Research On The Regulatory Mechanism Of Sodium Butyrate And Endothelial Cells On The Osteogenesis Of Mesenchymal Stem Cells

Nowadays,bone defeats gradually endanger people's health and life.Bone tissue engineering holds great promise for solving this problem.With the development of research,however,it is found that tissue engineered bone failed to completely repair bone defeats,especially the large bone defects or ununion.The limited osteogenic differentiation of bone-forming cells during in vitro culturing is one of the reasons,especially for amniotic membrane-derived mesenchymal stem cells(AMSCs),which are less capable of osteogenic differentiation than bone marrow-derived mesenchymal stem cells(BMSCs)in vitro,and the regulatory mechanism of osteogenesis of AMSCs is poorly understood.Another reason is the poor cell activities inside the engineered bones,which easily lead to cell necrosis.Although endothelial cell-based pre-vascularization strategy has been applied,the cellular and molecular bases of osteogenic-endothelial cell interactions,as well as their impact on endothelial vascularization and osteogenic differentiation,are not fully understood,which will affect the osteogenesis and angiogenesis of the engineered bones in vitro,and finally the repair of bone defeats in vivo.To address these key issues,small molecules were appilied to regulate the osteogenic differentiation of human amniotic membrane-derived mesenchymal stem cells(hAMSCs),and the regulatory mechanism of sodium butyrate(NaBu)with different concentrations and exposure time on osteogenesis of hAMSCs was investigated;the cellular and molecular bases of MSCs-ECs interactions,as well as their impact on endothelial vascularization and osteogenic differentiation in 2D/3D coculture model were also explored.Our research will provide data support and strategy guidance for regulating osteogenesis,angiogenesis and engineering pre-vascularized bone tissue in vitro.Firstly,by adding NaBu to a-MEM and osteogenic induced medium(OIM),the impacts of NaBu,a histone deacetylase inhibitor(HDACi),on cell proliferation and osteogenesis of hAMSCs were investigated.It was shown that NaBu at a low concentration(?1.0 mM)inhibited cell proliferation by arresting cell cycle at G0/G1 rather than inducing apoptosis;5.0 mM NaBu intended to induce cell apoptosis.When NaBu was supplemented at a concentration of 0.50 mM for 3 days during osteogenic induction,the osteogenesis of hAMSCs was significantly enhanced.However,a higher concentration(1.0 mM)and longer exposure time(14 days)of NaBu showed transcriptional inhibition during osteogenesis.Further studies showed that NaBu at lower concentration for a short exposure time could promote the phosphorylation of ERK,induce the hyperacetylation of H3K9(H3K9-Ace),reduce the expression of histone deacetylase 8(HDAC8),and thus enhance the levels of osteogenesis-related genes(ALP,Runx2,OPN,and OCN)and proteins(Collal,OPN,OCN,Runx2,and TAZ);while NaBu at higher concentration for a long exposure time inhibited osteogenesis of hAMSCs by down-regulation of H3K9-Ace and up-regulation of HDAC8,thus leading to the decrease of osteogenesis-related genes and proteins.Secondly,by constructing a 2D coculture model in vitro,cell interactions between MSCs and ECs were investigated.Rat bone marrow-derived mesenchymal stem cells(BMSCs)were isolated with the whole bone marrow adherence method.After subculture,cell surface antigens,cell growth and cell differentiation were identified,implying that the isolated BMSCs have typical characteristics of MSCs.Based on the different effects of cell ratios and culturing media on osteogenesis and angiogenesis,BMSCs and HUVECs were directly cocultured at various ratios in 4 different media,and then the coculturing medium(OIM+1%ECGS)and cell ratio(BMSCs:HUVECs=1:2)were confirmed.Based on different 2D coculture models,the effects of direct and indirect coculture on osteogenic differentiation and angiogenesis were investigated,and the roles of gap junctions,cytokines and exosomes in coculture system were explored.Results showed that direct coculture between BMSCs and HUVECs could promote the osteogenic differentiation of BMSCs in osteogenic induction environment,but indirect coculture using Transwell and conditioned medium failed to promote the osteogenic differentiation of BMSCs,which indicated the stimulating effects of HUVECs on osteogenic differentiation of BMSCs depended on the direct contact between the two cell types,not on cytokines or exosomes.Further experiment demonstrated HUVECs promoted osteogenic differentiation of BMSCs via stimulating a variety of signaling pathways,including BMP signaling pathway,Hedgehog signaling pathway and Wnt signaling pathway.As for the influence of BMSCs on the angiogenic pothelial of HUVECs,under the condition of osteogenic induction,the formation of capillary-like structures of HUVECs was seriously hindered no matter BMSCs were in direct contact with HUVECs or not.Moreover,the direct coculture between BMSCs and HUVECs induced the decreased expression of both angiogenic genes and proteins.In addition,direct coculture of BMSCs and HUVECs had no significant effects on the expression of cell surface antigens in both cell types,and CD 31 could be used as a marker for subsequent cytometric cell sorting in cocultures.Based on the above results,to further explore the molecular mechanism of MSCs-ECs interactions in cocultures,the effects of gap junctions and molecular transporting through gap junctions on osteogenesis and angiogenesis were investigated.Immunofluorescence staining and dye transfer experiments confirmed that BMSCs and HUVECs could form Cx43-mediated gap junctions which possess the function of substance transport.By using gap junction inhibitor 18GA and activator PTH,it was further proved that gap junctions formed between the two cell types could affect osteogenic differentiation and angiogenic potential of cocultures,and the expression of Cxc19 and VEGF in BMSCs were correlated with gap junction protein Cx43.Through numbers of literature research and bioinformatics software prediction,it was found that microRNAs 200b(miR-200b)may play a regulatory role in the coculture system.By detecting the expression of miR-200b in BMSCs and HUVECs monoculture and cocultures and cocultures treated with gap junction activator PTH,inhibitor 18GA and exosomes inhibitor GW4869,it was found that miR-200b could transfer from BMSCs to HUVECs via Cx43-mediated gap junctions to regulate osteogenesis and angiogenesis.As a negative regulator,the decrease in miR-200b level in BMSCs derepressed the expression of VEGF-A,leading to increased osteogenic differentiation.Once inside HUVECs,miR-200b reduced the angiogenic potential of HUVECs through downregulation of ZEB 2,ETS 1,KDR and GATA 2.Additionally,TGF-? was found to trigger the transfer of miR-200b to HUVECs.Upon adding the TGF-(3 inhibitor SB431542 or TGF-?-neutralizing antibody,the formation of capillary-like structures in coculture could be partially rescued.Finally,in order to investigate the MSCs-ECs interactions in 3D coculture system,BMSCs and HUVECs were simultaneously seeded on Cultispher S microcarriers and cultured in a"growth-induction" two-stage culture system in a spinner flask.The osteogenic differentiation and angiogenesis of cocultures in a 3D dynamic culture environment were investigated,and the comparison of levels of key genes between 3D dynamic culture and 2D static culture were made.Results showed that after 3D dynamic coculture,the osteogenesis of cocultures was significantly enhanced compared with BMSCs monocultures,while the angiogenesis of cocultures was compromised,which was consistent with the phenomenon in 2D static coculture.qRT-PCR experiment confirmed that the level of osteogenic differentiation markers in cocultures were significantly higher than those in BMSCs monocultures,and the changes of Cxcl9 and VEGF-A were the same as Cx43,all of which were basically the same as 2D static coculture except for the significant time point.Based on the flexibility of microcarriers,a preliminary attempt was made to construct pre-vascularized bone micro-tissues.It was found that the strategy of combination of micro-tissues cocultured in growth medium for 1 day and cocultured in osteogenic induced medium for 14 days at the ratio of 1:1 could effectively realize the function of osteogenesis and angiogenesis of micro-tissues simultaneously.Taken together,this study explored the effects of small molecule NaBu on the osteogenic differentiation of human amniotic membrane-derived mesenchymal stem cells and investigated the interactions between bone marrow-derived mesenchymal stem cells and endothelial cells in the 2D static and 3D dynamic culture environment,which provided theoretical basis and strategy guidance for regulating osteogenesis and angiogenesis in vitro and engineering pre-vascularized bone tissue.