This study was approved by the Ethics Committee of Biruni University (2019/34-12) and was conducted in accordance with the World Medical Association Declaration of Helsinki.
Dental materials tested in this study
This study examined the cytotoxic effects of a composite (Gradia Direct, GC Europe, Belgium), a HVGIC; Equia Forte, GC Europe, Belgium), and an alkasite restorative material (Cention N, IvoclarVivadent, Liechtenstein) on DPSC (Table 1).
Preparation of samples
In total, 12 specimens of each material were prepared under sterile conditions in a laminar flow chamber (Heal Force, China) and placed into cylindrical Teflon molds (5.0 mm diameter × 2.0 mm height). Thereafter, the lower and upper surfaces of the materials were covered with transparent matrix tape to prevent the formation of an oxygen inhibition layer; the polymerization phase was initiated by placing the Teflon molds between two glass coverslips to remove excess material and prevent air bubble formation. The materials were cured or set in accordance with the manufacturers’ recommendations. An amalgamator device (GC Europe, Belgium) was used to mix materials in capsule form, and a light device (Elipar™ S10; 3M ESPE, St. Paul, MN, USA) was used to polymerize light-cured restorative materials.Thebiomaterials were sterilized using ultraviolet light for 30 minutesbefore thestart of the experiments to prevent bacterial, fungal, or yeast contamination.
Cell culture and experimental design
Human DPSCs (CELPROGEN, 36086-01, USA) were supplied as a cell line and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Sigma Aldrich, St. Louis, MO, USA), 100 U / mL penicillin / streptomycin (Sigma Aldrich, St. Louis, MO, USA), 100 U / mL L-glutamine (Sigma Aldrich, St. Louis, MO, USA), and 100 U / mL sodium pyruvate at 37°C under 5% CO2 humidified air. The third passage DPSCs were detached using a 0.05% trypsin–EDTA solution (Sigma Aldrich, St. Louis, MO, USA) and a monolayer was cultured at a concentration of 5 × 105 in 25 cm flask containing DMEM medium.
A total of 12 samples of each material were divided into three subgroups containing four samples each. Freshly prepared samples were placed in 10 ml DMEM and incubated for 24 and 72 hours to obtain eluates.Cytotoxicity was assessed using MTT and xCELLigenceassays as described below, and DMEM was used as control.
Determination of cell viability using MTT assay
Cytotoxic effects of the three tested materials on cell viability and proliferation were evaluated using the MTTassay (Sigma Aldrich Inc., St. Louis, USA). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) is a stable tetrazolium salt. The production of NAD(P)H in the glycolytic pathways of living cellscan decrease and cause formation offormazan crystals, the concentration of which is directly proportional to the number of living cells at the end of the experiment.
In this study, 3× 104 cells were grown, placed on 96-well plates, and incubated at 37°C for 24 hours. The next day, 100 µl of different concentrations of the medium in which the biomaterials were stored for 24 and 72 hours were applied to the plates. To allow examination of the effects of these substances on cell viability over time, 10 µl of MTT was added 24 hours after the application of the medium and left to incubate for 4 hours at 37°C in the dark. Thereafter, 100 μl of thesolubilization solution was added to each well andthe plate was kept in the incubator overnight. The absorbance (optical density) of the samples was measured using a spectrophotometer (ELISA reader) at 590 nm, and the number of viable cells in the medium was determined.
Proliferation and cytotoxicity assay of DPSC cell lines usingxCELLigence assay
The xCELLigence system(RocheAppliedScience, and ACEA Biosciences)was used to assess the survival of DPSCs upon exposure to variousdental materials over time. Physiologic changes in the cells were identified and measured by the electronic impedance of the sensor electrodes. This real-time monitoring system provides quantitative information on the biological status of cells, including cell number, viability, and morphology;the relative changes in the electrical impedance are displayed by the cell index system.
Next, 200 μL of the cell suspensions were seeded intoa 16-well E-plate (30,000 cells/well; well volume:250 μL; base diameter of well: 5 mm)in a laminar flow cabinet, placed in the incubator at 37°C and 5% CO2, and monitored using the RTCA-DP system at 15-minute time intervals for up to 72 hours with or without dental materials. The control samples received only medium and, in accordance with the xCELLigence technical manual, at least three repeats of each experimental condition was performed to facilitate statistical evaluation.
Data were calculated using the RTCA-DP integrated software of the xCELLigence system and theGraphPad Prism 9.1.1 (GraphPad Software, Inc) program. Data from the proliferation experiments were statistically evaluated using thetwo-way ANOVAtest, and ap-valueof <0.05 was considered statistically significant.