REVIEW
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
ABSTRACT
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: [email protected]
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:
www.advancesintherapy.com
123
Adv Ther (2013) 30:858–869
DOI 10.1007/s12325-013-0065-9
sub-epithelial keratectomy; Keratoconus;
Ophthalmology; Riboflavin; Ultraviolet-A
INTRODUCTION
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 [1–4]. 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
Protocol)
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 8–9 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) 30:858–869 859
123
LASEK), or removal with a laser (transepithelial
CXL).
Riboflavin 0.1% solution is applied every
2–5 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
2–5 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) 30:858–869
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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 tonometers—GAT, 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) 30:858–869 861
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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].
Complications
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
perforation.
Finally, a few cases of infectious keratitis
post CXL have been reported in the literature
862 Adv Ther (2013) 30:858–869
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[37–39]. 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 [42–44]. 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
proposed.
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) 30:858–869 863
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use of several complementary steps to the CXL
procedure:
• Transepithelial phototherapeutic keratectomy
(t-PTK) [55–57]. 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
debridement.
• Topography-guided and other forms of
photorefractive keratectomy (PRK) [58–71].
The use of topography-guided PRK–CXL in
post-LASIK ectasias, such as the Athens
protocol described by Kanellopoulos and
coworkers [68], has been successfully
applied.
• Corneal implants, also known as intracorneal
ring segments [72–83]
• Phakic intraocular lens implantation [84–89].
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 Bunsen–Roscoe
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
[94–96]. 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
case–control 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) 30:858–869
123
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.
CONCLUSION
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.
ACKNOWLEDGMENTS
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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