The murine monocyte/macrophage cell line RAW264.7 and stromal cell line ST2 were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China). A four-point bending system was obtained from Sichuan University (Chengdu, China). The force-loading plates were made from the bottom part of the 75-cm² cell canted-neck culture flasks (Falcon #353135; Corning Life Sciences, Tewksbury, MA, USA), each 3 × 8 cm² in area and 1.15 mm thick. Rabbit anti-human ephrinB2 polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

All procedures involving animals were approved by the Animal Care and Use Committee of Jilin University. The animals used were 8-week-old male Wistar rats, with an average weight of 200 g. The rats were anesthetized by intramuscular injection with 0.2 mL·kg^-1 Sumianxin (Academy of Military Medical Sciences, Changchun, China). A nickel-titanium closed-coil spring exerting an orthodontic force of 50 cN was ligated unilaterally to the maxillary first molar and incisor (Figure 1). The maxillary first molar on the opposite side was not moved and was used as the control group. We examined a total of 10 teeth in five rats. Experimental tooth movement was conducted for 3 days (n = 5).

Rats were sacrificed by intracardial perfusion with 4% paraformaldehyde in 0.1 M of phosphate buffer (pH 7.4) under anesthesia. The maxillae were dissected and molar-bearing segments of alveolar bone were cut from each side and further fixed overnight in 4% paraformaldehyde buffer. The specimens were decalcified in 10% ethylenediamine tetraacetic acid solution (pH 7.4) for 8 weeks, and further dehydrated in ascending concentrations of ethanol and embedded in paraffin. Serial 5-µm-thick sections including the crestal areas mesial and distal to the maxillary molars were cut and mounted on L-poly-lysine-coated glass slides. Sections were stained with hematoxylin and eosin and immunohistochemistry was performed. For immunohistochemistry staining, endogenous peroxidase in the tissue section was blocked with 1% hydrogen peroxide for 10 minutes and then further blocked with 1% goat serum in phosphate-buffered saline for 30 minutes. The primary antibody used was polyclonal rabbit anti-human ephrinB2. An immunohistochemistry staining kit (Maixin Bio, Fuzhou, China) was used for further staining. Finally, sections were counterstained with hematoxylin and mounted. Negative controls were labeled using the same procedures, but omitting the incubation with primary antibodies. Staining density was analyzed using Image-Pro Plus (Media Cybernetics, Bethesda, MD, USA) and represented as the mean density.

RAW264.7 cells were maintained in low-glucose Dulbecco's modified Eagle medium (L-DMEM; Life Technologies, Gaithersburg, MD, USA) and ST2 cells in alpha-minimum essential medium containing 10% fetal bovine serum (Life Technologies), 100 U·mL^-1 penicillin, and 100 mg·mL^-1 streptomycin (Life Technologies) at 37℃ in a humidified atmosphere of 5% CO₂. The medium was changed every 2-3 days. The cells were seeded onto the force-loading plate at a density of 1 × 10⁵ cells·cm^-2. RAW264.7 cells on the force-loading plates were cultured in L-DMEM containing 50 ng·mL^-1 recombinant mouse RANKL (rmRANKL; R&D Systems, Minneapolis, MN, USA). RAW cells differentiated into tartrate resistant acid phosphatase-positive multinucleated osteoclastic cells in the presence of RANKL.19

The force-loading plates were subjected to cyclic uniaxial compressive strain by the four-point bending system20 at 0.5 Hz (Figure 2). Cells were loaded with compressive force at 2000 µε (µε is defined as deformation, with 1,000 µε corresponding to 0.1% deformation) for 1, 2, and 4 hours, respectively. Control cells were cultured on similar plates without any mechanical loading and kept in the same incubator.

Cells were harvested after 1, 2, and 4 hours of the application of compressive force. Total RNA was extracted using an EASYspin RNA extraction reagent kit (Biomed, Beijing, China) from cells cultured on the plates according to the manufacturer's instructions. RNA was reverse-transcribed using the PrimerScript® RT reagent kit (Takara, Dalian, China) with gDNA eraser to synthesize cDNA. qPCR (n = 3) was performed using SYBR Green Premix Ex Taq (Takara) and the MxPro Mx3005P real-time PCR detection system (Agilent Technologies, Santa Clara, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a normalization control. The primers used for qPCR were shown in Table 1. The cycling conditions were as follows: 95℃ for 30 seconds followed by 40 cycles of 95℃ for 5 seconds, 55℃ for 30 seconds, and 72℃ for 1 minute. The 2^-ΔΔCt method was used for calculating the relative expression levels, and values were the average of triplicate measurements.

All experiments were performed on at least three individuals. Results are presented as the mean ± standard deviation (SD). The significance of the differences was determined using the two-tailed Student's t-test and one-way analysis of variance (ANOVA). The difference was considered significant if p < 0.05. The IBM SPSS ver. 19.0 for Windows (IBM Co., Armonk, NY, USA) was used for statistical analysis.

Histological examination showed a gap between the first and second molar indicating that the loaded maxillary first molar was moved mesially. In the compression area, the surface of alveolar bone was rough and there were resorption lacunae on the bone surface (Figure 3). Multinucleated osteoclasts were seen adjacent to the alveolar bone surface. No osteoblasts were observed in the compression area.

Since no osteoblasts were found in the compression area, only ephrinB2 was examined by immunohistochemistry. EphrinB2 immunoreactivity was observed in fibroblasts in periodontal tissues from both the control and experimental groups (Figure 4A and 4B). In periodontal tissues of the control group without tooth movement, ephrinB2 was weakly and uniformly expressed in periodontal fibroblasts (Figure 4A). In periodontal tissues of the experimental group, ephrinB2 was strongly expressed in osteoclasts adjacent to alveolar bone in the compression area (Figure 4B). Negative controls showed no immunolabeling (Figure 4C). As shown in Table 2, the difference in ephrinB2 expression levels in periodontal tissues between the experimental and control groups was statistically significant (p < 0.05).

In order to investigate whether compressive force could directly stimulate osteoclast differentiation, we applied cyclic uniaxial compressive force produced by a four-point bending system to RAW264.7 cells. Following this, gene expression was assessed for two well-known markers of differentiated osteoclasts, nuclear factor of activated T cells cytoplasmic 1 (NFATc1)21,22,23 and calcitonin receptor (CTR).24 As shown in Figure 5A and 5B, the levels of NFATc1 and CTR mRNA were significantly upregulated in RAW264.7 cells after 1 hour and 2 hours of the application of compressive force (p < 0.05). Our data indicate that cyclic compressive force can promote the differentiation of osteoclasts.

To understand the relationship between compressive force and ephrinB2 expression in osteoclasts, we evaluated the expression of ephrinB2 in RAW264.7 cells. As shown in Figure 5C, there was a significant increase in the expression of the ephrinB2 gene after 1 hour and 2 hours in compressed RAW264.7 cells (p < 0.05).

To investigate the response of osteoblasts to compressive force, we performed an in vitro experiment using ST2 cells. The expression of the transcription factors Runx2 and Sp7 was assessed in these cells after the application of compressive force. Runx2 plays a key role in mediating osteoblast maturation and is necessary for normal osteogenesis,25,26 while Sp7 is believed to be downstream of Runx2 and may promote osteoblast maturation.27,28 As shown in Figure 6A and 6B, the mRNA levels of Runx2 and Sp7 were significantly decreased in ST2 cells after the application of compressive force (p < 0.05). These findings suggest that compressive force can inhibit osteoblast differentiation.

It has been reported that both EphB4 and ephrinB2 are found in osteoblasts. To understand whether EphB4 and ephrinB2 expression in osteoblasts is affected by compressive force, we evaluated the expression of these molecules in ST2 cells. As shown in Figure 6C and 6D, there was a significant decrease in the expression of the EphB4 gene in ST2 cells after 1 hour and 2 hours of exposure to compressive force (p < 0.05). A significant decrease in the expression of ephrinB2 was also detected after 4 hours of compressive force (p < 0.05).