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Static magnetic field-induced IL-6 secretion in periodontal ligament stem cells accelerates orthodontic tooth movement – Scientific Reports

Animals

All the animal experiments were approved by the Ethics Committee of the Animal Laboratory of Kunming Medical University (no. Kmmu20220137). The Laboratory Animal Centre at Kunming Medical University provided specific-pathogen-free, 8-week-old, healthy male Sprague–Dawley rats. The rats were maintained in a barrier environment throughout the experiments and fed conventional food and water. Moreover, frequent body weight measurements were made regularly. The testing was conducted in accordance with the ARRIVE recommendations, the U.K. Animals (Scientific Procedures) Act, 1986 and related regulations, EU Directive 2010/63/EU for animal experiments, or the National Institutes of Health’s manual for the use and care of laboratory animals (NIH Publications No. 8023, revised 1978).

OTM model

Rats were anesthetized with 3% sodium pentobarbital (0.1 mL/100 g) by intraperitoneal injection. Retention grooves approximately 0.1-mm deep were ground into the mesial surface of the left maxillary first molar and into both sides of the maxillary incisors. A 4-mm-long NiTi constant-force tension spring (50 g, SuHang, Shenzhen, China) was fixed between the left upper first molar and the maxillary incisors with a 0.20-mm metal-wire ligature. The tension spring was adjusted using a spring dynamometer to produce a horizontal force of 50 g before fixation. Light-cured resin (3M, St. Paul, MN, USA) was used to reinforce the upper-incisor ligature. The model did not demold during the experiments.

Localized magnetic-field exposure model

While the OTM model was being established, the OTM + SMF group received a 5-0 nylon suture (Lingqiao, Ningbo, China) to fix a NdFeB magnet (Yuexing Magnet Co., Shenzhen, China) to the left buccal mucosa of the rat opposite the first molar. The magnets were 4 mm in diameter and 1-mm high, with a 1.5-mm-diameter circular hole in the center and covered with a biosafe resin (3M, St. Paul, MN) to avoid possible cytotoxic damage. The OTM + sham group received 5-0 nylon sutures to fix a non-magnetic metal dummy with the same size and shape to the same position in the left cheek of the rats. The magnets and the non-magnetic metal-loaded dummy did not affect the normal functions of the rats, such as mastication. The magnetic field strength of the magnet was measured at 200 ± 20 mT using a Tesla Gaussmeter (Nohawk, Tianjin, China).

IL-6 inhibitor injections

Tocilizumab (GlpBio, Montclair, CA) is an anti-IL-6R neutralizing antibody that competitively binds IL-6R to inhibit the function of IL-6. Tocilizumab was diluted to 10 mg/mL with saline (Kelun, Sichuan, China) using a previously reported concentration45. The inhibitor group was injected with 50 μL of tocilizumab locally in the vestibular sulcus of the upper-left first molar, and the control group was injected with 50 μL of saline (Kelun, Sichuan, China) at the corresponding site. The injections were performed on days 1 and 4 of the experiment.

Micro-CT analyses

Seven days after the models were established, the maxillary jaws and teeth were picked up and fixed in a 4% formaldehyde solution for 24 h. The maxillary jaws and tooth were scanned with a micro-computed tomography (micro-CT) system (PINGSENG Healthcare Inc., Kunshan, China). Images were reconstructed and analyzed using Mimics software 21.0 (Materialise, Leuven, Belgium). In the micro-CT image, the distance between the crowns of the first left two molars in the maxillary jaws was measured in a direction parallel to the direction of OTM as the distance of tooth movement. The OTM distance was determined by investigators blinded to the group.

Histological analysis

After the CT scan, the rat maxillae were immersed in a 10% solution of EDTA (MVS-0098, MXB, FuZhou, China) for decalcification, and the solution was altered every 3 days. After 21–28 days, the tissue could be punctured without resistance with a sterile needle to establish the completion of decalcification. Afterward, the tissues were dehydrated in graded ethanols and embedded in paraffin. Four μm thick paraffin slices were cut and stained with hematoxylin and eosin (HE) and tartrate-resistant acid phosphatase (TRAP).

HE staining was used for histomorphological analysis. The sections were dewaxed and dehydrated, rinsed with distilled water, stained with hematoxylin for 15 min, rinsed with tap water, fractionated with 1% hydrochloric acid in alcohol for 2 s, rinsed, washed in phosphate-buffered saline (PBS), stained in eosin for 30 s, dehydrated, air-dried, and sealed with gum.

TRAP staining was used to identify and analyze osteoclasts. Sections were dewaxed, dehydrated, and incubated in TRAP solution (Sigma-Aldrich, St. Louis, MO) in accordance with the manufacturer’s instructions at 37 °C for 60 min, after which the osteoclasts appeared burgundy under the microscope. Sections were then stained with hematoxylin for 10 s, rinsed, fractionated with 1% hydrochloric acid in alcohol for 1 s, rinsed, dehydrated with 75% ethanol for 10 s, air-dried, and sealed with gum. The osteoclast area was analyzed using Image J-Pro plus 6.0 (National Institutes of Health, Bethesda, MD).

Immunohistochemistry

After dewaxing and dehydration, the antigens in the EDTA-treated samples were heat-repaired in an oven at 100 °C for 40 min. Three percent hydrogen peroxide (SP KIT-A2, MXB, Fuzhou, China) was used to block the sections for 10 min and in goat serum for 45 min (SP KIT-B1, MXB, Fuzhou, China). Sections were then incubated with IL-6 primary antibody (1:400; GXP263019, Genxspan, Alabama, USA) overnight at 4 °C for approximately 10 h. Slices were then washed with PBS (Hyclone, Cytiva, Marlborough, MA, USA) and incubated with a secondary antibody (KIT-5010, Maishin, Fuzhou, China) for 15 min at 37 °C. Nuclei were re-stained with hematoxylin (DAB0031, MXB, Fuzhou, China) for 30 s, dehydrated, and sealed in gum. For semi-quantitative analysis, the average optical density (AOD) of IL-6 in the stained tissues was calculated using Image J-Pro plus 6.0 (National Institutes of Health, Bethesda, MD). AOD = (integrated optical density)/area.

Isolation and culture of periodontal stem cells

This part of the experiment was approved by the Ethics Committee of the Affiliated Stomatological Hospital of Kunming Medical University (No. KYKQ2022MEC030), and informed consent was obtained from the patients in accordance with the Declaration of Helsinki. PDLSCs were isolated and cultured as previously described46. Briefly, PDLSCs were taken from the premolars of young patients 12–20 years old. These premolars had been removed for orthodontic treatment and were healthy teeth without dental or periodontal disease. Primary PDLSCs were isolated from these premolars and cultured after enzymic digestion in α-MEM medium (Hyclone, Logan, Utah) with 15% fetal bovine serum (FBS; BI, Israel) and 100 U/mL penicillin/streptomycin (Hyclone) for 7 days at 37 °C in a humidified environment with 5% CO2. When cell density reached ≥ 70%, the cells were purified, expanded, and subjected to limiting dilution analysis.

Cellular additive force model

P5-generation PDLSCs in good condition were inoculated into 6-well plates (Corning Incorporated, Corning, NY, USA) at 2 × 105 cells per well and pressure-loaded when cell confluence reached 80%. A 33-mm-diameter, 17 g transparent glass plate (Qingxing Glass, Shenzhen, China) was placed flat on the cell layer, in uniform contact with the cell layer, exerting a pressure of 2 g/cm2 on the cells. The PDLSCs were subjected to stress continuously for 24 h, followed by collecting the conditioned medium.

Cellular magnetic-field exposure model

Cells were stimulated by a customized NdFeB SMF device (Li Tian Magnetics Technology Co., Ltd., Sichuan, China), which could generate a magnetic field with a strength of 200 ± 50 mT. Cells were exposed to the magnetic field for 24 h, and then the conditioned medium was collected. All plates were incubated in the same incubator.

Extraction and culture of bone marrow macrophages (BMMs)

BMMs were extracted from 6-week-old C57 mice tibias and femurs. Monocytes were raised in α-MEM medium with 10% FBS and 25 ng/mL of M-CSF (CB34, Novoprotein, Shanghai), and nonadherent cells were washed after 2 days to obtain macrophages. Macrophages were cultured in PDLSC-conditioned medium and a-MEM containing 10% FBS (BI, Israel), and thereafter with osteoclast induction solution containing 100 ng/mL of RANKL (CR06, Novoprotein, Shanghai) and 25 ng/mL of M-SCF. TRAP staining was used to detect osteoclasts after 5–7 days.

Western blot

Before immunoblotting, protein content was determined using the assay kit (BL521A, White Shark, Hefei, China), and all samples were adjusted to the same protein concentration. To separate equal quantities of protein, 12.5% polyacrylamide-gel electrophoresis was used. After electrophoresis, polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) were used to transport proteins. Next, the membranes were incubated with various primary antibodies, including TRAP (1:1000; #13908, CST, Germany), and b-tubulin (1:20,000; Proteintech, Rosemont, IL, USA), following treatment with a secondary antibody labeled with horseradish peroxidase (goat anti-rabbit, 1:5000, CST, Germany). Protein bands were detected using an electrochemiluminescence detection kit (P0018, Biyuntian, Shanghai, China) and an electrochemical gel imaging system (ChemiDocTM XPS+; Bio-Rad, Hercules, CA, USA). b-Tubulin expression was used as an internal reference.

siRNA transfection

GenePharma (Shanghai, China) supplied the small interfering RNAs (siRNAs). The RNA sequences are provided in Supplementary Table 1. IL6-siRNA or siNC was transfected into hPDLSCs (70% confluent) in accordance with the manufacturer’s instructions (Lipofectamine™ 2000 Transfection Reagent, Invitrogen, USA). The transfection efficiency was calculated by observing the number of positively expressed cells under a confocal scanning microscope after 24 h. Subsequently, a pressure of 2 g/cm2 was applied to the cells for 24 h and the conditioned medium was collected. The effect of interference was determined using RT-PCR.

Real-time PCR

Cellular RNA was isolated using the Total RNA Extraction Kit (Tangan, Branford, CT, USA). We converted 1000 ng of RNA to cDNA using the PrimeScript™ RT reagent Kit with the gDNA Eraser (RR047, Takara, Shiga, Japan). Real-time quantitative PCR was performed with TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (RR820, Takara). Amplification conditions were as follows: 95 °C for 30 s, 40 cycles at 95 °C for 5 s, and 60 °C for 34 s. Relative gene expression levels were quantified using the 2−ΔΔCt method. The mRNA expressions of the target genes were normalized to that of b-actin. The primer sequences are provided in Supplementary Table 2.

RNA-seq analysis

The PDLSCs were exposed to an SMF and mechanical loading for 24 h. RNA was then obtained using the above technique. The quality of the RNA was evaluated on a 2100 Expert Bioanalyzer (Agilent, Santa Clara, CA, USA). Shanghai Majorbio Bio-Pharm Technology selected qualified RNA samples for library creation and sequencing on the NovaSeq 6000 platform (Illumina, San Diego, CA, USA). The Majorbio Cloud Platform (http://www.Majorbio.com), a free online platform, was used to analyze and visualize the data. Differential expression analysis was performed using the RSEM, DEGs with |log2FC| ≥ 1 and FDR < 0.05 (DESeq2) were considered to be significantly different expressed gene. We have uploaded the raw RNA-seq dataset to the NCBI, the accession number is PRJNA1099930.

Statistical analysis

SPSS 25.0 was used for the statistical analysis. The raw data were confirmed to have a normal distribution using a one-sample Kolmogorov–Smirnov test. The independent t-test was used to compare the two groups. To compare three or more groups, a one-way variance analysis was employed, followed by Dunnett’s post-hoc multiple comparisons. Each datum represents at least three independent experiments. The results are presented as means ± standard deviation (SD). Differences with P < 0.05 were considered statistically significant.

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