Study On The Fabrication Of Laminin Gradient Via Inkjet Printing And Its Effect On The Alignment Of Endothelial Cells

The alignment and migration of endothelial cells play an essential role in blood vessels'histological and pathological activities. The wound healing after clinical surgery, re-endothelization of endothelial injury areas and the formation of blood vessels (angiogenesis) of microcirculation after implantation in hosts, are all associated with the physiological migration and alignment of endothelial cells. Chemotaxis is one of the approaches to regulate this process. Chemotaxis refers to the characteristic movement or orientation of a cell along with a chemical concentration gradient.To investigate the influence of the protein surface density gradient on endothelial cell alignment, a novel approach for the fabrication of a laminin gradient on gold-coated substrates has been developed in this study. The fabrication process and the formation of protein gradient were well optimized and characterized in this work. Endothelial cells which were isolated from human umbilical vein were seeded onto gradient surfaces of substrates for investigating cells'alignment. This study contains three parts of detailed work as following:1. A novel approach for fabrication laminin gradient: firstly, programmed printing of an alkanethiol (11-mercaptoundecanoic acid, C10COOH, MUA) gradient onto gold-coated substrates via a modified inkjet printer; secondly, backfilling with 11-mercapto-1-undecanol (C11OH, MUD), thus leading to formation of self-assembly monolayer with functional MUA molecules gradients. The–COOH moieties were activated and then covalently linked with laminin. This treatment led to a surface density gradient of laminin. We employed contact angle measurement, X-ray photoelectron spectroscopy (XPS) and confocal laser scanning microscopy (CLSM) to optimize and characterize the self-assembled monolayers (SAMs) and protein gradient, respectively. The results confirmed that we successfully constructed continuous laminin gradient.2. Endothelial cells were digested and isolated from human umbilical vein, and cultured with RPMI1640 medium including 20% FBS and 0.35g/L glutamine. Cell morphologies were observed inverted microscope. Cells were identified by immunocytochemical staining assay. The results show that prepared cells expressed characteristicⅧfactor of endothelial cells with high cell viabilities. It thus suggests that it is an effective approach to acquire primary endothelial cells from human umbilical veins.3. Endothelial cells were seeded on the surface density gradient with length of 30 mm and uniform density of laminin, respectively. Cells were cultured for 24 hours and stained regarding their cytoskeletons and nuclei. CLSM observations demonstrated that the higher the laminin density the more cells were observed when comparing with those of uniform laminin surfaces. The result indicates that cell attachment is dependent on the surface density of laminin. We observed a strong alignment tendency in parallel to the gradient with polarization of more than 70% at various positions.This work broadens our methodology to investigate chemical stimuli induced cell directional alignment. It is potentially important for understanding cell alignment/ingrowth behavior for angiogenesis and implant technology including tissue-engineered structures.