Development and characterization of corneal substitutes from decellularized tissues

Abstract

Diseases affecting the cornea can lead to vision impairment and ultimately to blindness. It is estimated that 45 million people worldwide suffer from corneal blindness. Rejection in high-risk patients and donor shortage have triggered the development of alternative therapies to corneal transplantation. Decellularized tissues were used in this Thesis to fabricate tissue engineered corneal substitutes. Firstly, the role of media supplements to promote a keratocyte-like phenotype in serum-expanded stromal cells was studied. Keratocyte phenotype was partially recovered after serum expansion via serum removal and supplementation with ascorbic acid and insulin-transferrin-selenium. In the second study, scaffolds obtained from decellularized porcine corneas were repopulated with phenotypically correct stromal cells. When implanted in a rabbit model of anterior lamellar keratoplasty (ALK), they integrated well but no benefit was observed when recellularized constructs were implanted compared to their acellular counterparts. In the third study a different approach was used and anterior corneal equivalents were fabricated by stacking sheets of decellularized tissues with cell-laden hydrogels. Cells in these constructs presented high cell viability and keratocyte-like phenotype. Epithelial cells attached and stratified. Constructs were robust to be sutured onto a porcine cornea in an ALK ex vivo model and tissue integration and re-epithelialization was observed. Finally, cornea extracellular matrix-derived hydrogels were fabricated from corneas decellularized with different methods, which impacted in the gel characteristics. Decellularization with sodium dodecyl sulphate rendered cytotoxic gels and gels obtained by freeze-thawing presented the best characteristics overall. These hydrogels were successfully used to fill a defect in situ on an ex vivo porcine cornea and supported host epithelial re-growth. To conclude, this Thesis shows the versatility of using decellularized porcine corneas for the biofabrication of corneal substitutes and highlights the importance of culture conditions in the maintenance or recovery of native cell phenotype.