Construction And Evaluation Of Heparin-Fibronectin-Vegf Biofunctional Coating For Anticoagulation And Induced-Endothelialization On Titanium

In cardiovascular interventional therapy, restenosis is the most important factor which influences vascular stent curative effect. Restenosis is due to thrombogenesis, tissue dysplasia and smooth muscle cells (SMCs) proliferation. It is known in many studies that vascular stents and other cardiovascular materials contacted with blood in a long term should exhibit excellent hemocompatibility. Researches have showed that endothelial cells (ECs) were the best surface contacted with blood. Therefore, endothelializaiton of vascular stent rapidly in-situ is necessary. Nowadays, focus is made on implanting biofunctional biomaterial to capture EPCs in blood, differentiating to autogenous ECs. This work applied electrostatic adsorption to immobilize heparin (Hep), fibronectin (Fn) and vascular endothelial growth factor (VEGF) on titanium (Ti) surface to construct a biofunctional coating with favorable properties to accelerate induced-endothelialization and inhibit thrombosis formation simultaneously.Ti substrates were activated by NaOH solution to produce negative charged super hydrophilic surface, and then samples were combined with Poly-L-lysine (PLL) by electrostatic adsorption to obtain positive charged amino-rich surface. Subsequently, electrostatic adsorption and specific recognition between biomolecules were made use of to immobilize Hep, Fn and VEGF on amino-surface, constructing Hep/Fn/VEGF biofunctional coating. Fourier Transform Infrared Spectroscopy (FTIR) demonstrated that there were abundant hydroxyl groups and amino groups respectively on Ti surface after NaOH activation and after PLL immobilization. X-ray photoelectron spectroscopy (XPS), Toluidine Blue O (TBO) assay and immunohistochemistry results indicated that biomolecules were successfully immobilized on amino-Ti surface, constructing Hep/Fn/VEGF biofunctional coating. Atomic force microscope (AFM) and water contact angle measurement results stated that surface roughness on Hep/Fn/VEGF biofunctional coating was smaller than Ti surface and Hep/Fn/VEGF biofunctional coating diaplayed preferable hydrophily compared with Ti. Heparin release result showed that there was a sudden release for heparin immobilization on the surface after12hours, but the release rate slowed down as time passed; VEGF release result aslo stated that VEGF release slowed down during28days process. Therefore, the release results indicated that the biofuctional coating exhibited considerable stability although electrostatic adsorption had been taken to construct Hep/Fn/VEGF biofunctional coating.Blood compatibility evaluation in vitro results suggested that the Hep/Fn/VEGF biofunctional coating displayed higher AT Ⅲ binding activity, less platelets activation and aggregation, prolonged activated partial thromboplastin time (APTT) compared with Ti surface. Fibrinogen adhesion and degeneration on biofunctional coating was more serious than Ti, but the degeneration proportion was lower, indicating favorable hemocompatibility.Vascular cell compatibility evaluation in vitro results investigated that the Hep/Fn/VEGF biofunctional coating could significantly promote endothelial progenitor cells (EPCs), endothelial cells (ECs) attachment and proliferation, inhibit ECs apoptosis in some degreee and inhibit SMCs proliferation compared with Ti. Induced-endothelialiation evaluation in vitro showed that the Hep/Fn/VEGF biofunctional coating could preferably promote EPCs chemotaxis and ECs migration. Therefore, the Hep/Fn/VEGF biofunctional coating displayed preferable cytocompatibility and induced-endothelialiation ability.Then focus was made on the influence of material surface property, biomolecules, mutual competition and equilibrium among biomolecules on surface biocompatibility. Mechanism study indicated that increasing surface hydrophily and roughness in nanometer scope could improve biocompatibility on material surface. Heparin, Fn and VEGF could exhibit their own biofunction by mutual competition and equilibrium on the surface, enhancing surface biocompatibility.In conclusion, the Hep/Fn/VEGF biofunctional coating was successfully constructed on Ti surface. By mutual competitive adsorption and equilibrium among biomolecules, Hep/Fn/VEGF biofunctional coating maintained favorable anticoagulant and infuced-endothelial properties. This work may provide an important reference for achieving anticoagulant/induced-endothelial biofunction of cardiovascular materials.