Tissue Engineering of Self Organized Corneal Stromal Cell-Secreted Extracellular Matrix

Abstract

Purpose:: Fibroblasts of human cornea react differently to flat and structured surfaces on which they are cultured. In this work, we have evaluated the behaviour of human corneal fibroblasts and their cell-secreted extracellular matrix on nanostructured and microstructured polystyrene (PS) in order to reconstruct human corneal stroma by tissue engineering.

Methods:: PS samples were prepared by hot embossing, replicated and transferred from a Si master of different grating periods and linewidths to a PS substrate. The smallest samples were 200 nm period grating with a 100 nm linewidth made by interference lithography. The largest features on our Si master were a 10 um period grating with a 5 um linewidth. To study cell behaviour on these substrates, we used sterelized PS substrate in standard Falcon six wells plates. Cells were obtained by culturing corneal stroma explants. After trypsinization, they were seeded at a concentration of 8000cells/cm2 and cultured in 3ml DMEM with 10% FCS, 100U/ml of penicillin, 25 ug/ml of gentamicin and 50 ug/ml of ascorbic acid in each well. The culture medium was changed every other day and fresh ascorbic acid was added.

Results:: We observed a rapid adhesion of the cells to the nanostructured surface, as in the control. Interestingly, cell division occurs only in the longitudinal axis of the gratings instead of the random cell orientation occurring after cytokinesis in control plates. Cells of the first layer, directly on the substrate, aligned in the same direction of the gratings. We also observed that after 7 days in culture, the second cell layer forming on top of the first one aligned themselves at a 60 degree angle from the first one, thus giving an internal organization similar to the native stromal cornea. Confocal microscopy study also revealed that the collagen fibres secreted from the corneal fibroblasts are aligned in the same direction than the cells, either in the first or the second layer formed in culture.

Conclusions:: This new method provide a mean to produce the alignment of cells and collagen which is crucial for the optical properties of the cornea.

Keywords: cornea: stroma and keratocytes • extracellular matrix • cornea: basic science