The Case for Epithelium-on CXL

Standardization will help overcome objections to transepithelial CXL.

By Parag A. Majmudar, MD

Corneal collagen cross-linking (CXL; not approved for use in the United States) has gained worldwide popularity over the past several years as an effective means of strengthening the cornea and thereby reducing, if not eliminating, the progression of ectatic disorders of the cornea such as keratoconus, pellucid marginal degeneration, and post-LASIK ectasia.

Vitamin B2 (riboflavin), in the presence of ultraviolet A (UVA) light, has been purported to create links between collagen fibers in the cornea. With the creation of these links, the cornea theoretically becomes more rigid and resists further ectatic progression. As an additional benefit, corneal remodeling may result in improved refractive and visual outcomes, although patients must be counseled that refractive correction with contact lenses will still be required. Patients frequently become more contact lens tolerant following CXL.

An essential requirement for CXL is obtaining an adequate concentration of riboflavin within the corneal stroma. This step serves two main purposes: (1) Riboflavin acts as a photosensitizer so that UVA light can complete the CXL process, and (2) riboflavin absorbs excess UVA so that damage to deeper structures within the eye (the endothelium, lens, and retina) is minimized.

In the late 1990s, the first clinical studies of CXL were performed by Theo Seiler, MD, PhD, and his group in Dresden.^1,2 The photosensitizer used at that time was 0.1% isotonic riboflavin solution in 20% dextran. This formulation had limited ability to penetrate the intact epithelium. The Dresden protocol therefore required epithelial removal in order to allow better penetration of the riboflavin preparation. Their successful results became the basis for the rapid adoption of this procedure.

EPI-OFF OBSTACLES

Unfortunately, there are several major obstacles, at least from the patient's perspective, when epithelial removal is performed during the traditional epithelium-off (epi-off) CXL procedure. Patients experience considerable pain for up to 7 days after this procedure. Removing the epithelium also eliminates a natural barrier to infection, and therefore, the risk of infectious keratitis is increased. Wajnsztajn and colleagues³ conducted a retrospective review of ocular complications in 206 eyes of 180 patients treated according to the Dresden protocol between 2007 and 2012. The investigators observed 28 ocular complications in 23 eyes (11.2%) of 22 patients (12 men, 10 women). These included delay of epithelial healing for up to 30 days in four eyes, hypertrophic epithelial healing in four eyes, marked superficial punctate keratopathy for greater than 30 days in 11 eyes, corneal sterile infiltrates in four eyes, microbial keratitis in four eyes (culture-positive in two), and marked corneal edema with scarring in one eye.

During the period of delayed wound healing, most patients are unable to work or, in the case of children, attend school for a significant length of time. Moreover, contact lenses, which are so vital to a keratoconic patient's visual functioning, may not be worn for up to several weeks. Many of these concerns could be greatly reduced if CXL could be performed without removing the epithelium. Ocular discomfort would be minimal, and patients might be able to resume contact lens wear and normal work or school activities within several days in most cases.

EPITHELIUM-ON AS an ALTERNATIVE

The concept of epithelium-on CXL is not new. The main concerns with so-called transepithelial CXL (TCXL) are that riboflavin cannot penetrate an intact epithelium and that the presence of the epithelium will block approximately 20% of the UV-A light from reaching the stroma. Both of these concerns are valid, but these assumptions may not be entirely correct.

The major objection to TCXL stems from studies that have found a lack of efficacy. Wollensak and Iomdina found that corneal biomechanical stiffening was five times greater after epi-off CXL compared with TCXL in an animal model.⁴ Other clinical and laboratory studies have reported weaker or no effect of CXL using the epi-on method.^5-10 Touboul et al¹¹ compared conventional, transepithelial, and accelerated CXL in 24 patients. Confocal microscopy showed corneal changes in conventional and accelerated cross-linking (ie, when the epithelium was removed), but in TCXL, there were no observable changes in the cornea. They concluded that TCXL had no benefit.

These studies suggest that TCXL has a significantly weaker biomechanical effect. Part of this may be due to insufficient transepithelial riboflavin diffusion into the corneal stroma. A limitation of most of these studies, however, is that standard riboflavin formulations were used. This formulation has been shown to have minimal penetration through an intact epithelium due to the large molecular sizes of riboflavin and dextran. For this reason, newer formulations of riboflavin such as Ricrolin TE (Ofta hi-tech Innovazione Tecnico Chirurgica; not available in the United States) have been formulated using alternative vehicles, and these commercially available solutions are being used with greater frequency outside of the United States. Moreover, many earlier authors did not attempt to enhance the penetration of riboflavin using any of several methods, including alteration of the riboflavin formulation by using additives such as benzalkonium chloride or by changing tonicity.

Another area of concern with these studies involves the physical act of riboflavin loading. An eye with an intact epithelium requires a significantly longer time (60- 80 minutes) than one in which the epithelium has been removed (typically 30 minutes). This is the case even with nondextran-containing vehicles. Many studies that claim decreased effectiveness of TCXL used a fixed loading time of only 30 minutes, and typically, the cornea was not checked to determine whether there were sufficient corneal riboflavin saturation before UVA treatment. In cases in which the surgeon notes inadequate riboflavin loading, continued loading with topical riboflavin may eventually result in a sufficiently loaded cornea to proceed with the UVA treatment stage, but if a rigidly timed loading protocol is used, that may be the reason that suboptimal crosslinking is demonstrated.

NEWER STUDIES

On the other hand, more recent studies have shown support for the efficacy of TCXL. Pinelli and colleagues12 reported no significant difference in the analyzed parameters between TCXL and standard CXL. Filippello et al⁶ performed bilateral TCXL in 20 patients with progressive keratoconus. In treated eyes, there were statistically significant improvements in BCVA and UCVA, keratometry, cone apex power, and higher-order aberrations, compared with untreated control eyes. Their conclusion was that TCXL treatment appeared to halt keratoconus progression and provide statistically significant improvements in visual and topographic parameters.

Stojanovic et al¹³ evaluated the efficacy and safety of TCXL using a multifactorial approach to achieve proper stromal riboflavin saturation. The authors used a non-dextran- containing, hypotonic solution and employed superficial disruption of the epithelial surface in order to enhance penetration of riboflavin. Riboflavin saturation was confirmed via slit lamp, rather than using an arbitrary time limit. Their results showed that distance UCVA and BCVA improved significantly. No eyes lost lines of acuity, while 27.4% of eyes gained 2 or more lines. Mean spherical equivalent decreased by 0.74 D, and mean cylinder reduction was 1.15 D. Scheimpflug-based topography showed a significant decrease in irregularity and asymmetry.

Rubinfeld et al¹⁴ presented a retrospective evaluation of TCXL at 6 (147 eyes) and 12 (79 eyes) months. Reduction in maximum keratometry (Kmax) on Pentacam Comprehensive Eye Scanner (Oculus Optikgeräte GmbH) was 0.997 D at 6 months and 1.17 D at 12 months.

The presence of a demarcation line after CXL has been thought to indicate the efficacy of corneal crosslinking.¹⁵ In fact, the significance of the demarcation line is uncertain. The increased optical density seen on optical coherence tomography or confocal microscopy may represent keratocyte apoptosis and subsequent repopulation.¹⁶ While a demarcation line after epithelium-off CXL has been shown at 300- to 350-μm depth,¹⁷ keratocyte apoptosis has been demonstrated after TCXL.⁴ Filippello et al reported a demarcation line 2 weeks after TCXL that was located approximately 100 μm from the corneal epithelium. ⁶ Using RTVue (Optovue Inc.) anterior segment optical coherence tomography, Stojanovic et al¹² showed in 24 eyes that the mean demarcation line was located at the depth of 317 μm from the surface, which is similar to that seen in epithelium-off CXL.¹³

Another argument against TCXL is the concern that an intact epithelium blocks the transmission of UVA light by 20% and therefore may result in decreased efficacy. Some studies have shown that epithelial UV absorption occurs only with wavelengths of less than 310 nm.^18-20 Nonetheless, in the worst-case scenario, if we assume a 20% reduction in UVA absorption by the stroma, which might result in shallower crosslinking compared with epi-off CXL, the density of collagen fibers is much higher in the anterior portion of the corneal stroma, where we assume most of the collagen crosslinking would occur.^16,21

Steps that can be taken to minimize surface absorption of and energy loss of UVA light include washing the riboflavin from the corneal surface prior to initiating UVA application.¹³ Even with traditional epi-off CXL, the optimal UVA irradiance and duration of treatment have yet to be determined. Therefore, future research on this aspect of CXL, including alterations in the UVA irradiance level to overcome the physical barrier of an intact epithelium, may change our understanding of this procedure.

CONCLUSION

Modifications of traditional CXL have already begun to occur, and TCXL will undoubtedly continue to evolve. I firmly believe that the standardization of riboflavin loading of the cornea and further advances in UVA irradiation technology will overcome the main objections to TCXL. As greater numbers of patients are treated worldwide with TCXL, we will have a better understanding of this exciting but, to date, relatively poorly understood procedure. TCXL will allow younger patients to undergo CXL and will facilitate bilateral treatments. Although keratoconus may be a small fish in the big pond of eye diseases, we have the potential to significantly affect the lives of these patients while maximizing their comfort, convenience, and safety.

Parag A. Majmudar, MD, is an associate professor in the Cornea Service of Rush University Medical Center, Chicago Cornea Consultants, Ltd. He is an investor in CXL Ophthalmics, LLC. Dr. Majmudar may be reached at (847) 822-5900; pamajmudar@chicagocornea.com.

1. Spörl E, Huhle M, Kasper M, Seiler T. Increased rigidity of the cornea caused by intrastromal cross-linking [in German]. Ophthalmologe. 1997;94:902-906.
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. Wajnsztajn D, Frenkel S, Frucht-Pery J. Early complications after crosslinking for keratoconus. Poster presented at: American Academy of Ophthalmology Annual Meeting; November 12, 2012; Chicago, IL.
4. Wollensak G, Iomdina E. Biomechanical and histological changes after corneal crosslinking with and without epithelial debridement. J Cataract Refract Surg. 2009;35(3):540-546.
5. Samaras KE, O'Brart, DP, Doutch J, et al. Effect of epithelial retention and removal on riboflavin absorption in porcine corneas. J Refract Surg. 2009;25(9):771-775.
6. Filippello, M, Stagni E, O'Brart DP. Transepithelial corneal collagen crosslinking: bilateral study. J Cataract Refract Surg. 2012;38(2):283-291.
7. Hayes S, O'Brart DP, Lamdin LS, et al. Effect of complete epithelial debridement before riboflavin-ultraviolet-A corneal collagen crosslinking therapy. J Cataract Refract Surg. 2008;34(4):657-661.
8. Bottos KM, Dreyfuss JL, Regatieri CVS, et al. Immunofluorescence confocal microscopy of porcine corneas following collagen cross-linking treatment with riboflavin and ultraviolet A. J Refract Surg. 2008;24(7):S715-S719.
9. Samaras KE, Lake DB. Corneal collagen cross linking (CXL): a review. Int Ophthalmol Clin. 2010;50(3):89-100.
10. Leccisotti A, Islam T. Transepithelial corneal collagen cross-linking in keratoconus. J Refract Surg. 2010;26(12):942-948.
11. Touboul D, Efron N, Smadja D, et al. Corneal confocal microscopy following conventional, transepithelial, and accelerated corneal collagen cross-linking procedures for keratoconus. J Refract Surg. 2012;28(11):769-775.
12. Pinelli R, Al Marzouky MM, El-Shawaf HI. Tensioactive-mediated transepithelial corneal crosslinking—first laboratory report. European Ophthalmic Review. 2009;3(2):67-70.
13. Stojanovic A, Chen X, Jin N, et al. Safety and efficacy of epithelium-on corneal collagen cross-linking using a multifactorial approach to achieve proper stromal riboflavin saturation. J Ophthalmol. 2012;2012:498435. doi:10.1155/2012/498435
14. Rubinfeld RS, Trattler W, Talamo J, et al. Retrospective evaluation of epithelial-on collagen cross-linking. EuCornea meeting; September 8-12; Milan, Italy.
15. Seiler T, Hafezi F. Corneal cross-linking-induced stromal demarcation line. Cornea. 2006;25(9):1057-1059.
16. Wollensak G, Iomdina E, Dittert DD, et al. Wound healing in the rabbit cornea after corneal collagen cross-linking with riboflavin and UVA. Cornea. 2007;26(5):600-605.
17. Seiler T. Crosslinking in the year 20/20: What will the future hold? Paper presented at: American Academy of Ophthalmology Annual Meeting, Cornea Subspecialty Day; November 10, 2012; Chicago, IL.
18. Kolozsvari L, Nogradi A, Hopp B, et al, UV absorbance of the human cornea in the 240- to 400-nm range. Invest Ophthalmol Vis Sci. 2002;43(7):2165-2168.
19. Ringvold A. Corneal epithelium and UV-protection of the eye. Acta Ophthalmol Scand. 1998;76(2):149-153.
20. Podskochy A. Protective role of corneal epithelium against ultraviolet radiation damage. Acta Ophthalmol Scand. 2004;82(6):714-717.
21. 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;29(9):1780-1785.

The Case for Epithelium-off CXL

Epi-off CXL has shown evidence of success in halting the progression of keratoconus.

By Rebecca McQuaid, MSc; Arthur B. Cummings, MB ChB, FCS(SA), MMed(Ophth), FRCS(Edin); and Michael Mrochen, PhD

From the patient's perspective, there is no question that corneal collagen cross-linking (CXL; not approved for use in the United States) without removing the epithelium (epi-on) is more comfortable, safer, and generally preferable to CXL with the epithelium removed (epi-off). However, the reason for performing CXL is to stabilize keratoconus, and the reported clinical outcome and experimental results of epi-on treatments demonstrate reduced efficacy: that is, less biomechanical stiffening of the cornea. Patients' comfort is of secondary importance in this situation, and the primary outcome measure is whether or not CXL has been successful in stabilizing or even improving the corneal shape. This is the fundamental thought in the mind of the practitioner of epi-off CXL.

Clinically, epi-off CXL has been shown to stabilize corneal curvature in eyes with progressive keratoconus, with no significant change in the refractive index of the cornea.¹ Since its regulatory approval in the European Union in 2006, CXL has been widely practiced around the world as an effective procedure for halting the progression of keratoconus and corneal ectasia.²

Before the introduction of CXL, the only option to overcome keratoconus progression was keratoplasty. Due to the recent downturn in the economic climate, the high cost of surgical procedures, and long hospital waiting lists for corneal transplantation, a person's chance of receiving treatment is diminished,³ giving cross-linking a significant advantage among options. CXL is less invasive, more cost-effective, and less stressful for the patient. CXL has a recovery period of 5 days, whereas 1 year of follow-up care is needed following corneal transplantation. The corneal transplant patient also faces potential graft rejection, something that does not affect the CXL patient. These factors are important when considering an effective treatment plan for a progressive disease.

WHY EPI-OFF CXL?

The Dresden protocol⁴ was created in order to standardize the original CXL treatment in 2003. The protocol included epithelial removal before soaking the cornea with a dextran-based 0.1% riboflavin solution, followed by exposure with ultraviolet A (UVA) light for 30 minutes under an intensity of 3 mW/cm². The epithelium, approximately 50 μm in thickness, forms a barrier to both riboflavin and UVA penetration. Removing the epithelium allows proper absorption of riboflavin into the cornea and anterior chamber in order for the UV-A light to efficiently illuminate the cornea.

For current techniques of epi-on CXL, soaking time can be up to 50 minutes before illumination, increasing treatment time and the risk of epithelial disturbance. Only 80% of UVA exposure occurs in the stroma after the UVA light penetrates the epithelium, creating a limited crosslinking effect compared with epi-off CXL.⁵ Recently, clinical studies have investigated the use of higher-intensity UVA light, consequently shortening treatment time and reducing the risk of corneal dehydration.

Clinical studies at the Wellington Eye Clinic have shown that eyes treated with epi-off CXL exhibit a significant reduction in keratometry over time (Figure 1). An ongoing clinical trial (26 eyes) comparing two treatments— 30 minutes at 3 mW/cm² using the UV-X 1,000 lamp (IROC Innocross AG) versus 10 minutes at 9 mW/cm² with the UV-X 2,000 lamp (IROC Innocross AG)—has shown similar safety with the two devices but increased efficacy with the UV-X 2000 device.⁶ The observed difference in the two treatments may be due to the increased intensity with the UV-X 2000 lamp, but the device's optimized beam profile with additional depth in the peripheral part of the beam may be the primary cause for the increased efficacy.

In summary, both our own clinical experience over the past 6 years and a review of the literature⁴ provide ample evidence that epi-off CXL is effective with low failure rates (less than 5%) and proven safety (less than 1% loss of BCVA of more than 2 lines). Numerous studies^7,8 have shown that the cornea flattens and regularizes to preoperative levels by approximately 3 months after the treatment. Visual acuity usually increases by 1 to 2 lines, and the cornea may flatten further over time.

EPI-ON CXL

The potential advantages of the epi-on approach are significant, but,to date, there is not sufficient evidence that it is effective.

Other methods such as iontophoresis have been investigated to achieve riboflavin delivery to the cornea with little or no disturbance to the epithelium.⁹ This technique involves application of an electrochemical effect to the cornea, enabling a distribution of riboflavin similar to that with the epi-off technique and resulting in corneal strengthening after exposure to UVA light. Although this method looks promising, more research is needed.

Kanellopoulos¹⁰ has presented results with intrastromal riboflavin instillation via a femtosecond laser-created pocket along with application of higher-intensity UVA light (7 mW/cm²). With follow-up time of 1.5 years (10 cases), he reported a reduction of 2.10 D in patients with refractive astigmatism and reduction of keratometry readings by 2.70 D. These results suggest that higher-intensity CXL with use of a femtosecond pocket instead of epithelial removal has the potential to be as effective as standard CXL.

CLINICAL RESULTS

To date, most clinical studies have focused on the success and failures of epi-off CXL. Investigators have shown that standard epi-off CXL affects the biomechanical properties of the cornea, increasing corneal rigidity by approximately 70%.¹¹ Laboratory investigations in human and porcine corneas examined the best treatment parameters for epi-off CXL, including riboflavin concentration, intensity and wavelength of UVA light, and treatment duration.^7,12

The largest clinical study to date to investigate longterm effects of standard CXL has been the Siena Eye Cross Study,⁸ in which 363 eyes with progressive keratoconus were treated. In 44 eyes followed for at least 4 years, long-term corneal stability was seen without relevant side effects.

Epi-on or transepithelial CXL has been reported to be less painful for the patient and to reduce the risk of infection postoperatively by keeping the epithelium intact. Although the short-term effects of epi-on seem positive, results reported to date do not provide significant evidence to suggest long-term success in halting the progression of keratoconus. A study with 3 years of follow-up found a reduction in steepest keratometry to be more prominent in corneas after epi-off CXL compared with epi-on CXL.¹³

Hafezi¹⁴ found the stromal concentration of riboflavin to be lower by a factor of 40 during epi-on CXL compared with epi-off CXL, and the long-term effect of epion CXL on corneal shape was reduced. This corresponds with the findings of Wollensak and Iomdina al,¹⁵ who demonstrated a reduction in biomechanical changes with transepithelial CXL compared with standard epi-off CXL.

The Wellington Eye Clinic is conducting a comparative study of epi-on and epi-off CXL. New epi-on protocols currently under clinical investigation demonstrate encouraging results. Riboflavin diffusion occurs in the same amount of time as current epi-off techniques or less, and 1-month postoperative data demonstrate corneal flattening rather than the steepening typically seen at this interval with epi-off CXL. Visual recovery is very fast, with day 1 postoperative vision equal to preoperative vision. This level of recovery can take up to 3 months in epi-off CXL. As promising as epi-on may appear at this early stage, the final test will be the corneal shape at the 1-year postoperative interval.

CONCLUSION

It would be preferable to have epi-on CXL as a reliable surgical option, given its promise of faster recovery time and reduction in postoperative pain. In order to make epi-on CXL an effective and safe treatment method, longer-term results for a larger sample group will be required.

It has been suggested that epi-on CXL is worthwhile for eyes with thin corneas due to its inability to penetrate as deeply into the stroma as epi-off CXL. For the moment, however, epi-off CXL has shown evidence of success in halting the progression of keratoconus, and that is the most important factor for this disease. The efficacy of epi-on CXL still must be shown in a prospective clinical trial. For the moment, therefore, our preferred approach is epi-off CXL, a procedure with a proven track record.

This article is reprinted with permission from the March 2013 issue of Cataract & Refractive Surgery Today Europe.

Arthur B. Cummings, MB ChB, FCS(SA), MMed(Ophth), FRCS(Edin), is a consultant ophthalmologist with the Wellington Eye Clinic in Dublin, Ireland. He acknowledged no financial interest in the products and companies mentioned herein. Dr. Cummings may be reached at abc@wellingtoneyeclinic.com.

Rebecca McQuaid, MSc, is an ophthalmic technician/ research assistant at the Wellington Eye Clinic in Dublin, Ireland. She acknowledged no financial interest in the products and companies mentioned herein. Ms. McQuaid states may be reached at rebecca.mcquaid @wellingtoneyeclinic.com.

Michael Mrochen, PhD, is the CEO of IROC Science to Innovation in Zürich, Switzerland. Dr. Mrochen may be reached at +41 043 500 0850; info@irocscience.com.

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