Preparation Of Cell Template By Softlithography And Co-Culture Endothelial/Smooth Muscle Cells

The recovery of intimal function and integrity on biomaterials surface is one of the best ways to prohibit the thrombosis and embolism complications of cardiovascular implanted devices and improve long-term lumen patency. In this thesis, Microtransfer Molding was used to fabricate the hyaluronic acid (HA) micropattern (Cells template) on the activated titanium (TiOH) surface, which was based on the design concept of biomimetic surface engineering. The HA micropatterns were characterized by scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR), contact angle measur-ement, surface profiler and energy dispersive X-ray spectroscopy (EDX). Furthermore, the stability and anti-adhesion function of HA were investigated. The zymohy- drolysis of hyaluronidase, collagen and fibronectin (FN) self-assembly were used to switch the anti-adhesion function of HA which was evaluated enzyme-linked immunosorbent assay (Elisa) and cell adhesion experiments. The adhesion behaviors of platelet, endothelial cells and smooth muscle cells on the cells template were studied, respectively, and the co-culture of endothelial cells /platelet and endothelial cells/ smooth muscle cells on the cells template were also investigated.The results of SEM FTIR and contact angle measurement showed the changes of morphology, increases of roughness and hydrophilicity and detection of ophilic groups on the Ti surface after treatement of NaOH solution. Surface profiler and SEM results showed that polydimethyl-siloxane (PDMS) stamps with high fidelity were prepared. SEM and EDX results demonstrated that HA micropattems with HA and TiOH alternating stripes had been prepared accurately on the surface of TiOH. However, thickness of HA stripe was not uniform. XPS results demonstrated that HA micropattems were still distinct and HA adsorbed stably on the surface of TiOH after washing by phosphate buffer (PBS).The results of cells adhesion showed that the anti-adhesion function of HA can be effectively switched by enzymatic action, collagen and FN self-assembly. The three treatments displayed weak effect on the smooth muscle cells (SMCs) proliferation, which was proven by the results of CCK-8. The self-assembly of collagen used to the change the anti-adhesion function of HA could obviously enhance the adhesion of ECs compared with other two switch methodsThe results of single cell culture showed that the adhesion of platelet, endothelial cells (ECs) and SMCs were restricted by HA micropatterns. Furthermore, when the size of HA/TiOH alternating stripe was 20μm/30μm, which was comparable to the size of ECs, ECs and SMCs were elongated along the stripes. In addition, the shape index of ECs cultured on patterned surface was more similar to that of ECs grown in vivo compared with ECs on flat surfaces. The co-culture of two kinds of cells, such as ECs/platelet and ECs/ SMCs, showed that co-culture system with ordered cell distribution could be obtained on the surface of HA/TiOH alternating stripes. The pattern of platelets (which adhered on TiOH alternating stripes) did not affect the morphology of endothelial cells. However, the long axis of ECs could be regulated through SMCs alignment. But SMCs could stride over the ECs and would form staggered multi-layers structure during a long time co-colture period.Our results indicates the anti-adhesion function of HA will be effectively changed by enzymatic action, Collagen and FN self-assembly. The cell templates can be used to regulate cell adhesion and spreading on the patterns, and construct a short-term co-culture system of ECs/SMCs.