Desenvolvimento e caracterização de córneas humanas descelularizadas e recelularização com células-tronco visando regeneração do epitélio corneano anterior

Abstract

Numerous diseases can affect corneal structure, leading to visual impairment and even blindness. Currently, the only treatment for most corneal illnesses is transplantation of corneal allografts. However, graft rejection and the lack of donors are the two major disadvantages associated to this procedure. Generation of artificial corneas based on decellularization methods is a promising alternative to overcome these problems. Therefore, the present study aimed to develop and characterize decellularized human corneas, recellularize these decellularized corneas (CDs) with human stem cells and promote the differentiation of these cells into corneal epithelial-like cells (CECs) in culture medium supplemented with allogeneic human serum (SH) replacing fetal bovine serum. Two decellularization protocols were tested: 1- sodium chloride (NaCl) treatment, 2 - NaCl plus nucleases treatment. The success of each method on the removal of cells from the cornea and the integrity of the extracellular matrix were investigated by histology, electron microscopy, DNA quantification, immunofluorescence and nuclear staining with Hoechst. The CDs were recellularized with human orbital fat-derived stem cells (OFSCs), human embryonic stem cells (hESCs), or human induced pluripotent stem cells (hiPSCs) and the biocompatibility of the CDs was analyzed by calcein-AM staining. The ability of the OFSCs, hESCs and hiPSCs to differentiate into CECs when cultured on CDs or on laminin in culture medium supplemented with SH was evaluated. The results showed that corneas processed using NaCl resulted in incomplete removal of cellular material. In contrast, corneas decellularized with NaCl plus nucleases method resulted in total removal of the cellular components. This treatment also kept the epithelial basement membrane and stroma completely intact. Calcein-AM staining demonstrated the viability, adhesion and a normal morphology of the OFSCs, hESCs and hiPSCs seeded on CDs. OFSCs did not differentiate towards the CECs lineage, while hESCs and hiPSCs differentiated into terminally differentiated CECs when seeded on CDs. These results showed that NaCl plus nucleases treatment of human corneas generates an acellular and biocompatible matrix with adequate histologic properties which have potential applications in corneal tissue engineering.