Corneal collagen cross-linking (C3) with riboflavin (vitamin B2) (C3-riboflavin) is an innovative technique to treat patients with keratoconus and corneal ectasia. Thanks to the original idea and research of Theo Seiler, MD, PhD, from Zurich, Switzerland, the popularity of combining riboflavin and ultraviolet A (UVA) irradiation to strengthen the corneal tissue is growing.¹

The cross-linking mechanism is not new to medicine; the same biomechanical principle has been used for a long time in orthopedic surgery and dental curing. The main goals of Dr. Seiler's initial studies were to (1) obtain useful and reliable parameters (eg, the concentration of riboflavin), (2) discover the influence of UV on safety and depth of penetration and (3) create efficacy measurements with elastometry/extensiometry. With these vital parameters settled, Dr. Seiler published the first study on the cross-linking technique for the treatment of keratoconus.² The technique uses UVA light and riboflavin to create new bonds between the adjacent collagen molecules so that the cornea is one-and-a-half times thicker and less malleable.³

CROSS-LINKING: THE MECHANISM
Why does combining riboflavin with UVA irradiation modify the biomechanics of the corneal tissue? There is a chemical explanation: Application of riboflavin on the cornea — with penetration for approximately 200 µm — and irradiation of the riboflavin molecules through UVA. This procedure leads to loss of the internal chemical balance of the riboflavin molecules, producing oxygen free radicals. The riboflavin molecule becomes unstable and stabilizes only when it is linked to two collagen fibrils. A crossed bridge is created between the collagen fibrils (ie, cross-linking) to produce a general strengthening of the cornea.

The safety of the cross-linking treatment has been verified through years of research. Some well-respected figures in the ophthalmic world, such as Dr. Seiler and Eberhard Spoerl, of the University of Dresden, Germany, have contributed to the number of studies on cross-linking. Dr. Seller and colleagues have also done studies on cytoxicity and endothellal cell counts. Recently an official document edited by the International Commission on Non-Ionizing Radiation Protection (ICNIRP) and American Conference of Governmental Industrial Hygienists (ACGIH) stated that this procedure has absolute safety. In fact, its recommendation was to not exceed a limiting dose of 1 J/cm2 for UV-irradiation times more than 1,000 seconds.⁴ Although the application of radiant exposure of 5.4 J/cm2 for 1,800 seconds during cross-linking clearly exceeds both the ACGIH and ICNIRP guidelines, riboflavin absorption in a normal thickness cornea (ie, >400 µm), however, exposes the corneal endothelium and lens to radiant exposure levels lower than without the riboflavin absorption in a nontreated eye exposed to the recommended limit.

PERSONAL EXPERIENCE
At the Istituto Laser Microchirurgia Oculare, in Brescia, Italy, we started to perform C3-riboflavin 7 months ago. Clinical indications for this treatment are diseases where a biomechanical stabilization of the cornea is sought. In particular, they are keratoconus, corneal melting and iatrogenic keratectasia. There are other potential indications for C3-riboflavin when combined with surgical tools/procedures. They include intracorneal rings, conductive keratoplasty, laser thermokeratoplasty, orthokeratoplasty, prevention of the central island after extended PRK, epi-LASIK or LASEK (Figure 1).

We have treated 10 bilateral patients (20 eyes) with no epithelial removal. Exclusion criteria included aphakic eyes and pachymetry thinner than 400 µm. During a preoperative consultation, UCVA, BSCVA, manifest refraction spherical equivalent, IOP, pachymetry, topography and difference maps, K values and enhanced corneal compensation verified the qualification of the patient for the C3-riboflavin treatment. We do not debride the epithelium unless it is essential (ie, whenever the penetration of riboflavin appears insufficient).

Each eye is treated with proparacaine 0.5% for ≤30 minutes before UVA exposure (ie, approximately two drops every 5 minutes). Riboflavin is then applied on the cornea for ≤25 minutes before irradiation and penetrates in the stroma for ≤200 µm. The riboflavin is then activated by a 30-minute exposure to the UVA light (ie, 370 nm fluence at 3 mW/cm2) to the central 8 mm of the cornea with speculums in place (bilateral). The riboflavin solution must be reapplied on the cornea every 3 minutes during the UVA irradiation.

RESULTS OF THE STUDY
During the study, we realized that our results could be compared with international results obtained by Dr. Seiler⁵ and presented by Brian S. Boxer-Wachler, MD, from Beverly Hills, California, at the First International Congress on Corneal Cross-linking, in Zurich, Switzerland.⁶

Six months postoperatively, progression of keratoconus ceased in all 20 eyes treated. Seventy-five percent of eyes (n=15) had a regressed pathology, and 80% of eyes (n=16) recorded an improvement in visual acuity. Moreover, there was no loss in corneal thickness. On the contrary, 40% of eyes (n=8) showed an average increase in corneal thickness of 21.9 nm.

We found the treatment to be safe. There was no loss in endothelial cell density; no change in the transparency of the cornea and of the crystalline lens; no retinal change; and no change in IOP. Our results showed that epithelial removal is not to be considered mandatory for the transmission of riboflavin in the cornea for UVA protection, and it is a patient-friendly procedure, with no pain in the postoperatively phase (Figures 2, 3).

What is our mission for the research in corneal collagen cross-linking with riboflavin? In my opinion, the main goals are to change keratoconus from a disease state into a syndrome and to stop or slow the progression of the pathology. Also, the possibility to combine C3-riboflavin with other procedures (eg, intracorneal rings, conductive keratoplasty) may be an option to obtain stability of the corneal tissue. Communication is once more a key factor: The improvement in visual acuity must be considered — and explained to the patient — as a bonus instead of a goal. Then, the patient must also be carefully followed from the psychological point of view, guided in the discovery of a new era in the ophthalmologic sciences.

Roberto Pinelli, MD, is the scientific director of the Istituto Laser Microchirurgia Oculare, in Brescia, Italy. Dr. Pinelli is a member of the CRSToday Europe Editorial Board. He states that he has no financial interest in the products or companies mentioned. He may be reached at pinelli@ilmo.it; +39 030 2428343 (phone); or +39 030 2428248 (fax).

1. Seiler T, Spoerl E, Huhle M, Kamouna A. Conservative therapy of keratoconus by enhancement of collagen cross-links. Invest Ophthalmol Vis Sci. 1996;37:S1017.
2. Seiler T, Quurke AW. Iatrogenic keratectasia after LASIK in a case of forme fruste keratoconus. J Cat Refract Surg. 1998;24:1007-1009.
3. Spoerl E, Huhle M, Seiler T. Induction of cross-links in corneal tissue. Exp Eye Res. 1998;66:97-103.
4. The International Comission on Non-Ionizing Radiation Protection. ICNIRP Guidelines. Guidelines on limits of exposure to ultraviolet radiation of wavelengths between of 180 nm and 400 nm (Incoherent Optical Radiation). Health Physics Society Journal. 2004;87(2).
5. Spoerl E, Seiler T. Techniques for stiffening the cornea. J Refract Surg. 1999;15:711-713.
6. Hagele G, Boxer Wachler BS. Corneal Collagen Crosslinking with Riboflavin (C3-R) for corneal stabilization. Presented at the International Congress of Corneal Cross Linking (CCL). December 9-10, 2005. Zurich, Switzerland.