Inducing The Migration Behavior Of Endothelial Cells By A Density Gradient Of Poly(Ethylene Glycol)

The migration of endothelial cells(ECs) is crucially important for many biological processes, such as embryonic vasculogenesis, wound healing, tissue regeneration and tumor growth etc., which is regulated by biochemical signals. To investigate the influence of the surface density gradient on endothelial cells(ECs) adhesion and migration, we employed a micro-injection method to fabricate a continuous linear poly(ethylene glycol)(mPEG-CHO) gradient on silicon slides surfaces in this work. The experimental process and the formation of PEG density gradient were well optimized and characterized in this study. Then, the adhesion and migration behavior of endothelial cells in response to the PEG gradient was observed in vitro. The research contents of this study include the following three aspects:1. The fabrication and characterization of uniform PEG surface: Firstly, silicon slides were treated with 3-glycidoxypropyltrimeth oxysilane(GPTMS) solution at 80 °C for different times, thus forming substrates with various GPTMS densities. Next, the treated substrates were backfilled with 3-Aminopropyltriethoxysilane(APTES), leading to formation of a series of silicon slides with different amino ending groups(-NH2). PEG molecules were finally immobilized to the APTES treated surfaces by means of chemistry grafting. We employed X-ray photoelectron spectroscopy(XPS) and contact angle measurement to characterize the relationships of-NH2, PEG density and GPTMS reaction time. The results confirmed that the densities of-NH2 and PEG decreased when increasing the GPTMS reaction time.2. The fabrication and characterization of gradient PEG density surface: According to the inverse proportional relationship between PEG density and the reaction time of GPTMS as mentioned above, we employed a micro-injection method to fabricate-NH2 gradient density on silicon substrates by controlling the GPTMS injection velocity of a syringe pump. Then,-NH2 density gradient was conjugated with PEG, resulting in the PEG density gradient. The characterization with X-ray photoelectron spectroscopy(XPS) and spectroscopic ellipsometry confirmed the successful formation of gradient PEG density. The treated silicon slide displayed a gradient density of PEG ranging from 0.56 to 0.75 chains/nm2.3. The ECs migration inducing by gradient PEG density: Endothelial cells(5×103 cells/cm2) were seeded onto the uniform and gradient surfaces for in vitro culture. The F-actin and nuclei of cells were stained with fluorescence kits and the cells morphology was observed with a confocal laser scanning microscopy(CLSM). The results indicate that cells culturing onto uniform PEG samples displayed disordered cytoskeleton organization with various directions, whereas, cells on gradient PEG surfaces mainly oriented in that direction of reduced PEG density. Moreover, we employed a living cell station to observe and record the migration of ECs. Then, we analyzed the cells photos, re-constructed the cells migration trajectories on various surfaces. Furthermore, the migration parameters including net displacement, total migration distance, chemotactic index and percentage of cells move towards gradient regarding cell migration were statistically analyzed. The results confirm that most of the cells on the surface with high PEG gradient density moved towards the direction with a low PEG density, and cells adhering on surfaces with high/middle PEG density had higher cell motility than that of low PFG density surface.The study provides an alternative to investigate the effect of the surface PEG gradient density on the migration of ECs, which is essentially important for understanding cell migration/in growth behavior for angiogenesis involved in implant technology.