Development of a Biopolymer–Epithelium Complex for Ocular Surface Reconstruction

Abstract

Abstract: : Purpose: To investigate epithelial cell adhesion and proliferation on a newly developed elastin–like polymer that mimics the functional characteristics of extracellular matrix. The final goal is to develop a biopolymer–epithelial complex suitable for ocular surface transplantation. Methods: A genetically engineered elastin–like polymer with cell attachment sequences for epithelial cells, was assayed in two forms: a cross–linked, hydrogel matrix, and a water solution. The latest form was spread onto glass coverslips and allowed to adsorb as 1, 2, or 3 molecular films, during 1 h at 55ºC in vacuum. Epithelial cells from human conjunctiva (IOBA–NHC cell line) were plated onto three different cell substrates: the newly developed bioelastic polymer (prepared as described); glass coverslips; and Thermanox® (the latest two ones as controls). Different histologic stains were tested to identify cells attached to the polymer. Substrates capacity to sustain epithelial cell adhesion and proliferation was evaluated. To evaluate putative damage of enzymatic detachment methods in epithelial cells adhesion receptors, cells were plated after detachment with 0.61 mM EDTA or 0.25% trypsin–1mM EDTA. To test adhesion, cells were plated, incubated for 4 h, stained, and counted. To study proliferation, proliferative cells were identified (immunostaining with anti–Ki–67 monoclonal antibody) and counted after 1, 3, and 5 days in culture. Results: Cells plated onto the polymer hydrogel could not be observed, as the polymer stained with all assayed stains, making it impossible to distinguish plated cells from the polymeric substrate. Consequently, no further experiments were done with this polymeric form. Epithelial cell adhesion onto the glass plate with the adsorbed polymer (in either 1, 2 or 3 molecular films) were enhanced compared to that of control substrates, whereas proliferation index was similar. Detachment using 0.61mM EDTA proved to be more adequate. Conclusions: Elastin–like polymers produced as recombinant proteins resembling ocular surface extracellular matrix have been developed and preliminarily shown to be a suitable scaffold to sustain epithelial cell growing. These protein–based polymers could potentially be used as extracellular matrix substitutes for ocular surface reconstruction or trasplantation. SUPPORT. RTIC C03/13 Instituto de Salud Carlos III and FEDER–CICYT MAT2004–03484–C02–01 and –02, Spain.

Keywords: transplantation • conjunctiva • proliferation