This study was confirmed by the Ethics Committee of Istanbul University Faculty of Dentistry, protocol number 2017/47/202.
Preparation of extracts
This study was conducted in accordance with the standards of the International Organization for Standardization (ISO) 10993-5:20097 and 10993-12:2021.
This study aimed to assess the cytotoxicity and biocompatibility characteristics of Sealapex (SybronEndo, Romulus, MI, USA), Apexit Plus (Ivoclar Vivadent, Schaan, Liechtenstein), AH Plus (Dentsply DeTrey, Konstanz, Germany), Totalfill BC Sealer (FKG Dentaire, La-Chaux-de-Fonds, Switzerland), and MTA-Fillapex (Angelus Industria de Produtos Odontologicos S/A, Londrina, PR, Brazil) provides details regarding the manufacturers and compositions of these sealers (Table 1). The endodontic sealers were prepared following the instructions provided by the manufacturers, with each sealer weighing 0.5 g. Apexit Plus and TotalFill BC Sealer were directly applied from their containers, while Sealapex, AH Plus, and MTA-Filapex were mixed in a sterile petri dish under aseptic conditions and then transferred to 6-well plates. Following mixing, sealers were incubated at 37 °C under humidified conditions for 24 h. Subsequently, the materials underwent sterilization using ultraviolet irradiation for 20 min. Specimens were covered with 2.5 mL of cell culture Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% penicillin-streptomycin, 1% gentamicin, and 0.4% amphotericin B, and incubated in the dark for 24 h at 37 °C under humidified conditions. The extraction ratio used was 0.2 g of sealer per 1 mL of the culture medium23. After incubation, the original extracts (conditioned medium) were utilized for further analyses.
Cell culture
Primary HPDLF cells were used in this study. These cells were sourced from previously extracted primary teeth by Islam A., et al. with the Near East University Scientific Research Evaluation Ethics Committee, under decision number YDU/2015/34/243 24. The deciduous teeth used in this study were collected from 10 healthy children aged 6 to 11 years. Informed consent was obtained from a parent and/or legal guardian for the participation of all minors included in this study. All participants were provided with detailed information about the study, and consent was given voluntarily by the parents or legal guardians before the study commenced. The selection criteria ensured that the samples were obtained from non-decayed deciduous teeth, free from abscesses, fistulas, or periapical lesions. After derivation of HPDLF cells, they underwent two passages, and were preserved in the Histology and Embryology Department of Manisa Celal Bayar University in Manisa.
The cells were cultured in DMEM with high glucose (Capricorn Scientific, DMEM-HPXA, Ebsdorfergrund, Germany), containing 10% fetal bovine serum (Capricorn Scientific, FBS-12B, Ebsdorfergrund, Germany), 1% L-glutamine (Biochrom GmbH, K0283, Germany), 1% penicillin/streptomycin (Biosera, XC-A4122, Nuaille, France), 1% gentamicin (Gibco, 15710-064, Grand Island, USA), and 0.4% amphotericin-B (Capricorn Scientific, AMP-B, Ebsdorfergrund, Germany). The cells were maintained under culture conditions of 37 °C and 5% CO2 until they get confluent. Cell viability was assessed using the trypan blue staining protocol in a hemocytometer (Biological Industries, 03-102-1B, Kibbutz Beit-Haemek, Israel).
Characterization of HPDLF cells
Indirect immunoperoxidase staining was used to analyze the distributions of CD34, CD90, and collagen1 for characterization. Initially, cells were fixed in 4% paraformaldehyde (1.04004.0800, Merck) for 30 min, followed by triple washing with phosphate buffer saline (PBS). Subsequently, permeabilization was achieved by incubating the cells with a 0.1% Triton-X-100 (A4975,0100, Applichem) solution on ice for 15 min. A 5-minute treatment with 3% hydrogen peroxide (H2O2, 1.08597.2500, Merck) was performed after PBS washing. The cells were then blocked with blocking solution (TA-125-UB, ThermoFisher) for 1 h at room temperature and subsequently incubated overnight at 4 °C with anti-CD34 (Santa Cruz Biotechnology, sc-74499, Texas, USA), anti-CD90 (Santa Cruz Biotechnology, sc-53456, Texas, USA) and anti-Collagen 1 (Millipore, 08-115, Darmstadt, Germany) antibodies. After washing with PBS, the cells were incubated with a biotinylated rabbit anti-mouse secondary antibody (TP-125-UB, ThermoFisher) for 30 min. Following another PBS wash, streptavidin-hydrogen peroxidase (TP-125-UB, ThermoFisher) was added for 30 min. Immunocytochemical reaction development was initiated by applying diaminobenzidine (DAB, 38611, ScyTek Laboratories) for 5 min. Post-DAB treatment, counterstaining was performed using Mayer’s hematoxylin (Bio-Optica, 05-06002/L Milano, Italy), followed by mounting with a mounting medium (DMM-125, Spring Bioscience). Immunolabeling intensity was assessed by two investigators at various time points using light microscopy (BX40, Olympus). Immunoreactivities were categorized as negative (-), weak (+), moderate (++), and strong (+++).
In vitro cytotoxicity analysis
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test is a standard colorimetric test that measures cellular metabolic activity and provides information about cell viability, proliferation, and cytotoxicity. For the cell viability assay, adherent HPDLF cells in a logarithmic growth phase were seeded (100 mL/well) in 96-well flat-bottom microtiter plates at a concentration of 1 × 103 cells/well. Then, the sealers’ extracts were applied to the cells at 1:0, 1:1, 1:2, 1:4, and 1:8 dilutions (100 µL/well) for 24–72 h using the culture medium as the dilution material. Cells were cultured with 100 µL of culture medium, and it was used as a negative control. All experiments were performed at least in 6 replicates and 3 independent experiments. The MTT solution (Sigma, M5655, Darmstadt, Germany) was prepared with PBS (in 5 mg/mL) just before use. Next, 10 µl of MTT solution and 90 µl of cell culture medium were added to each well and incubated at 37 °C for 4 h. The process was stopped by adding 50 µl of dimethyl sulfoxide (DMSO, Applichem, A3672, 0250, Darmstadt, Germany) to each well. Finally, reaction was detected at 570 nm (Abs570, Biotek ELX800UV) absorbance. The absorbance value readings were normalized to untreated control cultures (100%).
Detection for apoptosis
The optimal concentration of each root canal filling material for the detection of apoptosis cells was determined by assessing concentrations that affect 50% of the cells via the MTT test. Subsequently, based on the dosage and timing outcomes of the MTT assay, extracts of root canal filling materials were administered to HPDLF cells. The culture medium was used as the control group.
The in situ apoptosis detection kit (ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit, Millipore, MA, USA) was used. The cells were fixed with 4% paraformaldehyde for 30 min and washed twice with PBS. They were then incubated for 10 min with a 0.1% Triton X-100 solution on ice for permeabilization, and endogenous peroxidase activity was inhibited with 3% H2O2. The cells were then incubated with equilibration buffer for 10–15 s and TdT-enzyme in a humidified atmosphere at 37 °C for 60 min. They were subsequently put into pre-warmed working strength stop wash buffer at room temperature for 10 min and incubated with anti-digoxigenin conjugate for 45 min. Each step was separated by careful washing in PBS. Next, DAB staining was conducted, followed by counterstaining with Mayer’s hematoxylin. All slides were then evaluated under a light microscope. To quantify the number of TUNEL positive (brown) cells, at least 3 random fields at 20 μm magnification, were counted and reported as percent of TUNEL positive cells.
Statistical analysis
IBM SPSS Statistics 22 (IBM SPSS, Turkey) program was used for statistical analysis. While evaluating the study data, Kruskal Wallis test was used to compare the parameters between groups, and Mann Whitney U test was used to determine the group that caused the difference. Mann Whitney U test was used for the comparison of parameters between two groups. Significance was evaluated at the p < 0.05 level.
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- Source: https://www.nature.com/articles/s41598-024-78344-z