Effect of Microtopographical Cue on Human Corneal Keratocytes Orientation

Abstract

Abstract: : Purpose: It is well known that many cells can orientate in a particular direction in response to a topographical cue. Such property has been explored in bioengineering of tissues which possess specific orientations such as in tendon. Most cells are capable of orientating themselves along narrow fibres as small as 5-50 microns, a phenomenon known as contact guidance first described in 1945. It is known that cells react to topographical cues by changing orientation, adhesion, movement, phagocytosis and cytoskeleton arrangement. In order to bioengineer such a structure, we explore the use of topographical cues for keratocyte orientation and the downstream influences on matrix gene expression. Methods: Human corneal keratocytes were obtained from discarded corneal-scleral ring after penetrating keratoplasty. Following the removal of epithelium and endothelium, the stroma was chopped in small pieces and put into tissue culture flask in reduced serum media. Polycaprolactone (MW 80,000) were used as a scaffold for this experiment. Two membranes were grooved (12.2 X 12.5 X 4 microns) and two were un-grooved. The grooved membrane was embossed using a silica master which is made by photolithography followed by dry-etching. The cellular orientations were viewed under scanning electron microscope, light and reflective microscope. Gene expression were analysed using SupeArray series. Results: Human derived corneal keratocytes respond to microtopographical cues by altering their cellular orientation along the direction of the grooves. An interesting finding is that the cells orientation changes with time in culture and the effects of multi-layering. Long term culture results in cells forming layers at right angles above the surfaces of the grooved structure. SEM images revealed orientation of the matrix being produced along the axis of the grooves. Changes in gene expression were also found to affected by microtopographic environment. Conclusions: Human corneal keratocytes respond to microtopographical cues by altering their cellular orientation along the grooves. The findings have implications not just for bioengineering of a corneal extracellular matrix but also the understanding of corneal wound healing. A combination of the multi-layering effects observed with a biodegradable matrix could be one way of potentially generating the complex arrangement of corneal matrices and ultimately transparency.

Keywords: cornea: basic science • cornea: stroma and keratocytes • gene/expression