Experimental groups
A light-curable resin matrix was made with 1:1 ratio of bisphenol A glycerolate dimethacrylate (Sigma-Aldrich, MI, USA) and triethylene glycol dimethacrylate (Sigma-Aldrich, MI, USA), and 0.3 wt.% of camphorquinone (Sigma-Aldrich, MI, USA) by mixing with ultra-sonication for 20 min. To this mixture, 0.6 wt.% of 2-(dimethylamino) ethyl methacrylate (Sigma-Aldrich, MI, USA) was added and stirred for 1 day without light disturbance. For the conventional glass filler, silane-treated filler powders (NanoFine® NF180, Schott, Landshut, Germany) composed of SiO2, BaO, B2O3, and Al2O3 was chosen. To prepare the hCS powder, a protocol from a previous study was used [11]. To mix the resin matrix and two types of fillers, hCS and silanized glass powder were added to the light-curable resin matrix and blended with a speed mixer (DAC 150.1 FVZ Speed mixer, Hauschild, Germany) at 3500 rpm for 5 min in a dark environment. Experimental groups were prepared using above method (Table 1), and Filtek Z250 (3M ESPE, St. Paul, MN, USA) was used as a commercial control (CC).
Table 1. Composition of the experimental groups (Wt.%) | |
Group | 0 wt.% hCS | 17.5 wt.% hCS | 35.0 wt.% hCS | 52.5 wt.% hCS |
Resin matrix | 30.0 | 30.0 | 30.0 | 30.0 |
Hydrated calcium silicate | 0.0 | 17.5 | 35.0 | 52.5 |
Silane-treated filler powders | 70.0 | 52.5 | 35.0 | 17.5 |
Depth Of Cure
According to ISO 4049 (2019), a metallic mould 4 mm in dimeter and 6 mm in height was positioned on a microscopic slide glass that was covered with a polyester film. The mould was slightly overfilled with the experimental material, without air bubbles, and then placed with a polyester film. On the top of the mould, a slide glass was placed and pressed to displace the excess material, and removed from the top of the mould. Then, the top surface was cured using a light-emitting diode curing unit (Elipar S10, 3M ESPE Co., Seefeld, Germany) for 20 s. Cured experimental material was separated from the mould immediately after irradiation, and the uncured materials were removed using a spatula. The height of the cured materials was examined at four random areas with a micrometer (Mitutoyo, Tokyo, Japan) and the value was divided by two.
Flexural Strength
The three-point flexural strength test was conducted according to ISO 4049 (2019). A stainless steel mould with dimensions of 25 mm × 2 mm × 2 mm was placed on a slide glass covered with polyester film, and the experimental materials were dispensed in it. Another slide glass covered with polyester film was positioned on the top of the mould, and the mould was clamped and pressed to remove excess material. The experimental resin was immediately irradiated using a light-emitting diode curing unit for 30 s with overlapping areas. The ten polymerized bar-shaped specimens from each experimental group were positioned in distilled water at 37 ± 1°C for 1 d before performing the three-point flexural strength test. The flexural strength value was obtained using a universal testing machine (Instron 5942, Instron, Norwood, MA, USA) with a crosshead speed of 1.0 mm/min until the specimens fractured. The maximum load (F) was observed, and the three-point flexural strength (σ) was obtained in megapascals using the formula: σ = 3Fl/2bh2, where F is the maximum load in Newtons, l is the 20 mm support distance, b is the specimen width, and h is the height of specimen.
Water Sorption And Water Solubility
Based on ISO 4049 (2019), six polymerized disk-shaped specimens from each group with a 15.0 ± 0.1 mm diameter and 1.0 ± 0.1 mm height were prepared. The diameter and height of specimens were measured to get volume (V), and the constant mass (m1) was obtained to an accuracy of 0.1 mg using a digital balance (XS105, Mettler Toledo AG, Greifensee, Switzerland). Then, the specimens were individually stored in 10 mL of distilled water at 37 ± 1°C. After 7 d, the disk specimen was taken from distilled water, and the surface water was blotted away, then weighed (m2) 60 sec after removal from the water. The specimen was then moved to a desiccator and weighed daily until a constant mass (m3) was observed. The water sorption (Wsp, µg/mm3) and water solubility (Wsl, µg/mm3) values were obtained using the formulas: Wsp = (m2-m3)/V and Wsl = (m1-m3)/V.
Ion Release
Eighteen polymerized specimens (25 mm × 2 mm × 2 mm) from each experimental group were prepared. To extract the ions from the specimen, a lactic acid solution was used after adjusting the pH to 4.0 by adding DL-Lactic acid (Sigma-Aldrich, Steinheim, Germany) to distilled water. Three specimens were then stored in a DL-Lactic acid solution at a ratio of 0.14 cm3/1 mL [12] for 1 h, 24 h, 7 d, 14 d, 30 d, 60 d, and 90 d, and the solution was exchanged at each point. After removing the specimens from the lactic acid solution at each time point, the storage solution was collected to conduct inductively coupled plasma-mass spectrometry (ICP-MS, Agilent 7900, Stockport, UK) followed by preconditioning with hydrofluoric acid to detect the calcium and silicon ions released from the specimens.
Antibacterial Effect
Strain cultivation and bacterial suspension preparation
Streptococcus mutans (S. mutans, KCTC No. 5365, KCTC, Jeollabuk-do, Korea) was incubated in the brain heart infusion broth (BHI, Difco, Sparks, MD, USA) at 37 ± 1°C for 2 days. Using an ELISA reader (Epoch, BioTek, Winooski, VT, USA), the S. mutans suspension was adjusted with culture media to establish an optical density value within the range of 0.4–0.6 at 600 nm.
Bacterial Attachment To The Specimen
Eleven polymerized disk-shaped specimens (10.0 ± 0.1 mm diameter and 1.0 ± 0.1 mm height) from each experimental group were prepared. The sterilized disk specimen was positioned on a 24-well plate (SPL Life Science, Gyeonggi-do, Korea), and 100 µL of the S. mutans suspension was dropped onto the specimen surface. The plates containing specimens were then stored at 37 ± 1°C. After 1 day, to remove loosely attached bacteria, the specimen was gently washed with distilled water for 10 s. The disk specimen exposed to S. mutans was then positioned in the new 24-well cell culture plate facing up.
Evaluation Of Bacteria Attachment On The Specimen
To visualize the attached S. mutans on the experimental resin surface after performing the process described in sub-section “Bacterial attachment to the specimen”, the specimen was fixed with Karnovsky’s fixative solution composed of 2% paraformaldehyde, 2% glutaraldehyde, and 0.1 M phosphate buffer solution (PBS) for 24 h. Specimen was then rinsed with 0.1 M PBS for 30 min and fixed with osmium tetroxide for 2 h. After then, the specimen was dehydrated by serial dilution using ethanol (50–100%) and dried for 2 h with a critical point dryer (Leica EM CPD300, Leica, Wien, Austria). Specimen was then coated with platinum using an ion sputter (Leica EM ACE600, Leica, Wien, Austria) and analyzed with a field emission scanning electron microscope (FE-SEM, Merlin, Carl Zeiss, Oberkochen, Germany) at an accelerating voltage of 15.0 kV.
Bacteria Viability By Live/dead Staining
After performing the process described in sub-section “Bacterial attachment to the specimen”, three specimens from each group were stained using a live/dead bacterial viability kit (Molecular Probes, Eugene, OR, USA). Equal volumes of propidium iodide and Syto 9 dye, which stain live and dead bacteria, respectively, were thoroughly mixed. Then, 1 mL of PBS was added to 3 µL of the propidium iodide and Syto 9 dye mixture in which each specimen attached with S. mutans was immersed for 20 min at 37 ± 1°C in the dark environment. The stained S. mutans on the experimental resin surface was observed using a confocal laser microscope (LSM700, Carl Zeiss, Thornwood, NY, USA). Live bacteria appear green, whereas dead bacteria appear red.
Antibacterial Activity By Colony Forming Units (Cfus)
After performing the process described in sub-section “Bacterial attachment to the specimen”, six specimens of each group was respectively placed in 1000 µL of culture media and sonicated for 10 min. After removing disk specimens, obtained bacterial suspension was then diluted, spread onto the BHI agar plates, and stored at 37 ± 1°C for 2 days before CFUs counting. The relative survival rate was calculated with CFUs using the equation: relative survival rate of S. mutans (%) = (1 - (CFUs of remaining bacteria on the specimen / CFUs of bacterial suspension applied on the specimen)) ⋅ 100.
Apatite Formation
Preparation of the samples
Twenty-four polymerized disk-shaped specimens (10.0 ± 0.1 mm diameter and 1.0 ± 0.1 mm height) from each experimental group were prepared. Artificial saliva was made by adding 0.4411 g CaCl2·2H2O (Sigma-Aldrich, Steinheim, Germany) and 0.245 g of KH2PO4 (Sigma-Aldrich, Steinheim, Germany) in 800 mL of distilled water. To this mixture, the pH was adjusted to 7.0 by dropping 0.5 M KOH (Duksan Reagents, Ansan, Korea) [13]. Each disk-shaped specimen was immersed in 10 mL of artificial saliva solution and kept at 37 ± 1℃ for 15 d, 30 d, 60 d, and 90 d. The artificial saliva was refreshed every 15 d.
Morphological And Chemical Analysis
Two disk-shaped specimens were randomly selected from the experimental groups at each time period, cross-sectioned, and polished using a polishing machine (Buehler, Lake Bluff, IL, USA) with a 1200-grit SiC abrasive paper (Deerfos, Incheon, Korea). The morphology and chemical components of the top surface and the cross section were analyzed using scanning electron microscopy-energy-dispersive X-ray spectrometry (SEM-EDS, Merlin, Carl Zeiss, Oberkochen, Germany) at a magnification of 500⋅ under an acceleration voltage of 15.0 kV.
Raman Spectroscopy Analysis
Two disk-shaped specimens were randomly selected from the experimental groups at each time period. The precipitates formed on the specimens were carefully harvested using a plastic spatula. The collected precipitates were positioned on a slide glass, and the Raman spectra were observed with a Raman spectrometer (LabRam Aramis, Horriba Jobin Yvon, France) under 50⋅, opening of 50 µm, laser wavelength of 532 nm, and Raman shift from 200 cm− 1 to 1400 cm− 1 [13, 14].
Xrd Analysis
Two disk-shaped specimens were randomly selected from the experimental groups at each time period. The precipitates formed on the specimen surface were obtained as previously described in sub-section “Raman spectroscopy measurement”. The peaks in the diffraction spectrum of the precipitates were observed by X-ray diffraction (XRD; Ultima IV, Rigaku, Japan) using Cu Kα radiation in the 2 theta range from 20 to 60°, with a step size of 0.02.
Statistical analysis
Acquired data on the depth of cure, three-point flexural strength, water sorption, solubility, and relative survival rate of S. mutans at the different hCS contents were analyzed with a one-way ANOVA test (SPSS 25, IBM Co., Armonk, NY, USA) followed by a Tukey’s post hoc test (p = 0.05).