Potential Application Of Electrospun Polycaprolactone Composite Fibers In Vascular Tissue Engineering

With the improvement of living standards,the aging of the social population has become a trend,and cardiovascular disease is one of the main causes of death among the aging population.Atherosclerosis is a common cardiovascular disease.It can lead to the formation of subintimal plaques,reduce the cross-sectional area of blood flow,and lead to the decrease of blood flow to the downstream tissues of plaques,which has great harm to human health.Drug therapy is mainly used in clinic,but stent and bypass grafting are needed in severe cases.Large-caliber vascular transplantation has achieved good clinical results,but small-caliber vascular transplantation still has some shortcomings,such as low long-term Patency rate and easy occlusion.The ideal vascular graft is autologous blood vessel,but the material is limited.Therefore,artificial blood vessels with biocompatibility and blood compatibility,which can meet the mechanical properties of natural blood vessels,have become the focus of research.At present,many kinds of vascular materials have been developed and studied by researchers.How to promote endothelialization,improve long-term patency and anti-thrombosis through functional modification is still a subject that needs to be explored and studied continuously.In recent years,electrospinning technology has come to the fore in vascular tissue engineering.Micro(nano)fibers prepared by electrospinning have been extensively studied and used to prepare tissue-engineered blood vessels because of their simple operation,high porosity and similar structure to natural extracellular matrix(ECM).Based on the above contents,this paper mainly takes polycaprolactone(PCL)as the research object,through the preparation of functional electrospun fiber reinforced endothelial and anticoagulant properties,and studies their physical and chemical properties,cell compatibility,blood compatibility and the ability to promote angiogenesis.The aim of this study is to prepare artificial blood vessel materials which can promote the proliferation and anticoagulation of endothelial cells and contribute to the development of new materials for vascular tissue engineering.The details are as follows:1.Polyaniline(PANI)is considered as a potential candidate for tissue engineering and regenerative medicine due to its excellent electrical conductivity,good redox stability,reversible oxidation and biocompatibility.PANI has a unique response to electrical stimulation,which regulates many functions of cells,such as adhesion,proliferation,migration and differentiation.Therefore,conductive PANI-PCL fibers were prepared by electrospinning to provide the basis for electrical stimulation,and to study the coupling effect of electrical stimulation and conductive PANI-PCL fibers on endothelial cell function.(1)PCL electrospun fibers were prepared by electrospinning technology.In order to improve the hydrophilicity of PCL,they were treated by plasma.After treatment,PANI was coated on the surface of PCL by in-situ oxidation polymerization to prepare conductive PANI-PCL fibers.The results of SEM,XPS and FT-IR showed that PANI was successfully coated on PCL surface with conductivity of 6.71×10~-33 S/cm,which provided a basis for the study of electrical stimulation responsiveness.Tensile tests showed that the tensile strength of PANI-PCL fibers increased to 3.51 MPa and the elongation at break was 110.45%,which could meet the requirements of vascular mechanics.In hydrophilicity test,PANI-PCL has better hydrophilicity than PCL,which is beneficial to cells.(2)In order to observe the cell compatibility and electrical stimulation responsiveness of fibers,human umbilical vein endothelial cells(HUVECs)were used as research objects.Cell viability was measured by living cell staining and MTT method.Cell proliferation and morphological changes were observed by fluorescence staining and SEM test.When PANI-PCL fibers were compared with PCL fibers,HUVECs showed strong adhesion and activity.The effect of proliferation is better under electric stimulation.The proliferation of HUVECs on PANI-PCL fibers is closely related to the intensity of electrical stimulation,of which 400 mV/cm stimulation is relatively good.The results provide a new idea for the study of conductive materials in vascular tissue engineering.2.In order to improve the anticoagulation and promote the proliferation of vascular endothelial cells,we chose ferulic acid(FA),which has both anticoagulant and endothelial cell proliferation effects,as the functional modified material,and prepared FA-PCL fibers by mixed electrospinning.The angiogenesis and anticoagulation properties of the functional fibers were observed.FA-PCL fibers were prepared by electrospinning with different mass fractions of FA(3%,5%,10%of PCL,respectively)blend with PCL.The morphology and properties of the prepared drug-loaded fibers were characterized.In terms of fiber morphology,FA-PCL still maintains the fiber structure,but the diameter uniformity of 3%FA-PCL and 5%FA-PCL fibers is not good,which also affects the tensile properties of the fibers,resulting in a decrease in the tensile strength of 3%FA-PCL and 5%FA-PCL fibers,but still meets the mechanical requirements of natural blood vessels.With the increase of FA quality,10%FA-PCL fibers are relatively uniform,and the tensile strength increases slightly compared with PCL,but there is no significant difference.The mechanical properties meet the requirements of practical application.In hydrophilicity test,the contact angle of FA-PCL fibers is slightly lower than that of PCL,but it still shows obvious hydrophobicity,which is unfavorable for cell adhesion.In vitro drug release,it was found that there was an obvious sudden release phenomenon at the initial stage of drug release,which may be due to drug attachment on the surface of the fiber.With the increase of FA mass fraction,the release rate accelerated,and the drug release mechanism was mainly diffusion release mechanism.(2)HUVECs cells were selected as the research object of cell compatibility.Calcein-AM was used to stain living cells,CCK-8 was used to test cell proliferation,fluorescence staining and SEM was used to observe cell adhesion,proliferation and morphology.PCL fibers loaded with ferulic acid are beneficial to the adhesion and proliferation of endothelial cells,and cell proliferation is closely related to the amount of FA.After 5 days of culture,5%FA-PCL has the most obvious effect on cell proliferation.The effect of FA-PCL fibers on angiogenesis was observed by matrix colloid tube forming test and scratch method in vitro migration test.The results showed that FA-PCL fibers could promote the formation of endothelial cell tubular structure and enhance cell migration ability compared with PCL.Among them,3%FA-PCL,5%FA-PCL had better effect,while 10%FA-PCL might be due to higher FA content.It inhibited the ability of cell growth and migration.(3)Hemolysis test,recalcification test,activated partial thrombin time(APTT)test,prothrombin time(PT)test and thrombin time(TT)test were carried out to investigate the blood compatibility of the prepared drug-loaded fibers.The fibers hemolysis rate of FA-PCL fibers accorded with the national standard,and the hemolysis rate was less than 5%.In anticoagulation experiment,FA-PCL fibers showed excellent anticoagulation performance,and the anticoagulation effect increased with the increasing of FA content.3.The results of the first two chapters show that PANI-PCL can promote cell proliferation under both electric and non-electric stimulation.Ferulic acid can improve anticoagulant performance and promote angiogenesis.However,in the process of drug release research,there is burst release.Based on the above experimental results,polyaniline and ferulic acid were simultaneously used to modify polycaprolactone in order to promote angiogenesis and anticoagulation,at the same time,PANI was coated to prolong the drug time.(1)FA-PCL fibers with different drug loadings were prepared by blending.After plasma treatment,PANI was coated on the surface of the fibers.Cell compatibility test,in vitro tube formation test,hemolysis test and anticoagulation test showed that PANI-10%FA/PCL fibers had obvious effects on promoting angiogenesis and anticoagulation.(2)The best drug-loaded conductive fiber was PANI-10%FA/PCL.According to the drug ratio,the tubular 10%FA-PCL vascular graft was prepared.The 10%FA-PCL vascular stent was still treated by plasma.Then,the conductive polymer PANI was coated on the surface to prepare the drug-loaded vascular graft.By replacing the common carotid artery in rabbits,the patency rate of the vascular graft and the changes of vascular tissue were observed.Fifteen days after in vivo vascular replacement,the patency rate of PANI-10%FA/PCL fibers was about 77%.Adhesions,materials and adherence could be seen in the wall of the tube.PANI-PCL usually has a rate of about 55%.Thrombosis can be seen in the lumen,surrounded by connective tissue,accompanied by inflammatory cell infiltration.In the detection of vascular endothelium,it was found that endothelialization was not ideal.Only a few cells in PANI-10%FA/PCL showed positive expression of CD31.