Multilayer Films Based On Layer-by-layer Assembly For Promoting Endothelialization

Promoting in situ endothelialization of cardiovascular implants has been regarded as an efficient solution to eliminate undesired post-implanting symptoms, such as thrombin, imflammatory reaction and restenosis. How to effectively achieve rapid-endothelialization and meanwhile guarantee long-term success of endothelialization still remains as a great challenge. Aiming at two key problems in endothelialization of cardiovascular implants:1. How to enhance competitiveness of endothelial cells (ECs) over smooth muscle cells (SMCs) for achieving rapid-endothelialization in the complex in vivo environment,2. How to maintain endothelial function of formed endothelial layer, a series of researches was carried out based on layer-by-layer assembly in this dissertation.1. Based on the characteristic of gene technology that ’transfection process is non-cell-specific and expressed protein is cell-specific’, the functional gene delivery multilayer films were constructed through layer-by-layer assembly of plasmid DNA encoding hepatocyte growth factor (HGF)(HGF-pDNA) and protamine sulfate (PrS). The (PrS/HGF-pDNA) multilayer films could transfect both ECs and SMCs and make them secrete HGF which was an EC-specific promoting protein. Consequently, only EC proliferation was promoted. Furthermore, (PrS/HGF-pDNA) multilayer films could enhance ECs’ competitiveness over SMCs’in EC/SMC coculture. The coronary stents modified by (PrS/HGF-pDNA) multilayer films could promote endothelialization and prevent restenosis in vivo. This study demonstrated functional gene delivery multilayer film was an efficient strategy for the purposes of enhancing ECs’ competitiveness in the environment with multi-type of cells, achieving rapid-endothelialization of implants and preventing restenosis.2. Based on the influence of substrate stiffness on cell adhesion, the stiffness-hybrid system ’upper soft multilayer film+underlying stiff substrate’ was established via fabricating multilayer films on glass substrates. The cell adhesion results showed EC and SMC adhesion could be regulated by tuning the bilayer number, namely, thickness of upper multilayer films. In EC/SMC coculture, the EC selective adhesion was achieved by increasing bilayer to certain number. The apparent stiffness cells sense was modulated by tuning the bilayer number of multilayer films, which might be the most probable factor for regulating cell adhesion. Furthermore, the EC selective adhesion could be also achieved by tuning bilayer number of upper multilayer films when employing other underlying substrates with different stiffness. This study provided a promosing strategy for enhancing ECs’ competitiveness over SMCs’ by regulating the apparent stiffness based on layer-by-layer assembly.3. The biopolymeric coatings made of poly(L-lysine)/hyaluronan (PLL/HA) multilayer films with different crosslinking degrees were constructed, serving as stiffness-controlled substrates. The growth of ECs and function of subsequent formed endothelial layers on substrates with different stiffness were investigated in the presence of soluble hepatocyte growth factor (HGF). The EC adhesion, migration and proliferation were all stiffness-dependent and were improved with HGF stimuli. ECs on substrates with low stiffness showed stronger response to soluble HGF in migration and proliferation than ECs on substrates with high stiffness. Finally, the formed endothelial layers on substrates with low stiffness exhibited better intergrity and higher level expression of endothelial nitric oxide synthase (eNOS) than on substrates with high stiffness, which demonstrated the substrates with low stiffness could benefit promotion of endothelial function. This study highlighted the influence of substrate stiffness on both EC growth and endothelial function.4. The (PLL/HA) multilayer films with low crosslinking degree were employed as the compliant substrates. Both adhesion and spreading of ECs and SMCs were remarkably improved on these compliant substrates by loading vascular endothelial growth factor (VEGF) in a matrix-bound manner. By modulating the density of loaded VEGF, the EC selective adhesion was achieved on these substrates. In addition, ECs could proliferate on these substrates while SMCs could not, which suggested ECs’competitiveness over SMCs’was enhanced. Compared to substrates with high stiffness, the formed endothelial layers on these compliant substrates could exhibit better integrity, higher releasing level of nitrix oxide (NO) and higher expression level of genes related to endothelial function, which highlighted substrates with low stiffness could benefit promotion of endothelial function. This study provides a promosing strategy for not only enhancing ECs’ competitiveness over SMCs’but also promoting endothelial function via the cooperation of compliant substrate and bioactive molecule.