The Effect Of Surface Gradient Biomaterial On Endothelial Cell Adhesion And Migration

To investigate the effect of surface gradient biomaterials on endothelial cells adhesion and motility, we developed two novel approachs for the fabrication of type I collagen gradient (collagen density gradient and the gradient of micro-radian width with various curvatures) onto substrate in this study. The first experiment involved sequential alkali hydrolysis of PDLLA films to produce–COOH density gradient along substrates, followed by covalently immobilizing collagen onto hydrolyzed PDLLA films. This treatment resulted in a surface-density gradient of collagen onto PDLLA surfaces. Contact angle measurement and confocal laser scanning microscopy were employed to characterize the–COOH gradient and collagen gradient, respectively. The collagen gradient onto PDLLA films was clearly visible by fluorescence microscopy observation. All results confirmed the feasibility of the fabrication of collagen gradient onto PDLLA films via alkali hydrolysis approach. Endothelial cells cultured on the low surface-density and moderate surface-density of collagen gradient areas displayed a strong motility (net displacement, chemotactic index, and migration rate, cell trajectories) tendency in parallel to the gradient. However, endothelial cells grown on the high surface-density of collagen gradient areas demonstrated a reverse response of motility to collagen gradient clues. The result suggests that cell motility is regulated by collagen gradient, however, with appropriate surface-density. The second experiment involves the micropatterning technology. Based on the principle of bionics, we designed some new patterns (the width of the micro-radian pattern is 20μm, 60μm, 100μm respectively) which similar to different blood vessels (from capillary to artery). From the patterns we can obtain a collagen substrate microenvironment with various curvatures. Scanning electron microscopy was employed to characterize the quality of PMDS stamp. Confocal laser scanning microscopy (CLSM) was utilized to characterize the absorption of FITC conjugated collagen solution. The staining of F-action and vinculin showed cells'adhesion on the substrates surfaces. Live Cell Imaging System was used to take cell's migration image recording of 6h. Software was used to analyze migration rate and cell's migration distance / net displacement between 20, 60 and 100μm on the protein substrates. The result suggests that Cell adhesion status is proportional to the width of the curvature, the smaller radius of curvatures,the faster of the migration rate. Meanwhile, it can make the cell motility strengthen. Cell movement ability on different width of the micropattern is: 100μm>20μm >60μm.These studies analyzed a series of migration parameters about endothelial cell's migration on various collagen protein gradient substrates, and obtained the optimal substrate for cell's movement and adhesion. This study provides proper substrates for investigating chemical stimuli that induced cell directional motility. It is potentially important for controlled angiogenesis for implantation of tissue-engineered devices and will provide new ideas to vascular remodeling and angiogenesis.