In Vitro Prevascularization And In Vivo Inosculation Research Of OECS-DBM Complex Via Dynamic Perfusion Culture

Objective:To investigate the adhesion,proliferation,differentiation and tube formation properties of human outgrowth endothelial progenitor cells(OECs)on OECs-DBM complex under dynamic perfusion culture in vitro,and to analyze the mechanism of in vitro prevascularization via dynamic perfusion culture and in vivo inosculation of recipient microvasculature with preformed microvessels of OECs-DBM complex.Methods:Human outgrowth endothelial progenitor cells from peripheral blood were isolated by density-gradient centrifugation and cultured with EGM-2 BulletKit medium.The cell morphology observation,properties of uptaking DiI-acLDL and binding FITC-Lectin,Fluorescence flow cytometry and tube formation assays were used together to define OECs.Then OECs were transfected with lentivirus-mediated enhanced green fluorescent protein(lenti-eGFP)to permit continuous observation after being seeded on decalcified bone matrix(DBM)scaffold in vitro.Transfection rate calculation and Methyl thiazolyl tetrazolium(MTT)assay of OECs under different Multiplicity of Infection(MOI)were used to optimize MOI for OECs.In parallel,proliferation activity,fluorescence flow cytometry and tube formation assay were conducted again to observe the influence of lenti-eGFP transfection on OECs.Then lenti-eGFP transfected OECs were statically seeded on DBM scaffold and randomly assigned to static culture group,dynamic perfusion group and inhabitor group,which were blank,transfered to dynamic perfusion system and added inhabitor respectively.The adhesion and proliferation activities of OECs on DBM scaffold were observed under fluorescence microscopy.OECs-DBM complex were harvested on 6th day for calculation of tube formation inside DBM scaffold under scanning electron microscope(SEM)and for RT-PCR and western blot analysis of CD34,VEGFR-1,VEGFR-2,VE-cadherin,Tie2 and VEGF mRNA and protein expression.Nude mice dorsal skin chamber model was prepared and texas red-labelled dextran was given i.v.via vena caudalis for visualizing and fluorescent observation of vascularization.After determination of time window for observation,OECs-DBM complex under static and dynamic perfusion culture were implanted into dorsal skinfold chamber respectively for the in vivo analysis of their vascularization on 1st,3rd,5th,7th,9th,12th day by calculating microvessels growing across the border of OECs-DBM complex and fluorescent integrated density of scaffold at both border and central area.To observe the inosculation of recipient microvasculature with preformed microvessels,OECs-DBM complex were harvested on 12th day for HE staining and fluorescent imaging.Results:OECs exhibited characteristic cobblestone morphology and ability to uptake acLDL and bind lectin as well as in vitro tube formation in Matrigel.Fluorescence flow cytometry showed that OECs express cell marker CD34 and VEGFR-2,but barely express CD 133.Cell marker profiles,proliferation activity and in vitro tube formation ability of OECs were not influenced when transfected with lentivirus-mediated enhanced green fluorescent protein effectively at MOI=50.Projected cell area,cell density and number of tube formation of OECs-DBM complex in dynamic perfusion group were greater than that in static culture group.The mRNA and protein expression level of the endothelial markers VEGF receptor 1(VEGFR-1)and-2(VEGFR-2),VE-cadherin,Tie2 increased,expression level of CD34 decreased in dynamic perfusion group when compared with static culture group.Inhibitor analysis showed that the phosphoinositide3-kinase(PI3K)and mammalian target of rapamycin(mTOR)inhabitor suppressed adhesion,proliferation,tube formation,and differentiation of OECs in response to shear stress.Injection of texas red-labelled dextran was conducive to define microvasculature when combined with dorsal skinfold chamber.And the results showed that ingrowth of recipient microvessels arcoss the border increased over time at both groups.More microvessels arcoss the border of scaffold were observed in dynamic perfusion group on 3rd,5th,7th day.Fluorescent integrated density of border in dynamic perfusion group were higher at all time points.The difference was more significant in central area.Fluorescent imaging of OECs-DBM complex slice showed that no eGFP-positive cells were found beyond the verge of DBM scaffold in both two groups.Conclusion:These novel findings demonstrate that shear stress generated by dynamic perfusion culture system increases adhesion,proliferation,differentiation into mature endothelial cells,and tube formation of human outgrowth endothelial progenitor cells and promotes prevascularization of DBM scaffold by activating the PI3K/Akt/mTOR signal transduction pathway.In addition,in vitro prevascularization by dynamic perfusion culture can increase and accelerate the blood perfusion of OECs-DBM complex obtained from recipient microvasculature by internal inosculation.Accordingly,this approach may markedly contribute to the future success of tissue engineering applications in clinical practice.