Corneal collagen crosslinking (CXL) has become widely used in recent years to treat early stages of keratoconus and iatrogenic corneal ectasia.¹ Riboflavin (vitamin B2) has a molecular weight of 376.36 g/mol and is a hydrophilic molecule. Because there are tight junctions between individual cells in the corneal epithelium, riboflavin cannot penetrate an intact corneal epithelium. The standard CXL treatment therefore includes mechanical debridement of the corneal epithelium within a 9-mm-diameter zone and subsequent application of 0.1% riboflavin every 3 to 5 minutes for 30 minutes before initiating ultraviolet-A (UV-A) irradiation (370 nm, 3 mW/cm2) for 30 minutes in combination with continual riboflavin application.²

During UV-A irradiation, stromal collagen and/or glycosaminoglycans are photochemically crosslinked via the natural lysyl oxidase pathway.³ Riboflavin acts as a photosensitizer for production of oxygen free radicals, which are necessary for the CXL process, but it also absorbs the UV-A irradiation and prevents damage to deeper structures such as the corneal endothelium, lens, and retina. The efficacy and safety of a CXL treatment depends on proper imbibition of the corneal stroma with riboflavin.

Most publications reporting short- and long-term safety and efficacy of CXL have used the original protocol for CXL as described above.^2,4-6 CXL is generally considered to be safe, but the epithelial debridement associated with the standard CXL treatment is followed by a few days of discomfort and pain and slow visual recovery. In order to reduce these side effects, various attempts to perform the CXL procedure with the epithelium on have been suggested.

Riboflavin penetrates readily into the anterior portion of a debrided cornea. Basic animal studies by Spoerl et al,⁷ however, indicate that a reasonably high riboflavin concentration is obtained only in the anterior 200 to 300 μm of the corneal stroma. Similar findings have been obtained with fluorescence microscopic measurements of intrastromal riboflavin concentrations. ⁸ Increasing the riboflavin concentration from 0.1% to 0.2% results in a higher intrastromal riboflavin concentration,⁸ but biomechanical tests suggest that similar stiffening of the corneal tissue is obtained with riboflavin concentrations ranging from 0.015% to 0.15%.⁷

Instead of complete debridement of the corneal epithelium, researchers have investigated whether superficial scratching of the epithelial surface manually or by excimer laser ablation would be sufficient to ensure penetration of riboflavin to the corneal stroma. These investigations have shown that, even if the tight junctions between superficial epithelial cells are removed with an excimer laser, the basal epithelial cell layers act as a barrier to riboflavin penetration.⁹ Similarly, superficial scraping with a thin needle, creating a grid pattern, was found to be insufficient to allow riboflavin penetration to the stroma.¹⁰

A number of chemical substances have a toxic effect on the corneal epithelium. Thus, benzalkonium chloride (BAK), tetracaine, pilocarpine, ethylenediaminetetraacetic acid (EDTA), gentamycin, oxybuprocaine, and tromethamine have been used to enhance riboflavin penetration through intact epithelium. Experimental studies in vitro have shown that 0.01% to 0.02% BAK in a hypoosmolar (0.44% NaCl) solution can increase the uptake of riboflavin to approximately one-third the concentration obtained in debrided corneas.^11,12

Clinically, using 0.005% BAK and riboflavin 0.1% in 20% dextran T-500, Wollensak et al¹³ found that CXL without epithelial debridement reduced the biomechanical effect to approximately one-fifth that of standard CXL. Clinical studies also suggest that 0.005% BAK is insufficient to promote riboflavin uptake through an intact epithelial layer.¹⁴

Tromethamine and EDTA can be used to enhance riboflavin uptake in corneas after superficial scraping, but the uptake is considerably less than in corneas with epithelium removed.¹⁰ In a noncomparative clinical study, riboflavin uptake enhanced with tromethamine and EDTA was, however, found to be effective in halting keratoconus progression.¹⁵ In vitro, tetracaine was shown to be inefficient to permit penetration of riboflavin into the corneal stroma.¹⁶

CONCLUSIONS

Penetration of riboflavin into the corneal stroma depends on the integrity of the corneal epithelium. Complete debridement of the epithelium most effectively ensures proper imbibition of the corneal stroma with riboflavin. Some of the published chemical modifications of riboflavin solutions for performing transepithelial CXL are promising but should not be used routinely until safety and efficacy have been studied in detail.

Jesper Hjortdal MD, PhD, is a Clinical Professor in the Department of Ophthalmology of Aarhus University Hospital in Denmark. Professor Hjortdal states that he has no financial interest in the products or companies mentioned in this article. He may be reached at +45 7846 3221; fax: +45 8612 1653; e-mail: jesper.hjortdal@dadlnet.dk.

1. Wollensak G. Crosslinking treatment of progressive keratoconus: new hope. Curr Opin Ophthalmol. 2006;17:356-360.
2. Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet-A-induced collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol. 2003;135:620-627.
3. Andley U. Photooxidative stress. In: Albert DM, Jakobiec FA, eds. Principles and Practice of Ophthalmology. Philadelphia: WB Saunders: 1992: 575-590.
4. Raiskup-Wolf F, Hoyer A, Spoerl E, Pillunat LE. Collagen crosslinking with riboflavin and ultraviolet A light in keratoconus: long-term results. J Cataract Refract Surg. 2008;34(5):796-801.
5. Hersh PS, Greenstein SA, Fry KL. Corneal collagen crosslinking for keratoconus and corneal ectasia: one-year results. J Cataract Refract Surg. 2011;37(1):149-160.
6. Ivarsen I, Hjortdal J. Collagen cross-linking for advanced progressive keratoconus. Cornea. In press.
7. Spörl E, Schreiber J, Hellmund K, et al. Studies on the stabilization of the cornea in rabbits. Ophthalmologe. 2000;97:203- 206.
8. Søndergaard AP, Hjortdal J, Breitenbach T, Ivarsen A. Corneal distribution of riboflavin prior to collagen cross-linking. Curr Eye Res. 2010;35:116-121.
9. Bakke EF, Stojanovic A, Chen X, Drolsum L. Penetration of riboflavin and postoperative pain in corneal collagen crosslinking: excimer laser superficial versus mechanical full-thickness epithelial removal. J Cataract Refract Surg. 2009;35:1363-1366.
10. Alhamad TA, O’Brart DP, O’Brart NA, Meek KM. Evaluation of transepithelial stromal riboflavin absorption with enhanced riboflavin solution using spectrophotometry. J Cataract Refract Surg. 2012;38:884-889.
11. Kissner A, Spoerl E, Jung R, Spekl K, Pillunat L, Raiskup F. Pharmacological modification of the epithelial permeability by benzalkonium chloride in UVA/riboflavin corneal collagen cross-linking. Curr Eye Res. 2010;35:715-721.
12. Raiskup F, Pinelli R, Spoerl E. Riboflavin osmolar modification for transepithelial corneal cross-linking. Curr Eye Res. 2012;37:234-238.
13. Wollensak G, Iomdina E. Biomechanical and histological changes after corneal crosslinking with and without epithelial debridement. J Cataract Refract Surg. 2009;35:540-546.
14. Koppen C, Wouters K, Mathysen D, Rozema J, Tassignon MJ. Refractive and topographic results of benzalkonium chlorideassisted transepithelial crosslinking. J Cataract Refract Surg. 2012;38(6):1000-1005.
15. Filippello M, Stagni E, O’Brart D. Transepithelial corneal collagen crosslinking: bilateral study. J Cataract Refract Surg. 2012;38(2):283-291.
16. Hayes S, O’Bart DP, Lamdin LS, et al. Effect of complete epithelial debridement before riboflavin-ultraviolet-A corneal collagen crosslinking therapy. J Cataract Refract Surg. 2008;34:657-661.