Roles of EphrinB2 and EphB4 in Alveolar Bone under Initial Compressive Mechanical Stress of Dental Implant Replacement

Research Article

J Dent & Oral Disord. 2018; 4(1): 1081.

Roles of EphrinB2 and EphB4 in Alveolar Bone under Initial Compressive Mechanical Stress of Dental Implant Replacement

AKitamura K¹*, Mine Y², Koto W¹, Wachi T¹, Shinohara Y¹, Makihira S¹ and Koyano K¹

¹Department of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Maidashi, Japan

²Department of Research and Development of Next Generation Medicine, Faculty of Medical Sciences, Kyushu University, Japan

*Corresponding author: Kazuyuki Kitamura, Section of Fixed Prosthodontics, Department of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Maidashi, Higashi-ku, Fukuoka, Japan

Received: December 01, 2017; Accepted: January 08, 2018; Published: January 15, 2018


The communication between ephrin/Eph families was recently shown to be involved in cell differentiation under mechanical stress. To understand the roles of EphrinB2 and EphB4 in osteoblast cells and alveolar bone under initial mechanical stress of dental implant replacement, first, MC3T3-E1 cells were subjected to cyclical compressive or extensive force in vitro. The expressions of EphrinB2 and EphB4 were examined by real-time RT-PCR or western blotting, together with expression of Osterix as an osteoblast differentiation marker. Next, the effects of mini-implant surfaces immobilised with EphrinB2 or EphB4 on the expression of the gene encoding Osterix in alveolar bone surrounding implants were examined by real-time RT-PCR. Osterix mRNA expression was decreased in MC3T3-E1 cells cultured under compressive conditions, compared with that under normal conditions. The mRNA and protein levels of EphrinB2 and EphB4 were increased in the cells cultured under compressive conditions. Initially, mechanical stress by torques decreased Osterix mRNA expression in alveolar bone surrounding mini-implants, compared with the control groups. Torque of 10 Nmm increased EphB4 mRNA expression compared with the control groups, while torques of 10 and 20 Nmm increased EphrinB2 mRNA expression. The mini-implant surface immobilised with EphrinB2-Fc recovered the suppression of Osterix. Taken together, the present results suggest that increased binding of EphrinB2 and EphB4 in osteoblast cells exposed to initial mechanical force, especially compressive force, may be involved in alveolar bone recovery, and may cause a gradual decrease of the primary fixation in a replaced dental implant and finally lead to stability.

Keywords: Compressive stress; EphrinB2-EphB4; Extensive stress; Implant


Mini-Implant: Titanium Mini-Implant; Ti-Disc: Titanium Disc; XPS: X-Ray Photoelectron Spectroscopy; CL: Cell Lysate


Recent investigations have demonstrated that molecules in the ephrin/Eph family regulate the differentiation of osteoblasts and osteoclasts under mechanical stress [1]. Ephrins and Ephs have a ligand–receptor relationship. Ephrins are transmembrane ligands, and Ephs are their tyrosine kinase receptors. Interactions between ephrinB- and EphB-expressing cells result in bidirectional signal transduction [2]. Activation of EphB receptors by ephrinB ligands is referred to as “forward signaling”, while activation of ephrinB ligands by EphB receptors is designated “reverse signaling” [2].. The communication between EphrinB2 and EphB4 was recently shown to be involved in the stimulation of osteoblast differentiation within the osteoblast lineage 2. In addition, EphrinB2 and EphB4 were reported to be regulated by mechanical forces in endothelial progenitor cells and periodontal ligament fibroblasts [1]. Meanwhile, ephrinA2-EphA2 interactions may be involved in the initiation phase of bone remodelling by enhancing osteoclast differentiation and suppressing osteoblast differentiation [3]. However, there have been no investigations on ephrin and Ephmolecules, including ephrinA2, EphrinB2, EphA2, and EphB4, in the surrounding bone under compressive mechanical stress environments after replacement of a dental implant.

Deformation and elastic deformation of the implant body after implantation, early osseointegration acquisition to the implant surface, and biological responses of surrounding structures to mechanical stresses related to a decrease in the primary fixation are involved in successful achievement of safe stability of an implant in alveolar bone [4]. In this study, we investigated the roles of EphrinB2 and EphB4, as known molecules related to osteoblast and osteoclast differentiation under mechanical forces, in remodelling of the bone tissue surrounding an implant under compressive and extensive mechanical stress environments arising from the implantation, to understand the biological behaviors’ of alveolar bone in the initial mechanical stress using a rat dental implant model.

Material and Methods


Recombinant rat EphrinB2-Fc and EphB4-Fc were obtained from R&D Systems (Minneapolis, MN). Pure wrought Titanium discs (Tidiscs; JIS, Japan Industrial Specification H 4600, 99.9 mass% Ti, 15- mm diameter; Kobelco, Kobe, Japan) were used to confirm the Tisurface modification of mini-implants by EphrinB2-Fc and EphB4- Fc. The Ti-discs were sandblasted (HI ALUMINAS; Shofu, Kyoto, Japan) before surface modification. Pure Titanium mini-implants (mini-implants; 2-mm diameter, 4-mm length; Kondo Technology, Tokyo, Japan) were used for the in vivo study (Appendix Figure 1). The Ti-discs and mini-implants were washed with acetone and ethanol in an ultrasonic bath for 30 min and autoclaved before use.

Cell culture under normal, compressive, and extensive conditions

The MC3T3-E1 cell line was purchased from the European Collection of Cell Cultures (ECACC, Wiltshire, UK). MC3T3-E1 cells were cultured in a-MEM supplemented with an antibiotic mixture (Thermo Fisher Scientific, Waltham, MA), 10% fetalbovine serum (Biological Industries, Haemek, Israel), and 50 μg/mL L-ascorbic acid (Merck, Darmstadt, Germany) at 37°C under 5% CO2/95% humidified air. During culture, the medium was refreshed at 3-day intervals, unless otherwise required for specific experiments. One group of MC3T3-E1 cells (normal conditions; Group-N) was maintained in the conventional environment described above, while two other groups (compressive conditions: Group-C; extensive conditions: Group-E) were cultured under cyclical compressive stress and extensive stress, respectively. A loading device (STB-140; STREX, Tokyo, Japan) was used to create the two types of stress.

Immobilisation of EphrinB2-Fc or EphB4-Fc on the surfaces of Ti-discs and mini-implants

Immobilisation of EphrinB2-Fc or EphB4-Fc on Ti surfaces was carried out as described previously [5]. Briefly, Ti-discs were immersed in 5% γ-aminopropyltriethoxysilane in acetone for 15 min at room temperature, washed with acetone, treated with 5% glyoxylic acid monohydrate for 2 h, and washed with ultrapure water. The surfaces of the specimens were then treated with 0.4% sodium borohydride for 24 h to reduce the imine groups to amine groups. After this series of pre-treatments, the specimens were washed with ultrapure water and autoclaved. The carboxyl groups on the specimen surfaces were activated with N-hydroxylsuccinimide/N-ethyl-N’- (3-dimethylaminopropyl)-carbodiimide (Biacore AB, Uppsala, Sweden), and then treated with 20 μg/ml EphrinB2-Fc or EphB4-Fc in sodium bicarbonate buffer (pH 8.0) for 30 min at 37°C and 16 h at 4°C to immobilise EphrinB2-Fc or EphB4-Fc on the surface, creating EphrinB2-Fc-Ti and EphB4-Fc-Ti, respectively. After washing with phosphate-buffered saline to remove any excess EphrinB2 or EphB4, the activated carboxyl groups were blocked by treatment with 1 M ethanolamine-HCl (Biacore AB) for 5 min. Untreated Ti-discs were used as control specimens (control-Ti). The body surfaces of the mini-implants were immobilised with EphrinB2-Fc or EphB4-Fc using the same method described above for the Ti-discs.

Implantation of mini-implants into the rat palatine process

Eight-week-old Wistar rats (Kyudo Co. Ltd., Fukuoka, Japan) were used for the animal study. The animal selection, management, anesthesia, surgery, and analysis procedures were approved by the Animal Care and Use Committee of Kyushu University (Approval No. A25-138). All in vivo experiments using the rat model were performed in accordance with the procedures allowed by the committee. Briefly, the rats were carefully anaesthetized with sevoflurane and pentobarbital. Prior to surgery, the implantation site was disinfected with 10% iodine. During the surgery, a mini-implant was placed into the palatine process of the maxilla using micro drivers, and the recipient gingival site was tightly sutured. The initial torques for implantation was 10, 20, and 30 Nmm. Each animal received only one implant. For real-time RT-PCR, the alveolar bone from the samples was collected after 3 h. Two independent experiments involving triplicate samples were performed.

Real-time RT-PCR analysis

Total RNA was extracted from homogenates using TRIzol (Thermo Fisher Scientific). First-strand cDNA was synthesized using ReverTra Ace (Toyobo, Osaka, Japan) with 100 ng of total RNA. The cDNA was amplified by BIOTAQ DNA polymerase (Bioline, Randolph, MA). Real-time RT-PCR analysis for Osterix, ephrinA2, EphrinB2, EphA2, EphB4, and β-actin was performed using a Rotor-Gene 6000 (Qiagen, Tokyo, Japan). β-actin was chosen as an internal control to standardize the variability in amplification arising from slight differences in the starting total RNA concentrations. The sequences of the primers and probes are listed in Appendix Tables 1 and 2. The sequences of the primers and probesfor β-actin were described previously [6].

Western blotting

Cell lysates (CLs) from cultured MC3T3-E1 cells were lysed in Laemmli buffer and stored overnight at -80°C. After centrifugation of the CLs at 15,000×g for 20 min at 4°C, the supernatants were transferred to new microtubes, boiled for 5 min, and stored at -20°C. The proteins present in the CLs were separated in SDS-PAGE gels and transferred to PVDF membranes (Immuno-Blot PVDF Membranes; Bio-Rad, Hercules, CA). The membranes were probed with primary antibodies against EphrinB2 (R&D Systems) and EphB4 (Proteintech, Wuhan, China), followed by incubation with antigoat IgG conjugated with horseradish peroxidase and anti-rabbit IgG conjugated with horseradish peroxidase (Abcam, Cambridge, UK) for detection of EphrinB2 and EphB4, respectively. Immune complexes containing horseradish peroxidase bound to the target molecules on the PVDF membranes were detected by an enhanced chemiluminescence detection kit (ECL plus Western Blotting Detection System; GE Healthcare, Buckinghamshire, UK).

X-ray photoelectron spectroscopy

To evaluate immobilized fusion proteins (EphrinB2-Fcand EphB4-Fc), the surface chemical compositions of treated samples were analysed by X-ray photoelectron spectroscopy (XPS; K-alpha; Thermo Fisher Scientific). Monochromatised Al Ka X-rays at 1486.6 eV were used for excitation under 2.0×10-7 Pa. The spot size of the incident X-rays was 400μm in diameter. The XPS machine was calibrated using Au 4f7/2 at 83.96 eV and Ag 3d5/2 at 368.21 eV. For survey scans, the detector was set at 200 eV as passing energy and 10 ms as dwell time. For narrow scans, the detector was set at 50 eV and 50 ms, respectively. All measurements were referenced by setting the hydrocarbon of C 1s to 284.8 eV. Detected elements were quantified using Avantage version 5.36 software (Thermo Fisher Scientific).

Data analysis

The significance of differences in the values of two groups was assessed by Student’s t-test. Differences among the mean values of the groups were subjected to one-way analysis of variance (ANOVA) and Tukey’s multiple-range test. SPSS version 20.0 software (IBM, Tokyo, Japan) was used for the statistical analyses.


mRNA expression levels of Osterix, ephrins, and Ephs in MC3T3-E1 cells stimulated by cyclic stress

The effects of cyclic compressive or extensive stress (5% and 10%) on osteoblast differentiation were monitored using the MC3T3-E1 osteoblastic cell line for up to 18 h. First, the expression levels of an osteoblast-specific gene transcription marker, Osterix, were analyzed to evaluate and standardize the differentiation stage of MC3T3-E1 cells under normal, compressive, and extensive conditions during culture periods of 1 to 18 h after the cells had reached confluence. β-actin was chosen as an internal standard to control for variability in amplification arising from differences in the starting total RNA concentrations.

The real-time RT-PCR results showed that the expression levels of Osterix mRNA were decreased in cells cultured under 5% compressive stress (Group-5C) for 1, 3, and 18 h, compared with cells cultured under normal conditions (Group-N) (Student’s t-test, p<0.05; Figure 1A). Similar results for the expression levels of Osterix mRNA were obtained for cells cultured under 5% extensive stress (Group-5E) compared with Group-N cells (Student’s t-test, p<0.05, p<0.01; Figure 1B).