Applying Tissue Engineering Nanofiber Controlling Sustainded-release With Nature Structure Of Cytokine In Angiogenesis

To construct a three-dimensional tissue structure,we should consider the important issues of early revascularization.There are three key elements of creating blood vessels in tissue engineering: vascular seed cells,tissue engineering scaffolds and cytokines.Therefore,it is a problem to be solved that how to load cytokines into scaffolds so that it could control sustained-release of cytokines.The research employed the technology of our laboratory protein sustained-release with nature structure,intending to construct tissue engineered fibers with high specific surface area、high porosity and controlling sustained-release with nature structureof cytokines and to study the potential of its application in angiogenesis.Firstly,we prepared BSA,the model drug,into protein-dextran nanoparticles by the frozen phase separation method,and then loaded them into nanofibers by electrospinning,we explored the process of electrospinning to determine the optimal prescription of beadedelectrospun fibers 、 smooth electrospun fibers 、 emulsion method fibers and characterized them in physical and chemical method.According to the optimal prescription of the model drug,VEGF was loaded into three fibers;SEM showed that they had porous structure,their average diameters were about 1μm and they had good mechanical properties;besides,the average diameter of beads among beaded fibers was 7μm,so they could wrap nanoparticles effectively.The encapsulation efficiency of nanoparticles、three fibers and the release kinetic of VEGF in three fibers in vitro were detected by ELISA method.The result showed the encapsulation efficiency of the beaded fibers was the maximum.During the process of releasing,VEGF in emulsion method fibers had most release of the first 5 days,so there was initial burst release phenomenon and the release was not completed;however for the electrospun fibers containing nanoparticles,VEGF was encapsulated into dextran particles in advance,then dispersed into the PLGA solution,during the process of release,the fibers were swelling,dextran particles were adhereing to form the diffusion channel and then releasing the protein,so sustained-release was reached.After 30 days,beaded and smooth fibers were still releasing.By CCK-8 method,the changes of active situation of VEGF during the preparing process of nanoparticles and different fibers were investigated.The result showed that compared to the negative control group,active situation from the highest to the lowest was as follows: stoste,nanoparticles,beaded fibers,smooth fibers,emulsion method fibers.So,if VEGF was prepared into dextran particles and then loaded into electrospun fibers,its activity could be protected effectively.At last,we implanted beaded fibers,smooth fibers,emulsion method fibers and blank fibers to hindlimb ischemic rats,after 14 days,rats were killed and the tissues were removed to make immunohistochemistry slices.The result showed that the capillary density of beaded fibers was the biggest,followed by smooth fibers group and emulsion method fibers group,and blank fibers group and control group were the smallest.It indicated that beaded fibers encapsulated more nanoparticles into beads,more VEGF with nature structure was released from beaded fibers in a better sustained-release manner,so it formed a more effective concentration to stimulate endothelial cell to proliferate,differentiate,then to promote the formation of small blood vessels,winning a stronger angiogenesis ability.