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Keratoconus is an inflammatory related progressive ectatic condition of the cornea due to focal biomechanical decompensation.^1,2 This focal biomechanical decompensation leads to progressive stromal thinning and ectasia with resulting topographic and visual abnormalities. Collagen crosslinking (CXL) is the only safe, effective procedure to halt the progression of keratoconus and other ectatic disorders. In this review, we discuss the principles and clinical applications of CXL.

The biomechanical stiffness of corneas with keratoconus is shown to be decreased by a factor of 0.7 (70 % lesser strength).  

The  total content of collagen in ectatic corneas is similar to healthy corneas but the arrangement of collagen fibrils and lamellae is widely different. The collagen fibrils at the apex of a keratoconus cornea form a wide layer, show no delimitation of the lamellae and almost absent interlacing among the fibrils. This disorganized arrangement and poor interlacing of the collagen lamellae reduces the biomechanical strength of the corneas with keratoconus.

The word crosslinking means the formation of chemical bonds or bridges between proteins and other large molecules. These crosslinks increase the tissue strength, stiffness, and resistance to degeneration.

Crosslinking is employed in multiple industries and manufacturing practices. It is utilized to harden materials in polymer industry, to strengthen filling  materials in dentistry and to stabilize tissues in bio-engineering.³ Among many applications in medical industry, crosslinking is used to polymerize intra-ocular lens materials, and to manufacture vascular graft materials.⁴

Enzymatic cross-linking is a natural post-translational modification of collagen by lysyl oxidase enzyme. Lysyl oxidase creates covalent cross links between the collagen fibrils through oxidative deamination of the lysine and hydroxylysine residues.⁵ Non enzymatic crosslinking occurs through glycation in diabetics as a natural ageing process.⁶

CXL is a photochemical reaction similar to photosynthesis where light energy (derived from UV-A) is converted into chemical bonds. As riboflavin absorbs UV-A light, it excites and transforms into singlet and then triple excited states. In the presence of oxygen (type-2, aerobic reaction), the excited riboflavin reacts with the triplet oxygen, and generates singlet oxygen radical. This highly reactive oxygen free radical reacts with the carbonyl groups on the amino-acids in the collagen peptides and forms crosslinks. In the absence of oxygen, (type-1, anaerobic reaction), the riboflavin free radicals interact with the collagen peptides and form the crosslinks. The type-1 crosslinking is less efficacious than type-2 crosslinking.

In the following section, we discuss the three major components of this photochemical reaction – ultraviolet light, riboflavin and oxygen. The discussion focuses on the mechanisms of involvement of these factors, the proposed modifications in their application to improve the technique.

UV light is the source of the energy for the crosslinking process. The absorption peak of riboflavin is at 370 nm, providing protection to the endothelium and internal ocular structures at this wavelength. In the absence of  a photosensitizer, cornea absorbs 35% of the incident UV A irradiation. In the presence of 0.1% riboflavin, corneal stroma of 400 microns thickness, absorbs 90% of the UV-A irradiation, thus less than 10% of the UV-A energy reaches the intraocular structures, which is absorbed by the lens. The resulting endothelial exposure is 0.18mW/cm2, which is lesser than the safety threshold of 0.35 mW/cm².

Another important parameter that determines the intra- ocular toxicity of UV-A irradiation is the vergence of the beam. Divergent beam from shorter distances such as given by  UV devices used during cross-linking has lesser energy density and is less deleterious to ocular structures.

Riboflavin acts as a photosensitizer in the process of CXL, and increases the stromal absorption of UV- A irradiation. As mentioned earlier, riboflavin has peak absorption at UV-A wavelength, hence protects the intraocular structures from the radiation toxicity. Riboflavin is a micronutrient, used as a food coloring agent and is safe in the event of systemic absorption.

Riboflavin has poor permeability through epithelial tight junctions, hence the necessity for epithelial removal during CXL. To improve the permeability, chemicals like Benzalkonium chloride (BAK),⁷ Ethylene-diamine-tetraacetic acid (EDTA), were added to riboflavin solution. Trans-epithelial CXL irrespective of the modifications, was inferior to the standard epithelium- off CXL in terms of biomechanical efficacy.^8,9

Physical and Biomechanical Effects of Collagen Crosslinking 

The structural effects of CXL are mentioned in (table1). 

The effects of cellular and extra-cellular ocular structures are mentioned in the (table 2). 

Wollensak et al reported the first in vivo use of UV-A irradiation with riboflavin in humans.¹⁴ The protocol named after the place of study (Dresden, Germany) involves debridement of central 7 mm of the epithelium, application of 0.1% riboflavin solution 30 minutes before the procedure and every 5 minutes during the UV-A irradiation at a dose 3mW/cm² for 30 minutes. The standard CXL has been demonstrated to effectively stabilize keratoconus and improve the topographic and biomechanical outcomes.^15–17

The Bunsen-Roscoe law of reciprocity states that the photochemical effect is directly proportional to the total dose of the irradiation(W/cm²) irrespective of the dose and duration of the exposure over certain range.18 The law has been named after R. Bunsen and H.E. Roscoe for their pioneering work in photochemistry.19 The Dresden protocol involves the irradiation time of 30 min with 3mW/cm2 dose amounting to a total UV-A energy of 5.4J/cm². To reduce the irradiation time, multiple combinations of irradiation dose and time, 9mW/cm2 for 15 min, 10 mW/cm2 for 9 min, 18 mW/cm2 for 5min , 30 mW/cm2 for 3 min, and 45 mW/cm2 for 2 min have been studied with the total UV-A energy of 5.4J/cm2.20–22 These techniques of CXL with shorter irradiation time are termed as accelerated CXL (ACXL).23 The accelerated protocols are introduced to clinical practice due to the observation that the corneal stiffening effect of the higher UV-A fluences over shorter durations were comparable to the original 3mW/cm2 irradiation. However, it is essential to understand that the Bunsen- Roscoe law of reciprocity is applicable only till certain irradiation intensities. Any accelerated CXL protocol should strike a balance between the irradiation intensity, exposure duration, the biomechanical strengthening effect and the safety profile.

A meta-analysis of eleven trials comparing ACXL with SCXL reported that SCXL resulted in greater reduction in steep keratometry (Kmax) compared to ACXL.24 However, there are reports of ACXL showing equally good topographic outcomes as SCXL.25,26 The variance between the topography outcomes can be explained by the different exposure times followed by the authors.20 Improvement in visual acuity is reported to be similar between ACXL and SCXL.24 Among the different ACXL protocols, lower irradiance and shorter exposure time protocols may result in better visual outcomes.27 The depth and latency of onset of the demarcation line is shown to be shallow and delayed in eyes treated with ACXL protocols than SCXL protocol.

Safe and effective cross-linking in eyes with thin corneas faces two challenges. First is the safety threshold of UVA energy at the endothelium. Wollensak et al, in their original article on collagen cross-linking, reported that in a 500 µ thick cornea with the 3mW/cm2 irradiance at the surface and 0.1% riboflavin, the UVA energy reduces by 95% and the energy at the endothelial level is 0.27J/cm2, leaving a twofold margin for toxic irradiance(0.65J/cm2).¹⁴ However, the total fluence employed in classic Dresden protocol, if used in corneas thinner than 400µ (after de-epithelialization), the toxicity threshold of 0.65J/cm2 could be reached at the endothelial level. Hence, the authors cautioned the use of Dresden protocol in corneas thinner than 400µ. Second is post collagen cross-linking stromal haze, which is seen more often in eyes with advanced keratoconus, and thin corneas.28 Since significant proportion of patients with keratoconus have eyes with thinnest pachymetry < 400µ, modifications in the technique of collagen cross-linking to make it a safer tool in these corneas is necessary. To ensure safety of cross-linking in thinner corneas, one has to ensure shallow depth of UVA treatment so that the endothelium is not exposed to the UV-A energy beyond the safe threshold level. To achieve this, one can increase the thickness of the cornea, or place a layer of biological or synthetic origin above the cornea. Apart from this, the total fluence of the UVA irradiation can be reduced by reducing the irradiation dose, exposure time or customize the irradiation dose as per the thickness parameters. The newer techniques are mentioned in the (table 3).

NXT-UVA calculator is a freely available online calculator, that helps customize  the UV-A ‘on’ time to the thickness of the treated cornea.(Figure 4) The  UV-A ‘on’ time was calculated based on Lambert- Beer equation. Since, the total irradiation dose is within the limits of endothelial toxic exposure, there is no risk of endothelial toxicity or decompensation.^29,30

Keratoconus (KC), being a progressive ectatic condition with asymmetric corneal steepening, causes corneal surface (anterior and posterior) irregularities and induces both lower and higher order aberrations (HOA) in many magnitudes higher than a normal eye.³⁸ The higher order aberrations are shown to be approximately 5.5 times higher in eyes with KC than in normal eyes.³⁸ Coma like aberrations including vertical coma are the dominant HOA in these eyes.^38,39 

Before discussing the laser based treatment approaches in KC, we need to understand the concept of surface normalization. The laser based ablation in eyes with KC is not aimed at refractive correction rather to regularize the corneal surface, to create better aspheric profile of the cornea, and to reduce the higher order aberrations. The location of the ectatic cornea affects the corneal asphericity and the magnitude and pattern of higher order aberrations.

Concept of Topography guided surface normalization 

The topography guided surface normalization usually follows a specific ablation profile. The ablation pattern is planned in such a way that simultaneously flattens the ectatic cone area and an arcuate area of cornea in the periphery away from the cone usually in the superior nasal location. This peripheral flattening induces steepening adjacent to the cone similar to a hyperopic treatment. The combination of flattening in cone area and adjacent steepening regularizes the corneal surface thereby reducing the HOA.⁴⁰

Let us discuss the planning technique in the two popular laser based platforms employed globally in  keratoconus eyes

Multiple authors have used different permissible upper limits for the stromal ablation in  topography guided PRK in ectatic eyes. The maximal permissible ablation depths by various authors are : Kymionis et al – 50 µ⁴¹, kanellopoulos et al – 50 µ⁴², Camellin et al – 55µ⁴³, Shetty et al- 40µ⁴⁰. 

For this platform, the topography examinations are done by the ALLEGRETO WAVE Topolyzer Vario, with the T-CAT (Topography guided custom ablation treatment) software for treatment. The platform also enables the clinician to choose the post ablation aspheric profile of the treated cornea.__42

The asphericity of the cornea, location of the cone (centered vs decentered) and the refractive error is taken into account while planning the topography guided custom ablation in keratoconic eyes.

The ectatic cone area is considered central if more than 50% cone area is with in the central 3mm zone on posterior elevation map.  In these eyes, cornea has more negative asphericity (high negative Q value) and a myopic refractive error due to central steepening. We target either the reduction of Q by 20-30% or partial refractive correction, where both approaches would induce central flattening and regularization making the cornea achieve a more physiological aspheric profile. To achieve this, the authors optimize the Zernike polynomials to achieve the equivalency between the defocus (C4) and the spherical aberration (C12) by a specific spherical error input in the targeted refraction tab. The choice to partially correct the refractive error is taken based on the baseline refractive error (<6D ) and thinnest pachymetry (>45µ).

Eyes with decentered cone, have a less negative Q value but other dominant higher order aberrations, including coma and trefoil. During the attempted surface regularization and reduction of HOA, the Q value may reduce significantly to a more negative values. If no refractive correction is attempted, the preoperative Q can be selected as zero. Partial refractive correction can be targeted without breaching the 40µ stromal ablation thickness rule. 

We initially assess the plano treatment ablation profile planned by the software, then apply Zernike polynomial optimization (to target C4 (Defocus) and C12 (Spherical Aberration) equivalency, and then finally attempt partial refractive correction based on the baseline refractive error and thinnest pachymetry.

The AMARIS is a flying spot laser platform that performs reversed single step wave-front based customized Trans epithelial PRK. The ablation profile is planned using ORK-CAM software. The step wise planning of  topography guided trans PRK is mentioned in detail in (figure 5). 

Demarcation line is a transition zone, between the crosslinked anterior stroma and untreated posterior stroma.⁴⁴ (Figure 7) Ultrastructurally , the treated stroma shows keratocyte apoptosis, increased density of the extracellular matrix and collagen fibre shrinkage. It is still debatable whether the depth of demarcation line is a valid structural marker of efficacy of CXL.

 

Newer advances in CXL have reduced the procedural duration, improved the patient comfort without significant reduction in the efficacy outcomes. The techniques of crosslinking in thin corneas have improved the safety profile of crosslinking in thin and ultra-thin corneas where standard CXL could lead to endothelial complications. Individualized CXL is a newer modality with the novel approach of modifying the irradiation times and total UV-A energy based on the pachymetry profile. Topography guided CXL shows superior visual outcomes compared to other forms of CXL. 

Future directions in the research on CXL should focus on improving predictable refractive outcomes of the procedure. Customized crosslinking with/without laser ablation protocols will make CXL a truly therapeutic and refractive procedure.