Studies On Reconstruction Of Corneal Tissue Engineering And The Cutivatived Cellular Induced Transformation

Objective:(1) To investigate the feasibility of kidney fiber capsule as carrier for cultivating corneal stromal cells and study the characteristics of the cultured cells.(2)To investigate the role of aqueous humor for the growth of corneal stromal cells.(3) Human umbilical vein endothelial cells were cultured in vitro and were transformed on kidney fiber capsule through the ways of double-surface culture with corneal stromal cells,adding 10%aqueous humor and 10ng/ml bFGF. The feasibility of umbilical vein endothelial cells as seeding cells for reconstruction of corneal endothelial layer was investigated.(4) Human peripheral blood endothelial progenitor cells were cultured in vitro and were transformed on kidney fiber capsule through the ways of double-face culture with corneal stromal cells,adding 10%aqueous humor and10ng/ml bFGF.The feasibility of peripheral blood endothelial progenitor cells as seeding cells for reconstruction of corneal endothelial layer was investigated.METHODS:(1) Rabbit corneal stromal cells were seeded on culture plates or kidney fiber capsule in vitro.Cultured cells were stained with vimentin,acridine orange,propidiumiodide and Hoechst and were observed under microscopes.(2)There were two groups,one group of supplementary aqueous humors,in which 10%aqueous humors were added in the experimental groups,one group of conventional culture.Using the technique of gelatin zymography,matrix metalloproteinases were detected.The cell proliferations of the two groups were examined by the CCK8 assay.The absorbance values of the CCK8 assay were analyzed through statistical approach.(3) Human umbilical vein endothelial cells were isolated with enzyme digestion method.The isolated cells were transformed through the methods as below.The cells seeded on one surface kidney fiber capsule and corneal stromal cells were on another surface.10%aqueous and 10ng/ml bFGF were added in the culture medium of endothelial cells.The changes of cultured cells were observed with H&E staining and immunofluorescence under microscope and scanning electron microscopy.(4) Endothelial progenitor cells were isolated from adult peripheral blood with lymphocyte separation medium.The isolated cells were seeded on one surface kidney fiber capsule and corneal stromal cells were on another surface.10%aqueous and 10ng/ml bFGF were added in the culture medium of endothelial cells.The changes of cultured cells were observed with H&E staining and immunofluorescence under microscope and scanning electron microscopy.RESULTS:(1) Corneal stromal cells seeded on the kidney fiber capsule grew well and had normal stromal cell morphology.Cultured cells attached firmly on the surface of kidney fiber capsule.Stromal cells showed polygonal or dendritic morphology with many intercellular joints. Corneal stromal cells seeded on the culture plate showed long spindle.The growth and proliferation of cells on the kidney fiber capsule were better than the cells on the culture plate.(2) Clear MMP2 and MMP9 electrophoretic bands of gelatin zymography analysis were observed. The absorbance values of the CCK8 assay of the group of supplementary aqueous humors were significantly higher(P＜0.05) than that of the conventional group from 1 to 5 days of culture.(3) Human umbilical vein endothelial cells attached onto kidney fiber capsule in 6 hours and cells extended pseudopods.Cells formed monolayer in 7 days and had a close arrangement.Human umbilical vein endothelial cells of conventional cultures were positively immunostained withⅧfactor.The cells were positively immunostained with neurofilament after 14 days of transformed cultures.(4) Endothelial progenitor cells of conventional cultures were spindle or round and positively immunostained with CD34.The cells were positively immunostained with Aquaporin 1 after 14 days of transformed cultures.CONCLUSION:(1)The kidney fiber capsule has good scaffold property,which facilitates the growth of corneal stromal cells.The cultured cells showed nature typical characteristics.This study provides a simple and efficient way for the cells cultured in vitro.The kidney fiber capsule is an ideal biomaterial for layering tissue engineered cornea stroma.(2) Human umbilical vein endothelial cells and endothelial progenitor cells appear some transformation through the ways of double-face culture with corneal stromal cells,supplementary 10%aqueous humor and 10ng/ml bFGF.They may be used as the seeding cell of reconstruction of corneal tissue engineering.