The Mechanism Research Of EphB4 In Promoting The Osteogenesis Of CTLA4 Modified MSCs Via Periostin

BackgroundThe functional reconstruction of large bone defects continues to be a c hallenge in orthopedic medicine,and using autogenic mesenchymal stem cells(MSCs)based tissue engineering bone(TEB)to deal with the issue has been successful in clinical practice.However,the limited availability of autogenic MSCs due to expansion time and individual differences in patients presents a great challenge to clinical application.Therefore,to explore the potential of allogeneic MSCs for using as seed cells in TEB becomes the research focus in this field.In addition,the immunogenicity of allogeneic MSCs is still poorly understood,but some researchers have reported that allo-MSCs can elicit an immune response in allotransplantation and cause graft failure.Previously,we successfully transfected MSCs with the(cytotoxic T lymphocyte associated antigen-4 immuoglobulin,CTLA4-Ig)gene and showed that these cells possess a superior ability for osteogenic differentiation in xenotransplantation.However,the underlying mechanisms are not clear.Recently,Erythropoietin-producing hepatocyte receptors(Ephs)and its ligand Eph receptor interacting proteins(ephrin)have been reported to play a crucial role in regulating bone metabolism.Among them,Eph B4/ephrin B2 axis has attracted much interest for the bidirectional signaling transduction.The reverse signaling through ephrinB2 into osteoclast precursors,which suppresses osteoclast differentiation,and forward signaling through Eph B4 into MSCs/pre-osteoblasts,which enhances osteogenic differentiation.Meanwhile,the tissue microenvironment consists of cell and extracellular matrix(ECM)is also important for tissue regeneration.ECM is vital to the regulation of cell adhesion,migration,and differentiation via cell?ECM interactions.In bone tissue,periostin(POSTN)is present in bone ECM and plays a crucial role in cell?ECM interactions through binding to Integrin to activate biosignal transduction.From the above,we speculate that the immune activation conditions may have negative impact on Eph B4/ephrinB2 axis and can destroy the biosignal transduction between POSTN and cells.This study uses different animal models to verify the osteogenesis effects in vivo,and reveal the impact of immune activation conditions on Eph B4 and POSTN expression.Then we make further exploration on the relationship between Eph B4 and POSTN,and try to find out the osteogenesis mechanism between Eph B4 and POSTN.This study will provide novel insights into the osteogenic mechanism of MSCs-CTLA4 in allotransplantation.Part One The function of Eph B4 in ectopic osteogenesis of CTLA4 modified MSCsMethods:1.MSCs from human bone marrow were isolated by density gradient centrifugation.TEB was structured by using the MSCs seeded on a two-sided scaffold consisting of DBM.Then,the TEB was cultured with osteogenic medium for 14 days.Before implan tation into adult male BALBc mice,TEB were infected with Ad-EGFP-CTLA4.2.TEB was implanted in a muscle bag near the femur in mice.The morphology of new bone formation was assessed using an animal Micro-CT.Femur samples with TEB were embedded in paraffin,and sectioned to 5 ?m for hematoxylin and eosin(H&E)and Masson staining.Immunohistochemistry was performed to detect the CTLA4 and Eph B4 expression.3.An immune activation condition was established by treating peripheral blood mononuclear cell(PBMCs)with phytohaemagglutinin(PHA).MSCs or MSCs-CTLA4 were added to structure the co-culture system.4.ELISA was performed to detect the IL-2 and IFN-? expression.Q-PCR and Western blotting were performed to detect the change of Eph B4 expression in MSCs or MSCs-CTLA4 under the immune activation condition.5.Q-PCR,immunofluorescence and special stain were performed to detect the related osteogenesis markers after the EphB4 was activated by ephrin B2-FC.6.In order to explore the osteogenic mechanism of Eph B4,Western blotting were performed to detect the related singal protein after the EphB4 was activated.Eph B4 over-expression and siRNA were used as control.Results:1.Membrane structure consist of MSCs can be found in DBM+MSCs under a light microscope,and green fluorescence were observed in DBM+MSCs-CTLA4 under a fluorescence microscope.This proves that the TEB was constructed successfully.2.In the histologic sections,CTLA4-positive cells were only found in DBM+MSCs-CTLA4 group.And the EphB4 expression level in DBM+MSCs-CTLA4 group was significantly higher compared with other groups.HE,Masson staining and micro-CT reconstruction all showed the new bone area was obviously detected in DBM+MSCs-CTLA4 group.Assessments of bone mass including BV/TV,Tb.N,and Tb.Th were significantly higher in DBM+MSCs-CTLA4 group compared with other groups,and Tb.Sp was significantly lower(P<0.05).3.PBMCs were dispersion in the culture medium.After PHA was added,the T lymphocyte were blastoformation and gather into cluster.The level of cluster in MSCs-CTLA4 group was less than that in MSCs group under a light microscope.ELISA showed that the IL-2 and IFN-? expression in MSCs-CTLA4 group were significantly lower than MSCs group(P<0.05).4.Western blotting and Q-PCR showed that the levels of EphB4 expression in MSCs group decreased significantly under the immune activation condition(P<0.05),but not in MSCs-CTLA4 group.5.Q-PCR showed that the levels of Runx2,COL1,OCN and ALP expression were significantly increased in ephrinB2-FC-treated groups(P<0.05).But under the immune activation condition,that phenomenon can only be found in MSCs-CTLA4 group.Immunocytofluorescense of OCN,ALP staining and calcium node staining both showed that the ephrin B2-FC treated can up-regulate the stain intensity only in MSCs-CTLA4 group under the immune activation condition.Analyze of integrated option density(IOD)was accordance with the observation(P<0.05).6.Western blotting showed that ?-catenin,Runx2 and COL1 expression in MSCs were significantly increased after ephrinB2-FC treated.We further detected the levels of p-GSK-3? expression in ephrin B2-FC-treated controls,empty cells,and MSCs over-expressing EphB4,but not MSCs expressing EphB4-siRNA,which were significantly higher compared with control(P<0.05).In the meantime,that phenomenon also can only be found in MSCs-CTLA4 group under the immune activation condition.Part Two Activation of EphB4 increase the POSTN expression in MSCsMethods:1.TEB was implanted in a muscle bag near the femur in mice.Immunohistochemistry was performed to detect the POSTN expression.2.Immunocytochemistry was performed to detect the POSTN expression in MSCs.Western blotting,Q-PCR and ELISA were performed to detect the POSTN expression after the EphB4 was activated.3.Q-PCR,special stain and Western blotting were performed to detect the related osteogenesis markers and singal protein after the Eph B4 was activated by ephrinB2-FC.NVP-BHG-712,Integrin ?v?3 blocking,Eph B4 over-expression and siRNA were used as control.Results:1.In the histologic sections,the POSTN expression level in DBM+MSCs-CTLA4 group was significantly higher compared with other groups.Analyze of integrated option density(IOD)was accordance with the observation(P<0.05).2.Immunocytochemistry showed that the positive stain of POSTN was located in cytoplasm in MSCs.Western blotting,Q-PCR and ELISA all showed that the POSTN expression level was significantly increased both in MSCs and Eph B4 over-expression groups after ephrin B2-FC-treated,but not in Eph B4-siRNA and NVP-BHG-712 groups(P<0.05).3.Q-PCR showed that the levels of Runx2,Osterix,COL1 and ALP expression were significantly increased in ephrin B2-FC-treated,EphB4 over-expression and POSTN-treated groups(P<0.05).But that phenomenon was not observed in NVP-BHG-712,Integrin ?v?3 blocking and Eph B4-si RNA groups.ALP staining,calcium node staining and the analysis of IOD was accordance with the m RNA expression.Western blotting showed that the levels of p-GSK-3?,?-catenin,Runx2 and COL1 expression in ephrinB2-FC-treated and Eph B4 over-expressing groups were significantly higher compared with control(P<0.05).And that phenomenon was not observed in NVP-BHG-712,Integrin ?v?3 blocking and Eph B4-siRNA groups.Part Three The function and mechanism research of POSTN in bone repair of CTLA4 modified MSCsMethods:1.TEB was used to repair the critical-sized segmental radius defects in rabbit model.The bone reparation was assessed using X-ray and Micro-CT.Histological patterns in histologic sections were observed by H&E and Masson staining.Immunohistochemistry was performed to detect the POSTN and ?-catenin expression.2.Immunocytochemistry was performed to detect the POSTN expression in MSCs and MSCs-CTLA4.Q-PCR and Western blotting were performed to detect the change of POSTN expression in MSCs or MSCs-CTLA4 under the immune activation condition.3.ALP staining and calcium node staining were performed to detect the osteoinduction of POSTN in MSCs or MSCs-CTLA4 under the immune activation condition.4.Western blotting was performed to detect the osteogenic mechanism of POSTN.Results:1.X-ray and Micro-CT showed that the plastotype of new bone in repair region in DBM+MSCs-CTLA4 group is obviously better than other groups.And the transverse section results showed that the marrow cavity in repair region in DBM+MSCs-CTLA4 group had been a marked reperfusion compared with other groups.Assessments of bone mass including BMD,BV and BV/TV in DBM+MSCs-CTLA4 group were significantly higher compared with other groups(P<0.05).Immunohistochemistry showed that the POSTN and ?-catenin expression level in DBM+MSCs-CTLA4 group was significantly higher compared with other groups.Analyze of IOD was accordance with the observation(P<0.05).2.Immunocytochemistry showed that the positive stain of POSTN was both located in cytoplasm in MSCs and MSCs-CTLA4.Western blotting and Q-PCR showed that the levels of POSTN expression in MSCs group decreased significantly under the immune activation condition(P<0.05),but not in MSCs-CTLA4 group.3.ALP staining and calcium node staining showed that the stain intensity can be increased with POSTN-treated both in MSCs and MSCs-CTLA4,but it can be suppressed by blocking the Integrin ?v?3.Under the immune activation condition,the increase effect of stain intensity was only found in MSCs-CTLA4,not in MSCs.Analyze of IOD was accordance with the observation(P<0.05).4.Western blotting showed that the levels of p-LRP6,p-GSK-3?,?-catenin and Runx2 expression in POSTN-treated groups were significantly higher compared with control,but not in NVP-BHG-712 group(P<0.05).And that phenomenon was also only be found in MSCs-CTLA4 group under the immune activation condition.Conclusion:1.The MSCs-CTLA4 based TEB achieved good results in ectopic osteogenesis in mice,and the Eph B4 expression in implanted region was found significantly higher compared with other groups.The immune activation condition could down-regulate the Eph B4 expression in MSCs but not in MSCs-CTLA4.Eph B4 could promote the osteogenic differentiation of MSCs through cross-talk with Wnt signal pathway indirectly.2.Activation of EphB4 can increase the POSTN expression in MSCs.3.The MSCs-CTLA4 based TEB achieve excellent results in repairing the critical-sized segmental radius defects in rabbit model,and the POSTN expression in implanted region was found significantly higher compared with control.The immune activation condition could down-regulate the POSTN expression and suppress the osteoinduction effect of POSTN in MSCs,but not in MSCs-CTLA4.4.POSTN could cross-talk with Wnt signal pathway by activating the Wnt/LRP6/GSK-3?/?-catenin to promote osteogenic differentiation of MSCs.