EMF device
The EMF generator, designed by the Naval Engineering University (Wuhan, China), consists of a waveform generator, an amplifier, Helmholtz coils, and an oscilloscope. This device is capable of producing SEMFs with adjustable frequencies ranging from 1 to 100 Hz and magnetic field intensities from 0 to 4 mT. During operation, the electromagnetic signals generated by the waveform generator are amplified before being output to the Helmholtz coils, which are wound with 0.8-mm coated copper wire. The Helmholtz coils are placed inside an incubator maintained at a constant temperature of 37 °C and 5% CO2, ensuring optimal experimental conditions for research and testing purposes.
BMSCs harvest and culture
BMSCs were harvested from 8-week-old male Sprague Dawley (SD) rats. The experimental procedures have been reviewed and approved by Ethics Committee of Experimental Animal Center of Huazhong University of Science and Technology (No. 3091, Wuhan, China). We have complied with all relevant ethical regulations for animal use. After the rats were sacrificed, their femurs and tibiae were removed under sterile conditions and immersed in sterile phosphate-buffered saline (PBS; Boster Bio Tech, Wuhan, China). The epiphyses were then removed, and each bone marrow cavity was thoroughly washed with Dulbecco’s modified Eagle medium (DMEM)/F12 culture medium containing 10% fetal bovine serum (FBS; Gibco, Grand Island, USA), 100 units/mL penicillin and 100 μg/mL streptomycin (Gibco). The cells were resuspended in the above complete medium and cultured in an incubator (37 °C, 5% CO2, and 100% humidity) for subsequent experiments. The culture medium was changed every other day.
BMSCs passage and stimulation
When the BMSCs reached about 90% confluence, they were detached using 0.25% Trypsin/EDTA (Gibco, Grand Island, USA) and passaged at a ratio of 1:3. The cells from passage 3 were counted, and then 2.5–3 × 105 cells were seeded into 6-well plate. After 24 h, the culture medium was replaced with osteogenic medium (OM; Cyagen, Shanghai, China). The specific arrangement of SEMF exposure on cells is shown in Fig. 1a. Briefly, the cells were individually exposed to SEMFs stimulation at a fixed frequency (15 Hz) and different intensities (0.4 mT, 0.7 mT, and 1 mT) for 7 or 14 days, 4 h per day. On the 7th day, RNA and protein expression levels were detected, and ALP staining was performed. On the 14th day, ARS staining was conducted.
RNA extraction and real time-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted using E.Z.N.A.® total RNA Kit I (Omega Bio-Tek, USA). The extracted RNA was then assessed for concentration and purity using a microplate reader (Bio-Tek USA). Subsequently, 1 μg RNA was transcribed to cDNA using a one-step RT-qPCR kit (Vazyme, China). The cDNA, SYBR Green Master mix and primers were mixed proportionally, amplified, and measured in RT-qPCR detection device. Relative mRNA levels were normalized for quantification compared with β-Actin levels. The primer sequences used in the experiment were listed in Table 1.
Alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining assay
BMSCs were seeded into 24-well plates for ALP staining after 7-day SEMF stimulation while ARS staining was performed after 14 days of intervention. The cells were fixed with 4% paraformaldehyde (Sigma, USA) for 30 min and then washed with PBS. Subsequently, each well was stained with 350 μL of ALP or ARS staining solution for 10 min. After three rinses with PBS, the stained cells were observed and photographed under a fluorescence microscope. Quantitative analysis of the staining intensity was carried out using Image J software.
Western blotting analysis
Cultured BMSCs were lysed with enhanced RIPA lysis buffer (Boster) containing 1% EDTA, protease, and phosphatase inhibitor cocktails. Following the determination of the protein concentration of the cell lysates, equal amounts of samples were separated by 10% SDS-PAGE and the protein bands were then transferred to polyvinylidene fluoride (PVDF; Millipore, USA) membranes. After being blocked with 5% bovine serum albumin (BSA; BioFroxx, Germany) for 1 h, the membrane bands were incubated at 4 °C overnight with their corresponding primary antibodies (anti-β-catenin, anti-Active-β-catenin, anti-OPN, anti-RUNX2, anti-ALP, anti-STAC2 and anti-β-Actin, Cell Signaling Technology, USA). The blots were subsequently incubated with secondary antibodies (Boster) for 1 h the next day. The target protein bands were visualized using Image Lab System (Bio-Rad, USA). Relative target protein levels were normalized for quantification compared with β-Actin.
Cell transfection
Approximal 2 × 105 BMSCs were seeded into 6-well plates. When the cell confluences reached around 80%, Lipofectamine 3000 reagent (Invitrogen, USA) was used for transfection. In each well, Opti-MEM (250 μL) and lipofectamine 3000 (5 μL) were added along with either miR-34b-5p mimic or inhibitor (20 μM, 50 μM, and 100 μM, Tsingke, China) or STAC2 siRNA (50 Μm, Tsingke). The cells were then incubated for 48 h. In order to maintain intracellular levels of miR-34b-5p and STAC2 during the long culture period of BMSCs (7 or 14 days), cell transfection was performed every 4 days. The transfection efficiency was visualized using fluorescence microscopy. The miRNA sequences used in the study were listed in Table 2.
miRNA sequencing
The total RNA was extracted from the samples using the miRNeasy Micro Kit (Qiagen, Germany). Subsequently, miRNAs were extracted and purified using the miRNA isolation kit (Sigma). Next, the complemental DNA (cDNA) library was prepared following the manufacturer’s instructions of the RNA Library Kit (Vazyme, China). The constructed library was processed on the Illumina HiSeq 4000 platform and low-quality reads were removed using Skewer 0.2.2. Principal component analysis (PCA) was employed to assess the transcriptome differences between each group. Differentially expressed miRNAs (DE-miRNAs) were determined based on the criteria of False Discovery Rate (FDR) < 0.0552. The cutoff for identifying DE-miRNAs was set at an adjusted p < 0.05 and a |log2 fold change (log2FC)| ≥ 0.585. TargetScan database (http://www.targetscan.org/mamm_31/) was used to predict the target genes of miR-34b-5p.
RNA sequencing and bioinformatic analysis
The total RNA was extracted as described above. Subsequently, 500 ng of RNA was utilized to construct cDNA libraries, which underwent high-throughput sequencing on the Illumina HiSeq 4000 platform. Quality assessment of the raw reads was performed using FastQC (v0.10.1). Following this, the acquired data underwent preprocessing to remove low-quality reads, quantify gene expression, and compare against a reference genome. The screening criteria of differentially expressed genes (DEGs) was set at an adjusted p-value of less than 0.05 and |log2FC| ≥ 1. The pathways associated with calcium channels were investigated using Gene Ontology (GO) database. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was conducted with R package (v3.11).
EdU assay
EdU assay (beyotime, China) was used for assessing cell proliferation. BMSCs were seeded in a 96-well plate and incubated with 100 μL EdU (10 μM) for 2 h. After fixed with 4% paraformaldehyde for 20 min and permeabilized with 3% Triton-X100 for 15 min, the cells were incubated with a mixed staining solution according to the manufacturer’s protocol for 1 h. Hoechest 33342 was added to incubate for 10 min. The stained cells were visualized under a fluorescence microscope (Nikon, Japan).
Luciferase reporter assay
TargetScan and RNAInter web were used to analyze the potential binding sites between miR-34b-5p and STAC2. The wild-type (WT) and corresponding mutant sequences of STAC2 were cloned into the pMIR-report vector. BMSCs were then seeded into 24-well plates and co-transfected with either the WT or mutant (MUT) plasmids, along with miR-34b-5p mimic, miR-34b-5p inhibitor, or their respective negative controls. Following 48 h of culture, cells were lysed, and the luciferase activity was measured using the Luciferase Reporter System (Promega, USA).
OVX rat model
In this study, all 12-week-old female SD rats were obtained from SJA Laboratory Animal Company (Certificate No. SCXK, Hunan, 2021-001, China). The methods were performed in accordance with relevant guidelines and regulations and approved by the Ethics Committee of Experimental Animal Center of Huazhong University of Science and Technology (No. 3091, Wuhan, China). A total of 18 rats were randomly divided into three groups (n = 6 per group). The groups were designated as follows: sham, OVX, and OVX + miR-34b-5p inhibitor. In the sham-operated group, only the adipose tissue surrounding the ovaries was removed. Bilateral ovariectomy was performed under anesthesia in both OVX control and OVX + miR-34b inhibitor groups. After a 2-week recovery period, a 2 mm diameter hole was drilled in the proximal femur of each rat. In the sham and OVX group, rats were intramedullary injected 5 μL of normal saline, while rats in OVX+miR-34b-5p inhibitor group received injections of 5 μL miR-34b-5p inhibitor solution (20 mmol/L) every 2 weeks. The holes were sealed with bone wax following each injection. After 10 weeks of injections, all rats were euthanized, and their femurs were harvested.
Micro-CT scanning
The collected femurs were fixed using 4% paraformaldehyde solution for 24 h. Subsequently, the microstructure of the bone was analyzed using the Viva CT 40 microcomputed tomography (micro-CT) system (Scanco, Switzerland). Our analysis primarily focused on the cancellous bone, and several trabecular morphometry parameters, including trabecular bone volume per tissue volume (BV/TV), trabecular number (TB.N), trabecular thickness (TB.Th), and trabecular separation (Tb.Sp).
Hematoxylin and Eosin (H&E) staining
The femur samples were fixed and decalcified in 10% EDTA solution for 4 weeks and embedded in paraffin. Following embedding, they were cut into sections and deparaffinized using xylene. Subsequently, the sections were incubated with hematoxylin for 5 min, followed by washing with acid ethanol. Next, the sections were incubated with 0.5% eosin, dehydrated stepwise, and blocked using Permount (Absin, China). The sections were observed and photographed under a light microscope (Olympus, Japan).
Masson staining assay
The Masson staining assay was conducted using the Trichrome Stain Kit (Sigma) following the provided protocol. After deparaffinization with xylene, the sections were immersed in Bouin’s Solution overnight. The slices were sequentially incubated with Hematoxylin Solution, Biebrich Scarlet-Acid Fuchin Solution, and Phosphotungstic/Phosphomolybdic Acid Solution for 5 min each. Then, the sections underwent staining with Aniline Blue solution for 8 min, followed by dehydration. Finally, the slides were blocked with a drop of Permount and observed using a light microscope.
Statistics and reproducibility
All experimental data were analyzed using GraphPad Prism V.8 software (GraphPad Software, USA), and the results were shown as mean ± standard deviation (SD). Animal experiments and cell experiments were independently replicated six times and three times, respectively. The differences among multiple groups were analyzed using one-way analysis of variance (ANOVA). A value of p < 0.05 was considered statistically significant.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
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- Source: https://www.nature.com/articles/s42003-024-06866-3