Nanofiber Film Growth Behavior Of Human Umbilical Vein Endothelial Cells

Scaffolds fabrication is one of the most important issues in tissue engineering and regenerative medicine. In the recent years, world wide researchers have made great efforts on the design and construction of scaffolds with various structures by mimicking the natural extracelluar matrix (ECM).In natural tissues, cells are surrounded by a complex mixing of nonliving materials that make up the ECM. It is composed of complex mixtures of proteins and proteoglycans, made up of nanofibers. And those fibers organized to form a 3D network with interconnected pores. ECM takes a important role in cell growth, migration and differentiation. In the recent years nanofibrous scaffolds with 3D structure have attracted intensive interests because of providing the micro-environment mimicking the structure of natural extracellular matrix. Usually three ways are applied to fabricate nanofibrous scaffolds, including self-assembly, phase separation and electrospinning. Among those electrospinning technique is the simplest way to make continuous nanofibers in various orientation with many composition.There are three main kind of materials used in electrospinning, natural ECM-based polymer, synthetic polymer and composite materials. Synthetic polymer is the most wide use material in tissue engineering scaffold, because they are easy to gain and usually have good mechanical properties. However, synthetic polymer are lack of bioactive signal. Composite materials, combining the mechanical character of synthetic polymer and biocompatibility of natural polymer, are becoming a promising direction in tissue engineering scaffold research.Carbon exists in all life forms, it has a stable physical property and good biocompatibility. Carbon nanotube (CNT) is a new member of carbon family. It can be considered as a nano-scale tube of graphitic carbon with outstanding properties of conductivity and mechanics. In this study, we have fabricated a novel nanofibrous scaffold from the multiwalled carbon nanotubes/polyurethane composite (MWNT/PU) with aligned fibrous architecture and nonwoven architecture electrospinning technique respectively; nonwoven and aligned nanofibrous scaffolds of PU, planar casting film of PU were prepared and used as control. The effects of nanofibrous architecture and carbon nanotube content on the growth behaviors and anticoagulation function of the human primary endothelial cells have been investigated. The molecular mechanism has been explored and discussed as well.The morphology of nanofibrous scaffolds were observed and characterized through scanning electron microscopy (SEM). The cells growth behaviors including proliferation, cytoskeletal development were investigated by cell viability assay and confocal microscope analysis respectively. The bioactive molecules secreted by the cells including tissue factor, plasminogen activator inhibitor-1, NO and collagen of IV type were analyzed by ELISA.The experimental results indicated that the diameter of the generated fiber made by electrospinning was around 300nm～500nm. Most of the fibers in the aligned nanofibrous scaffold were arranged in one direction. The aligned nanofibrous scaffold presented a significant enhancement to the cells proliferation. The cells growing in the nanofibrous scaffolds exhibited more relevant phenotype than those on the planar casting film of PU. And the cells growing in the aligned nanofibrous scaffolds synthesized and secreted more extracelluar matrix molecules than those growing on the control substrates. The confocal images showed that majority of the cells on the aligned nanofibrous scaffolds arranged in an orientation parallel to the direction of nanofibers in the scaffolds, displaying extending morphology as that in natural tissues. It was also found that adding MEK inhibitor to the culture medium could disrupt the orentation of the cells growing on the aligned nanofibrous scaffolds, and the cells lost their extending mophorlogy as well. The induced cytoskeletal changes implied that MAPK might play an important role in the cell migration.