Effect Of β-TCP\HIF-1α Control-released Composite On Endothelial Network Formation Of The Pre-vascularized Cell Sheet

The aim of this study was to explore the effects of HIF-1α on HUVECs and endothelial network formation based on the pre-vascularized cell sheet technology(CST).Methods:(1)The construction of β-TCP\HIF-1α control-released composites: first,using HIF-1α protein to compound different gradients of solutions(50,100,200mg/L)which were sterile filtered and subpackaged for further use.Then put the aseptic β-TCP scaffolds into the different gradients of HIF-1α solutions(soaked for 3days).ELISA Kit was used to test the releasing concentrations ofβ-TCP\HIF-1α control-released composites at different time points in vitro.(2)HUVECs migration test and the construction of pre-vascularized cell sheet: cell culture of fetal bone marrow mesenchymal stem cells(fBMSCs)and HUVECs;Transwell assay tested the recruitment and migration of HUVECs under different groups,and then the samples were stained by gentian violet,taken photos,and analyzed by Image J software;fBMSCs were seeded into six-well-plates at a density of 9×104cell/cm2 to form fBMSCs sheets for 14 days;then HUVECs were seeded onto the fBMSCs sheets at a cell density of 5×104cell/cm2 to form the pre-vascularized cell sheets under continuously cultured.We observed the general view and morphology of pre-vascularized cell sheet.(3)Effect of β-TCP\HIF-1α control-released composites on endothelial network formation of pre-vascularized cell sheets: this section was divided into five groups(plain β-TCP scaffold,50,100,200mg/L β-TCP\HIF-1α control-released composites,and 100mg/L HIF-1α),and immunofluorescent staining of CD31 was used to detect the networks formation of pre-vascularized cell sheets at 1,3,7,14 days.Results:(1)The releasing concentrations of eachβ-TCP\HIF-1α control-released composites peaked at day 3,then fell down gradually;the accumulated-releasing rates of each groups arrived more than the average of 85% at day 21.(2)fBMSCs were spindle shaped spreading and HUVECs were oval and displayed a typical cobblestone-like morphology;a thin layer of fBMSCs sheet was formed after continuously cultured for 14 days,which margins curled inward and could be lifted up by tweezers.(3)Transwell assay suggested that HIF-1α had the ability to recruit and migrate HUVECs,especiallywith the concentration of 100mg/L HIF-1α(P<0.05).(4)As for the results of effect on endothelial network formation of pre-vascularized cell sheets: 100mg/L HIF-1α at day 3 and 200mg/L HIF-1α\β-TCP control-released composite at day 7 formed more significant networks than the other groups(P<0.05).Conclusion:(1)β-TCP\HIF-1α control-released composite has sustained releasing effects;(2)HIF-1α has the ability to recuit and migrate HUVECs;(3)HIF-1α has effect on networks formation of the pre-vascularized cell sheet.