| Thrombosis formation and restenosis after coronary artery stent implantation continue to be problematic. Although various platforms and paramets of stents have been designed and optimized over years, postoperative complications are hard to avoid. The vascular endothelialium layer is always provide a nonthrombogenic surface as well as prevent intimal overproliferation, thereby, the presence of a confluent endothelium layer on stent surface have been widely considered as an ideal approach to prevent the occourance of thrombus and restenosis. Nowadays, accelerate endothelialization by surface biomimetic microenvironment construction have become a new hotspot. However, researchers have not yet identified a right combination of surface physicochemistry and biofunction to meet the biocompatibility requirement of coronary artery stent for clinical use. In this study, on the basis of endothelium regeneration mechanism, laminin/heparin/SDF-la microenvironment was constructed on Ti surface using the method of covalent immobilization and nanoparticle loading, respectively. The aim of this study was to induce and accelerate endothelium regeneration on Ti surface on the basis of favorable surface hemocompatibility and anti-renstenosis property.For covalent construction of laminin/heparin/SDF-1α microenvironment, Ti was firstly activated by NaOH to creat a high negatively charged surface for electrostatic binding of positively charged poly-1-lysine; then laminin/heparin complex was covalent immobilized on poly-1-lysine coated surface via EDC/NHS/MES crosslinking; after that, take the advantage of the specific interaction between SDF-1α and heparin, SDF-la was assembled onto laminin/heparin modified surface. Due to the specific binding characteristic between laminin and heparin, the alteration of laminin concentration ratio may not only affect surface laminin binding density, but also have influence on heparin binding amount and bioactivity. In this study, thereby, different concentration combinations of laminin and heparin were setup for detailed surface physicochemistry property characterization and biocompatibility performance comparison, and the results suggested that the modified surface present most favorable biocompatibility when the laminin/heparin concentration ratio reached 0.2:5.0 mg/ml. In vivo animal test further confirmed that the optimized laminin/heparin microenvironemt provide significant effect in preventing intimal hyperplasia and accelerate endothelialization. The introduction of SDF-1α seems have no significant effect on vascular cells and endothelial progenitor cells proliferation, but possess adequate effect on endothelials migration and endothelial progenitor cells homing.For the construction of nanoparticle loading type laminin/heparin/SDF-la microenvironment, laminin loaded heparin/poly-1-lysine nanoparticle was firstly prepared via the electrostatic interation between negatively charged laminin loaded heparin and positively charged poly-1-lysine; then the nanoparticle was immobilized on dopamine coated Ti surface through a Schiff base reaction; finally SDF-1α was assembled onto nanoparticle modified surface by specific binding to heparin. In this study, the alteration of heparin/poly-1-lysine concentration ratio was found may affect the particle size, stability and binding density. According to in vitro biocompatibility evaluation results, the heparin/poly-1-lysine nanoparticle modified surface possesses the biofunction of selectively inhibit thrombosis and smooth muscle cells over-proliferation, but promote endothelial cells and endothelial progenitor cells growthing when the heparin and poly-1-lysine concentration ratio was above 7.0:0.5 mg/ml. On this basis, laminin was loaded to optimized nanoparticles, however, it was found in this study the laminin loading may facilitate the interaction between nanoparticle and dopamine coating, and thereby cause the increasing of particle binding density. According to the further biocompatibility investigation, heparin/poly-1-lysine nanoparticle with the concentration ratio of 10.0:0.5 mg/ml was finally identified as the optimized technique. The further assembling of SDF-la seems have no significant influence on endothelial cells proliferation, but could effectively promote endothelial cells migration and endothelial progenitor cells homing, as well as prevent thrombus formation.The biomolecules that used in above two types of microenvironment was totally the same, but possess different construction method and technique parameter. To further investigate and screen out the reasonable biomimetic microenvironment construction method, in vitro and in vivo biocompatibility comparsion of these two kinds of optimized microenvironments was prepared. According to the results, biomolecules immobilized with covalent method always present high stability, while nanoparticle loading type microenvironment exhibit controlled and continued release kinetic of function molecules; compared to nanoparticle microenvironment, the covalent microenvironment exhibit higher laminin exposed density, but lower heparin bioactivity and SDF-la binding density; the two types of microenvironment exhibit comparable property on in vitro endothelial cells and endothelial progenitor cells growthing, but the nanoparticle microenvironment given superior property on anti-thrombosis, anti-inflammation and induce endothelial progenitor cells homing; in vivo aminla test also suggested that the nanoparticle microenvironment could effectively prevent intimal hyperplasia.Based on the comparison results of above two types of microenvironments and the knowledge of pathological evolution at implantation site after PCI sugery, a tentative idea of surface modification with time-ordered function was developed in this study. The core content of time-ordered function concept was taking the pathological evolution into consideration and construct reasonable microenvironment to apply a stage-adjusted remedy. In this study, nanoparticle 5.0 (NP5.0) was finally selected for surface time-ordered function investigation. Through in vitro dynamic release model and biocompatibility evaluation, the study demonstrated that the NP5.0 modified surface presented favorable biomolecules release kinetic and could meet the time-ordered function requirements of:inhibit thrombosis, inflammation and smooth muscle cells over-proliferation within 1-7 days; selectively promote endothelium regeneration but prevent thrombus and smooth muscle cells over-proliferation within 7-14 days; maintain adequate hemocompatibility and endothelial cells compatibility within 14-28 days.In conclusion, laminin, heparin and SDF-1α were selected in this study for surface biomimetic microenvironment construction. Take the advantage of the specific intermolecular interaction, the construction method was optimized and finally screened out two types of laminin/heparin/SDF-la microenvironment. According to the in vitro and in vivo biocompatibility evaluation and comparison, this study further provide a novel concept of surface modification with time-ordered function, and the feasibility was initially investigated by in vitro and in vivo evaluation. This study provides important guidance for reasonable surface microenvironment construction and potential means for suitable stent platform designing. |
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