Tooth collection and sample preparation
The extracted tooth samples were obtained from third molars scheduled for removal for preventive reasons at the School and Hospital of Stomatology, Fujian Medical University. Patients aged between 18 and 25 years provided informed consent for the use of their extracted teeth. Exclusion criteria comprised teeth with immature roots, caries, those subjected to root canal treatment, restorations and visible chalky demineralization of enamel, and cracks observed under a stereomicroscope (10×) (ZEISS; Stemi508; Carl Zeiss AG; Oberkochen; GER).
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This study received approval from the local Ethics Committee (Ethics Committee of the School and Hospital of Stomatology, Fujian Medical University, approval number 2021-FJMUSS-062). Also, written informed consent was obtained from participants.
A total of 36 teeth were collected and cleaned on their outer surfaces before being immersed in normal saline (0.9% NaCl; Kelun Co. Ltd.; Chengdu, China) and stored in a refrigerator at 4 °C, for no longer than 1 month post-extraction. The samples were randomly allocated into three groups (n = 12): Group 1 (control group), Group 2 (single radiation 70 Gy group), and Group 3 (fractionated radiation 70 Gy group).
Sample size calculation
The sample size for the study was calculated using G*Power 3.1.9.7 (University of Düsseldorf, GER), based on an a priori analysis of variance (ANOVA) for fixed effects, omnibus, one-way design. The calculation aimed to detect a large effect size (f = 0.65) with an alpha level of 0.05 and a power of 85%. This effect size was derived from previous studies21, which quantified the impact of radiation on dentin microhardness, indicating significant decreases in microhardness compared to control. The analysis indicated that a total sample size of 30 was required to achieve these parameters, assuming equal distribution across the three groups. Considering potential sample loss and to ensure a robust analysis, a total of 36 samples (12 per group) were ultimately collected.
Radiation model of isolated teeth
Following fixation of the isolated tooth with silicone rubber (3M Deutschlan GmbH; MDSA-V02; Neuss; GER), the buccal side of the tooth was positioned upward, and normal saline was applied to soak approximately 1 cm above the tooth surface to simulate the moist oral environment (shown in Fig. 1). The tooth samples were directly exposed to radiation using a linear accelerator (Varian Clinac 23EX; Clina; California; USA) to replicate clinical therapy for head-and-neck cancer patients.
Schematic diagram of radiation mode of isolated teeth.
Radiation approaches: In the single radiation group, a radiation dose of 70 Gy was administered. In the fractionated radiation group, a dose of 2 Gy/day was continuously delivered for 5 days, followed by a 2-day rest period, for a total of 7 weeks. The cumulative radiation dose reached 70 Gy.
Scanning electron microscopy (SEM)
Teeth were sectioned with a water cooled diamond disc (Buehler; IsoMet low speed; Buehler An ITW Company; Chicago; USA) to obtain specimens sized 4 mm × 3 mm × 2 mm. Following ultrasonic cleaning (Supmile; KQ-500DE; Kunshan Jiang, China) for 20 min, the samples were sequentially dehydrated using ethanol gradients of 30%, 50%, 70%, 80%, 90%, and 100%, and subsequently gold-coated using an ion sputtering coater (Gevee; GVC-1000; Beijing KYKY Co. Ltd.; Beijing, China) after air drying. Surface morphology of the specimens was examined using scanning electron microscopy (Quanta 250; FEI; Hillsboro; USA).
Roughness detection
The isolated teeth of each group underwent ultrasonic cleaning with deionized water for 20 min. Following drying, a roughness meter (Kosaka; EF680; Kosaka Laboratory Ltd.; Tokyo; JPN) was utilized to assess the surface roughness of each group. The cut-off value is 0.8 mm, the evaluation length is 3.2 mm, and the measurement speed is 0.1 mm/s. Three positions were randomly measured for each sample, and the average value was recorded as the roughness value of the sample.
Surface microhardness test
Teeth were sectioned along the buccal and lingual aspects using a water cooled diamond disc, fixed with acrylic resin (New century; Shanghai New century Dental Materials Co. Ltd.; Shanghai, China), and the microhardness of enamel on the buccal or lingual sides, dentin at the dentin–enamel junction, and dentin at the cemento-enamel junction was measured post-ultrasonic swaging. The microhardness tester (Buehler; VH1202; Buehler An ITW Company; Chicago; USA) was set with a test force of 200 gf, a loading time of 15 s, and diamond indentation was observed under 600× magnification, adjusted to the center of the field of view. The surface of the sample was clearly imprinted, and the diagonal length of the diamond (d1, d2) was measured using a measuring instrument. The Vickers microhardness value (HV) of each sample was automatically calculated by the instrument. Three points were measured for each sample, approximately 200 μm apart, and the average HV value was recorded as the Surface Microhardness (SMH) value of the sample.
Bacterial culture
Streptococcus. Mutans (S. Mutans) ATCC 25175 strains were cultured on Brain–Heart Infusion (BHI; OXOID; CM1135B; Thermo Fisher; Hampshire; UK) agar (Biofroxx; 8211GR500; NeoFroxx; GER) and incubated at 37 °C in CO2 incubator (Jing Hong; DNP-9082; Shanghai Jing Hong Laboratory Instrument Co. Ltd.; Shanghai, China).
Specimen preparation
Teeth were sectioned using a water cooled diamond disc to obtain specimens sized 4 mm × 3 mm × 2 mm. The back and sides were coated with silicone rubber (3M Deutschlan GmbH; MDSA-V02; Neuss; GER), leaving only the radiated side exposed. After ultrasonic washing (Supmile; KQ-500DE; Kunshan Jiang, China), ethylene oxide sterilization (SAN QIANG MEDICAL; SQ-H40; Henan, China) was performed, and specimens were stored in sterile normal saline. Sterilized dental blocks were placed into 24-well plates (Corning; DWG01261; New York; USA) with one block per well, radiated side facing up. Diluted S. mutans solution (1 × 106 CFU/mL) was inoculated into the wells at 1 mL per well and incubated in a constant temperature incubator at 37 °C for 48 h.
Adhesion test
After removal, teeth were gently washed three times with PBS buffer (Beijing Dingguo Changsheng Biotechnology Co. Ltd.; Beijing, China). Each tooth was placed in a 1.5 mL centrifuge tube (Axygen; MCT-150-C; New York; USA), one sample per tube, and 200 μL PBS was added to each tube. Teeth were vortex-shaken for 2 min with Vortex oscillator (Maxi Mix; Thermo Fisher Scientific; Massachusetts; USA). The bacterial suspension in the centrifuge tube was diluted to 10−4 using the double dilution method. Twenty microliters of the diluted solution were inoculated onto BHI (OXOID; CM1135B; Thermo Fisher; Hampshire; UK) agar (Biofroxx; 8211GR500; NeoFroxx; GER) plates and incubated at 37 °C for 48 h. The number of bacterial colonies was counted, and the concentration of the original bacterial solution was calculated based on the colony count.
Crystal violet staining
After removal, teeth were gently washed three times with PBS buffer. PBS was blotted, and 1 mL of 4% paraformaldehyde was added per well for fixation for 15 min. Then, 1 mL of 0.1% crystal violet (Beyotime; C0121; Beyotime Biotechnology; Shanghai, China) was added for staining for 15 min. The tooth block was removed from the silicone rubber, rinsed with PBS, and dried. Each well received 1 mL of 95% ethanol for decolorization, placed on a shaker (MIX-1500; Shenzhen Huinuo Biotechnology Co. Ltd.; Shenzhen, China) for complete dissolution for 30 min, and 200 μL from each well was transferred to a 96-well plate for optical density measurement at a wavelength of 595 nm with microcoder (SpectraMax iD3; Molecular Devices; Sunnyvale; USA).
Artificial enamel caries formation
The selected teeth were immersed in demineralizing solution (2.2 mM KH2PO4, 2.2 mM CaCl2, 50 mM acetic acid, pH 4.422) at 37 °C for 96 h to produce artificial enamel caries under continuous, incubator shaker (100 rpm; MIULAB; ES-60; MIULAB; Hanzhou, China). The tooth surface was painted with 2 layers of acid-resistant nail varnish, leaving an enamel-exposed window of only 4 mm × 3 mm. Then the demineralization of tooth surface was observed with stereomicroscope (ZEISS; Stemi508; Carl Zeiss AG; Oberkochen; GER), and test Vickers microhardness value (HV) calculate △SMH% = (SMH0 − SMH1)/SMH0 * 100%.
Detection of demineralization by confocal microscopy
The dental plates were stained with 0.1 mmol/L Rhodamine B (Macklin; R817226; Shanghai Macklin Biochemical Technology Co. Ltd.; Shanghai, China) solution, and the fluorescence penetration was observed under laser confocal microscopy (Olympus; FV3000; Olympus Corporation; Tokyo; JPN) (the red part represents the demineralized penetration area). Keep the shooting conditions of the measured images consistent, image-J software (National Institutes of Health; USA) was used to measure the average fluorescence density.
Statistical analysis
Statistical analysis was performed using SPSS 25.0 software (IBM; New York; USA). If the data followed a normal distribution and variances were equal, One-way ANOVA was employed for inter-group comparison, with the LSD test used for multiple comparisons. If the data did not follow a normal distribution or variances were unequal, the non-parametric Kruskal–Wallis H test was used for between-group comparisons. The significance level for all tests was set at bilateral α = 0.05.
