Corneal Collagen Crosslinking with Riboflavin and UVA for Keratoectasia and Beyond

By Karolinne Maia Rocha, MD, PhD and Ronald R. Krueger, MD, MSE

Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH

Collagen Crosslinking with Riboflavin as a Photosensitizer

The corneal crosslinking (CXL) procedure increases the formation of collagen covalent bonds by photosensitized oxidation, which leads to a biomechanical stabilization of the cornea [1-3]. Experimental studies have demonstrated that the photosensitizer riboflavin and UV irradiance lead to corneal tissue strengthening by increasing collagen covalent bonds, similar to photopolymerization in polymers [1]. CXL leads to a significant increase in collagen fiber diameter. Immunofluorescence confocal microscopy has shown a pronounced compacting of collagen fibers in the anterior stroma of porcine corneas after riboflavin and UVA exposure [4]. However, the compacting of the collagen fibers was observed only in the absence of the epithelium, due to a limited penetration of riboflavin through the epithelial tight junctions into the stroma.

Corneal collagen crosslinking has been used to stop the progression of keratoconus and may reduce the need for penetrating keratoplasty [5]. Progressive corneal ectasia includes primary keratoconus, keratoconus after corneal refractive surgery and pellucid marginal degeneration. In these conditions, the corneal stroma is structurally weakened and biomechanically unstable. CXL has been shown to improve the biomechanical properties of the cornea as demonstrated by ex vivo stress-strain experiments in porcine and human corneas [6]. Corneal stiffness has been shown to increase by 300% in some of these experiments [6]. The stiffening effect of the riboflavin and UVA treatment is similar to formaldehyde-induced tissue stiffening and fixation in pathologic specimens, also caused by CXL [7].

Clinical Outcomes

The first landmark article on this therapy, published 5 years ago, demonstrated clinically significant stiffening of the corneal stroma after CXL in patients with keratoconus, reporting a mean keratometric regression of 2 D over the course of 23 months after 30 minutes of exposure to ultraviolet A (UVA) light and topical application of riboflavin [8]. This study included 23 eyes with moderate or advanced progressive keratoconus. CXL was effective in halting the progression of keratoconus for as long as 4 years following the procedure. A mean preoperative progression of keratometry (max K) by 1.42 D in 52% of eyes over a 6-month period was followed by a postoperative decrease in 70% of eyes, revealing a reduction of mean keratometry by 2.01 D. Moreover, the postoperative spherical equivalent refraction was reduced by an average of 1.14 D; additionally, 22% of the untreated fellow control eyes had a postoperative progression of keratectasia of 1.48 D [8].

In a randomized, controlled trial of 49 patients performed in Australia, the 1-year outcome of 33 eyes with documented progression of keratoconus treated with riboflavin/UVA crosslinking procedure showed a flattening of the steepest simulated keratometry by a mean value of 1.45 D (P = 0.002) [9]. An improvement in BSCVA and stability of endothelial cells density were also noted.

CXL has also been used successfully to stop the advancement of ectasia in eyes following excimer laser ablation. In a published German study of CXL for patients with ectasia after LASIK, the biomechanical status of the cornea was stabilized with a halting of the refractive and topographic progression of ectasia over a period of 18 months [10]. Furthermore, intrastromal corneal ring segments (Intacs) implanted within an externally created channel in the cornea followed by CXL treatment may represent a novel combined treatment for postoperative ectasia [11].

Surgical Technique

Under topical anesthesia, the central 7 mm of the corneal epithelium is removed using a blunt knife, or 30 seconds of application of 20% alcohol. As a photosensitizer, riboflavin 0.1% solution in the glucose polymer Dextran T500 20% solution (Pharmacosmos, Holbaek, Denmark) is applied 15 minutes before the irradiation and every 2 to 3 minutes during the irradiation. Alternatively, a smaller area of epithelium may be removed, provided that the time of application of riboflavin is increased and the presence of riboflavin in the anterior chamber is confirmed by the presence of a yellow flare during slitlamp examination prior to the application of UVA light. Using a UV-X radiation system (Peschke Med, Huenenberg, Switzerland), 370 nm UVA light is applied at a 1-cm working distance for 30 minutes using a 3 mW/cm2 irradiance (approximately 5.4 J/cm2) (Figure 1 and 2). After the treatment, an antibiotic eyedrop is applied, and a bandage contact lens is fitted to the corneal surface until reepithelialization.

New Components and Future Applications

Beyond the standard riboflavin-based crosslinking methods and dosing for ectatic disease, a new rapid method of crosslinking called flashlinking has been introduced using a customized photoactive crosslinking agent (Figure 1) [12]. The flashlinking agent is a multicomponent (>3) photochemical mixture, which is activated with UVA exposure in a non-thermal reaction to form a hydrogel that can absorb 80% water. In contrast to riboflavin-UVA crosslinking, it is oxygen-independent and requires little UVA exposure (photochemical reaction, not photosensitizing reaction). It also has augmentation properties (forms hydrogel material within the cornea) and is not purely creating intrastromal crosslinks. In comparison to standard crosslinking, similar efficacy with flashlinking has been demonstrated in stiffening the cornea, when measured with surface wave elastometry [12]. The advantage of the flashlinking over riboflavin is the reduction of UVA exposure time from 30 minutes to 30 seconds. However, further studies are necessary before it can be introduced into the clinical practice.

In addition to tissue stiffening, the authors of this tutorial also presented the novel mixture of riboflavin and type-1 collagen for crosslinking of a clear collagen gel into an adherent and resilient anterior stromal substitute for corneal reconstruction after lamellar graft dissection (Figure 2) [13]. Photochemical corneal augmentation by the crosslinking of a collagen gel mixture in porcine corneas has demonstrated good optical clarity and strong tissue adhesive properties [13].

Beyond keratoectasia, CXL can also be used in treating corneal melting conditions or infectious keratitis, as the crosslinking strengthens a collagenolytic cornea and UVA irradiation sterilizes the infectious agents. Furthermore, UV radiation with riboflavin has been shown to stop the keratolytic process in eyes with progressive corneal ulceration. In four patients suffering from various melting ulcerations of the cornea, CXL led to a halting of the progression in three patients [14].

Intrastromal delivery of riboflavin after creating two femtosecond laser pockets at 350 µm and 150 µm depth followed by CXL with high irradiance UVA (15 mW/cm2 for 7 minutes) was clinically helpful in reducing the corneal thickness and symptomatology of corneal edema secondary to bullous keratopathy [15]. The procedure postponed a corneal transplant in an 84-year-old woman with bullous keratopathy.

Conclusion

Based on the principle that increasing the number of covalent bonds between the collagen fibers might enhance the biomechanical stability of ectasic corneas, the chief goal for performing the cornea CXL procedure is to halt the procession of ectasia; indications include keratoconus and pellucid marginal degeneration. CXL may also be effective in the treatment and prophylaxis of keratectasia after laser in situ keratomileusis. Sufficient peer-reviewed evidence has supported the safety and efficacy of CXL. Future applications of CXL in infectious keratitis, bullous keratopathy and as a stromal substitute in corneal augmentation procedures expand the potential uses for this technology.

Figure 1. Experimental procedure in porcine corneas using the flashlinking component and UVA (370 nm) double diode light source delivering an irradiance of 3.0 mW/cm2 when held at a 1.2-cm distance from the cornea for 30 seconds.

Figure 2. Clear collagen liquid gel mixed with a customized flashlinking agent is applied to the corneal lamellar dissection site in porcine corneas. UVA light at 3mW/cm2 irradiance and 30 seconds exposure facilitates flashlinking of the liquid collagen into an adherent solid of similar clarity and consistency as the surrounding cornea.

References

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