Characterization of Corneal Epithelial Cell Growth Over Chitosan-Gelatine Biopolymers

Abstract

Purpose: : To investigate corneal epithelial cell adhesion, proliferation, viability, and characterization on biopolymers based on chitosan-gelatine (C-G) to be further used to reconstruct the damaged ocular surface.

Methods: : Membranes were obtained using 85% deacetylated chitosan and different concentrations of pig skin gelatine derived from collagen (0%, 20%, 50% and 80%), in order to mimic extracellular matrix composition. Glutaraldehyde (0.50% wt) was added as chemical crosslinker. Only those polymers that could resist surgical stitches were further studied. Corneal epithelial cells were cultured onto the different membranes for several days. Plastic and amniotic membrane were used as control substrata. To test cell adhesion, cells were incubated for 1/2, 1, 2, 4, and 6 hr, stained with calcein, and counted. To study cell proliferation, the medium culture was collected after 1, 2, 4, and 8 days and the Alamar blue test was used. Cell viability was assessed after 6 hr and 1, 2, and 4 days using the Calcein/EthDIII kit. Cells in 5 fields (10x) were counted and the results were given like percentage of live cells. Immunostaining and real time PCR for several cytokeratins (K3, K7, K12, and K19) and adhesion proteins (E-cadherin, ZO-1, desmoplakin, connexin43 and sprr2) were performed.

Results: : C-G 80:20 membranes could not be sutured and cells did not grow on it. Cell adhesion and proliferation were satisfactory for the C-G 50:50 and 20:80 formulations. The highest cell viability was obtained with the C-G 50:50 membranes after 1, 2, and 4 days (99%). Epithelial cells grew and proliferated on all systems and they expressed all the corneal and adhesion markers. The C-G 50:50 membranes maintained the adhesion marker expression (mainly ZO-1 and sprr2) for 22 days whereas the C-G 20:80 membranes did so with the corneal expression markers (K3, K7 and K19).

Conclusions: : C-G (20:80 and 50:50) membranes were suitable substrates to support epithelial cell attachment and growth. Additionally, ocular surface epithelial phenotype was maintained. These biopolymers may be appropriate for the development of new tissue engineering scaffolds to restore vision by reconstructing the ocular surface.

Keywords: cornea: epithelium • cornea: basic science • gene/expression