Translational biomaterials and engineering approaches forcorneal regeneration: targeting stromal stabilization andlimbal stem cell deficiency

Cornea is a transparent and avascular tissue essential for maintaining clear vision. However, corneal ectatic disorders, such as keratoconus, lead to progressive thinning and biomechanical weakening, causing the cornea to lose its regular shape. While riboflavin/ultraviolet-A (UVA) corneal crosslinking (CXL) is widely used to enhance corneal rigidity in clinics, it presents challenges such as cytotoxicity, patient discomfort, and treatment failure in some cases. In addition to biomechanical instability, damage to the limbal region can lead to limbal stem cell deficiency (LSCD), a condition characterized by the loss or dysfunction of limbal stem cells responsible for regenerating the corneal epithelium. LSCD results in impaired wound healing, chronic inflammation, neovascularization, and vision loss, making stem cell-based therapies crucial for corneal surface restoration. This research presents two innovative therapeutic strategies: (1) Ruthenium-blue light mediated CXL as a safer and more effective alternative to Riboflavin-UVA based CXL for corneal stabilization, and (2) FasL-conjugated GelMA nanogels incorporated into human limbal stem cell heterospheroids to enhance transplantation outcomes by inducing apoptosis in immune cells and thereby reducing immune rejection. To overcome the limitations associated with clinical treatments, the first part of this thesis explores an alternative CXL approach utilizing tris(bipyridine)ruthenium(II) ([Ru(bpy)₃]²⁺) and sodium persulfate (SPS) with blue light (430 nm) to induce collagen crosslinking via dityrosine bond formation. The study evaluates the safety and efficacy of ruthenium mediated CXL in ex vivo bovine corneas and in vivo Wistar albino rat models, comparing its structural and histological effects with conventional riboflavin/UVA CXL. The findings indicate that ruthenium mediated CXL effectively enhances corneal stiffness while minimizing UV-induced cellular damage, presenting a promising clinical alternative for keratoconus treatment. The second part of this thesis addresses limbal stem cell deficiency (LSCD). Current limbal stem cell transplantation methods are hindered by immune rejection, necessitating systemic immunosuppression. To mitigate this issue, we propose a novel strategy leveraging the Fas/Fas ligand (FasL) pathway for localized immune modulation. By functionalizing gelatin methacryloyl (GelMA) nanogels with FasL, we developed an immune-protective biomaterial to enhance limbal stem cell survival post-transplantation. These advancements contribute to the development of clinically viable therapies for corneal disorders, improving treatment outcomes for keratoconus and LSCD.