Biofunctionalization of titanium surface midified by heparin-VEGF-fibronectin LBL to improve endothelial cell proliferation and blood compatibility

摘要

Introduction: In-stent restenosis and subsequent thrombosisremain a significant complication following the implantation of coronary stents. Different approaches have been used in developing novel coronary stents to protect against thrombosis and minimize restenosis. In the present study, we designed a biomacromolecular layer-by-layer (LBL) coating with heparin, vascular endothelial growth factor (VEGF), and fibronectin onto nickel-free titanium surface to improve blood compatibility and endothelial cell proliferation. Materials and Methods: The cleaned p-titanium disks (15 mm in diameter and 2 mm thick) titanium disks were immersed in PEI solution for 30 min to obtain a stable positive charge on the surface, then gently rinsed with a large amount of deionized water for 1 min to consolidate the charged state of the substrates and dried with a flow of nitrogen. Titanium disks were then dipped in heparin and VEGF solutions for 20 min alternatively, followed by the same rinsing and drying procedures. The deposition process mentioned above was repeated 11 times to achieve complete biofunctionalization of the surface. Finally, Fn was introduced for 20 min to obtain an Fn-terminated functionalized multilayer film. Moreover, PEI/Fn modified titanium disks, PEI/Heparin modified titanium disks, and PEI(Heparin/VEGF)_5 modified titanium disks were similarly achieved for further cell experiments. Results and Discussion: The multilayer assembling process was monitored by water contact angle and surface plasmon resonance, respectively. With increasing the number of layers, the deposition of polyelectrolyte as self-assembled ultrathin multilayer films showed linear growth of absorbance. In vitro blood compatibility results revealed that the fabricated layers prolonged activated partial thrombin time and prothrombin time, reduced platelets activation and aggregation, and reduced blood hemolysis rate. Cell adhesion and growth results showed that the assembled multilayer films significantly promoted cell attachment and growth, and the endothelialization property of the multilayer films was preferable compared with the untreated titanium disk. Conclusions: These results suggest that titanium surface modification using biofunctional multilayer films composed of heparin, VEGF, and fibronectin may serve as a potential approach to inhibit thrombosis and promote re-endothelialization of cardiovascular stents.