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BASIC INVESTIGATIONS
Collagen Fiber Diameter in the Rabbit Cornea After Collagen
Crosslinking by Riboflavin/UVA
Gregor Wollensak, MD,* Michaela Wilsch, PhD,† Eberhard Spoerl, PhD,* and Theo Seiler, MD, PhD‡
Objective: Collagen crosslinking of the cornea has been developed
recently as a quasiconservative treatment of keratoconus. Biome-
chanical in vitro measurements have demonstrated a significant in-
crease in biomechanical stiffness of the crosslinked cornea. The aim
of the present study was to evaluate the effect of this new procedure
on the collagen fiber diameter of the rabbit cornea.
Methods: The corneas of the right eyes of 10 New Zealand White
albino rabbits were crosslinked by application of the photosensitizer
riboflavin and exposure to UVA light (370 nm, 3 mW/cm
2
) for
30 minutes. The left fellow control eyes were either left untreated
(rabbits 1–4), deepithelialized (rabbits 5–7), or deepithelialized and
treated with riboflavin/dextran solution (rabbits 8–10) to exclude an
influence of epithelial debridement or hydration changes on the fiber
diameter. On ultrathin sections of samples from the anterior and pos-
terior cornea, the collagen fiber diameter was measured semiauto-
matically with the help of morphometric computer software.
Results: In the anterior stroma, the collagen fiber diameter in the
treated corneas was significantly increased by 12.2% (3.96 nm), and
in the posterior stroma by 4.6% (1.63 nm), compared with the control
fellow eyes. In the crosslinked eyes, the collagen fiber diameter was
also significantly increased by, on average, 9.3% (3.1 nm) in the an-
terior compared with the posterior stroma within the same eye.
Conclusions: Collagen crosslinking using riboflavin and UVA
leads to a significant increase in corneal collagen diameter. This al-
teration is the morphologic correlate of the crosslinking process lead-
ing to an increase in biomechanical stability. The crosslinking effect
is strongest in the anterior half of the stroma because of the rapid
decrease in UVA irradiance across the corneal stroma as a result of
riboflavin-enhanced UVA absorption.
Key Words: collagen fiber diameter, crosslinking, cornea, UVA, ri-
boflavin
(Cornea 2004;23:503–507)
We have recently developed a technique of collagen
crosslinking in the cornea using UVA and the photosen-
sitizer riboflavin to stiffen the cornea. In a prospective clinical
pilot study including 22 patients with moderate or advanced
progressive keratoconus and with a follow-up time of up to 4
years, the progression of keratoconus could be stopped in all
treated eyes. Regression with a reduction of the maximal kera-
tometry readings by 2 diopters was achieved in 70% of pa-
tients. Corneal and lens transparency as well as endothelial cell
density remained unchanged.
1
Collagen crosslinking might
therefore become a new way to stop the progression of kera-
tectasia in keratoconus patients. By this means, the need for
penetrating keratoplasty in keratoconus might be significantly
reduced in the future. Given the simplicity and minimal costs
of the treatment, it might also be well suited for developing
countries. Another possible clinical use of collagen crosslink-
ing lies in the field of refractive surgery, corneal ulcers, and
stromal melting and thinning.
In extensive experimental studies including biomechan-
ical stress-strain measurements, we could show a significant
increase in corneal rigidity by about 70% in rabbit and porcine
corneas after the crosslinking treatment
2–4
and an increased
resistance to enzymatic digestion by collagenases.
5
Crosslinking is a widespread phenomenon and can also
be found in the aging and cataractous lens, where crosslinking
of the lens crystallins leads to increased rigidity of the lens and
to an increase of the molecular weight of the crystallin proteins
from 20,000 to over 50,000.
6
The aim of the present study was to elucidate the mor-
phologic correlate of the new treatment by evaluating the in-
fluence of the crosslinking treatment on the corneal collagen
fiber diameter.
MATERIALS AND METHODS
Animals
Ten female New Zealand White albino rabbits weighing
2–2.5 kg were used for the experiment. The right eyes of rab-
bits 1–10 were crosslinked with riboflavin and UVA. The left
fellow eyes served as intraindividual controls, remaining com-
pletely untreated, in rabbits 1–4. In the left eyes of rabbits 5–7,
epithelial debridement alone, and in rabbits 8–10 epithelial de-
Received for publication June 11, 2003; revision received October 3, 2003;
accepted October 7, 2003.
From the *Department of Ophthalmology, Universitätsklinikum CGC,
Dresden, Germany; †Max-Planck-Institute for Molecular Cell Biology
and Genetics, Dresden, Germany; and ‡Institut fu¨r Refraktive Chirurgie
und Ophthalmo-Chirurgie (IROC), Zurich, Switzerland.
Reprints: Dr Gregor Wollensak, Wildentensteig 4, D-14195 Berlin, Germany
(e-mail: gwollens@hotmail.com).
Copyright © 2004 by Lippincott Williams & Wilkins
Cornea • Volume 23, Number 5, July 2004 503
bridement plus the application of dehydrating dextran/ribo-
flavin solution was performed to exclude an influence on fiber
diameter of epithelial abrasion or associated hydration
changes. All animal procedures were approved by the ethics
comittee and conformed to the ARVO Resolution on the Use
of Animals in Ophthalmic and Vision Research.
Crosslinking Treatment
Ten rabbits were anesthetized with subcutaneous injec-
tion of a mixture of 1.5 mL ketamine hydrochloride 10% (35
mg/kg) and 0.5 mL xylazine hydrochloride (5 mg/kg). For pre-
medication diazepam (10 mg) and atropine (0.5 mg) were
used. After anesthesia, the central 5 mm of the cornea were
deepithelialized mechanically using a blunt hockey knife. Af-
ter the debridement, riboflavin photosensitizer solution con-
taining 0.1% riboflavin-5-phosphate and 20% dextran T-500
was dropped onto the cornea 5 minutes before the irradiation
and every 5 minutes during the irradiation. The UVA irradia-
tion (370 nm) was applied using a double UVA diode (Roith-
ner Lasertechnik, Vienna, Austria) with an irradiance of
3 mW/cm
2
for 30 minutes ata1cmdistance from the cornea
(Fig. 1). The animals were killed 4 hours after treatment in
general anesthesia using an overdose of pentobarbital.
Transmission Electron Microscopy
After enucleation, the whole eye globes were fixed in
2.5% glutaraldehyde in 0.1 M PBS buffer at 4°C. After fixa-
tion, the globes were bisected, and 4 mm
2
samples of the cen-
tral cornea were dissected at 50 µm depth for the anterior and at
350 µm corneal depth for the posterior stroma (Fig. 2) and em-
bedded in epon. Ultrathin epon sections 50–70 nm thick were
cut and contrasted with uranyl acetate and lead citrate and
evaluated morphometrically using the electron microscope
Morgagni 268D (Philips, Eindhoven, The Netherlands) at
×89,000 magnification. The EM pictures were transferred to a
computer screen by an attached MegaView II camera. The di-
ameters were marked manually with a computer mouse (Fig.
3) and calculated with the help of the semiautomatic software
program Analysis (Soft Imaging System GmbH, Münster,
Germany).
In each case, the diameters of 80 to 160 contiguous fiber
section profiles were measured for the anterior and posterior
stroma (Fig. 3). Only fiber profiles with clearly defined bor-
ders of high contrast were included; profiles with low contrast
and indistinct borders were discarded. These were fewer than
1% of the total fiber count.
In some sections with a slightly ellipsoidal section pro-
file (Fig. 4) as a result of oblique sectioning, the minimal trans-
verse diameter of the collagen fibers was measured because in
ellipsoidal section profiles the shortest diameter is equal to the
diameter of the corresponding circular section profile.
Statistics
The outcome variables were compared statistically us-
ing one-way ANOVA followed by Sidak post hoc test and ex-
FIGURE 1. Irradiation of rabbit eye using a double UVA diode
(370 nm, 3 mW/cm
2
, 30 minutes) at a 1-cm distance.
FIGURE 2. Schematic illustration of the anterior (a) and posterior (p) sample localization in the center of the cornea at 50 µm and
350 µm depth.
Wollensak et al Cornea •Volume 23, Number 5, July 2004
504 ©2004 Lippincott Williams & Wilkins
pressed as mean ± standard deviation. All statistical analyses
were performed with the Statistical Package for the Social Sci-
ences (SPSS GmbH, Munich, Germany).
RESULTS
ANOVA testing showed statistical significance at the
level of significance 0.001 between the treated and untreated
corneas in their anterior and posterior localization. In brief, the
following observations were made:
1. In the anterior stroma, the collagen fiber diameter of the
crosslinked corneas was significantly increased by 3.96 ±
2.5 nm (12.2%) on the average (Sidak test, P= 0.008) com-
pared with the untreated control fellow eyes (Table 1).
2. In the posterior stroma, the collagen fiber diameter of the
crosslinked corneas was increased by 1.63 ± 1.45 nm
(4.6%) on the average (Sidak test, P= 0.023) compared
with the untreated control eyes (Table 2). Only in the pos-
terior stroma of cases 5 and 6 was the collagen fiber diam-
eter not statistically significantly increased.
3. In the crosslinked eyes, the collagen fiber diameter was also
significantly increased by on the average 3.12 ± 2.1 nm
(9.3%) in the anterior compared with the posterior stroma
within the same cornea (Table 3). The control eyes only
showed a tendency for an increased diameter in the anterior
stroma with no statistically significant difference compared
with the posterior collagen fiber diameter in the same eye.
DISCUSSION
This study has shown a statistically significant increase
in corneal collagen fiber diameter, by an average of 12.2%
(3.96 nm) in the anterior and by 4.6% (1.63 nm) in the posterior
stroma, as a result of riboflavin/UVA-induced collagen
crosslinking. In addition, the collagen fiber diameter was also
significantly increased by an average of 9.3% (3.1 nm) in the
anterior compared with the posterior stroma in the crosslinked
corneas.
Similar to our results, a statistically significant increase
in the collagen fiber diameter of the cornea by 4.5% with age-
related crosslinking has been shown by others.
7,8
In photosen-
sitized reactions as occurs with riboflavin and UVA treatment,
an excited so-called triplet state of the sensitizer is induced by
FIGURE 4. Rectangular section with circular section profiles (B). Oblique sections with ellipsoidal section profiles (C). The mini-
mum profile diameter is identical in both section profiles (A).
FIGURE 3. Measurement of collagen fiber diameter using mor-
phometric computer software. The numbers indicated corre-
spond to the number of measured profiles (n = 160), the
drawn lines to the minimum diameter. TEM, ⳯89,000.
Cornea •Volume 23, Number 5, July 2004 Collagen Fiber Diameter After Collagen Crosslinking
© 2004 Lippincott Williams & Wilkins 505
the absorption of UVA light. So-called reactive oxygen spe-
cies (ROS) or free radicals are generated that can cause, on the
one hand, photooxidative damage of cells and, on the other
hand, physical crosslinking of collagen, thereby increasing the
fiber diameter and the mechanical stiffness of the collagen in-
volved.
9
An increase in collagen fiber diameter caused by
crosslinking induced by aging
8
or diabetes mellitus is a general
phenomenon and has been measured in various other collag-
enous tissues.
10
It has been elegantly demonstrated in vitreous
samples of diabetic patients using scanning electron micros-
copy.
11
The reason for the increased fiber diameter is that the
induced crosslinks push the collagen molecules apart, result-
ing in an increased intermolecular spacing and diameter of the
collagen fibers.
12
Interestingly, also in cataract formation
crosslinking mediated by endogenous riboflavin has been
found to lead to a massive increase in the molecular weight of
crystallin proteins and increased lens hardness.
6,13
The relative increase in the collagen fiber diameter com-
pared with the untreated eyes was more pronounced in the an-
terior than in the posterior stroma. In addition, the relative dif-
ference in collagen fiber diameter between anterior and poste-
rior stroma of the same eye was statistically significant in the
treated eyes. This finding can be explained by the rapid loss of
UVA irradiance across the cornea because of the increase in
UVA absorption by the photosensitizer riboflavin. In an earlier
experiment, we measured a 95% reduction of UVA irradiance
at the endothelial level
2
after riboflavin/UVA treatment, which
could explain the smaller degree of crosslinking in the poste-
rior portion of the cornea. In the untreated eyes, only a nonsig-
nificant tendency for a greater collagen fiber diameter in the
anterior compared with the posterior stroma of the same eye
was found, which has already been described by others.
14
The
anterior localization of the crosslinking effect is a great advan-
tage in the clinical application because the corneal endothe-
TABLE 2. Difference in Collagen Fiber Diameter in Posterior Stroma Between
Crosslinked and Control Fellow Eyes
Rabbit
Treated Eye
(nm)
Control Eye
(nm)
Rel. Difference
(nm)
Significance
(P)
1 30.69 ± 2.88 29.49 ± 2.81 1.2 0.018
2 33.48 ± 2.94 32.07 ± 2.49 1.41 0.003
3 34.27 ± 3.42 32.13 ± 3.41 2.14 0.0001
4 34.40 ± 2.86 29.71 ± 2.63 4.69 0.0001
5 33.51 ± 2.62 33.86 ± 2.17 −0.35 0.922
6 34.72 ± 2.46 33.81 ± 2.37 0.91 0.092
7 31.90 ± 3.31 28.49 ± 3.12 3.41 0.0001
8 32.54 ± 2.45 31.67 ± 2.67 0.87 0.005
9 33.13 ± 2.89 32.34 ± 3.21 0.79 0.006
10 33.76 ± 2.93 32.54 ± 2.87 1.22 0.003
Mean diff.: 1.63 ± 1.45
TABLE 1. Difference in Collagen Fiber Diameter in Anterior Stroma Between
Crosslinked and Control Fellow Eyes
Rabbit
Treated Eye
(nm)
Control Eye
(nm)
Rel. Difference
(nm)
Significance
(P)
1 36.78 ± 2.94 27.65 ± 2.19 9.13 0.0001
2 36.96 ± 2.81 33.40 ± 2.77 3.56 0.0001
3 37.11 ± 3.49 34.26 ± 3.20 2.85 0.005
4 36.76 ± 3.08 32.67 ± 2.60 4.09 0.0001
5 33.15 ± 2.43 31.64 ± 2.82 1.51 0.009
6 40.43 ± 2.52 33.15 ± 2.28 7.28 0.0001
7 33.23 ± 3.24 31.73 ± 3.24 1.50 0.036
8 37.89 ± 2.33 33.67 ± 2.56 4.22 0.0001
9 35.04 ± 2.67 31.78 ± 3.14 3.26 0.0003
10 36.21 ± 2.54 33.97 ± 2.43 2.24 0.005
Mean 3.96 ± 2.47
Wollensak et al Cornea •Volume 23, Number 5, July 2004
506 ©2004 Lippincott Williams & Wilkins
lium is therefore not affected by photooxidative damage and is
spared.
The increase in collagen fiber diameter by 3.96 nm after
riboflavin/UVA-induced corneal crosslinking should not lead
to a loss of corneal transparency because the induced inhomo-
geneity is much lower than the critical threshold value for cor-
neal opacification of 150 nm (one third of the wavelength of
white light).
15–17
Accordingly, loss of transparency has never
been observed so far in our 4-year clinical study of ribo-
flavin/UVA treatment.
1
The influence of fixation on corneal collagen fibril di-
ameter has been examined systematically by others using x-ray
diffraction measurements of unfixed cornea as the gold stan-
dard. They found an increase of the collagen fiber diameter
through crosslinking induced by the fixative glutaraldehyde
and a reduction in fiber diameter by the embedding resin. The
two opposite effects cancel each other out.
18
In our series, all
the specimens underwent the same fixation and processing so
that the relative differences between the specimens cannot
have been influenced by tissue processing anyway. In all but
the untreated control eyes of cases 1–4, a slight postoperative
corneal edema was observed. The collagen fiber diameter,
however, is not affected by corneal hydration as demonstrated
in the control eyes of cases 5–10 where epithelial debridement
with and without dehydrating dextran solution was compared
and not significantly different, which has also been shown by
others similarly.
19,20
In conclusion, riboflavin/UVA-induced collagen
crosslinking causes an increase of the corneal collagen fiber
diameter that is most pronounced in the anterior portion of the
stroma. This feature is the main morphologic alteration under-
lying the increased biomechanical stiffness of the cornea after
collagen crosslinking using riboflavin and UVA.
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TABLE 3. Anterior-posterior Difference of Collagen Fiber
Diameter in the Crosslinked Eyes
Rabbit
Rel. Difference
(nm)
Significance
(P)
1 6.09 0.0001
2 3.48 0.0001
3 2.84 0.0001
4 2.36 0.0001
5−0.36 0.943
6 5.71 0.0001
7 1.33 0.113
8 5.35 0.0001
9 1.91 0.008
10 2.45 0.0001
Mean 3.12 ± 2.07
Cornea •Volume 23, Number 5, July 2004 Collagen Fiber Diameter After Collagen Crosslinking
© 2004 Lippincott Williams & Wilkins 507
