Experimental Research On Construction Of Tissue Engineering Trachea

Objective To isolate, harvest, culture and identify bone marrow mesenchymal stem cells(BMMSCs) from human ribs. BMMSCs characteristic was observed in vitro so that to explore the possibility of BMMSCs as seed cells for tissue engineering.Methods BMMSCs were harvested from ribs and isolated, purified by the method of density gradient centrifugation and adhering to the culture plastic. Cells of P2and P4were examined by flow cytometry for the presence or absence of special hematopoietic and endothelial markers (CD29, CD34, CD44, CD45, CD105). Cell growth curve of P0, P4, P8of human BMMSCs was drawn according to cells counting.Results Methods of density gradient centrifugation and adhering to the culture plastic succeed to harvest mononuclear cells with specific characteristics. The cells showed presence of CD29, CD44, CD105and absence of CD34, CD45. Growth curve showed that there was difference of latency, activity and flat periods between primary and passage culture.Conclusion1. Methods of density gradient centrifugation and adhering to the culture plastic can harvest high purified BMMSCs.2. The excellent growing ability of BMMSCs makes it suitable as seed cells for tissue engineering.3. BMMSCs showed high percent presence of CD29, CD44, CD105and absence of CD34, CD45. Objective To investigate the in vitro induction and differentiation of BMMSCs into human airway epithelial cells. The cells induced were observed and identified by morphology to explore the possibility as seed cells for tissue engineering trachea.Methods Cells derived from BMMSCs were cultured in the medium with cytoplasm of lysated rabbit tracheal epithelial cells. HE stain and Gimsa stain was employed to identify the BMMSCs differetionated to tracheal epithelial-like cells.Results Human bone marrow-derived mesenchymal stem cells significantly changed from long spindle shape to polygon shape after3days. HE stain and Gimsa stain show the obvious difference between the BMMSCs and the cells cultured in the medium with cytoplasm of lysated rabbit tracheal epithelial cells.Conclusion The induction of differentiation from BMMSCs to the tracheal epithelial-like cells in vitro is possible. Objective To investigate the morphologic, physiochemical and cytotoxity of the polyepoxy compound and glutaraldehyde-fixed rabbit trachea. Methods Twelve rabbit tracheas were divided into three groups and treated with polyepoxy compound (PC group, n=4), glutaraldehyde(GA group, n=4), and untreated group(fresh group, n=4)respectively. Morphologic and physiochemical properties of three groups, including colors, wall thickness and elastics were studied before and after fixed. Samples of each group were taken out at various elapsed fixation periods, the fixation index were calculated. The moisture content for each sample was determined. Tissue stability was studied by collagenase â…¡ digestion in vitro. The cytotoxicity of PC-fixed rabbit tracheas and GA-fixed rabbit tracheas was studied by co-culturing with BMMSCs, another method is fixation the BMMSCs with different gradient PC or GA fluid and then compare the count of survival cells.Results There were no difference in wall thickness, colors and elastics between PC group and fresh group but there were significant difference between PC group and GA group. During the program, it was learned the rate of the crosslinking with the glutaraldehyde fixation was faster than that of the polyepoxy compound fixation. The moisture content for both studied groups was lower than the fresh one, while the polyepoxy compound-fixed rabbit tracheas were relatively higher than its glutaraldehyde-fixed counterparts. Also, the mechanical properties of PC group were better than the glutaraldehyde-fixed counterparts. Both of two were better than the fresh group. The tissue stability for both studied groups was comparable and higher than the fresh one. The results showed that the co-culturing BMMSCs with PC-fixed rabbit trachea survived and grew, but the BMMSCs co-culturing with the GA-fixed trachea were necrosis within3days. As the fixation fluid, the PC and GA were both high cytotoxicity, less than15%BMMSCs can survive if they were treated with the origin gradient PC or GA, but when the fixation fluid gradient was released by8times, more than70%BMMSCs can survive and grow.Conculsion The polyepoxy compound can effectively preserve the structure of the rabbit trachea and improve the tissue stability and enhance the tension of rabbit trachea. Polyepoxy compound fixation can significantly reduce the cytotoxicity of the trachea than glutaraldehyde.