Collagen Cross-linking: The Ties That Bind

An update on the safety and stability of this innovative procedure to strengthen the cornea.

By Yaron S. Rabinowitz, MD

There is increasing evidence from review of the literature, and preliminary data from the ongoing prospective clinical trial in our practice, that collagen cross-linking is emerging as an effective means of stopping the progression of corneal thinning in patients with keratoconus, pellucid marginal degeneration and ectasia after LASIK.^1-5 Other emerging potential applications include treatment of infectious corneal ulcers, corneal edema in bullous keratopathy, augmenting corneal flattening after surgically inserting intracorneal rings and strengthening the cornea prior to PRK treatment in mild or "forme fruste" patients with keratoconus.^6-9 There are now over 40 peer-reviewed articles supporting its efficacy in halting the progression of keratoconus (Table 1) and isolated reports supporting its use for the latter-described emerging indications.¹⁰

How is the Treatment Performed?

The procedure is carried out in sterile conditions, preferably in an operating room. After applying topical anesthesia and alcohol to the cornea, the central 7 mm of corneal epithelium is removed. The surface is then treated by the application of riboflavin (vitamin B2) 0.1% solution (10mg riboflavin-5-phosphate in 10 ml dextran 20% solution) every two minutes for 30 minutes. The patient is then examined under a slit-lamp to check for flare in the anterior chamber to ensure adequate riboflavin penetration, and ultrasonic pachymetry is performed to ensure the cornea is at least 400 μm thick prior to commencing treatment. UVA radiation of 370 nm wavelength and an irradiance of 3mw/cm² at a distance of 5 mm from the cornea are applied for a period of 30 minutes delivering a dose of 5.4J/cm².

During treatment, riboflavin drops continue to be instilled every two minutes. At the completion of the surgery, antibiotics are put in the eye and a bandage lens is inserted, which is removed once there is total epithelial healing.

A UV lamp with modifications designed to deliver a homogenous illumination has been developed and is commercially available (UV-X, IROC, Zurich, Switzerland). The manufacturer claims that in contrast to the LED lamps that are dependent on their exact distance from the cornea to ensure that ineffective treatment (too far) or endothelial damage (too close) does not happen, their device uses the Koehler's beam path that is less sensitive to changes in treatment distance. In addition, because of this optical design, the radiant energy diverges behind the cornea, reducing the risk to the retina.¹⁰

This device, distributed by Peschke Meditrade, is not FDA-approved in the United States; as such, US physicians cannot now use this device without an investigational device exemption (IDE) from the FDA. Our practice has an IDE to use this device for our ongoing clinical studies to determine the safety and efficacy of this new treatment in halting the progression of keratoconus.

How Do UV Light and Riboflavin Enhance Collagen Cross-linking?

The primary role of collagen in the cornea is to act as supporting tissue. Aggregated forms of the collagen monomers are strengthened by intermolecular cross-links. This process happens as part of maturation, and also in aging and in diseases such as diabetes.

Collagen fibrils naturally cross-link as part of their maturation process. The cross-links are formed by oxidative deamination of the e-amino group of a single lysine or hydroxylysine in the amino and carboxytelo peptides of collagen by the enzyme lysyl oxidase. The aldehyde thus formed reacts with a specific lysine or hydroxylysine to help form divalent bonds that link the molecules head to tail and spontaneously convert during mat uration to trivalent cross-links.

Cross-linking also occurs during the aging process by a nonenzymatic reaction called glycation. Prolonged exposure to monosaccharides results in a spontaneous bond between the reducing sugar and the amino group of a protein. Increased stiffness of the cornea with age has been demonstrated by studying its stress strain behavior. An increase in the cross-sectional area of the collagen molecule from 3.04 to 3.46 nm² attributed to this process has been shown in human corneas with age using high-angle and low-angle X-ray diffraction pattern studies.¹⁰

Oxidation is the third mechanism whereby cross-links are formed in collagen. This is distinct from the other two types of cross-linking and can occur after the process of photo-oxidation (UV mediated). A monomer substrate in the presence of a photo initiator (riboflavin) can polymerize by way of cross-linking in the presence of a UV light source. This process is routinely used in industry, resulting in highly cross-linked material suitable for applications such as in dental materials.¹⁰

This method of cross-linking was found by Wollensak and Spoerl to be the most effective in increasing corneal rigidity. In their initial laboratory work, they demonstrated that an increase in stress in porcine corneas (by 72%) and human corneas (by 329%) and a Young's modulus by a factor of 1.8 in porcine corneas and 4.5 in human corneas.¹¹ Their studies also showed that there was more effect in the anterior stroma than posterior stroma, attributable to greater degree of UV penetration in the anterior stroma with decay in the posterior layers possibly due to riboflavin shielding.

Clinical Studies

Wollensak et al. published their first report on humans in January 2003, using riboflavin and UVA to induce crosslinking in 16 eyes of 15 patients with progressive keratoconus. A subsequent publication reported on 22 eyes of 24 patients with a follow-up time of between three months and four years. They reported that in all treated eyes the progression of keratoconus was stopped. In 70% of eyes, there was a regression with a mean reduction of the maximal keratometry readings by about two diopters and refractive error of approximately one diopter. Visual acuity improved slightly in 65% of eyes.¹²

Since that pilot study, there have been eight studies reporting their results, summarized in Table 1. The study methodologies are variable and as such are not directly comparable; however, all studies report varying degrees of im prove ment in visual acuity and reduction in keratometry with a progressive trend of improvement in the duration of follow up. The longest study to date by Raiskup-Wolf et al. reported seven-year results at the university of Dresden. They noted a decrease in maximum keratometry of 2.7 D in year one, 2.2 D at two years and 4.8 D at three years. Visual acuity improved by one line per year in 54% of patients in the first three years. Two patients had continued progression and had to undergo repeat cross-linking procedures.³

In the only randomized prospective controlled clinical trial of collagen cross-linking in progressive keratoconus published to date, Wittig-Silva et al. reported on 66 eyes of 49 patients with documented progression of keratoconus. Interim analysis of treated eyes showed a flattening of the steepest simulated keratometry (K-max) by an average of 0.74 D at three months, 0.92 D at six months and 1.45 D at 12 months.

A trend toward improvement of BSCVA was also observed. In the control eyes, mean K-max steepened by 0.60 D in after three months, 0.60 D after six months and by 1.28 D after 12 months. BSCVA decreased by a logmar of 0.003 over three months, 0.056 over six months and 0.12 over 12 months. No statistically significant changes were found for spherical equivalent or endothelial cell density.⁴

There are only two other prospective randomized clinical trials in the US using this methodology, for which the data have yet to be published: the multicenter US FDA trial on 160 patients, for which one-year data have been collected but not yet analyzed or published, and our clinical trial at the Cornea Eye Institute (www.laser-prk.com) in which we have enrolled 76 patients to date using the UV-X device.

Figure 1. Preop topography OD in progressive keratoconus. (See the case report above for additional details.)

Figure 2. Dense central stromal opacity three weeks postop. (See the case report above for additional details.)

Safety Concerns and Suggestions for Improvement

A significant concern has been the effect of UV light on the cornea and other ocular structures. Experiments to validate the procedure's safety has been exhaustively performed in preclinical experiments by Spoerl et al. The potential cytotoxicity of UVA light and the UVA/riboflavin exposure on keratocytes and endothelial cell function have been characterized in a series of in vitro experiments. In each, UVA exposure (370 nm, 30 minutes) and riboflavin (0.025% solution) were administered to mimic conditions of clinical usage. Irradiance levels were varied to determine the irradiance for catatonic effects. Keratocyte toxicity was evaluated in por cine keratocyte cultures after exposure to riboflavin alone, UVA light alone (irradiance range 2 to 9 mW/cm²), and UVA plus riboflavin.

Riboflavin alone had no cytotoxic effect on keratocytes. The cytotoxic threshold for inducing cellular necrosis or apoptosis was 5mw/cm² for UVA light alone and 0.5mW/cm² for the UVA/riboflavin treatment. Using the Lambert-Beer equation, in human corneas the cytotoxic keratocyte UVA irradiance of 0.5mW/cm² stopped at 300 microns.¹³

Endothelial cell damage with UVA is believed to be due to the oxidative damage caused by the oxygen reactive-free radicals that are generated by UV light. The lower cytotoxic thresholds observed in the UVA/riboflavin combination in endothelial and keratocyte toxicity studies is consistent with increase in UVA absorption in the presence of riboflavin. These studies suggest that this treatment is safe in corneas that have been adequately saturated with riboflavin and are at least 400 μm in diameter.¹⁴

In their pilot study, Wollensak et al. reported that corneal and lens transparency, and endothelial cell density, remain unchanged after treatment.¹² In their review of the safety of the procedure, Spoerl et al. have highlighted the importance of homogenous irradiance of UVA in the field of application. They advise that hotspots should be avoided as it may mean local exceeding of safety limits, causing focal endothelial cell damage.¹⁴

Based on analysis of complications from the literature in publications that have long-term data, the following recommendations are suggested to potentially avoid complications:

1. Exclude eyes with K readings of greater than 58 diopters. Studies have shown that such patients have an increased risk of progression after treatment and also a significant risk of increase in haze after treatment.¹⁵

2. Treat patients with keratoconus only if they are less than 35 years old. Studies have shown that an age of 35 or more is associated with a higher complication rate.¹⁶ This does not apply to patients with pellucid marginal degeneration who start progression at an older age or patients with progressive ectasia after LASIK.

3. Corneas thinner than 400 μm should be excluded from cross-linking because of the potential for endothelial toxicity.^17,18

4. Don't treat patients with visual acuities of 20/25 or better. Treatment of patients with good acuity is a significant risk factor for both unhappy patients and potential complications.¹⁵

5. Continue instilling riboflavin drops and BSS to avoid stromal dehydration during UV treatment — this will lead to less risk for endothelial damage and stromal haze.^16,19

Complications

Considering the number of cases performed and the fact that this is a very new procedure, the complication rate is extremely low, attesting to the safety of the procedure. There have been some reports of bacterial, herpetic and Acanthomoeba keratitis, which is not surprising considering there is an epithelial defect that is managed with a bandage contact lens for a while.^10,19 There is no evidence that the immune system of the cornea is lowered by the treatment.

There are several reports of focal haze, which can persist for several months but ultimately cleared up without affecting visual acuity. Mild stromal haze, which eventually disappears, is not uncommon in many patients with cross-linking. In fact, in our study we try to document evidence of corneal haze by OCT as evidence that the treatment has had a cross-linking effect on the cornea (see Figures 3 and 4).

Figure 3. This OCT scan demonstrates mid-stromal opacity. (See the case report above for additional details.)

Figure 4. OCT at six months demonstrates mild mid-stromal haze barely detectable on slit lamp evaluation. (See the case report above for additional details.)

There have been no reports of total corneal decompensation requiring a corneal transplant but several series do report a loss of best-corrected vision in a very small number of patients. Much longer-term data are needed to determine if there are any significant long-term complications, although seven-year data suggest that this is a safe procedure at least over that period of time. The long-term effect of prematurely aging the cornea by stiffening it remains to be seen.

Epithelial Removal vs. Epi-on Procedures

There are a handful of physicians who believe it is better to leave the epithelium on vs. removing the epithelium. It has been our experience that leaving the epithelium on significantly diminishes the effect of the treatment; there are scientific studies to support this.^20,21 We have seen a significant number of patients in the past year in our practice who have requested retreatment after having received the epi-on procedure and OCT pictures have not demonstrated the haze or demarcation lines we have seen with the epi-off procedure. Until it can be proven scientifically and in the laboratory that epi-on is safe and effective, we will not offer this mode of treatment to our patients.

Clinical Trial at the Cornea Eye Institute in Beverly Hills

Our clinical trial is still ongoing and we are still actively recruiting patients. This is the only trial that I am aware of in the US where patients can be treated using this proven methodology supported by publications from the Dresden group with at least seven years follow up. Additional trials using other devices are also ongoing but the instrumentation and methodology differs from that published by the Dresden group (see www.clinicaltrials.gov for further info).

Inclusion criteria for our trial are patients with keratoconus, pellucid or ectasia after LASIK who have demonstrated at least one diopter of progression within the past year by refraction or videokeratography. Patients are randomized to two treatment groups: (1) cross-linking with UV light only or (2) Intacs implantation followed by crosslinking with UV light three months later. The purpose of our study is to demonstrate which is more efficacious in reducing the progression of keratoconus: cross-linking alone or the combination of Intacs and cross-linking.

Exclusion criteria for entry into the study include K readings in excess of 58 diopters, pachymetry thinner than 400 μm and central corneal scarring. Since this study is not supported by industry, patients are charged for treatments provided. In some instances, insurance will cover the cost of the Intacs but because cross-linking is still considered investigational, we are not aware of any insurance company that will cover the cost of this treatment.

For those interested in referring patients for this study, you can either email me at rabinowitzy@cshs.org or call our office at 310-423-9640 and ask for the cross-linking study coordinators Betty or Martha.

Our preliminary data are very encouraging and results appear to be in line with previously reported data, with the vast majority demonstrating a lack of progression. Even though we have only limited one-year follow up, we are surprised by the number of keratoconus patients who report a definite improvement in their quality of vision and are even more surprised by several who have demonstrated an improvement in measured smelled acuity.

One such patient with one-year follow up had what appeared to be an unusual complication but ended up with a superb visual outcome (see Case Report, above). Although I commenced this study with a high degree of skepticism and some trepidation, interim analysis of our data has made me very excited about this new technology, which I believe will ultimately play a future role in managing our keratoconus patients. In the meantime, we will continue to collect data to demonstrate efficacy and prove long-term safety. OM

References

1. Hoyer A et al: UU Collagen Cross-Linking with Riboflavin and UVA Light. Klin Monbl Augenheilkd. 2010 Jul 2. [Epub ahead of print]
2. Caporossi A et al: T.Long-term results of riboflavin ultraviolet a corneal collagen cross-linking for keratoconus in Italy: the Siena eye cross study. Am J Ophthalmol. 2010 Apr;149(4):585-93.
3. Raiskup-Wolf et al: Collagen cross-linking with riboflavin and Ultraviolet-A light in keratoconus: long term results. Journal of Cataract and Refractive Surgery. 2008. May 34: 796-801.
4. Wittig-Silva C et al: A randomized clinical trial of collagen cross-linking in progressive keratoconus: preliminary results: J Refract Surg. 2008: Seot 24:S720-725.
5. Hafezi F et al: Corneal collagen cross-linking Riboflavin and Ultraviolet A to treat induced keratectasia after laser in situ keratomileiusis. J Cataract Refract Surg. 2007:Dec 33:2035-2040.
6. Iseli et al: Ultraviolet A/riboflavin corneal collagen cross-linking for infectious keratitis associated with corneal melts. Cornea. 2008:27:590-594.
7. Bottós KM et al. Effect of collagen cross-linking in stromal fibril organization in edematous human corneas. Cornea. 2010 Jul;29(7):789-93.
8. El-Raggal TM. Sequential versus concurrent KERARINGS insertion and corneal collagen cross-linking for keratoconus. Br J Ophthalmol. 2010 Jun 28.
9. Kanellopoulos AJ, Binder PS. Collagen cross-linking (CCL) with sequential topography-guided PRK: a temporizing alternative for keratoconus to penetrating keratoplasty. Cornea. 2007 Aug;26(7):891-5.
10. Ashwin PT, McDonnel PJ. Collagen cross-linkage: a comprehensive review and directions for future research. British Journal of Ophthalmology. 2010:94:965-970.
11. Wollensak G, Spoerl E, Seiler T. Stress-strain measurements of human and porcine corneas after riboflavin-ultraviolet-A-induced cross-linking. J Cataract Refract Surg. 2003 Sep;29(9):1780-5.
12. Wollensak G et al: Riboflavin/Ultraviolet-a-induced collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol. 2003: May 125:620-627.
13. Wollensak G et al: Endothelial cell damage after riboflavin – ultraviolet -A treament in the rabbit. J Refract Surg. 2003:29:1786-90.
14. Spoerl et al: Safety of UVA-riboflavin cross-linking of the cornea. Cornea. 2007 May 26385-89.Review
15. Koller T et al: Complication and failure rates after corneal crosslinking. J Cataract Refract Surg. 2009:35(8):1358-1362.
16. Raiskup F et al: Permanent corneal haze after riboflavin-UVA-induced crosslinking in keratoconus. J Refract Surg. 2009;25(9):S824-828.
17. Wollensak G et al: Significance of the riboflavin film in corneal collagen crosslinking. J Cataract Refract Surg. 2010;36(1):114-120.
18. Wollensak G et al: Corneal endothelial cytotoxicity of riboflavin/UVA treatment in vitro. Ophthalmic Res. 2003;35(6):324-328.
19. Kymonis GD et al: Additional complications of corneal crosslinking. J Cataract Refract Surg. 2010;36(1)185; author reply 186.
20. Wollensak G et al: Biochemical and histological changes after corneal crosslinking with and without epithelial debridement. J Cataract Refract Surg. 2009. Mar,35(3)540-6.
21. Bottos KM et al: Immunoflourscence confocal microscopy in porcine corneas following collagen cross-linking after treatment with riboflavin and Ultraviolet A. J Refract Surg. 2008 Sept:24:S715-719.

Yaron S. Rabinowitz, MD, is director of the Cornea Eye Institute, a private practice specializing in corneal diseases in Beverly Hills, Calif. He is also director of ophthalmology research at Cedars-Sinai Medical Center and a clinical professor in ophthalmology at UCLA School of Medicine. He has been a recipient of an NIH grant to study the genetics of keratoconus for the past 17 years. He can be reached at rabinowitzy@cshs.org.