Construction Of Endothelial Cell Selective Surface Via Layer-by-layer Self-assembly

Surface induced thrombosis and restenosis constitute major clinical failures of cardiovascular stent implantation. Previous researches have demonstrated that the occurrences of thrombosis and restenosis are ultimately due to the endothelium injury or delayed/uncompleted endothelialization on the stent. The in situ rapid endothelialization of the implants may be the ultimate solution to the thrombosis and restenosis. Two kinds of natural polyelectrolyte multilayer coatings were designed and coated onto biomedical devices in this dissertation, to realize the high selective attachment of the endothelial cells (EC) and rapid endothelialization on the biomedical material surfaces.1. The study of cell-resistant polysaccharide multilayer and its functionalization for EC selective attachment.—Two natural polysaccharides, antithrombogenic heparin and cytocompatible while thrombogenic chitosan, were chosen as the polyanion and polycation, respectively, to construct polyelectrolyte multilayer on the poly(ethylene terephalate) (PET) surface. Considering that the diffusion property of heparin in the multilayer and the swollen hydrogel-like property of the polysaccharide multilayers, we hypothesis that the multilayer which is fabricated of heparin and chitosan may have the synergic properties of excellent hemocompatibility and non-specifically cell-resistant. Quartz crystal microbalance with dissipation (QCM-D) and spectroscopic ellipsometry results indicate the successful alternate deposition of heparin and chitosan onto the substrate. The analysis of the data from the QCM-D and ellipsometry reveal that the heparin/chitosan multilayer preserves swollen and hydrogel-like property. The QCM-D and Ultraviolet spectroscopy (UV) measurements show that the heparin/chitosan multilayers are stable under PBS incubation and flushing conditions. The in vitro hemocompatibility tests and human vascular EC/smooth muscle cell (SMC) culture results reveal that a surface coating with synergic properties of excellent antithrombogenicity and non-specifically cell-resistant was obtained via layer-by-layer deposition of the heparin and chitosan. Furthermore, the QCM-D analysis and the cell culture results of the heparin/chitosan multilayer before and after chemical crosslinking demonstrate that the swollen and hydrogel-like property of the native heparin/chitosan multilayer is the essential reason to the cell-resistant of this coating.The vascular EC specific adhesive peptide sequence REDV and the vascular EC targeting antibody anti-CD34 were immobilized onto the cell-resistant heparin/chitosan multilayer subsequently. The in vitro human vascular EC/SMC culture results reveal that the functionalization of the REDV and anti-CD34 antibody onto the cell-resistant heparin/chitosan multilayer can achieve specifically selective attachment of vascular EC.2. The study of cell-compatible natural polyelectrolyte multilayer and its functionalization for rapid endothelialization.—Two natural polyelectrolytes, antithrombogenic heparin and cytocompatible while thrombogenic collagen, which is one of the extracellular matrix proteins, were chosen as the polyanion and polycation, respectively, to construct polyelectrolyte multilayer on the 316L stainless steel disk surface. The results of QCM-D, ellipsometry, UV and atomic force microscopy (AFM) results indicate the successful fabrication of heparin/chitosan multilayer. The QCM-D and ellipsometry measurements show that the heparin/collagen multilayers are stable both under PBS incubation and flushing conditions. The in vitro hemocompatibility tests and human vascular EC culture results reveal that an antithromboresistant and EC compatible surface coating was obtained via layer-by-layer deposition of the heparin and collagen. The heparin/collagen multilayer was also performed onto the stainless steel stent surface. The fluorescent photographs show that a smooth and homogenous stent surface coating was obtained via heparin/collagen multilayer modification. The results of scanning electronic microscopy (SEM) and confocal laser scanning microscopy (CLSM) show that good EC affinity of the heparin/collagen multilayer modified stent is also obtained. These results indicate that antithrombogenic and EC favorable surface coating was obtained via layer-by-layer deposition of heparin and collagen onto stent.The vascular EC targeting antibody anti-CD34 was subsequent immobilized onto the cytocompatible heparin/collagen multilayer after glutaraldehyde crosslinking. The ellipsometry and QCM-D results demonstrate that the antibody functionalized multilayer coatings is stable both in static incubation and flushing conditions. The in vitro hemocompatibility tests and EC/SMC culture results indicate that both heparin/collagen multilayers with or without the anti-CD34 antibody functionalization not only preserve good hemocompatibility, but also promote cell attachment and growth notably. The heparin/collagen multilayer coatings show no selectivity in promotion of EC and SMC, whereas the attachment and growth of the vascular EC can be specifically enhanced on the anti-CD34 antibody functionalized heparin/collagen multilayer. The metabolic activity assessment and the NO secretion measurements further indicate that the EC adherent to the anti-CD34 antibody functionalized multilayer surface have better viability and possess the specific function of the natural vascular EC. In vivo experiments indicate that the anti-CD34 antibody can enrich and accelerate the attachment of the vascular cells onto the stent and rapid endothelialization is realized. Compared with bare stents and heparin/collagen multilayer modified stents, the neointimal hyperplasia on the anti-CD34 antibody functionalized multilayer modified stents is significantly inhibited. The success of the anti-CD34 antibody functionalized heparin/collagen multilayer coating in rapid endothelialization and anti-restenosis might indicate that the immobilization of EC specific ligands onto a cytocompatible matrix can be a good approach for in situ endothelialization and a possible solution to in stent restenosis.