Corneal cross-linking: intrastromal riboflavin concentration in iontophoresis-assisted imbibition versus traditional and transepithelial techniques

Am J Ophthalmol. 2014 Mar;157(3):623-30.e1. doi: 10.1016/j.ajo.2013.11.018. Epub 2013 Dec 7.


Purpose: To determine differences in riboflavin concentration in the anterior, intermediate, and posterior stroma after 3 corneal cross-linking imbibition techniques (standard epithelial [epi]-off, epi-on, and iontophoresis-assisted administration) of 0.1% riboflavin.

Design: Experimental laboratory investigation of human cadaver corneas not suitable for transplantation.

Methods: Ten corneas underwent imbibition with epi-on (n = 3), epi-off (n = 3), iontophoresis (n = 3), and saline exposure (control; n = 1). Femtosecond laser was used to produce 3 8-mm discs of the superficial (0-150 μm), intermediate (150-300 μm), and deep stroma (>300 μm). Riboflavin concentration was measured with high-performance liquid chromatography. The main outcome measure was riboflavin concentration at the 3 evaluated depths.

Results: The overall stromal concentration of riboflavin was 34.1 ± 7.1 μg/g in epi-off, 7.2 ± 3.7 μg/g in epi-on, and 15.0 ± 5.1 μg/g in iontophoresis. The mean riboflavin content in the superficial slice in the epi-off group was about 2-fold greater than that of the iontophoresis group (50.5 ± 5.3 μg/g and 23.6 ± 2.5 μg/g, respectively) and 4-fold greater than that of the epi-on group (11.7 ± 3.3 μg/g). Similar differences among the 3 groups were observed for the intermediate and posterior stromal slices, presenting an evident reduction of riboflavin concentration with increasing depth in all groups. Slice depth-dependent decrease in riboflavin concentration was statistically significant (general linear model (GLM); F1,6 = 62.265, P < .001), as was the group-dependent variation (GLM; F2,6 = 20.268, P = .002) and the slice depth group interaction (GLM; F2,6 = 18.004, P = .002).

Conclusions: Corneal cross-linking transepithelial iontophoresis imbibition yielded greater and deeper riboflavin saturation with respect to conventional epi-on, while maintaining the advantages of avoiding epithelial removal and shorter procedure time, but did not reach concentrations obtained with standard epi-off.

Publication types

  • Comparative Study

MeSH terms

  • Aged
  • Chromatography, High Pressure Liquid
  • Corneal Stroma / metabolism*
  • Cross-Linking Reagents / pharmacokinetics*
  • Epithelium, Corneal / metabolism
  • Humans
  • Iontophoresis*
  • Middle Aged
  • Photosensitizing Agents / pharmacokinetics*
  • Riboflavin / pharmacokinetics*


  • Cross-Linking Reagents
  • Photosensitizing Agents
  • Riboflavin