RESUMEN
Purpose: To investigate the impact of tissue derived biological particles on enzyme-mediated weakened corneas. Methods: Rabbit corneas were treated with enzymes to create an ex vivo ectatic model that simulated representative characteristics of keratoconus (KC). Porcine cornea, cartilage, and lymph node tissues were processed to remove most cellular components and cryomilled into microparticles. The KC corneas were cultured in medium containing the tissue-derived biological particles (TDP) overnight. The mechanical, thermal, ultrastructural changes, and gene expressions of corneal stromal cells were characterized to evaluate the effects of the TDP treatment. Results: The enzyme treatment significantly reduced corneal mechanics and thermal stability, and also disrupted the extracellular matrix ultrastructure. After culturing with TDP medium, the Young's modulus of the modeled KC corneas increased by ~50%, comparable to normal cornea controls. Similarly, the thermal denaturation temperature of the corneas was restored. These findings also corresponded to a significant increase in collagen fibril density after TDP treatment. Furthermore, corneas cultured in TDP medium significantly downregulated expression of the pro-inflammatory gene Tnfα, and restored the expression of the key keratocyte markers Aldh, keratocan, and biglycan. Conclusions: Tissue-derived biological particles reinforce mechanical and thermal properties of corneal tissue in an ex vivo model of KC. Through this study, we demonstrate and characterize the previously unexplored impact of tissue-derived biological scaffolds on corneal biomechanics, thermal stability, and gene expression, presenting a potential new therapy for ocular disease.
RESUMEN
Purpose: To evaluate the crosslinking effect of functionalized chondroitin sulfate (CS) in an ex vivo rabbit cornea model. Methods: Chondroitin sulfate molecules were chemically modified with the N-hydroxysuccinimide (NHS) group. Enucleated rabbit eyes were crosslinked with 2, 5, or 10 mg/mL CS-NHS solution for 30 or 60 minutes. The CS-NHS penetration, corneal swelling ratio, Young's modulus, and ultrastructure of the crosslinked corneas were characterized. In addition, rabbit corneas were further treated with a collagenase-chondroitinase solution to create an ex vivo keratoconus (KC)-like model. The KC model corneas were crosslinked with a standard riboflavin-ultraviolet (UV) method or alternatively with CS-NHS. Corneal mechanics, ultrastructure, and keratocyte gene expression were evaluated after UV and CS-NHS crosslinking. Results: CS-NHS effectively penetrated into the corneal stroma within 60 minutes of treatment initiation. CS-NHS crosslinking reduced the swelling ratio by 35%, increased Young's modulus by 20%, and increased collagen fibril diameter and density. CS-NHS crosslinking improved corneal mechanics of KC model corneas to levels comparable to those with UV crosslinking. Moreover, CS-NHS crosslinking demonstrated significant downregulation of proinflammatory gene expression of keratocytes, indicating a potential protective effect imparted by CS-NHS during crosslinking. Conclusions: Our results demonstrated that CS-NHS can reinforce normal and KC model corneal mechanics, and restore collagen density and alignment in KC model corneas without causing extensive keratocyte apoptosis and proinflammatory gene upregulation. Therefore, CS-NHS crosslinking can potentially provide an effective, safe, and biocompatible means of corneal reinforcement.