nanoparticles were mounted on aluminum stubs and then coated with a thin layer of gold by a sputter apparatus.
2.4.3. Cytotoxicity assay
Mouse Fetal Fibroblast (MFF)is used to evaluate the in vitro toxicity of HAp nanoparticles and CeO2 nanozymes by using MTT assay. The studied cells were cultured in DMEM-high glucose medium supplemented with 10% of fetal bovine serum (FBS) and 1% of penicillin-streptomycin solution in incubator with a humidified atmosphere of 5% CO2 and 37ºC for 24 h before seeding in 96-well culture plates at the density of 5 ×105 per well. Then, the cells were treated at different concentrations (0, 20, 40, 80, 160 and 200 µg/ml) of CeO2 nanoparticles, PBS as solvent of nanoparticles and a blank (empty wells, no cells) to measure the background for tow incubation time of 24 and 48 hours.
At each interval, the medium was removed and the wells were washed twice with PBS, then, each well was replenished with fresh culture medium with 10 µl of MTT (5 mg/ml) The culture plates were incubated for 4 hours at 37 ºC. Subsequently, the culture solution was removed and 100 ml of DMSO was added to each well and plate kept at room temperature in dark for 1 to 2 hours until intracellular purple formazan crystals became visible under the microscope. Subsequently, the absorbance of cells was measured by Microplate reader scanning spectrophotometer Model 680 (Microplate reader, BioTek Synergy™ H1) at 570 nm. The experiments were measured in triplicate.
Relative Cell viability was calculated using the equation below
Where Abssample is the absorbance of the cells incubated with nanoparticles and Abscontrol is the absorbance of the cells incubated with PBS and wells treated with MTT without the presence of any cell as a blank.
2.5. Designing of three-component dental bleaching system
In the current project, a three-component dental bleaching system was designed that not only could dental stains but also could improve the enamel microhardeness. Briefly, the mentioned system includes 3 gels with different performance:
- Gel 1: this gel contains the hydrogen peroxide (15%), carbomer 941 (2% w/w), menthol (0.2% w/w), saccharin (0.1% w/w).
- Gel 2: this gel composed from nanozyme CeO2 (0.2% w/w), carbomer 941 (2% w/w), menthol (0.2% w/w), saccharin (0.1% w/w).
- Gel 3: this gel comprises from hydroxyapatite (0.2% w/w), sodium fluoride (0.5% w/w) carbomer 941 (2% w/w), menthol (0.2% w/w), saccharin (0.1% w/w).
It is noteworthy that the gel 1 as a whitening agent, gel 2 as an activator agent and gel 3 as a supplemental gel were considered.
2.6. Sample preparation and staining procedure
The 18 human extracted teeth were randomly divided into two groups of 9 teeth, which teeth of each group were separately stored into coffee and tea for 72 hours (Figure S2).
Accordance with a standard protocol for artificial staining, the staining solutions were prepared as follow:
- Coffee solution: 12 g coffee in 200 mL boiling water
- Tea solution: 2 g tea in 100 mL boiling water
Then, the prepared teeth were immersed into the filtered solutions over a 3 days-period at room temperature; coffee and tea solutions were renewed daily. After the embedding period, the teeth were washed and placed into distilled water until required time. To evaluate staining, various approaches are reported in the literature 12; in this study, we used a standard Vita shade guide (Vita Zahnfabrik, Bad Säckingen, Germany), which the shade tabs were arranged in a numeric value based on the manufacturer suggestion (B1, A1, B2, D2, A2, C1, C2, D4, A3, D3, B3, A3.5, B4, C3, A4, C4). After artificial staining of teeth, only teeth with shade C4 were selected for bleaching process (14 teeth).
To evaluate the effectiveness of the prepared gel, first, the teeth were brushed and washed with distilled water before applying of bleaching gel onto them to avoid the false-positive results. In the following, the dried teeth mounted separately in plaster, so that the root of each tooth was placed in a plaster cast and the crown of the tooth was visible at the plaster outside. Besides, pumice powder accompanied with brush was applied onto the teeth surface to verify the gel performance (Figure S3).
2.7. Bleaching process
To scrutinize the whitening efficacy of the synthesized gel in comparison with commercial bleaching gel, the prepared teeth were divided into four experimental groups, as follows:
Group 1) the teeth bleached with the prepared bleaching gel
Group 2) the teeth bleached with the prepared bleaching gel accompanied with supplemental gel
Group 3) the teeth bleached with the bleaching gel BOOST (Germany, 35% hydrogen peroxide)
Group 4) the teeth bleached with the bleaching gel FGM (Brazil, 35% hydrogen peroxide)
It should be noted, which in this study for more accurate assessment, the crown thickness, for each tooth, was vertically sectioned in half; after cutting of each tooth into two equal halves of A and B, the teeth were mounted in acrylic. Then, bleaching gels were separately applied to only one half of each tooth and the other half was considered as the control.
2.8. Microhardness assessment
Fourteen days post-bleaching, a microhardness tester (Kentron Microhardness tester, Torsion Ballance Company, Clifton, NJ, USA) was employed to determine
Knoop microhardness (KHN; kg/mm2), at a load of 200 g with the indentation time of 15 seconds.
2.9. Ethical aspects
For the clinical studies, all methods are performed according to the guidelines approved by Kermanshah University of Medical Sciences. The current project has succeeded in receiving the code of ethics with the number 1399.946.56 from Kermanshah University of Medical Sciences. All the ethical issues have been monitored during the project in accordance with the existing protocols.
2.10. Statistical analysis
The reported data in this study, are expressed as mean ± standard deviation (SD). The statistical differences between values was assessed through on way ANOVA with P-value> 0.05.