An Overview of Corneal Collagen Cross-Linking (CXL)

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    An Overview of Corneal Collagen Cross-Linking (CXL)

    George D. Kymionis Dimitrios G. Mikropoulos

    Dimitra M. Portaliou Irini C. Voudouragkaki

    Vassilios P. Kozobolis Anastasios G. P. Konstas

    To view enhanced content go to www.advancesintherapy.comReceived: September 24, 2013 / Published online: October 30, 2013 Springer Healthcare 2013


    Corneal collagen cross-linking (CXL) was first

    described over a decade ago and is now

    considered to be one of the most important

    surgical innovations of modern

    ophthalmology. Prior to its introduction, no

    interventions were available to arrest, or slow

    down ectatic disease progression, with corneal

    transplantation required in the majority of

    cases. Unlike earlier treatments of corneal

    ectasias that attempted to only improve the

    consequences of the disease, CXL aims to

    address the corneal biomechanical weakening

    itself. The long-term safety and efficacy of CXL

    have been established in several studies that

    have documented significant improvements in

    all outcome measures (visual acuity, spherical

    equivalent, astigmatism, and keratometric

    findings). The emerging combination of CXL

    with other interventions (termed CXL plus)

    optimizes the visual and topographic outcomes.

    This, along with the expansion of the

    techniques indications for other clinical

    conditions, such as microbial keratitis,

    highlights the continuous improvement of the

    initial technique and confirms its wide

    acceptance. Overall, CXL has already

    demonstrated much promise and has several

    clinical indications, representing a clear

    example of recent advances in ocular therapy.

    Keywords: Corneal collagen cross-linking;

    CXL plus; Ectatic disorders; Laser-assisted

    G. D. Kymionis D. M. PortaliouFaculty of Medicine, Institute of Vision and Optics,University of Crete, Heraklion, Greece

    D. G. Mikropoulos A. G. P. Konstas (&)3rd University Department of Ophthalmology,Aristotle University of Thessaloniki, Thessaloniki,Greecee-mail:

    I. C. Voudouragkaki A. G. P. Konstas1st University Department of Ophthalmology,Aristotle University of Thessaloniki, 1 KyriakidiStreet, 546 36 Thessaloniki, Greece

    V. P. KozobolisDepartment of Ophthalmology, University Hospitalof Alexandroupolis, Alexandroupolis, Greece

    V. P. KozobolisEye Institute of Thrace, Alexandroupolis, Greece

    Enhanced content for Advances in Therapy

    articles is available on the journal web site:


    Adv Ther (2013) 30:858869

    DOI 10.1007/s12325-013-0065-9

  • sub-epithelial keratectomy; Keratoconus;

    Ophthalmology; Riboflavin; Ultraviolet-A


    Corneal collagen cross-linking (CXL)

    constitutes a minimally invasive surgical

    intervention employed for the management of

    ectatic corneal disorders, such as keratoconus,

    pellucid marginal corneal degeneration and

    post-laser in situ keratomileusis (LASIK)

    corneal ectasia [14]. It has been previously

    demonstrated that in keratoconus, the number

    of diagonal links of collagen fibrils is

    significantly reduced [5]. These fibrils provide

    the cornea with mechanical stability. When

    they are lacking, the cornea gradually becomes

    destabilized due in part to thinning of the

    central and para-central areas, which in turn

    causes irregular astigmatism, myopia and

    reduction in visual acuity.

    The CXL principle is based on the formation

    of chemical bonds (cross-links) among stromal

    collagen fibrils, thereby strengthening and

    stabilizing the diseased cornea. The use of

    riboflavin, also known as vitamin B2, in

    conjunction with ultraviolet-A (UV-A)

    irradiation facilitates the formation of cross-

    links between collagen fibrils in the corneal

    stroma, providing a stiffening effect capable of

    halting progression of the ectasia [1, 2].

    Prior to the introduction of CXL, the

    possible treatment options for ectatic corneal

    disorders included spectacle correction, contact

    lenses, intrastromal corneal ring segment

    implantation [6] and, in advanced cases,

    lamellar or penetrating keratoplasty [7]. All

    these options had a single goal of

    symptomatic treatment and did not aim to

    stabilize the ectatic disorder per se. In contrast,

    CXL arrests the progression of the

    primary disorder, thereby addressing the

    pathophysiology of the disease rather than just

    its symptoms.

    Original Surgical Technique (Dresden


    The term cross-linking in the biological

    sciences is used to express the formation of

    chemical bridges following chemical reactions

    between proteins or other molecules. Usually,

    cross-links can be formed by chemical reactions

    that are initiated by heat, pressure, or radiation.

    The result of such reactions is the change in the

    biological molecules physical properties.

    Natural enzymatic cross-linking is part of the

    post-translational modification of collagen.

    During the aging process of the human body,

    both enzymatic and non-enzymatic cross-

    linking occur in various parts, such as the skin

    or the arteries. A key observation that resulted

    in the introduction of CXL for the management

    of keratoconus is the fact that diabetics often do

    not show progression of corneal ectatic

    disorders due to naturally occurring non-

    enzymatic cross-linking [1].

    The standard CXL protocol was first

    described by Wollensak and colleagues [1] and

    is often referred to as the Dresden protocol.

    This treatment protocol constitutes the

    benchmark of the CXL procedure and has set

    the foundation for the evaluation of safety and

    efficacy of the technique. CXL is always

    conducted under sterile conditions in the

    operating room. After application of topical

    anesthesia, the central 89 mm of the

    epithelium is removed. It is now possible to

    perform mechanical removal of the epithelium

    with a blade (or more recently employing a

    rotating brush), removal with the use of alcohol

    (laser-assisted sub-epithelial keratectomy,

    Adv Ther (2013) 30:858869 859


  • LASEK), or removal with a laser (transepithelial


    Riboflavin 0.1% solution is applied every

    25 min for approximately 30 min to facilitate

    penetration of the corneal stroma, until the

    stroma is completely penetrated, as indicated by

    yellow flare in the anterior chamber. Different

    commercially available UV-A light sources can

    be used. The role of riboflavin in CXL is

    twofold. Not only does it work as a photo

    sensitizer for the induction of cross-links, but by

    acting as a selective filter, it also protects the

    underlying tissues from the harmful influence

    of UV-A. It has been shown by Wollensak and

    coworkers [8] that the cytotoxic irradiance

    level stands at 0.5 mW/cm2 for keratocytes

    after UV-A irradiation combined with the

    photosensitizer riboflavin, which is 10-times

    lower than the cytotoxic irradiance of

    5 mW/cm2 after UV-A-irradiation alone.

    Before treatment, the intended 3 mW/cm2

    surface irradiance (5.4 J/cm2 surface dose) can

    be confirmed using a UV light meter. In a

    previous investigation, Wollensak [2] proposed

    a pre-operative corneal thickness of 400 lm as a

    minimum safety limit to avoid posterior corneal

    tissue damage during CXL. In rabbits, corneal

    endothelial toxicity was reached by irradiance

    of 0.36 mW/cm2, while this level of radiation

    exposure in human corneas reached a depth of

    less than 400 lm. Spoerl and coworkers [9] also

    reported that a safety threshold of 400 lm

    corneal pachymetry in the presence of

    riboflavin was necessary to limit UV-A

    irradiance to less than 1 J/cm2 at the level of

    the corneal endothelium, anterior chamber,

    lens and retina. Unquestionably, the presence

    of riboflavin enhances the safety profile. The

    cornea is exposed to the above stated level of

    UV-A energy for a total of 30 min. During

    treatment, riboflavin solution is applied every

    25 min to ensure saturation of the tissue. After

    treatment, a bandage contact lens is applied

    until the epithelium is completely healed and

    is combined with the application of topical

    corticosteroids, antibiotic, and non-steroidal

    anti-inflammatory agents.

    Efficacy, Safety and Clinical Outcomes

    Several clinical studies of CXL have now been

    conducted in Europe and the USA, all of which

    provide information on the efficacy and safety

    of the procedure in the short, medium and long

    term. Even though CXL has become common

    practice in Europe, in the USA, the US Food

    and Drug Administration (FDA) has not

    yet approved this treatment modality.

    Nevertheless, there are two ongoing clinical

    trials, the results of which may lead to FDA

    approval. This will positively impact availability

    and the cost of treatment options, and also

    liability issues in the USA.

    The first study in human eyes was conducted

    in 2003 by Wollensak and coworkers and

    included 23 cases [1]. This study included

    follow-up data for up to 4 years and

    demonstrated topographic stability and

    improvement of the mean keratometric

    (K) values in approximately 70% of treated

    patients. Furthermore, 65% of treated patients

    also showed a small improvement in visual

    acuity [1].

    Caporossi and coworkers [10] presented

    preliminary results on CXL, including ten

    cases with 6 months follow-up. Refractive

    results demonstrated a reduction of about 2.5

    diopters (D) in the mean spherical equivalent,

    topographically confirmed by the reduction in

    mean K values. A second study by the same

    research group detected stability of the corneal

    ectatic disorder in 44 cases after a minimum of

    48 months of follow-up [11]. Corneal symmetry

    improvement was seen in 85% of patients.

    860 Adv Ther (2013) 30:858869


  • In a subsequent comparative study,

    Coscunseven and coworkers [12] confirmed

    the initial findings reported by Wollensak and

    coworkers [1]. Following CXL, this group

    detected a mean decrease in spherical

    equivalent refraction of 1.03 2.22 D and an

    increase in uncorrected distance visual acuity

    (UDVA) and corrected distance visual acuity

    (CDVA) of 0.06 0.05 and 0.10 0.14 D,

    respectively, for the group treated. In contrast,

    they documented progression for all tested

    parameters in the eyes that were not treated.

    Agrawal [13] presented his results in a series

    of Indian eyes showing that 1 year after CXL

    treatment, 54% of eyes gained at least one line

    of CDVA. The K value of the apex decreased by a

    mean of 2.73 D in 66% of eyes and remained

    stable (within 0.50 D) in 22% of eyes. Similar

    1-year follow-up results confirming the efficacy

    of the procedure were reported by Asri and

    coworkers [14] in a series of 142 eyes with a

    greater than 2-D difference in K readings in

    21.3% of cases and stability in another 68.8%.

    Similar statistically significant improvements in

    all tested parameters after 12 months have been

    reported for two other studies [15, 16].

    Vinciguerra and coworkers [17] detected

    similar gains in keratometric and refractive

    findings but also showed that corneal and

    total wavefront aberrations were reduced

    1 year after CXL treatment in their series of 28

    eyes. Koller and coworkers [18, 19] made an

    interesting observation during the 12-month

    follow-up period of their patients treated for

    keratoconus. They observed corneal flattening

    [18] with regularization of the corneal shape

    [19], as captured by means of Scheimpflug

    imaging. Thus, the authors concluded that the

    CXL effect causes a progressive topographic

    improvement throughout the follow-up period.

    In another study, Goldich and coworkers

    [20] observed a significant improvement in

    CDVA (0.21 0.1 to 0.14 0.1; p = 0.002)

    and stability in UDVA (0.62 0.5 to

    0.81 0.49; p = 0.475). There was a

    significant decrease in the steepest-meridian

    keratometry (53.9 5.9 to 51.5 5.4 D,

    p = 0.001) recorded 24 months after CXL in

    eyes with keratoconus. Similar long-term,

    successful results (with 3-year follow-up)

    have been published by Raiskup-Wolf and

    coworkers [21], who conducted a study in

    241 keratoconus cases. Only two patients

    needed a second CXL treatment because of

    apparent progression of the ectasia.

    Kymionis and coworkers [22] established a

    significant increase in intraocular pressure

    (IOP) measurements by Goldman applanation

    tonometry (GAT) at 6 months (from 9.95

    3.01 to 11.40 2.89 mmHg) and then at

    12 months (from 9.95 3.01 to 11.35

    3.38 mmHg) following CXL (both p\0.001).The authors attributed this pressure rise to the

    increased corneal rigidity and stiffness, which

    came about due to the formation of cross-links

    in the corneal stroma of the treated cases.

    Gkika and coworkers [23] evaluated IOP with

    three different tonometersGAT, Pascal

    dynamic contour tonometer (PDCT) and

    ocular response analyzer (ORA) tonometer

    before and after CXL, and concluded that

    PDCT had greater accuracy in keratoconus

    patients before and after CXL. The same

    research group tried to assess corneal

    hysteresis and corneal resistance factor in

    keratoconic eyes before and after CXL,

    proving that CXL exerts a non-significant

    impact on ORA measurements [24].

    Several studies in the literature have

    investigated the use of CXL in post-LASIK

    corneal ectasia with up to 25-month follow-up

    [3, 25, 26]. These studies have demonstrated no

    progression of ectasia in conjunction with

    visual and topographic improvement.

    Adv Ther (2013) 30:858869 861


  • There are now a number of published

    investigations that have evaluated the safety of

    the CXL technique [20, 27, 28]. The first study

    [20] evaluated the corneal endothelium by

    specular microscopy and the retina by

    comprehensive fundus examination and

    optical coherence tomography analysis. The

    investigators concluded that no morphologic

    abnormalities were detected after CXL, and that

    the endothelial cell density and foveal thickness

    remained unchanged [20]. A subsequent study

    [27] reported no changes in crystalline lens

    density and foveal thickness 12 months after

    CXL, while the third study [28] also confirmed

    the absence of retinal morphologic changes

    after CXL.

    A recent study investigated CXL specifically

    in pediatric patients and reported encouraging

    preliminary results [29], but these observations

    must be confirmed in large controlled trials, and

    the technique must be applied with caution in

    children. Chatzis and Hafezi [30] documented

    visual, refractive and topographic stabilization

    and improvements after pediatric CXL similar

    to those reported for adult treatment outcomes

    over 2 years. Nevertheless, they did observe

    some keratometric progression at 3 years of

    follow-up. The findings suggest that pediatric

    CXL may not provide long-term stability

    comparable to adult treatment and these

    younger patients may require re-treatment,

    especially in a subset of those patients with

    persistent eye rubbing.

    Corneal Collagen Cross-Linking in Thin

    Corneas (Under 400 lm)

    It should be noted that there are many cases

    with keratoconus and post-LASIK ectasia who

    exhibit corneal stromal thickness less than

    400 lm, and who achieve a satisfactory visual

    acuity by means of spectacles or contact lenses.

    In these cases, the current CXL treatment

    protocol prohibits the surgical procedure due

    to inadequate corneal thickness. Nevertheless,

    two groups have now proposed an alternative

    treatment protocol targeting thin corneas

    [31, 32]. They employ hypo-osmolar riboflavin

    solution with overall satisfactory results. Still, it

    is essential to bear in mind that since these

    ectatic corneas with less than 400 lm corneal

    thickness are outside the range of the Dresden

    protocol, the risk of the procedure is greater and

    a higher rate of complications may occur. For

    example, a significant postoperative decrease in

    endothelial cell density has been documented

    by Kymionis and coworkers in a few of these

    cases [33].


    To date, few complications have been reported

    during and after CXL. Therefore, CXL is now

    generally considered a safe and effective surgical

    procedure. In some cases, stromal edema is

    detected immediately after CXL surgery, but

    this is transient and, fortunately, without

    clinical significance.

    A case report of herpetic keratitis with iritis

    after CXL [34] has led to the belief that cross-

    linking can induce herpetic keratitis with

    inflammation in rare cases, even in patients

    with no history of herpetic disease. Another

    case of diffuse lamellar keratitis developing after

    CXL in a patient with post-LASIK ectasia has

    also been reported [35]. This case was

    successfully managed with intensive topical

    corticosteroids. Labiris and coworkers [36]

    published a case of acute inflammatory

    response after CXL resulting in corneal

    melting and descemetocele, which led to


    Finally, a few cases of infectious keratitis

    post CXL have been reported in the literature

    862 Adv Ther (2013) 30:858869


  • [3739]. These resulted in corneal ulceration

    and scarring. Another single case of ectasia

    progression despite CXL treatment in a

    pregnant woman has been published [40]. In

    this last case, one possible hypothesis could be

    that the high estrogen levels associated with

    pregnancy may have adversely affected the

    rigidity of the cornea resulting in failure of the

    CXL procedure [41].

    New CXL Indications

    A new promising line of indications for CXL in

    other types of corneal pathology is currently

    under investigation.

    Infectious Keratitis

    The treatment of microbial keratitis with the

    use of CXL has recently raised interest among

    the scientific community. To date, CXL has

    been shown clinically to be beneficial in cases of

    resilient pathogens, such as drug-resistant

    Streptococcus pneumoniae and Gram-negative

    Escherichia coli [4244]. Martins and coworkers

    [43] have proven the in vitro antimicrobial

    efficacy of riboflavin/UV-A (365 nm)

    combination for bacterial and fungal isolates.

    In all published studies, there was a rapid

    decrease in pain and the corneal re-

    epithelialization process was accelerated

    following CXL. In a pertinent published case

    series of five patients with infectious keratitis

    and corneal melting, Iseli and coworkers [45]

    employed CXL surgery after topical and

    systemic antibiotic treatments had failed.

    Encouragingly in all cases, corneal melting

    ceased and emergency corneal transplantation

    became unnecessary [45].

    It goes without saying that CXL should not

    be seen as the procedure of choice for infectious

    keratitis and should only be applied with

    caution as it may have toxic effects on these

    susceptible diseased corneas. Moreover, not all

    pathogens will respond positively to CXL

    treatment and this especially applies in the

    case of herpes simplex virus because the use of

    UV light may act as a stimulus for virus

    replication, exacerbating the infection and

    potentially leading to corneal perforation

    [46, 47].

    Ulcerative Keratitis

    CXL seems to have an anti-edematous effect

    on the cornea and, therefore, it has been

    successfully applied in cases of bullous

    keratopathy [48]. Two reports by Kozobolis

    and coworkers [49] and Ehlers and coworkers

    [50] have presented the results after CXL in

    patients with combined ulcerative keratitis and

    bullous keratopathy that was unresponsive to

    conventional treatment regimens. In both

    reports, the patients ulcer, visual acuity and

    corneal edema were significantly improved.

    Recently, CXL has also been investigated for

    modifying donor tissue prior to keratoplasty

    [51] and as an adjunct to orthokeratology [52].

    Finally, the use of CXL for prophylaxis in

    patients whose corneas are deemed to be at a

    high risk for developing corneal ectasia after

    LASIK surgery for myopia [53] has been


    CXL Plus

    The term CXL plus was introduced in 2011 and

    refers to several combined procedures aimed at

    enhancing the success of CXL [54]. It is well

    documented that when performed on its own,

    the CXL procedure is not intended to improve

    vision. However, at our disposal, there are now

    additional interventions to the original CXL

    protocol that can improve visual acuity and

    thus optimize the final surgical outcome. To

    date, controlled clinical evidence exists for the

    Adv Ther (2013) 30:858869 863


  • use of several complementary steps to the CXL


    Transepithelial phototherapeutic keratectomy(t-PTK) [5557]. Kymionis and coworkers

    [56] proved that epithelial removal using

    t-PTK (Cretan protocol) during CXL resulted

    in better visual and refractive outcomes in

    comparison with mechanical epithelial


    Topography-guided and other forms ofphotorefractive keratectomy (PRK) [5871].

    The use of topography-guided PRKCXL in

    post-LASIK ectasias, such as the Athens

    protocol described by Kanellopoulos and

    coworkers [68], has been successfully


    Corneal implants, also known as intracornealring segments [7283]

    Phakic intraocular lens implantation [8489].Labiris and coworkers [90, 91] investigated

    the effect of keratoconus, CXL and CXL

    combined with topography-guided

    photorefractive keratectomy (t-PRK) on self-

    reported quality of life (QOL) by means of

    the 25-item National Eye Institute Visual

    Function Questionnaire (NEI-VFQ 25) and

    concluded that keratoconus has a significant

    impact on patients QOL, even in its early

    stages, with functional best-spectacle-

    corrected visual acuity. Moreover, CXL, and

    especially CXL combined with t-PRK,

    appeared to exert a beneficial impact on

    self-reported QOL.

    CXL Extra

    In an attempt to accelerate the time required

    for CXL treatment utilizing the Dresden

    standard protocol (usually 1 h of surgical

    time), investigators have explored two

    different research avenues: riboflavin

    application by iontophoresis aiming at rapid

    stromal saturation, and the use of high fluence

    irradiation of UV-A light [92, 93].

    The Dresden protocol relies on the

    application of UV-A light (365 nm) at the

    intensity of 3 mW/cm2 for 30 min, delivering

    a total of 5.4 J/cm2 energy onto the cornea

    [1]. In accordance with the BunsenRoscoe

    photochemical law of reciprocity, if the

    intensity and time change while the total

    energy is maintained, the effects of any

    photochemical reaction (in the current

    context, the CXL procedure) are similar.

    This implies that the total energy delivered

    and amount of cross-linkage induced in a

    standard CXL session should be similar to

    irradiation at 9 mW/cm2 for 10 min, 15 mW/

    cm2 for 6 min, and 30 mW/cm2 for 3 min,

    with all ultimately delivering the same

    energy (5.4 J/cm2) [93]. These new treatment

    protocols are referred to as accelerated CXL

    or CXL extra.

    The main concerns for accelerated CXL are

    its repercussions on the safety of the

    procedure, given that despite a similar total

    energy being applied in CXL extra, the

    intensity of irradiation is higher and may

    have a harmful effect on the corneal

    endothelium. Initial accounts of CXL extra,

    however, report results comparable to those

    obtained with the standard Dresden protocol

    [9496]. Epithelial healing occurs uneventfully

    and there are no detectable alterations in

    endothelial cell density as documented by

    confocal microscopy. In contrast, Cingu and

    coworkers [97] reported transient corneal

    endothelial changes following accelerated

    CXL (18 mW/cm2 for 5 min) for the

    treatment of progressive keratoconus in a

    casecontrol study. In this investigation,

    which employed corneal specular microscopy,

    a decrease in endothelial cell density was

    observed postoperatively at 1 month, which

    864 Adv Ther (2013) 30:858869


  • returned to pre-operative values after 6 months

    of follow-up.

    Overall, accelerated CXL protocols seem to

    be a promising alternative in minimizing the

    duration of the treatment and lessening patient

    discomfort. Future large, controlled studies are

    needed to confirm the immediate and long-

    term safety of the procedure.


    CXL has marked a new, less invasive era in the

    management of corneal ectatic disorders.

    Since the first pilot studies over a decade

    ago, many modifications and several

    improvements to the original protocol have

    been successfully carried out. These steps

    maximize the cross-linking effect and by

    doing so, halt progression and postpone

    or even avoid the need for corneal

    transplantation, as well as improving

    functional vision in patients with ectasias.

    The CXL plus and CXL extra protocols may

    represent the future of this procedure, but

    more research is needed before these steps

    are widely adopted. The clinical utility of

    CXL has already been well demonstrated

    even though there is, to date, no adequate

    knowledge regarding long-term, unforeseen

    consequences. Future research will further

    elucidate and consolidate the place of CXL

    among the most innovative surgical therapies

    in ophthalmology.


    No funding or sponsorship was received for this

    study or publication of this article. Dr.

    Anastasios Konstas is the guarantor for this

    article, and takes responsibility for the integrity

    of the work as a whole.

    Conflict of interest. George Kymionis,

    Dimitrios Mikropoulos, Dimitra Portaliou, Irini

    Voudouragkaki, Vassilios Kozobolis, and

    Anastasios Konstas declare that they have no

    conflict of interest in any of the materials or

    methods described herein.

    Compliance with ethics guidelines. The

    analysis in this article is based on previously

    conducted studies, and does not involve any

    new studies of human or animal subjects

    performed by any of the authors.


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    3. Hafezi F, Kanellopoulos J, Wiltfang R, Seiler T.Corneal collagen crosslinking with riboflavin andultraviolet A to treat induced keratectasia after laserin situ keratomileusis. J Cataract Refract Surg.2007;33:203540.

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    Adv Ther (2013) 30:858869 869


    An Overview of Corneal Collagen Cross-Linking (CXL)AbstractIntroductionOriginal Surgical Technique (Dresden Protocol)Efficacy, Safety and Clinical OutcomesCorneal Collagen Cross-Linking in Thin Corneas (Under 400 microm)ComplicationsNew CXL IndicationsInfectious KeratitisUlcerative Keratitis

    CXL PlusCXL Extra



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