Preparation of Specimen
The ten specimens were designed in sizes of 10 X 10 X 3 mm, a size capable of use in cell culture (Fig. 2).
For this study, a DLP 3D print was designed using a CAD design program (Exocad; GmbH, Darmstadt, Germany) and then this was converted to a stl file using DLP 3D modeling software (Zenith D ZD200 V1.0.5). UV curing was applied to a temporary restorative resin material with a layer thickness of 120㎛, printing speed 20minutes and a light source of 405㎛ LED using a 3D printer(Zenith D; Dentis, South Korea) and this was printed out to manufacture a specimen. Dental materials that are commercially used to fabricate temporary restorations were used and the materials were MODEL (ZMD-1000B MODEL; Dentis, South Korea), CASTABLE (ZMD-1000B CASTABLE; Dentis, South Korea), CLEAR-SG (ZMD-1000B CLEAR-SG; Dentis, South Korea), TRAY (ZMD-1000B TRAY; Dentis, South Korea), and TEMPORARY (ZMD-1000B TEMPORARY; Dentis, South Korea). Existing temporary resin materials fabricated in self-curing method used were Jet Tooth Shade Acrylic Resin (Lang REF 1430; Lang Dental Mfg, USA), Alike (ALIKE 81; GC America, USA), Milky blue (MILKY BLUE; Nissin Dental, Japan), Tokuso Curefast (CUREGRACE; Tokuyama Dental, Japan), and Uni-Fast Ⅲ (A3; GC Dental Product co, Japan).
In this study, Self-curing resin specimens for each material were fabricated and 2g of the powder of the standard mixing ratio and approximately 1 ml of the liquid that is exclusive for each resin powder were used for the self-curing resin materials. After pouring the liquid into a rubber cup, the powder was added and mixed with a spatula for approximately 10 seconds. The specimens were fabricated when the mixture adopted a dough state, which is free of adhesiveness and allows easy shaping. After the final setting, fabrication of all specimens were completed using the polishing process standard in dental restoration fabrication.
The specimens were polished using a carbide bur, silicone point, and finishing wheel and cleansed. Polishing procedure for cell culture was conducted for both of study groups using SiC paper to eliminate the effect of roughness which was generated during fabrication.
In-vitro Cell Culture
A fibroblast cell line (L929; derivative of strain L, Mus musculus mouse, ATCC, CRL-2593) was used in this experiment. Fibroblast cells were cultured at 37℃ in a humidified incubator containing 5% CO2. The culture medium used was minimum essential medium (α-MEM; Welgene Co., Ltd., Seoul, Korea) containing 10% fetal bovine serum (FBS), 1% penicillin streptomycin, 10 mM β-glycerophosphate (Sigma), and 10 μg/mL ascorbic acid. Cell culture maintenance was performed by washing the cells with Dulbecco’s phosphate-buffered saline (DPBS) followed by cell detachment using trypsin-EDTA. The detached cells were then suspended in culture medium, centrifuged, counted using trypan blue dye, plated in culture plates (10 mL, 3 × 104 cells/mL), and cultured at 37 ℃.
Cell attachment analysis
The surface and edge of the 10 specimens made in each of the 10 materials were trimmed in the shape of a plate of a size sufficient for cell culture. Cell morphology was compared using confocal laser scanning microscopy (CLSM; C1 Plus, Inverted IX81, Olympus, Japan) and scanning electron microscopy (SEM; JSM-6360; JEOL Techniques, Tokyo, Japan). Fibroblast cells with a density of 3 × 104 cells/mL were cultured for 1 day on each of the specimens sterilized with 70% ethanol. To prepare for CLSM observation, cells were then fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X, and blocked with 1% bovine serum albumin. The specimens were then immersed in phalloidin and 4',6-diamidino-2-phenylindole to stain the cellular actin and nuclei, respectively. Prior to SEM characterization, attached cells were rinsed with DPBS and fixed with 2.5% glutaraldehyde for 10 min. Thereafter, sequential dehydration was conducted by 5 min immersions in 75%, 95%, and 100% ethanol, and the specimens were treated with 1,1,1,3,3,3-hexamethyldisilazane for 10 min.
Analysis of Cell Proliferation
For this experiment, cell proliferation was attempted and observed from the specimens fabricated using the 3D printing method and resin specimens fabricated using the self-curing method. The plates were then placed into the wells and fibroblast cells were cultured. After 3 and 5 days of culturing, the specimens were rinsed with DPBS. For the methoxyphenyl tetrazolium salt (MTS) assay, FBS-absent medium containing 10% of MTS was added to each of the specimens and incubated at 37℃ for 2 h. 200 μl of the medium was then placed into a 96-well plate and absorbance was measured at 490 nm using a Micro-reader (Model 550; BioRad, USA).
Statistical Analysis
Tests for normality were performed using a Kolmogorov-Smirnov test and Shapiro-Wilk test. Levene’s test was performed to test homogeneity of variances. After performing Kruskal-Wallis test using non-parametric statistics and statistically significant differences were presented. Pair-wise comparison was performed, and inter-group comparison was done within the confidence interval of 95%. Statistical significance was indicated as *p<.05 and **p<.01, the sample size (n=3). Statistical analyses were performed using IBM SPSS (IBM SPSS 25.0 Inc., Chicago, IL, USA).