Sample size calculation and tooth selection
This study protocol was approved by the Ethics Committee (HREC-DCU 2020-008) and Institutional Biosafety Committee (DENT CU-IBC 007/2020).
Currently, no study has been done on the efficacy of LC on remineralization of dentin carious lesions after SDF application. Therefore, the sample size determination was carried out based on the authors' pilot study before this one (5 samples/groups, a total of 4 groups), giving a partial eta squared of 0.304. Using the G*power program, a two-way ANOVA statistical test determined that at least 6 samples/group would be required in this study (α = 0.05, β = 0.20). Due to the small sample size, the sample size was increased to 10 specimens/group to compensate for potential specimen preparation and data analysis errors. Therefore, 40 carious primary molars were used in this study.
Furthermore, we studied the SEM-EDS from the 40 specimens. The sample size calculation for elemental profiling (EDS) was performed based on an ex vivo study by Mei et al. [6], determining 16 carious primary molars; hence, 4 teeth per group were required. Surface morphology (SEM) was studied on 50% of the EDS samples.
The inclusion criterion for both analyzes was primary molars with either occlusal or occluso-proximal natural caries from 4- to 6-year-old children. The exclusion criteria were teeth with restorations, developmental defects, arrested caries, craze lines, or radiographic dental caries involving more than the middle 1/3 of the dentin.
Specimen preparation
Forty primary molars with carious dentin (10 samples/group), extracted according to the patient’s treatment plans, were collected from the dental department at Nongjik hospital in Pattani, Thailand, and stored in 0.9 % sodium chloride at 4ºC until used. Subsequently, the teeth were cut along the buccolingual axis using a slow-speed cutter (Isomet 1000; Buehler Ltd., Lake Bluff, Illinois, USA). The samples were rinsed with deionized water in an ultrasonic bath and dried. Clear nail polish was applied on the specimens except for the dentin lesion. Finally, the specimens were fixed in 1x1x0.9 cm (WxLxH), self-curing acrylic resin blocks, and polished with 600-, 800-, and 1,000-grit silicon carbide papers (Fig. 1).
Radiographic assessment
Using an X-ray alignment system, baseline digital radiographs were taken to generate reproducible projection geometry (Fig. 2). A phosphor digital imaging plate No. 0 (CS 7600; Carestream Health, Rochester, New York, USA) in a protective sleeve was stabilized in a customized film holder. Each dentin block was put over the imaging plate with the buccal aspect of the tooth facing toward the X-ray tube. A 15-mm thick, rectangular, plexiglass plate was placed over the plexiglass supporter, acting as a soft tissue equivalent [13]. A customized device for locking the collimator was used to position the X-ray collimator perpendicular to the tooth and receptor [12]. The radiographs were taken using an intraoral X-ray system (Kodak 2200; Carestream Health, Rochester, New York, USA) with settings of 70 kVp, 7 mA, and 0.119 s exposure time. The same machine and settings were used throughout the experiment by the same investigator (J.K.).
Permuted block randomization
After the digital radiographs were taken, they were saved as Tagged Image File Format with a resolution of 792 dots per inch. Image-Pro Plus software version 7.0 (Media Cybernatics; Rockville, Maryland, USA) was used to evaluate the baseline lesion depth (LD) and mineral density (MD) of the samples from groups divided. The specimens were excluded if the occlusal or occluso-proximal caries involved more than the middle 1/3 of the dentin. LD was determined by dividing the distance from the dentinoenamel junction (DEJ) to the deepest part of dentin lesions by the total depth of the dentin as measured parallelly from the DEJ to dentin-pulp junction. MD was measured by identifying two areas of interest (AOIs) at the same lesion depth on each specimen; the deepest part of the dentin lesion and the adjacent sound dentin as a control. The sizes of the measurement windows were set at 15x15 pixels for the outer 1/3 dentin lesion and normal dentin, and 30x30 pixels for the middle 1/3 dentin lesion to cover a larger area (Fig. 3). The MD measurement was described as mean density/intensity value. The baseline MD of each specimen (MDbaseline) was determined by the difference between the MD at the deepest part of the lesion (MDlesion) and the MD at the adjacent normal dentin (MDcontrol).
The specimens were sterilized with hydrogen peroxide gas and divided into 4 groups according to their baseline LD and MD using permuted block randomization to ensure equal distribution of different LDs and MDs in all groups:
Group 1: SDF applied for 10 s (10SDF)
Group 2: SDF applied for 60 s (60SDF)
Group 3: SDF applied for 10 s + LED light curing 20 s (10SDF+LC)
Group 4: SDF applied for 60 s + LED light curing 20 s (60SDF+LC)
The specimens were immersed in artificial saliva (KCl, MgCl2, CaCl2, K2HPO4, KH2PO4, sodium carboxymethylcellulose, sorbitol, sodium benzoate, and deionized water) of pH ~ 7 for 1 h before SDF application to create the pellicle on the carious surface mimicking oral condition. Each application was performed in a dark, sterile laminar flow cabinet, using a microbrush soaked with premeasured 5 µL of 38% SDF (Saforide; Toyo Seiyaku Kasei Co., Ltd., Osaka, Japan). An LED dental curing light with an output intensity of 520 mW/cm2 and a wavelength range of 450–470 nm (Demi Plus; Kerr, Orange, California, USA) was used in the LC groups. The light intensity was calibrated before curing 20 specimens and the light source contacted the tooth surface when light curing.
Bacterial pH-cycling
Due to the antibacterial property of SDF, the demineralization solution was prepared with two cariogenic bacteria, Streptococcus mutans (S. mutans) ATCC 25175 and Lactobacillus casei (L. casei) IFO 3533 as previously described [14, 15]. They were cultured on tryptic soy agar plates at 37°C, 5% CO2 for 24 h. One colony of each culture was transferred to tryptic soy broth containing 0.5% yeast extract and incubated at 37oC, 5% CO2 for 16 h. The incubated broths of each bacteria were diluted in tryptic soy broth containing 0.5% yeast extract, 2% sucrose, and 1% glucose to achieve optical densities of 0.1 as measured by a spectrophotometer at 540 nm. The cultures were mixed at a 1:1 ratio (≈1.37 x 108 CFU ml-1 of S. mutans and ≈7.8 x 107 CFU ml-1 of L. casei) to make a dentin demineralizing solution with the average pH of 6.31 ± 0.1[16].
The specimens were immersed in the demineralizing solution for 4 h and in artificial saliva without fluoride for 20 h. The specimens were rinsed using deionized water before changing the solution. The specimens were sterilized with hydrogen peroxide gas after bacterial-pH cycling for 7 d [15] and re-assessed for post-treatment MD using DSR. The study flowchart is presented in Fig. 4.
Digital subtraction radiographic analysis
The samples were radiographed post-bacterial pH cycling as described for the post-treatment radiographs. They were superimposed with their baseline radiographs using Image-Pro Plus software to create digital subtraction radiographs (Fig. 5). At baseline, the two recorded AOI windows on the dentin lesion and normal dentin were later used to measure the MD of the same area on the subtraction radiographs. To compare the MD between samples, the MD of the subtraction radiographs at the normal dentin were adjusted (adjusted MDS-control) to an intermediary pixel grey value of 128 [17], and the MD of the subtraction radiograph at the dentin lesion was calculated (adjusted MDS-lesion) using the rule of three. The MDsubtraction was the difference between the adjusted MD of the lesion and the normal dentin. A positive MDsubtraction value represented remineralization, whereas a negative value represented demineralization.
MDsubtraction = adjusted MDS-lesion – adjusted MDS-control
Before the subtraction analysis, J.K. was calibrated for measuring mineral density on the images by an oral and maxillofacial radiologist (S.P.) using a randomized 20% of all the subtraction radiographs to ensure reproducibility and reliability by intraclass correlation coefficient (ICC). Each investigator separately measured the MDsubtraction of all the samples. When there was any difference in the measurement, the two investigators discussed and agreed on the measurement. The mean MDsubtraction in each group was calculated, and this value was referred to as the mean mineral density difference (mMDD).
Statistical analysis
Statistical analysis was performed using SPSS software version 22.0 (IBM, Armonk, New York, USA). The Shapiro-Wilk test was used to assess the distribution of the data. One-way ANOVA was used to compare the baseline LD and MD between the groups. The mMDD between the groups determined by the subtraction method was analyzed using two-way ANOVA, generalized linear models (GLMs) with Bonferroni post hoc test. The significance level was set at p < 0.05.
Surface morphology and elemental profiling
The sterilized post-treatment blocks were taped to an aluminum stand to undergo elemental profiling with SEM-EDS after 7 d of bacterial pH-cycling. Four samples were randomly selected from each group for assessing the post-treatment elemental profile using an INCAEnergy Energy Dispersive X-ray Spectroscopy (EDS) system (Oxford Instruments, High Wycombe., UK) with a scanning electron microscopy (SEM, JSM-IT300, JEOL, Japan) at a 15 kV operating voltage. Two of the four samples were randomly selected to assess their surface morphology by SEM (Quanta 250; FEI Company, Netherlands). Hence, a total of eight specimens were kept dry in a desiccator cabinet before being evaluated. After the elemental profile of the surface lesion was assessed, the specimens were coated by gold sputtering and taped to the aluminum stand to evaluate the surface lesion morphology after treatment at magnifications of 1,000x, 10,000x, and 30,000x with 20kV.