Resumen de Generación de córneas humanas artificiales por ingeniería tisular para su utilización como medicamentos de terapias avanzadas
Corneal diseases are one of the most important causes of visual impairment in the world. Thus, many patients affected by this pathology finally need a corneal transplant. This treatment implies two major problems: the lack of donors and the possibility of graft rejection. Therefore, the generation of a corneal substitute in the laboratory has emerged as a necessity. In order to accomplish this challenge, tissue engineering appears as a promising science whose aim is to generate artificial tissues and organs that can replace damaged tissues and organs in the human body. Moreover, construction of artificial organs and tissues by tissue engineering is strongly dependent on the availability of viable cells. For that reason, the viability and the physiological status of cells kept in culture must be evaluated before the cells can be used for clinical purposes. Regarding tissue engineering of the cornea, different approaches have been attempted. In this thesis, two different models of artificial corneas are developed together with the characterization of corneal cell cultures and the establishment of a clinical translational approach.
Both models are based on the combination of corneal cells and scaffolds: one based on fibrin-agarose scaffolds and the other one based on acellular xenografts. Corneal keratocytes were obtained from sclerocorneal limbus donated by human cadavers after digesting corneal stroma using collagenase. Epithelial corneal cells were obtained applying an explants culture technique. Because of the difficulty of culturing human corneal endothelial cells, the characterization of endothelial cells in culture was carried out using rabbit corneal endothelial cells, without using them to generate the tissue engineering corneal contructs. The human artificial cornea based on fibrin-agarose scaffold was developed seeding these cells into an artificial matrix generated using a mixture of agarose VII and fibrin obtained from human donors. The acellular xenograft model was created applying a decellularization process to pig corneas, based on NaCl. After obtaining an acellular corneal stroma from pig corneas, the human keratocytes were seeded over it. Once both models were developed, histological and optical analyses were carried out.
In both models, a well-developed stroma was observed based on the presence of collagen and proteoglycans. The keratocytes proliferated and spread, migrating across the corneal matrix. Some immunohistochemical assays were performed, showing the differentiation and characteristic expression of corneal proteins. Optical analyses revealed the high transparency level that both models presented, observing that UV-light was mostly absorbed by the corneal substitutes. Regarding corneal endothelial cells, a remarkable decrease in the K/Na ratio from the fourth to the fifth subculture was found, suggesting that cells of the first four subcultures would be better candidates for tissue engineering, together with the necessity of achieve cell confluence to maintain the endothelial cell pattern. All these results suggest that corneal substitutes made by tissue engineering show similar characteristics to human corneas. Thus, artificial corneas could represent a promising treatment for many corneal diseases which do not have a proper and adequate established treatment.
