Identification of Treatment Protocols for Effective Cross-Linking of the Peripheral Cornea: An Experimental Study

This experimental ex vivo study evaluated how modifying corneal collagen cross-linking (CXL) parameters can improve treatment efficacy in the peripheral cornea (9–11 mm zone), a region implicated in recurrent keratoconic progression and transplant tilt after keratoplasty. Human donor corneas were treated using continuous UVA irradiance of 9 mW/cm² while varying total delivered energy (5.4, 7.2, or 10 J/cm²) and oxygen availability during treatment (normoxia at 21% O2 vs hyperoxia at 100% O2). Biomechanical stabilization was assessed by stress–strain testing to a defined strain endpoint and by collagenase digestion time, while cellular effects were evaluated by TUNEL assay quantification and H&E histology to estimate depth and magnitude of stromal cell damage. Increasing total energy generally increased resistance to deformation and enzymatic digestion, and supplemental oxygen further increased biomechanical stability and digestion resistance within protocols. Standard fluence (5.4 J/cm²) produced limited biomechanical improvement in the peripheral cornea, whereas 7.2 J/cm² with 100% O2 and 10 J/cm² without supplemental oxygen provided the most robust improvements, although higher fluence with hyperoxia produced deeper cellular effects that raised concerns about endothelial safety margins. Overall, the results support adapting pCXL protocols for the thicker peripheral cornea to improve long-term transplant stability, with 7.2 J/cm² + 100% O2 or 10 J/cm² (normoxia) emerging as the most effective compromise between biomechanical benefit and safety considerations in this ex vivo model.