MC3T3-E1 cells were maintained in high glucose medium (HyClone, Logan, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; BBI, Shanghai, China), 100 U/ml penicillin and 100 µg/ml streptomycin (HyClone, Logan, USA) and cultured with 5% CO2 at 37 °C. For osteogenic differentiation, the cells were cultured in high glucose medium supplemented with 5% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 10− 8 mol/L dexamethasone (Sigma, St, Louis, MO) and 10 mmol/L β-glycerophosphate (Solarbio, Beijing, China). The medium was replaced every two days.

Referring to past studies, EphrinB2-fc (Sino Biological, Beijing, China) was used to stimulate ephrinB2-Ephb4 signaling in MC3T3-E1 cells at a concentration of 4 µg/ml(Wang et al. 2018). NVP-BHG712 (Selleck, Shanghai, China) was used to inhibit Ephb4 signaling, and we used it at a concentration of 200 nM(Mundy and Elefteriou 2006).

MC3T3-E1 cells were seeded onto plastic cell culture plates (Miri Technology, Chengdu, China) at a density of 1 × 105 per dish and cultured in normal culture medium in cell culture dishes (Fig. 1a1). The cells grew adherently on the plastic plates. For TF stimulation, the plastic plates were transferred to the tensile force dish (Fig. 1a2), and the four-point bending force device system (Sichuan University, Chengdu, China) was used to apply TF to the cells on the plastic plates (Fig. 1a3-a4). The TF applied to the cells was 2000 µε (µε is defined as deformation, with 1000 µε corresponding to 0.1% deformation). A schematic diagram of applying TF is shown in Fig. 1b.

MC3T3-E1 cells were stimulated with TF for 0 h, 2 h, 6 h and 12 h, digested with trypsin, seeded in a 96-well plate at a density of 5000 cells per well, and incubated at 37 °C for 24 h. At the time point of 24 h, 200 µl AM/PI was added to the wells and incubated at 37 °C in the dark for 15 min. Then, a fluorescence microscope was used for observation.

MC3T3-E1 cells were inoculated onto cell culture dishes (bottoms were covered by plastic plates) at a density of 1 × 105 per dish. TF stimulation was loaded on cells for 0 h, 2 h, 6 h and 12 h. Then, the cells were digested with trypsin, seeded in a 96-well plate at a density of 5000 cells per well, and cultured for 24 h at 37 °C. At the time point of 24 h, 10 µl Cell Counting Kit reagent (New Semi Biotechnology, Suzhou, China) was added to each well and incubated at 37 °C for 1–2 h, and then a microplate reader was used to measure the absorbance at 450 nm.

MC3T3-E1 cells were cultured in the osteogenic induction medium and stimulatedwith TF for 0 h, 2 h, 6–12 h each day, after 7 days, 4% paraformaldehyde (PFA) was used to fix the cells, and an ALP staining kit (Beyotime Biotechnology, Shanghai, China) was used to stain the cells. Following staining, the cells were visualized by a microscope.

Approximately 2 × 104 cells /well were seeded in a 24-well culture plates and stimulated with TF for 0 h, 2 h, 6–12 h each day, after 21 days, 4% paraformaldehyde (PFA) was used to fix the cells and 2% ARS (Beyotime Biotechnology, Shanghai, China) was used to stain the cells. Following staining, the cells were visualized by a microscope.

After stimulation with TF, total mRNA in MC3T3-E1 cells was extracted using an RNA extraction kit (Forge Biotechnology, Chengdu, China) according to the manufacturer’s instructions and reverse transcribed using a transcription kit (CWBIO Biotechnology, Beijing, China). QPCR was performed using a SYBR kit (CWBIO Biotechnology, Beijing, China). Each sample was prepared in triplicate, and each experiment was repeated at least 3 times. GAPDH was used as control. The sequences of the primers for mouse RUNX2, OPN, OCN, Ephb4, ephrinB2 and GAPDH are shown in Table 1. The relative gene expression levels were calculated using the 2−ΔΔCT method.

After stimulation with TF, proteins were extracted from MC3T3-E1 cells using RIPA buffer (Beyotime Biotechnology, Shanghai, China) containing 1% phenylmethylsulfonyl fluoride (PMSF; Beyotime Biotechnology, Shanghai, China) for 30 min, and the protein concentrations were measured using a bicinchoninic acid (BCA; New Semi Biotechnology, Suzhou, China) protein assay kit. Then, the protein samples were mixed with 5 × loading buffer (CWBIO Biotechnology, Beijing, China) and heated at 100 °C for 10 min. The samples were then separated on 12.5% SDS–PAGE gels and transferred to nitrocellulose membranes for 90 min at 200 V. The membranes were subsequently blocked with 5% bovine serum albumin (BSA) for 1 h at 4 °C and incubated with primary antibodies at 4 °C overnight. The anti-mouse primary antibodies used in this study were as follows: RUNX2 (1:1000, catalog no. 12556 S; CST), OPN (1:1000, catalog no. A19092; ABclonal), OCN (1:500, catalog, no.93876; Abcam), ephrinB2 (1:1000, catalog no. ab131536; Abcam) EphB4 (1:1000, catalog no. ab254301; Abcam), p-EphB4 (1:1000, catalog no. PA5-64637; Thermo Fisher), ERK1/2 (1:1000, catalog no. 4695 S; CST), p-ERK1/2 (1:1000, catalog no. 4370 S; CST), P38 (1:1000, catalog no. 8690; CST), p-P38 (1:1000, catalog no. 4511; CST), JNK1/2/3 (1:1000, catalog no. 9252; CST), p-JNK1/2/3 (1:1000, catalog no. 4668; CST), GAPDH (1:1000, catalog no. AF1186; New Semi Biotechnology) and β-actin (1:1000, catalog no. 66009; Proteintech). After being washed with Tris-buffered saline containing 0.05% Tween-20 (0.05% TBST buffer), the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000, catalog no. A0208; Beyotime Biotechnology) for 1 h at 4 °C. Protein bands were visualized using enhanced chemiluminescence (ECL; New Semi Biotechnology, Suzhou, China), and the band intensities were analyzed by ImageJ (Fujifilm, Tokyo, Japan).

Twenty SD male rats aged 6 weeks (200–220 g) were used to establish rat OTM models, which were purchased from the Experimental Animal Center of Jilin University. The animal experiments were performed using a previously described method [43]. Nickel-titanium tensile springs were used to connect the incisors and first molars of the maxillary (Fig. 6a). The TF of the spring was 50 g and the treatment lasted for 7 days. The rats were randomly divided into 4 groups (5 rats/group): OTM group (OTM treated, injected with PBS 20 µl/day), ephrinB2-fc group (OTM treated, injected with ephrinB2-fc 2 mg/kg/day in 20 µl), NVP-BHG712 group (OTM treated, injected with NVP-BHG712 5 mg/kg/day in 20 µl), ephrinB2-fc + NVP-BHG712 group (OTM treated, injected with ephrinb2-fc 2 mg/kg/day in 10 µl and NVP-BHG712 5 mg/kg/day in 10 µl). Rats were given local injection once a day. The site of administration was on the surface of the meital and distal alveolar bone of the maxillary first molar, and 10 µl medicine was injected at each side. On the 7th day, all rats were sacrificed by overdose injection of 1% sodium pentobarbital, and the upper jaw was dissected and fixed in 4% paraformaldehyde (PFA) for 24 h.

Micro CT (HiScan XM Micro CT) was used to scan the maxillary alveolar bones of the rats. The scanning parameters were as follows: 80 kV, 100 µA, single exposure time 50 ms, scanning resolution 25 μm, scanning interval 0.5 degrees. AMIRA software was used for reconstruction and analysis. The distance between the first molar and second molar represented the tooth moving distance. The alveolar bone on the distal side of the first molar was selected as the ROI, and the trabecular bone parameters were measured. The main measurement parameters were BV/TV, Tb. Th., and Tb. Sp.

The samples fixed with 4% PFA were decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 12 weeks. After dehydration with an ethanol gradient, the samples were embedded in paraffin and sectioned at a thickness of 3 μm. The sections were stained with hematoxylin-eosin (H&E) staining solution and observed under a microscope.

After baking and dewaxing the sections, they were placed in 1% sodium citrate buffer at 62 °C overnight for antigen repair. The sections were then closed with endogenous catalase by adding 100 µl of 3% hydrogen peroxide dropwise to each section and incubated for 15 min at room temperature away from light. After washing 3 times using PBS, the sections were sealed with drops of sealing solution and incubated at 37 °C. 1 h later, the sections were incubated with primary antibodies (anti-EphB4 and ephrinB2 antibodies) for 12 h at 4 °C. After rinsing, the samples were incubated with biotin-labeled goat anti-mouse/rabbit IgG polymer secondary antibody for 2 h. The color was developed by DAB colorant, and the staining was restained by hematoxylin.

The data are expressed as the mean ± standard deviation. Comparisons between two groups were performed with two-way t tests, and comparisons among multiple groups were performed with one-way ANOVA followed by Bonferroni post hoc tests. P values < 0.05 were considered significant.