To date, corneal transplantation has been the only therapeutic choice for corneal failures, yet the global donor shortage makes limitation for this therapy. Tissue engineering strategies can be promising to compensate for this limitation and production of HCEC grafts. The purpose of this study was to produce a transparent and transplantable membrane made of Nanochitosan (NCh)-polycaprolactone (PCL) blends for carrying corneal endothelial cells.

Chitosan nanoparticles were prepared using ionic-gelation method. NCh solution were added to PCL solution to achieve the final ratio of 3:1 (NCh-PCL). Membranes were made using molding method. Biocompatibility and Biomechanical features of the membrane were examined after its preparation including cytotoxicity as well as transparency. HCECs were isolated and cultured on the NCh-PCL membrane. The culture medium contained 5µm Y-27632. The cells phenotype was evaluated by immunostaining against ZO-1, Na+/K+ ATPase. Proliferative activity and cytotoxicity were examined using MTT assay. Related genes expression was evaluated by PCR.

Replacing of the chitosan with nanochitosan in the NCh-PCL membranes was found to be appropriate scaffold, for its desired biomechanical and safety characteristics as well as improving cell behaviors such as cell adhesion and expansion. Immunostaining against ZO-1, Na+/K+ ATPase and actin filaments showed that treatment with Y-27632 not only promoted the cells adhesion and proliferative activity of the endothelial cells but also maintained the cells shape and phenotype while the irregular cell shapes was obtained in the control cultured HCECs. Investigation of gene expression showed that expression of ZO-1, Na+/K+ ATPase, and HTERT remarkably affected by culturing HCECs on NCh-PCL membrane and treating with Y-27632 with compared to control.

NCh-PCL membrane has favored effects on cultured HCECs behaviors such as cell adhesion and proliferation, so, it can be used as a suitable carrier for corneal endothelial cells transplantation. Y-27632 has an appropriate effect on HCECs proliferation, shape and phenotype.