The system used for the experiments consisted of two identical exposure chambers, one for active field and one for sham exposure. The active ELF-EMF contained 1mT rms of a sinusoidal 50-Hz field produced by an electromagnetic generator (Agilent model, Santa Clara, CA, USA) connected with a power amplifier (model 216; NAD Electronics Ltd, London, UK), an oscilloscope (ISO‐TECH model ISR658; Vicenza, Italy) and a Gaussmeter (MG‐3D; Walker Scientific Inc., Worcester, MA, USA). A current flow of 42 mA passed through a 160-turn solenoid coil (22 cm length, 6 cm radius, copper wire diameter 1.25 × 10 − 5 cm), with a 98% of field homogeneity in the central region. The sham chamber was characterized by the dual-winded coils turned in the opposite direction with identical currents to generate the same thermal load (~ 1 W at 1 mT) but without generation of an electromagnetic field. The magnetic field (MF) lines internally follow the longitudinal path of the solenoid, going in the opposite direction externally and forming a loop. The MF flux density was continuously monitored using a Hall-effect probe connected to the gaussmeter. Both exposure systems were computer controlled and analyzed by SW-U801-WIN software for automated and continuous generation monitoring of coil currents. Temperatures were recorded by a two-channel thermometer (TM-925, Lutron, Coopersburg, PA, USA). No significant temperature changes were observed to be associated with the application of the ELF-EMF field (∆T = 0.1 °C) (Patruno et al. 2020). Figure 1 shows the experimental device used for cell exposure.
Preliminary experiments have been conducted to identify the propagation areas where the cell culture is uniformly covered by the generated ELF-EMF. In the central area of the solenoid, where the field lines are parallel to its length, a field uniformity of 98% was achieved. Therefore, the cell cultures were placed within this region of the solenoid.
The exposure systems were placed in two different commercial CO2 incubators (HeraCell 240i, Thermo-Fisher Scientific, Waltham, MA, USA) maintained at 37 ± 0.3 °C in a humidified atmosphere of 5% CO2 for the same times and conditions.
Cell culture establishment
Periodontal ligament tissue biopsies were carried out from five human premolar teeth removed for orthodontic reasons. Selected patients were in a healthy general condition without oral and systemic diseases. Biopsies were collected from the third coronal area of the periodontal ligament using a Gracey curette to remove only the small tissue fragments. Patients enrolled in the present study had signed the informed consent. The present study was approved by the Ethics Committee of G. d’Annunzio University (266/2014). Tissue samples were placed in a petri dish with TheraPEAK™ MSCGM-CD™ Chemically Defined Mesenchymal Stem Cell Medium’s formulation (Lonza, Basel, Switzerland). The culture medium was changed twice a week for 15 days until the hPDLSCs started to migrate spontaneously from tissue fragments. Then, cells were collected by adding 0.1% trypsin-EDTA solution (Lonza, Basel, Switzerland) and subsequently sub-cultured until passages 2 and 3 for the following experiments (Trubiani et al. 2019).
The hPDLSC characterization was performed by evaluation of mesenchymal marker expression and their ability to differentiate toward adipogenic and osteogenic lineages. Stem cell markers were identified by flow cytometry. Briefly, the hPDLSCs at passage 2 were detached by 0.1% EDTA trypsin (Lonza, Basel, Switzerland) and resuspended in PBS (Lonza, Basel, Switzerland). Subsequently, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD45 and CD105 (Ancell Corp., Stillwater, MN, USA), phycoerythrin (PE)-conjugated anti-CD73, FITC-conjugated anti-CD90 and PE-conjugated anti-CD34 (Beckman Coulter, Fullerton, CA, USA). This labeling led to analyzing the cells using the FACSCalibur flow cytometer (Becton-Dickinson, Mountain View, CA, USA) and CellQuestTM software (Becton-Dickinson). To prevent non-specific fluorescence, isotopes were used as controls. At the end of the analysis, the data obtained were analyzed using the specific software FlowJoTM (Becton-Dickinson) (Diomede et al. 2021).
The osteogenic and adipogenic differentiation ability of hPDLSCs was evaluated by both colorimetric assay and gene expression analysis. Concerning colorimetric detection for osteogenic differentiation, the cells were cultured for 28 days with hMSC Osteogenic Differentiation Medium (Lonza, Basel, Switzerland). At the end of 21 days, osteogenic differentiation was assessed by Alizarin Red S solution staining (Sigma-Aldrich, Milan, Italy) able to identify calcium deposits. The adipogenic differentiation was carried out by culturing the hPDLSCs for 28 days with hMSC Adipogenic Differentiation Medium (Lonza, Basel, Switzerland). Subsequently, Oil Red O solution (Sigma-Aldrich, Milan, Italy) was used to show the presence of lipid droplets at the cytoplasmic level. The completed osteogenic and adipogenic differentiation was highlighted by Leica DMIL inverted light microscopy (Leica Microsystem, Milan, Italy). Subsequently, gene expression analysis was performed through RT-PCR set-up using TaqMan Gene Expression Assays and Taq-Man Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA). This analysis allowed to identify some osteogenic and adipogenic markers, confirming that the hPDLSCs were correctly differentiated. Specifically, Runt-related transcription factor-2 (RUNX-2 Hs00231692_m1) and alkaline phosphatase (ALP Hs01029144_m1) were evaluated for osteogenic differentiation, while fatty acid-binding protein 4 (FABP4 Hs01086177_m1) and peroxisomal proliferator-activated γ receptor (PPARγ Hs01115513_m1) were evaluated for adipogenic differentiation. In both cases, beta-2 microglobulin (B2M Hs99999907_m1) (Applied Biosystems) was used to normalize the obtained results (Marconi et al. 2021).
All the experiments were performed in duplicate using hPDLSCs at passage 3 (p3). Cells were cultured in the presence of osteogenic medium (OM) or not (without OM) for two different times of treatment, 10 and 28 days. For each treatment, cells were exposed to ELF-EMF or to sham system for 6 h a day (Fig. 2).
Cell viability and proliferation assay
hPDLSCs were seeded in 96-well flat-bottom plates at a density of 1 × 103 cells/well. After 24 h, cells became confluent, the medium was replaced, and cells were incubated for 6–24 and 48 h in presence of ELF-EMF. Cell viability and proliferation were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay according to the manufacturer's recommendations (Merck KGaA, Darmstadt, Germany). At the end of incubation, MTT reagent was added for 2 h and incubated at 37 °C, and absorbance was assessed at OD590 nm using a Glomax multi-detection reader spectrophotometer (Promega, Milan, Italy). Absorbance data were normalized to the unexposed control group, which was considered 100% of viability.
Osteogenesis process evaluation in the presence of experimental conditions
The evaluation of calcium deposition and extracellular matrix (ECM) mineralization was obtained by Alizarin Red S staining assay performed at 10 and 28 days of culture in all considered conditions. Samples were washed with PBS, fixed in 10% (v/v) formaldehyde (Sigma-Aldrich) for 30 min and then washed twice with abundant distillated (d) H2O prior to the addition of 0.5% Alizarin Red S solution in H2O, pH 4.0, for 1 h at room temperature. After incubation under gentle shaking, cells were washed with dH2O four times for 5 min. For staining quantification, 800 μl 10% (v/v) acetic acid was added to each well. Cells were incubated for 30 min and were scraped from the plate, transferred into a 1.5-ml vial and vortexed for 30 s. The obtained suspension, overlaid with 500 μl mineral oil (Sigma-Aldrich), was heated to 85 °C for 10 min and then transferred to ice for 5 min, carefully avoiding opening of the tubes until fully cooled, and centrifuged at 20,000×g for 15 min. Then, 500 μl supernatant was placed into a new 1.5-ml vial, and 200 μl 10% (v/v) ammonium hydroxide was added (pH 4.1–4.5); 150 μl of the supernatant obtained from the cultures were read in triplicate at 405 nm by a spectrophotometer (Synergy HT, BioTek, Bad Friedrichshall, Germany).
hPDLSCs were cultured in a chamber slide with eight wells (IBIDI, Gräfelfing, Germany) and maintained in all considered conditions. At 10 days, the samples were fixed for 30 min at room temperature with 4% paraformaldehyde in PBS, pH 7.4, and permeabilized with 0.1% Triton-X100 in PBS for 10 min, followed by blocking with 5% skimmed milk in PBS for 30 min. Samples were incubated with primary monoclonal antibody, anti-RUNX-2 (1:200, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), followed by anti-rabbit Alexa Fluor 488 (Molecular Probes, Life Technologies, Monza, MI, Italy). All samples were incubated with Alexa Fluor 568 phalloidin red fluorescence conjugate (1:400) as a marker of the cytoskeleton actin and with TO-PRO staining to highlight cellular nuclei. Samples were observed using a confocal laser scanning microscopy system (Zeiss LSM800, Zeiss, Jena, Germany), equipped with a Plan Neofluar oil-immersion objective (40 ×/1.3 NA). Images were collected using an argon laser beam with excitation lines at 488 nm and a helium-neon source at 543 and 633 nm. Line profile and co-localization analyses were performed offline on images acquired at a resolution of 1024 × 1024 pixel at 12 bit (4096 gray values) using Zen2010 software (Zeiss). The relative fluorescence intensities of RUNX-2 were quantified using NIS-Elements AR imaging software (Nikon). For the counting statistics of immunofluorescence-positive nuclei for ERK and pERK, ten views (100×) were randomly chosen in each experimental group and analyzed using NIS-Elements AR imaging software (Nikon). All the experiments were repeated at least three times. Data are presented as the mean and standard error of the mean (mean ± SEM). The comparison analysis of different groups was done using a one-way analysis of variance followed by a post hoc Bonferroni evaluation using GraphPad Prism5. Differences were termed statistically significant at p < 0.05.
RNA isolation and real-time PCR analysis
hPDLSCs were seeded at 8 × 104 cells/well in six-well plates. Reaching 80% of confluence, culture plates were divided into two groups: (1) OM or (2) no OM for 10 and 28 days. Both plates were exposed or not to ELF-EMF. Cells in each condition were maintained in culture for 28 days, and, after harvesting, total RNA was isolated using the classic phenol–chloroform method. Total RNA was quantified at 260 nm using NanoDrop 2000 ultraviolet-visible (UV–Vis) spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA); 1 μg of RNA was reverse transcribed to cDNA for 15 min at 42 °C and 3 min at 90 °C to inactivate Quantiscript Reverse Transcriptase, according to the protocol of QuantiTect-Reverse Transcription Kit (Qiagen, Hilden, DE). Real-time PCR was performed in a Mastercycler ep realplex real-time PCR system (Eppendorf, Hamburg, Germany), using PrimeTime Gene Expression Master Mix (IDT, Coralville, IA) to evaluate the gene expression of RUNX-2 and COL1A1, using GAPDH as housekeeping gene. The amplification program consisted of a pre-incubation step for cDNA denaturation (3 min 95 °C), followed by 40 cycles consisting of a denaturation step (0.05 s 95 °C) and an annealing step (0.30 s 60 °C). RT-PCR was performed in two independent experiments, and triplicate determinations were carried out for each sample. Expression levels for each gene were performed according to the 2−ΔΔCt method.
Western blot analysis
The proteins were collected after 6 h, 24 h and 48 h of culture under sham and ELF-EMF conditions. Moreover, at the end of the osteogenic differentiation, the proteins were isolated from the cellular cultures following the procedure: after 10 and 28 days of culture OM/ELF-EMF, OM/sham, without OM/ ELF-EMF and without OM/sham, the hPDLSCs were lysed with RIPA buffer (Thermo Fisher Scientific) and collected by scraping. At this point, the lysate was centrifugated at 16,000×g × 15 min at 4 °C allowing the elimination of debris. The obtained lysate was quantized by BioSpectometer (Eppendorf, Germany) and a concentration of 50 µg was used for sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis (Bio-Rad V3 Western Workflow™, Milan, Italy). The transfer procedure was realized with semidry technique on PVDF membranes (Immobilon‐P transfer membrane; Millipore). The saturation was performed incubating the membranes with 1× PBS, 5% milk and 0.1% Tween for 2 h at RT. The primary antibodies anti-Ki-67 (1:500 mouse; BioGenex, Fremont, CA, USA), anti-COL1A1 (1:1000 rabbit; Invitrogen, MA, USA), anti-RUNX-2 (1:500 mouse; Santa Cruz Biotechnology,CA, USA) and anti-OPN (1:500 mouse; Santa Cruz Biotechnology) were inserted overnight at 4 °C. Respectively, the peroxidase-conjugated anti-mouse secondary antibody and anti-rabbit secondary antibody (1:5000; Bethyl Laboratories, Montgomery, AL, USA) were finally incubated at RT for 1 h. Eventually, the membranes were read using the Alliance 2.7 system (Uvitec Ltd, Cambridge, UK), which allowed identifying and quantifying the bands obtained. The data achieved were normalized with the protein expression of β-actin (1:750 mouse, Santa Cruz Biotechnology).
All results were expressed as mean ± standard deviation. For repeated measures, ANOVA was performed to compare means between groups.
The ‘fold change’ of gene expression levels was calculated with the 2−ΔΔCt method. The hypothesis that the fold change between exposed and not exposed was equal to 1 was tested with the Student’s t-test for unpaired data.