In Vitro And In Vivo Study Of Alginate Hydrogel Conglutinating Cells To Polycaprolactone Vascular Scaffolds Fabricated By Electrospinning

Cardiovascular disease(CVD)is one of the major causes of death worldwide.And bypass surgery is one of the most commonly used strategies for treating CVD since it was first used in 1949.For example,coronary artery bypass grafting(CABG)has been the gold standard for the revascularization of patients with coronary artery disease(CAD),particularly in high-risk patients with multivessel disease.In the United States,1.4 million arterial bypass operations are performed for treating ischemic heart disease and peripheral vascular disease annually(including approximately 500,000 CABG procedures).Autografts have a better primary and secondary patency than artificial small-diameter vascular grafts(SDVGs)(<6 mm internal diameter).Nevertheless,a large number of patients do not have suitable autologous arteries or veins due to pre-existing vascular disease,amputation,or previous harvests.Therefore,alternative grafts are clearly needed.Prior studies showed that seeding cells on the surface of synthetic or natural graft scaffolds before implantation is an effective strategy to achieve the ideal tissue engineered vascular graft(TEVG).However,the time lag before cell maturation is still an obstacle in the clinical application of preseeded grafts.In this study,we applied an innovative cell-seeding technology for seeding cells on artificial vascular scaffolds,and fabricated new-type TEVG by this method.The rat adipose-derived stem cells(rADSCs)were harvested from adult male Sprague-Dawley rats and cultured and differentiated rADSCs into rat endothelial progenitor cells(rEPCs)as seed cells for TEVG.Differentiated rEPCs were identified via immunofluorescence and flow cytometry.Scaffolds were fabricated by electrospinning polycaprolactone(PCL),and then investigated their morphology,mechanical properties and biocompatibility using in vitro and in vivo experiments.We seeded rEPCs on PCL scaffolds via alginate hydrogel conglutinating cells(AHCC)method.And we successfully fabricated TEVG by this method.Then,we seeded rEPCs on the surface fo PCL scaffolds through AHCC method and observed it for demonstrating the feasibility of this method.Meanwhile,we built the rat infrarenal abdominal aorta vascular graft implantationn model for observing the patency rate,endothelialization situation on the surface of graft,histological situation,immunohistochemical situation and morphology of the tissue on the surface on graft.The results of the studies showed as following.The immunofluorescence and flow cytometry results showed that the CD34,CD133,VWF surface antigen was positive expression obviously.And the differentiated rEPCs had advantages such as rapidity of expansion in culture,keeping the cell type in rEPCs in a relatively long time and growing well after being frozen,which showed that the rEPC was the ideal seed-cell for consisting TEVG.The in vitro and in vivo experiments in a rat model indicated that the electrospinning PCL scaffolds had submicron fiber diameters,favorable mechanical properties for vascular implantation,non-cytotoxicity,and favorable biocompatibility.The in vitro and in vivo experiments in a rat model indicated that the electrospinning PCL scaffolds had submicron fiber diameters,favorable mechanical properties for vascular implantation,non-cytotoxicity,and favorable biocompatibility.The characters meant that the electrospinning PCL scaffold was a ideal vascular scaffols,which could be applied on TEVG consisting.The in vitro study of AHCC method showed that more adhesion and proliferation was noticed in AHCC scaffolds at the same time point.And AHCC method had properties such as timesaving,high efficiency property,non-toxity,and higher expected patency rate.It demonstrated that AHCC method could be an effective method for seeding cells.Accoding to the observing results of impanted TEVG in rat infrarenal abdominal aorta vascular graft implantationn model at 1w,2w and 4w time point,at the same time point,compared with control groups(blank PCL(BP)scaffolds;blank alginate hydrogel coating(BAHC)scaffolds;natural sedimentation seeding cells(NSSC)scaffolds),AHCC scaffolds had a better regeneration and configuration of the endothelium tissue,and the morphology of the tissue on the inside surface of the AHCC scaffolds was more similar to the morphology of the natural endangium.These results indicated that AHCC scaffolds had the higher patency rate compared with control groups.Through AHCC method,seed cells could adhere directly to the surface of the vascular scaffolds,and we could save the time for waiting cell adhesion.According to the results of this research,AHCC could be an effective method to seed cells on the vascular scaffolds.We could use AHCC method to quickly produce an TEVG that could endothelialize rapidly in a short time.AHCC method has advantages such as timesaving property and higher patency rate.