MC3T3-E1 rat pre-osteoblastic cell lines obtained from the Military Medical University’s Orthopedic Laboratory were cultured in alpha-modified Eagle medium (α-MEM, Hyclone, USA) containing 10% fetal bovine serum (FBS; Gicbo). , USA) and an antibiotic and an antibiotic 1% solution. Cells were incubated at 37 °C, 5% CO2the atmosphere O2. In the experiments, cells were seeded into six dishes at a density of 300,000 cells per well, and the media volume was kept at 3 ml. Media change was performed the next day after seeding before electrical stimulation was applied.
DC Stable Electrostimulation System Setup
The DC stimulation chamber was used in this study based on the original design described by Mobini and Leppik et al.13. The current output terminal was powered by six 1.5V batteries. The circuit integrates an adjustable buck regulator power supply (LM2596S) to maintain a constant output voltage and increase or decrease the resistance component through the required current. The current output terminal connects to the wiring terminal installed on the side of the 6-slot cover. The center line of each hole was drilled on the 6-hole plate cover so that the distance between the two holes was 30 mm. A titanium alloy wire (0.8 mm in diameter) was used as the implanted electrode, and the effective length of the implanted electrode was 20 mm. The electrode was parallel to a silver-plated copper cable, and the loose end of the copper wire was attached to the end of the wires on the left side of the 6-slot panel cover. The six-hole plate was processed to impregnate the cell for communication between parallel holes. The device schematic and physical schematics are shown in Figure 1.
MC3T3-E1 cells were stimulated with direct current for 24 and 48 h. A control group of cultured cells was also maintained without electrical stimulation. Cells in the anode and cathode and cells of the control group were collected separately; Relevant cellular suspensions were mixed with 0.4% Trypan blue (catalog No. 15250061; Gicbo, USA) in a 9:1 ratio, and 20 μl of the suspensions were added to a Countstar counting plate and observed under a microscope. Dead cells were stained blue, while live cells were colorless and transparent, and the number of live cells was counted within 3 min.
After 24 and 48 h of electrical stimulation, cells of each group were collected separately and the cell concentration was adjusted to 1 × 106/ Ml. According to the procedure of the Apoptosis Assay Kit (BD Biosciences, USA), cells were suspended in 5 ml flow tubes; 5 μL of fluorescein isothiocyanate (FITC)-Annexin V and 5 μL of propidium iodide (PI) staining solution were added to the flow tubes; The tubes were gently mixed and incubated for 15 min at room temperature and protected from light; 500 µL of 1× ligation solution was added. The FITC fluorescence level in the FITC channel and the PI fluorescence level in the PI channel were detected instantly using the method recommended in the kit. Data analysis was performed using FlowJo VX. Each experiment was repeated three times.
Real time quantitative PCR
After 7 days of cell culture, cells in the positive and negative electrodes as well as cells in the control group were collected. Total RNA of cells was extracted by SimplyP Total RNA Extraction Kit and reverse transcribed into cDNA. The expression levels of osteocalcin, osterix and osteonectin were assessed by quantitative real-time PCR. Experiments were repeated three times, with GAPDH as the internal reference, and the relative expression levels of osteocalcin, osterix and osteonectin were calculated by the 2-Ct method. The primer sequences used were as follows (Table 1).
Animals and Fractions Model
A total of 12 New Zealand white rabbits (male, weight, 2.5–3 kg) used in the experiment were provided by a commercial vendor (Animal Certificate Number: SCXK (Yu) 2021-0010). This study was conducted with the approval of the Experimental Animal Ethics Committee of the Children’s Hospital of Chongqing Medical University, China (CHCMU-IACU20210114003), and was carried out according to ARRIVE guidelines. All methods were performed in accordance with the Animals (Scientific Procedures) Amended Act 1986 in the United Kingdom and Directive 2010/63/EU in Europe. All animals were reared at room temperature (20°C to 25°C), 40%-60% RH, and 12–12 h daily cycle. Food was provided uniformly by professional breeders, and water was served advertising. Before the operation, rabbits were fasted for food and water, and 3% pentobarbital sodium was injected along the vein of the ear margin at a dose of 1 ml/kg for sedation. Rabbits were maintained supine and immobilized on the operating table; The right hind foot is revealed. A sterile towel was applied and the surgical site was disinfected with iodophor three times. The skin was cut longitudinally between the first and third phalanges with a scalpel, and the muscle and fascia were separated to expose the phalanges. The phalanges were cut with a bone saw to tighten them completely; Excess bone fragments were cleaned up; The fracture site was fixed with a 0.8 mm Kirschner wire; The wound was sutured layer by layer with 3-0 nylon sutures; The rabbit double fracture model was completed by external plaster fixation (Fig. 2). For 3 days after the operation, all rabbits were injected intramuscularly with penicillin (80,000 units) once daily to prevent infection, and the wound was monitored for signs of infection. The animals were randomly divided into experimental and control groups, with six rabbits in each group. Rabbits in the experimental group underwent DC stimulation. Thus, immediately after modeling, the DC negative electrode was connected to the first Kirchner phalanx wire, the positive electrode was connected to the third Kirchner phalanx wire, and continuous stimulation was applied using 10-µA DC. The DC supply device used in the experiment was the same as in the cell experiment.
The fracture site was photographed in all rabbits at one and a half days and one month after modeling to monitor fracture healing, the position of the fissure pins, and to assess fracture line changes and bone scab formation after fracture.
In the first month, the animals were euthanized. Toe bones were dissected and fixed in 4% paraformaldehyde and then decalcified in JYBL-I decalcification solution (catelog #G2470; Solarbio). Decalcified bones were paraffin-embedded and cut into 5 μm slices, followed by HE staining and Goldner’s trichrome staining.
The results were statistically analyzed using SPSS (version 20.0) and GraphPad Prism V.8.00. Data were expressed as mean and standard deviation and analyzed using a t-test and one-way analysis of variance (ANOVA), excluding histological data. The stained sections were photographed and analyzed for histology using Aperio ImageScope v184.108.40.20629 (Leica, USA). Statistical significance was defined by p < 0.05.
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