Research On Regulatory Mechanism Of Osteogenesis And Angiogenesis Of BMSCs-HUVECs Co-culture Under Static And Fluid Shear Stress Condition

Pre-vascularized bone tissue constructed in vitro could reduce mass transfer limitations in grafts and promote bone repair.In endothelial cell-based pre-vascularization strategies,mesenchymal stem cells(MSCs)and endothelial cells(ECs)are co-cultured in vitro,to generate osteogenic differentiation and vascular network structures.However,the cellular interactions and molecular mechanisms between MSCs and ECs in the co-culture system is still unclear,especially under osteogenic induction conditions.Moreover,there are mechanical stimulation in bone tissues in vivo.Among them,fluid shear stress(FSS)is an important factor in regulating osteogenic differentiation of MSCs and angiogenesis of ECs in bone tissue.However,the researches on the effects of FSS stimulation on osteogenesis and angiogenesis of co-cultures in vitro have just begun.In this study,rat bone marrow-derived mesenchymal stem cells(BMSCs)and human umbilical vein endothelial cells(HUVECs)were used to explore the osteogenesis and angiogenesis of co-cultures in growth medium(GM)or osteogenic induction medium(OIM).Moreover,the effect of BMSCs on angiogenic capability of HUVECs in OIM and FSS on osteogenic differentiation of BMSCs-HUVECs co-cultures,as well as the related molecular mechanisms were also investigated.Results shown that BMSCs and HUVECs co-cultured in OIM had higher osteogenesis ability but failed to form capillary-like structure while the results of co-culture in GM were the opposite.Further research found that the chemokine(C-X-C motif)ligand 9(Cxcl9),secreted by BMSCs in the OIM co-culture,could interact with vascular endothelial growth factor(VEGF)and prevent its binding to HUVECs,which abrogated angiogenesis of BMSCs-HUVECs co-culture.In addition,our study found that the expression of Cxcl9 in BMSCs was related to the mTOR-STAT1 signaling pathway.Therefore,blocking this signaling pathway via rapamycin inhibited the expression of Cxl9,thereby improving angiogenesis in the OIM co-cultures.A rocking see-saw platform was used to apply FSS to the cells cultured in the well plate.Results showed that the cytoskeleton of BMSCs monocultures and co-cultures was rearranged along the direction of shear force under FSS stimulation.In addition,fluid shear stress significantly promoted the migration of HUVECs,but had no significant effect on angiogenesis of HUVECs monocultures and BMSCs-HUVECs co-cultures.Results demonstrated that fluid shear stress promoted the proliferation of co-cultures when stimulated for 1?2 or 3 h per day,while no significant difference on proliferation was observed in BMSCs monocultures for different stimulated time.Furthermore,BMSCs monocultures had the higher osteogenic differentiation when FSS stimulated for 1 or 2 hours per day;while BMSCs-HUVECs co-cultures had the highest osteogenesis when FSS stimulated for 2 hours per day.The co-cultures had the higher osteogenesis compare with the BMSCs monocultures under FSS stimulation.Further research found that FSS stimulation and co-cultured with HUVECs synergistically promoted the expression of Integrin ?1 in BMSCs in co-cultures.FSS stimulation could activate the downstream FAK-ERK1/2-Runx2 signaling pathway through Integrin ?1 activation,thereby promoting osteogenic differentiation of BMSCs in co-cultures.In summary,this study explored the cellular interactions and related mechanisms of osteogenesis and angiogenesis in BMSCs-HUVECs co-culture systems under static condition and FSS stimulation,which provided the theoretical basis for the regulation of pre-vascularized bone tissue in vitro.