Immobilization Of SEMA4D/heparin On Ti Surface To Construct A Microenvironment For Antithrombosis And In Situ Endothelialization

Currently the therapeutic stents for percutaneous coronary intervention(PCI)are mainly non-biodegradable metal stents.With the increase of patients with cardiovascular disease year by year,in the near future and for a longer period of time to come,metal based drug eluting stents(DES)will still be widely used.Although the complications of implanted DES have been reduced after continue optimization,some complications such as late thrombus and restenosis are still exist and become hidden dangers after DES implantation,directly influencing the quality of life for patients and even causing the patients death.To address these complications,developing the performance of anti-coagulation and fast endothelialization is the key point of further optimizing DESThis thesis focuses on the modification of anti-coagulation and in situ endothelialization on titanium based cardiovascular stent materials.Biomolecules heparin and SEMA4D were introduced onto the Ti based biomaterial surface.To further developing the speed of in situ endothelialization and simulating the tissue repair that regulated by the co-participation and synergistic effects of multiple chemotactic factors,chemotactic factor CXCL12(also called SDF-la),which can mobilize and capture endothelial progenitor cells(EPC),and promote the differentiation and proliferation of EPC,was introduced onto the modified surfaces after the successful construction of SEMA4D/heparin microenvironment.The mechanisms of SEMA4D/heparin microenvironment capturing endothelia cells(EC)and macrophages(MA)under dynamic state were explored.Furthermore,the mechanism of in situ endothelialization promoted by multiple chemotactic factors was also discussedFirst,SEMA4D/heparin microenvironment was constructed on Ti surface,and characterised by multiple test facilities,such as Fourier transform infrared spectrometer(FTIR),X-ray photoelectron spectroscopy(XPS),atomic force microscopy(AFM),water contact angle(WCA)and particle size and potential analysis.The results of FTIR and XPS showed that with the increase of constructed SEMA4D,the quantity of surface heparin increased,at the same time the amount of SEMA4D/heparin complex and the total loading heparin increasedSecondly,the blood compatibility,cell compatibility and immunity compatibility were further evaluated.The blood tests showed that SEMA4D/heparin modified microenvironments possess a favorable blood compatibility of preventing platelet adhesion and activation,inhibiting fibrinogen adhesion and degeneration,and effectively prolonging aPTT and TT.The cell experiments showed that SEMA4D/heparin modified materials can promote EC chemstaxis and haptotaxis migration and enhance EC proliferation.A higher constructed density of SEMA4D has a better EC migration and proliferation.Moreover,SEMA4D/heparin microenvironments had a favorable effect of positive immunoregulatory that promote MA secrete immunosuppressive factor IL-10 and inhibit secretion of proinflammatory factor IL-6 and TNF-a,thus facilitate wound healing and in situ endothelialization.In vivo experiments showed SEMA4D/heparin material has a favorable in situ endothelializationThirdly,to simulate tissue repair which regulated by the synergistic effects of multiple chemotactic factors,CXCL12 was introduced onto the SEMA4D/heparin modified microenvironment to optimize the in situ endothelialization.The physicochemical properties and biocompatibility of constructed SEMA4D/CXCL12 were further studied.Although the activation of platelets was depressed,in the mass the blood compatibility of SEMA4D/CXCL12 microenvironments was slightly worse than that of SEMA4D/heparin microenvironments.The EC migration was weakened after the introduction of CXCL12,but the EC adhesion and proliferation,and the EPC capture were improved.In vivo tests indicated that in situ endothelialization was developed on SEM A4D/CXCL 12 microenvironments.Last,the mechanisms of EC and MA dynamicly adhering and spreading on SEMA4D/heparin surfaces,and the mechanisms of different structural transformation of SEMA4D/heparin complexes regulating the cellular physiology were studied by the quartz crystal microbalance with dissipation(QCM-D).Under dynamic state,the SEMA4D/heparin microenvironment with high heparin and low SEMA4D promotes the EC adhesion and spreading.However,the SEMA4D/heparin microenvironment with high SEMA4D but low heparin would enhance the MA adhesion and more spreading.Different conformations of SEMA4D/heparin complexes cause cellular physiology change,thus leading to a difference of in situ endothelialization.The SEMA4D/heparin complexes in sample 4D50 mainly mediated the cellular physiology relating receptor CD72,and the complexes in sample 4D100 mainly regulated the cellular physiology of receptor PLEXINB1.The in situ endothelialization regulated by multiple chemotactic factors is determined by the activity of mobilized cells.Under the synergistic effects of SEMA4D and CXCL12,the SEMA4D/CXCL12 co-modified microenvironment realized a faster and more efficient in situ endothelialization,at which process the chemotactic factor CXCL12 played a more important roleThe design idea of anti-coagulation and in situ endothelialization promotion in this thesis comes from tissue repair that needs the co-participation and synergistic effects of multiple chemotactic factors,having theoretical value and practical meaning for the construction of stents and blood-contacting vascular devices that preventing the thrombus formation.