Study On The Fabrication Of Collagen Gradient Via Microcontact Printing And Its Effects On The Adhesion And Migration Of Endothelial Cells

To transfer proteins with various concentrations to the same substrates via a single step, we tried to modify the conventional microcontact printing technique in this study. Microcontact printing technique was employed to fabricate template materials with four different concentrations of collagen (50, 100, 200, 300μg/ml) on the same substrate. Scanning electron microscopy was employed to characterize the quality of PMDS stamp. Confocal laser scanning microscopy (CLSM) was utilized to characterize the absorption of different concentrations of FITC conjugated collagen (50, 100, 200, 300μg/ml) on the substrates surfaces. Software was used to analyze the fluorescence intensity of adsorbed protein on the substrates. Albumin was then used to block the substrates for cell culture of human umbilical vein endothelial cells (hUVECs). Substrates with no collagen adsorption were used as control samples. The influence of different concentrations of collagen on the proliferation of hUVECs within 6, 24, 48 and 72 hours was investigated via MTT assay. The cytoskeletal structures of cells were characterized by CLSM. Path measurement of single cell after 24 h culture indicates the cell's migration speed and absolute displacement. Experiment of observation and quantitative analysis found the results. Fabricated PDMS stamps with complete pattern are flat. Template substrates were fully covered with evenly distributed collagen protein. The fluorescence intensities of adsorbed FTIC conjugated collagen were 38.51±1.63, 55.21±3.88, 73.17±3.59, and 80.95±1.12 in corresponding with four different concentrations of 50, 100, 200 and 300μg/ml, respectively. Endothelial cells spread well on various substrates coated with collagen than those of control samples. The proliferation of endothelial cells on collagen coated substrates is higher (P<0.05) than that of control. With collagen concentration increasing from 50μg/ml to 300μg/ml, the proliferation abilities and absolute displacements of endothelial cells increased (p<0.05) between groups. Except for the group with concentration of 300μg/ml, the migration speed of endothelial cells on collagen coated substrates was lower (P<0.05) than that of control. However, the migration speed of endothelial cells on collagen coated substrates with different concentrations increased (P<0.05) along with collagen concentration increasing from 50μg/ml to 300μg/ml.To sum up, microcontact printing technology is available for fabrication different concentrations of protein domains on the same substrate surface for regulating the adhesion and migration of endothelial cells. It thus provides useful information for understanding the chemotaxis.