Quantitative Measurements of Stromal and Epithelial Riboflavin With a Novel Transepithelial High-Concentration Riboflavin Soak-and-Rinse Protocol

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Purpose: To enrich stromal riboflavin concentration and reduce epithelial riboflavin in transepithelial corneal collagen crosslinking (CXL). Methods: Ex vivo experiments on rabbit corneas were performed. The control group followed the standard (epi-off) Dresden CXL: a 30-minute epi-off application of 0.1% riboflavin and 20% dextran. The transepithelial riboflavin solutions consisted of various riboflavin concentrations in 1% hydroxypropyl methylcellulose in 0.45% saline, with or without 0.01% benzalkonium chloride (BAK). The novel soak-and-rinse protocol consists of 0.8% riboflavin with 0.01% BAK and 1% hydroxypropyl methylcellulose (hypotonic) applied for 20 minutes, followed by a 10-minute saline rinse. Stromal and epithelial thicknesses were measured by optical coherence tomography; riboflavin concentrations were quantified by spectrophotometry on 3-mm stromal buttons and epithelial eluates. Statistical analysis employed one-way analysis of variance, linear regression, and one-tailed unpaired t-tests. Results: The 20-minute soak increased stromal riboflavin compared to the 10-minute soak. A 76% higher stromal concentration was achieved by adding BAK to the transepithelial 0.8% riboflavin solution (P < 0.05). The 10-minute rinse achieved a Dresden-equivalent stromal riboflavin level and reduced epithelial riboflavin by 5.9-fold compared to a 20-second rinse (P < 0.0001). Conclusions: Stromal riboflavin concentrations equivalent to those achieved with the epi-off protocol can be achieved by transepithelial application of the novel high-concentration riboflavin formulation. The additional 10-minute rinse effectively reduced epithelial riboflavin levels, facilitating the delivery of ultraviolet light and oxygen into the stroma during CXL. Translational Relevance: The novel transepithelial riboflavin soak-and-rinse protocol may potentially enhance the efficacy of transepithelial CXL by reducing epithelial consumption of ultraviolet light and oxygen and increasing the stromal CXL reaction.