Cell Behaviors and Its Gene Analysis of Nerve-Like Cell on Three-Dimensional Fibrous Matrix

It is an important factor for a scaffold design for tissue engineering uses that biodegradable polymer is utilized as a matrix suitable for cell growth and influences various cell behaviors, and it will also affect further gene expression of target cell. So, the goal of this research grant will establish an experiment model for development of tissue engineering to find out the influence of cell behaviors and gene expression via control on signal factors or/and scaffold design by a model matrix for nerve regeneration which we will do. The experiment will finishes with two years including (1) fabrication of three-dimensional porous membrane and its coated with functional natural biopolymer, (2) measurement and characterization of the obtained electrospun membranes in physico-chemical properties, (3) the cytotoxicity and in vitro study of the obtained membrane, and (4) the gene expression and the immunocytochemistry of cultured cell on the membranes. In practical, dichloromethane and N,N-dimethyl formamide will be used as a binary solvent system to dissolve poly(D-lactide) [PLLA] or/and poly(butylene sucinate-co-adipate) [PBSA] to fabricate two series of electrospun mats with nearly 10-folds micro-/nanometeric diameter, respectively. Furthermore, collagen type I and poly(-glutamic acid) will be used to coat the electrospun membranes. The thickness, contact angle, porosity and pore size of the membranes will be measured, and morphology of membranes will also be performed by epi-fluorescent microscope and scanning electron microscope. NIH3T3 will be utilized to investigate the cytotoxicity against membranes, and pheochromocytoma of the rat adrenal medulla (PC12) with/without nerve growth factor (NGF) will be cultured on these fibrous membranes and its cell adhesion and cell proliferation will be measured by WST-1 kits. The green-fluorescence expressed EGFP plasmid will be transcript into the PC12 to observe the morphology of the cells on the membranes. And, Gap43, Map2, Bcl-xL, Bcl-2 and FAS will be performed by gene analyses of PC12 cells after cultivation on the various membranes. We expect these obtained results could establish an optimized solution of nerve-guiding cell model to develop the further artificial peripheral nerve system.