Purpose: The purpose of this study was to determine the effects of the Photoactivated Chromophore for Keratitis Corneal Cross-Linking (PACK-CXL) protocol modifications on corneal resistance to enzymatic digestion and treatment depth.
Methods: Eight hundred one ex vivo porcine eyes were randomly divided into groups of 12 to 86 corneas, treated with various epi-off PACK-CXL modifications, including acceleration (30 > 2 minutes, 5.4 J/cm2), increased fluence (5.4 > 32.4 J/cm2), deuterium oxide (D2O) supplementation, different carrier types (dextran versus hydroxypropyl methylcellulose [HPMC]), increased riboflavin concentration (0.1 > 0.4%), and riboflavin replenishment during irradiation (yes/no). Control group eyes did not receive PACK-CXL. A pepsin digestion assay was used to determine corneal resistance to enzymatic digestion. A phalloidin fluorescent imaging assay was used to determine the PACK-CXL treatment effect depth. Differences between groups were evaluated using a linear model and a derivative method, respectively.
Results: PACK-CXL significantly increased corneal resistance to enzymatic digestion compared to no treatment (P < 0.03). When compared to a 10 minute, 5.4 J/cm2 PACK-CXL protocol, fluences of 16.2 J/cm2 and higher increased corneal resistance to enzymatic digestion by 1.5- to 2-fold (P < 0.001). Other protocol modifications did not significantly change corneal resistance. A 16.2 J/cm2 fluence also increased collagen compaction in the anterior stroma, whereas omitting riboflavin replenishment during irradiation increased PACK-CXL treatment depth.
Conclusions: Increasing fluence will likely optimize PACK-CXL treatment effectiveness. Treatment acceleration reduces treatment duration without compromising effectiveness.
Translational relevance: The generated data help to optimize clinical PACK-CXL settings and direct future research efforts.