Regulation Of The Migration Of Endothelial Cells By A Gradient Density Of Vascular Endothelial Growth Factor

The migration of endothelial cells(ECs) is essentially important for angiogenesis, which is involved in many p hysiological and pathological processes, including embryonic development, wound healing, tissue regeneration, and tumor growth etc. Chemotaxis is one of the approaches to regulate this process. Gradient biomaterials could affect cells behaviors, such as ad hesion, spreading and migration.To investigate the influence of the growth factor surface density gradient on endothelial cells(ECs) adhesion and migration, an approach to fabricatea gradient density of vascular endothelial growth factor(VEGF) onto silicon slides has been developed in this study.The fabrication process and the formation of growth factor gradient were well optimized and characterized in this work. Endothelial cells were seeded onto gradient surfaces of substrates for investigating cell’s adhesion and migration. The study contains three parts of detailed work as following:1. The fabrication and characterization of uniform VEGF density surface: Firstly, silicon substrates were immersed with 3-glycidoxypropyltrimeth oxysilane(GPTMS), resulting in self-assembly monolayer(SAM) with hydroxyl ending groups; Next, the treated substrates were backfilled with 3-triethoxysilylpropylsuccinicanhydride(TESPSA), leading to formation of SAM with carboxyl ending groups(-COOH).To adjust the immersion time of GPTMS, a series of surface with different –COOH density could be obtained. The-COOH moieties were then activated for the immobilization of VEGF, which leading to a surface uniform density of VEGF. We employed toluidine blue staining(TBO), contact angle measurement and VEGF enzyme- linked immune detection kits(ELISA) to characterize the relationships of –COOH, VEGF density and GPTMS reaction time. The results confirmed that the density of –COOH and VEGF reduced linearly with GPTMS reaction time extended.2. The fabrication and characterization of gradient VEGF density surface: Based on the liner relationship of VEGF density and GPTMS reaction time, the VEGF gradients were fabricated using an injection method. Gradual injection of GPTMS and then back filling with TESPSA, a gradient density of –COOH onto the silicon slides produced. The-COOH moieties were then activated for the immobilization of VEGF, which leading to a surface gradient density of VEGF. The successful formation of VEGF gradient densities were confirmed by confocal laser scanning microscopy(CLSM) and X-ray photoelectron spectroscopy(XPS), respectively. The treated silicon slide displayed a gradient density of VEGF from 54 to 132 ng/cm2 with a slope of 7.8 ng/cm2/mm.3. The ECs migration induced by gradient VEGF density: The F-actin and nuclei of cells were stained with immunochemistry and cytoskeleton morphology of ECs was observed with CLSM. Cells adhered to samples with uniform VEGF surfaces displayed random cytoskeleton organization with various orientations. In comparison, most cells cultured onto gradient surfaces demonstrated an actin orientation tendency along gradient direction of increasing VEGF density. The EC were monitored with a live cell station. The cells migration trajectories on various surfaces were rebuilt by analyzing the sequential images. Various parameters such as migration trajectories, net displacement, total migration distance, chemotactic index and percentage of cells move towards gradient regarding cell migration were calculated. ECs cultured on the surface gradient density of VEGF demonstrated preferential orientation and an enhanced directional migration behavior. Up to 72% of cells migrated towards the region with high surface density of VEGF. However, the grad ient density of VEGF had no significant effect on the cell migration rate.The study provides an alternative to explore chemical-directing cells migration, which is essentially important for understanding cell migration/ in growth behavior for angiogenesis involved in implant technology.