Experimental Study Of Dual Anti-CD34, VEGFR-2Biological Engineering Stent Combined With Local Endothelial Progenitor Cells Transplantation For The Management With Late In-stent Restenosis

Part Ⅰ Rabbit endothelial progenitor cells isolation,culture,identificationand in vitro dual anti-(CD34,VEGFR-2) biological engineering stentfabricationObjective: To explore the method of endothelial progenitor cells (EPCs) isolation,cultureand amplification from rabbit peripheral blood and in vitro dual anti-CD34,VEGFR-2biological engineering stent preparation.Materials and Methods: Mononuclear cells (monocytes,MNCs) layer was obtained fromrabbit peripheral blood by density gradient centrifugation.EPCs were screened from theadherent cells,cultured,differentiated,and amplified in vitro.The EPCs morphology wasobserved by microscope, EPCs markers CD34and VEGFR-2(Vascular endothelial growthfactor receptor-2) were tested by immunohistochemical staining,EPCs function wasconfirmed by uptaked Dil labeled acetylated low-density lipoprotein(Dil-ac-LDL)andbinding FITC-labeled Ulex lectin-1(FITC-UEA-1).Transplant CM-Dil labeled EPCs weretraced in vivo. And using fibronectin (fibronectin,FN) as the carrier,with repeat dippinginto dual anti-(CD34,VEGFR-2) antibodies solution and drying in vitro,the biologicalengineering stent was consturcted.Result: New Zealand white rabbit peripheral blood MNCs were cultured in vitro.Theshape changed from round-polygonal-fusiform under light microscopy,cell surface markersCD34and VEGFR-2expression,phagocytosis of both Dil-ac-LDL and FITC-UEA-1,suggested that these cells were putative EPCs.Dual anti-CD34,VEGFR-2biologicalengineering stent surface could adhere EPCs,the cells washed down from the stent surfacehave functional properties of EPCs. Conclusion: EPCs can be cultured,differentiated and proliferated from rabbit peripheralblood through the separation of MNCs in vitro,with the characteristics of stem cells.Usingfibronectin as a carrier,the anti-CD34,VEGFR-2are adhered to the surface of the metalstent struts and enhancing the efficiency of EPCs target homing. Part Ⅱ Experimental study of dual anti-CD34,VEGFR-2biologicalengineering stent combined with local endothelial progenitor cellstransplantation for the management with late in-stent restenosisObjective: To establish a rabbit model of atherosclerosis.Place double anti-(CD34,VEGFR-2) biological engineering stents and local transplant EPCs,and evaluate theprevention effect of the stent for late in-stent restenosis.Materials and Methods: Sixteen male New Zealand rabbits were fed with high fat diet tobuild model of atherosclerosis.They were randomly divided into experimental group (n=8)and control group (n=8),the control group was placed with drug-eluting stents (sirolimus)stents into the common iliac or aorta arteries,the experimental group was deployed withdual anti-(CD34,VEGFR-2) biological engineering stents and local transplant EPCs, whileblocking the blood flow to improve EPCs uptake.Follow-up for two months.CTangiography was used for detected vascular patency.DSA was performed to assess thedegree of in-stent restenosis.Specimens of artery with stent in were checked with HEstaining,software for image analysis,and quantitative were used for analysis of the degreeof restenosis.Results: Sixteen cases of rabbit artery stenting were all technicallly successful.Theexperimental stent lumen was almost patent,including one case of restenosis,the restenosisrate was1/7.The control group was significantly thicker of the intima,one case of completeocclusion,one cases of restenosis,the restenosis rate2/7was significantly higher than theexperimental group. The percent of area stenosis(PAS) in experiment group is (11±5.5)%,is significantly lower than the control group’s PAS(31.2±16.7)%(x2＝18.6,P<0.05).Dualantibody stent group’s neointimal area,the maximum neointimal thickness and lumen stenosis were significantly less than the DES stent group’s,Dual antibody stent group’sluminal area was significantly larger than the control group’s.The difference werestatistically significant (P<0.05).Conclusion: The dual antibody CD34,VEGFR-2biological engineering stents improvehoming number of EPCs,prompt rapid re-endothelialization of the stent,reduce intimalhyperplasia,decrease the late in-stent restenosis rate.The engineered stent has betterefficacy and safety than DES stent.