EphB Signaling Participate In Axial Compression Modulated Osteogenesis Of MSC

IntroductionCompression of various intensities may exert physiological or pathological effects onto skeleton, according to clinical practice and lab experiments. However, the mechanism was not clear. It is important to study the mechanism of compression on skeleton physiology and pathology. Fully understanding in promoting or inhibitory effects of compression will help doctors to apply therapeutic compression, and may introduce a new cue for skeleton disease therapy. It is difficult to achieve repeatable results in biomechanics due to the complicated stress environment in vivo. Therefore, bioreactors were produced to mimic stress environment in vitro.Nevertheless, precise and bionic stress environment and long-term stable biochemical environment were not achieved in present bioreactors, either commercialized or DIY. It is urgent to breakthrough this technical bottleneck. Ephrins and ephrin receptors(Eph) were found to participate in skeleton development, remodeling and diseases, thus the ephrin and Eph family may be a new cue for understanding the mechanism of compression on skeleton. This study produced a novel bioreactor for viscoelastic tissues and biomaterials, and then explored the mechanism of compression modulated osteogenesis of mesenchymal stem cells(MSC) cultured in three dimensional scaffolds, including the efforts of Eph signals and mechanotransduction.Methods1. This study aimed to promote precision and bionic efforts of compression in bioreactors. Elastic component was introduced into the stress generating device, thus minimized the stress mutation and overshoot in compression applying for viscoelastic objects. Stress environment of cancellous bone and achilles tendon were analyzed in finite element model, then the data was introduced to progame bionic compression control software. For long-term stable biochemical environment, medium biochemical environment control system was upgraded, including optimized hollow fiber semipermeable membranes, medium pathways, and control programe.2. To identify the effort of bionic compression on mesenchymal stem cells(MSC), tissue engineered bones were culture in the novel bioreactor and divided into three groups: static cultured, bionic compression, dynamic cultured(bionic compression and cont roled biochemical environment).3. Ephrins and Ephs were detected with PCR in osteogenesis of MSC. Treatments of ephrin B1, si RNA, overexpression of recombined Eph B6 were applied to study the effort of Eph B6 on MSC.And then Eph B6 signals were detected, focused on Rho A-ROCK pathways.4. Define optimal compression and overloaded compression in this bioreactor for further biomechanical study. Detected the potential connects of Eph B4 and Eph B6 between integrin β1 and β3 by co-immunoprecipitation and immunofluorescence; other proteins such as FAK and vinculin were detected as well. The potential binding sites of Eph B6 between integrin β3 were predicted in computer model.Results1. The elastic component was introduced into the stress generating device, thus produced stable precisely compression for viscoelastic biomaterials. Bionic compression control software was achieved based on finite element analysis of stress environment of cancellous bone and achilles tendon. The upgraded substance exchanger could produce long-term stable biochemical environment according to pre-determined parameters.2. This prompted bioreactor could produce bionic compression to prompt proliferation and osteogenesis of MSC in three dimensional(3D) cultures. Moreover, cells lined in synotropy and cytoskeleton stretching in same direction.3. Real time PCR and immunofluorescence suggested that Eph B6 was down- regulated in osteogenesis of MSC. Treatment of ephrin B1-Fc decreased ALP activity and Runx2 m RNA. si RNA of Eph B6 upregulated Runx2 m RNA, ALP activity, and increased alizarin red stained nodules, while overexpression of recombined Eph B6 leaded to conversed results. However, it is interesting to find that transfections of si RNA and recombined Eph B6 seem not interference chondrogenesis and adipogenesis of MSC.4. Phosphorylation of Rho A was decreased in osteogenic MSC, and was increased in Eph B6 si RNA infected MSC. Exposed in ROCK inhibitor Y-27632, MSC expressed increased Runx2 m RNA, as well as that in cells infected with si RNA and recombined Eph B65. Compression of 0.5Hz, peak deformation of 10% and bionic control curve was defined as optimal compression, while compression of 0.5Hz, peak deformation of 20% and bionic control curve was defined as overloaded. MSC exposed to optima l compression was detected with increased ALP activity and upregulated Runx2 m RNA.6. Exposed to optimal compression, Eph B4 and integrin β1 complex was detected in co-immunoprecipitation, and Eph B6 and integrin β3 were detected when overloaded. By laser confocal microscopy, Eph B4 and integrin β1were located together on partial plasmalemma, while Eph B6 and integrin β3had similar distribution. Moreover, Eph B4 and Eph B6 were recruited to the initial part of F-actin.7. FAK, Vinculin and Paxilin were detected by co-immunoprecipitation with Eph B4, both in optimal and overloaded compression, while none of them formed complex with Eph B6.8. Eph B6 and integrin β3 may assemble complex by direct connection, according to computer prediction. The potential binding sites of Eph B6 were 230-290, 362-393, 555-580,610-635, and integrin β3 of 189-204, 284-313.Conclusion1. This novel bioreactor could produce precisely bionic compression onto tissues and 3D scaffolds, and provide long-term stable biochemical environment.2. Eph B6 may inhibit osteogenesis of MSC via prompting Rho A phosphorylation. Si RNA or overexpression of Eph B6 didn’t interference chondrogenesis and adipogenesis of MSC, suggesting that Eph B6 was a specific modulator for osteogenesis, but not attenuated the differentiation potential of MSC.3. Optimal and overloaded compressions were defined with parameters of this bioreactor. In different compression, Eph B4 and Eph B6 connected with integrin β1 and β3, respectively, and were recruited to F-actin. This result suggested a mechanism of Eph B4 and Eph B6 involved inmechanotranduction via forming complexes with integrins.4. FAK, Vinculin and Paxilin were detected in complex of Eph B4 and integrin β1, suggested that these adaptors may amplify signals or initial some signal pathways.5. Eph B6 and integrin β3 may assemble complex by direct connection, according to computer prediction. One of the potential binding site of Eph B6 was located in the FNⅢ repeats.