Ruthenium-induced Corneal Collagen Crosslinking under Visible Light

Abstract

Purpose : Corneal collagen crosslinking (CXL) is a commonly used minimally invasive surgical technique to prevent the progression of corneal ectasias, such as keratoconus. Unfortunately, riboflavin and UV-A light-based conventional CXL procedures have not been successfully applied to all patients, and they usually have frequent complications, such as corneal haze and endothelial damage. This study proposes a new method for corneal crosslinking by using ruthenium (Ru) based photoinitiator solution and visible light (430 nm).

Methods : As a ruthenium photoinitiator solution, we applied tris(bipyridine)ruthenium(II) ([Ru(bpy)₃]²⁺) and sodium persulfate (SPS) mixture covalently crosslinks free tyrosine, histidine, and lysine groups under visible light exposure (400-450 nm). Firstly, we demonstrated the effects of the Ru/visible blue light procedure on the viability and toxicity of the human corneal epithelium, limbal, and fibroblast cells via flow cytometry, immunofluorescence staining, and cell toxicity assays. After that, bovine corneas crosslinked with ruthenium mixture and visible blue light were characterized, and their biomechanical properties were compared with the conventional riboflavin/UV-A crosslinking approach.

Results : [Ru(bpy)₃]²⁺ and SPS did not show toxicity under 1 mM in any human corneal cell lines. The crosslinked corneas with the non-toxic ruthenium-based visible light CXL method showed significantly higher Young modulus in the hybrid rheometer, higher mechanical stiffness in uniaxial stress-strain test, and more bonding between collagen fibrils under scanning electron microscopy compared to conventional riboflavin, and UV-A light-based method applied corneas. Additionally, other materialistic characteristics (hydrodynamic behavior, optical transparency, and enzymatic resistance) of ruthenium-based crosslinked corneas were significantly better than riboflavin-based crosslinked corneas.

Conclusions : In all biomechanical tests, the visible light-based crosslinked corneas with non-toxic doses of ruthenium photoinitiator solutions showed better results than conventional UV-A crosslinked corneas. In this way, we prevent UV-A light-induced cytotoxicity in a faster and more effective collagen crosslinking procedure. We foresee that Ru/visible light-based crosslinking treatment could become the standard therapy for all ectatic corneal pathologies because of its superior biocompatibility, safety, and efficiency very soon.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.