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Focus On Corneal Collagen Cross-linking | May 2012

The Drug Science of Cross-linking

Research is elucidating how the combination of riboflavin and ultraviolet light strengthens the cornea.

The use of chemical cross-linking in materials science as a means of corneal stiffening is well described. Ten years ago, surgeons began crosslinking collagen to stabilize progressive corneal ectatic disorders. During this time, much has been elucidated about the drug science behind this treatment.


Riboflavin, also known as vitamin B2, is a naturally occurring photosensitizer. It is the precursor of flavin mononucleotide and flavin adenine dinucleotide, coenzymes that are crucial for the metabolism of carbohydrates, fats, and proteins into energy. Riboflavin is an essential constituent of all living cells. It is water soluble and nontoxic, and it is used as a coloring agent in food and pharmaceuticals. The intake of riboflavin from food and dietary supplements ranges from 4 to 10 mg per day. No adverse effects have been associated with high intakes of riboflavin from food or supplements. In the cornea, collagen cross-linking occurs naturally with aging due to an oxidative deamination reaction that occurs within the end chains of the collagen. The standard concentration of riboflavin used in the riboflavin-ultraviolet A (UVA) treatment for ectatic disorders is 0.1%. The maximum amount of riboflavin to which a patient is exposed during corneal collagen cross-linking with the standard Dresden protocol is estimated to be 1.6 mg, if the volume of one drop is 0.05 mL and one drop is being instilled every 2 minutes for 30 minutes before and 30 minutes during UVA light irradiation (32 drops total). This amount is considered to be well within a safe dosage.1


A series of in vitro experiments has characterized the potential cytotoxicity of UVA light and the UVAriboflavin exposure on keratocytes and the function of endothelial cells. Riboflavin alone has not been shown to have a cytotoxic effect on keratocytes or endothelial cells. The cytotoxic threshold for inducing cellular necrosis or apoptosis was 5 mW/cm2 for UVA light alone and 0.5 mW/cm2 for the UVA-riboflavin treatment. Using the Lambert-Beer equation, in human corneas, the cytotoxic keratocyte UVA irradiance of 0.5 mW/cm2 is reached at a stromal depth of 300 μm.2 Researchers believe that damage to endothelial cells is due to oxidative injury caused by the reactive oxygen free radicals (singlet oxygen, superoxide anion, hydrogen peroxide) that are generated by the interaction of riboflavin with UV light. Ninety-four percent of incident UVA light is absorbed by the anterior 400 μm of the corneal stroma in the presence of riboflavin (Figure), whereas only 32% is absorbed within that depth in the absence of riboflavin.3,4 Riboflavin therefore serves the dual purpose of sensitizing the stroma to the effect of UV light and protecting the corneal endothelium from UVA damage.


Researchers have evaluated the use of riboflavin for the inactivation of blood-borne pathogens through the production of reactive oxygen species (ROS),5 which can be created by the triplet excited state of riboflavin.6 In the cornea, ROS can create advanced glycation end products,7 which, in turn, increase collagen diameter.8 Investigators have suggested that the advanced glycation end products may stiffen the cornea9 and, in so doing, may serve as the mechanism underlying the relatively protective effect that diabetes has on the development of keratoconus.10

The majority of cross-linking occurs between collagen a and b chains. These cross-link into g chains and larger aggregates. Corneal proteoglycans also cross-link with themselves to form larger polymers, but collagen and proteoglycans show only limited cross-linking with one another.11 This suggests that cross-linking creates collagen-to-collagen cross-links as well as proteoglycanto- proteoglycan cross-links but only a small amount of collagen-to-proteoglycan cross-links. Although the concept is poorly understood, investigators suspect that ROS induce stabilization, either between collagen fibers or to their surrounding matrix. Corneal collagen cross-linking is the first surgical procedure that appears to halt the progression of corneal ectatic disorders such as keratoconus and ectasia after refractive surgery.1,12 The corneal epithelium permits the passage of small, lipophilic molecules, whereas the stroma forms a barrier against the passage of lipophilic compounds while readily allowing the movement of hydrophilic molecules. 13 Riboflavin is a highly hydrophilic molecule that absorbs UV light at a wavelength of 370 nm.14 Intact, tight junctions of the corneal epithelium block the absorption of riboflavin and the transmittance of UVA light into the corneal stroma.15,16 Investigators have developed numerous techniques for the transepithelial delivery of riboflavin, although the efficacy of the traditional epithelium-off technique is currently better supported by the literature.17

The aforementioned studies characterizing the nature and interaction of riboflavin and UVA light have revolutionized the treatment of corneal ectatic disorders and paved the way for the next generation of cross-linking treatment modalities.

Derrick A. Huey, MD, is a resident physician in the Department of Ophthalmology at the Medical University of South Carolina in Charleston and at Magill Vision Center in Mt. Pleasant, South Carolina. He acknowledged no financial interest in the material presented herein.

George O. Waring IV, MD, is the director of refractive surgery at the Storm Eye Institute and assistant professor of ophthalmology at the Medical University of South Carolina in Charleston. Dr. Waring is also the medical director of Magill Vision Center in Mt. Pleasant, South Carolina. He is a consultant to Topcon Medical Systems, Inc., and serves as chairman of the scientific advisory board for the company's cross-linking technologies. Dr. Waring may be reached at georgewaringiv@gmail.com.

  1. Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet-A-induced collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol. 2003;135:620-627.
  2. Wollensak G, Spoerl E, Reber F, Seiler T. Keratocyte cytotoxicity of riboflavin/UVA treatment in vitro. Eye. 2004;18:718-722.
  3. Cho KS, Lee EH, Choi JS, Joo CK. Reactive oxygen species-induced apoptosis and necrosis on bovine corneal endothelial cells. Invest Ophthalmol Vis Sci. 1999;40:911-919.
  4. Spoerl E, Schreiber J, Hellmund K, et al. Studies on the stabilization of corneas in rabbits [in German]. Ophthalmologe. 2000;97:203-206.
  5. Goodrich RP, Gilmour D, Hovenga N, Keil SD. A laboratory comparison of pathogen reduction technology treatment and culture of platelet products for addressing bacterial contamination concerns. Transfusion. 2009;49:1205-1216.
  6. McCall AS, Kraft S, Edelhauser HF, et al. Mechanisms of corneal tissue cross-linking in response to treatment with topical riboflavin and long wavelength ultraviolet radiation (UVA). Invest Ophthalmol Vis Sci. 2010;51:129-138.
  7. Brummer G, Littlechild S, McCall S, et al. The role of nonenzymatic glucagon and carbonyls in collagen crosslinking for the treatment of keratoconus. Invest Ophthalmol Vis Sci. 2011;52:6363-6369.
  8. Wollensak G, Wilsch M, Spoerl E, Seiler T. Collagen fiber diameter in the rabbit after collagen crosslinking by riboflavin/UVA. Cornea. 2004;23:503-507.
  9. Hager A, Wegscheider K, Wiegand W. Changes of extracellular matrix of the cornea in diabetes mellitus. Graefes Arch Clin Exp Ophthalmol. 2009;247:1369-1374.
  10. Seiler T, Huhle S, Spoerl E, Kunath H. Manifest diabetes and keratoconus: a retrospective case-control study. Graefes Arch Clin Exp Ophthalmol. 2000;238:822-825.
  11. Zhang Y, Conrad A, Conrad G. Effects of ultraviolet-A and riboflavin on the interaction of collagen and proteoglycans during collagen cross-linking. J Biol Chem. 2011; 286(15):13011-13022.
  12. Hafezi F, Kanellopoulos J, Wiltfang R, Seiler T. Corneal collagen crosslinking with riboflavin and ultraviolet A to treat induced keratectasia after laser in situ keratomileusis. J Cataract Refract Surg. 2007; 33:2035-2040.
  13. Prausnitz M, Noonan J. Permeability of cornea, sclera, and conjunctiva: a literature analysis for drug delivery to the eye. J Pharm Sci. 1998;87(12):1479-1488.
  14. Wu M, Eriksson L. Absorption spectra of riboflavin-a difficult case for computational chemistry. J Phys Chem A. 2010;114:10234-10242.
  15. Bottos KM, Schor P, Dreyfuss J, et al. Effect of corneal epithelium on ultraviolet-A and riboflavin absorption. Arq Bras Oftalmol. 2011;74(5):348-351.
  16. Podskochy A. Protective role of corneal epithelium against ultraviolet radiation damage. Acta Ophthalmol Scand. 2004;82:714-717.
  17. Baiocchi S, Mazzotta C, Cerretani D, et al. Corneal crosslinking: riboflavin concentration in corneal stroma exposed with and without epithelium. J Cataract Refract Surg. 2009;35(5):893-899.
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