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Original Article

Effects of riboflavin/UVA corneal cross-linking onkeratocytes and collagen fibres in human cornea_ 49..56

Rita Mencucci MD,1 Mirca Marini PhD MD,2 Iacopo Paladini MD,1 Erica Sarchielli PhD,2 Eleonora SgambatiPhD,3 Ugo Menchini MD1 and Gabriella B Vannelli PhD MD2

1Department of Oto-Neuro-Ophthalmological Surgical Sciences, Eye Clinic, 2Department of Anatomy Histology and Forensic Medicine –University of Florence, Policlinic of Careggi, Florence, and 3Department of Health Sciences, University of Molise, Campobasso, Italy

ABSTRACT

Purpose: To evaluate the effects of corneal cross-linking on keratocytes and collagen fibres in humancorneas.

Methods: Fifteen corneal buttons were examined. Tenwere from patients with keratoconus submitted topenetrating keratoplasty and five of them weretreated with cross-linking 6 months before penetrat-ing keratoplasty. Five normal corneal buttons fromhealthy donors were used as controls. All sampleswere prepared for TUNEL assay and Western blotanalysis for the detection of keratocyte apoptosis andimmunohistochemical analysis for the morpholo-gical evaluation of keratocytes and collagen fibrediameter.

Results: Normal corneas exhibited no TUNEL-positivekeratocytes and keratoconic and cross-linkedcorneas showed moderate apoptotic cells mainly inthe anterior part of the stroma. This apoptotic trendwas confirmed by the cleavage of poly (ADP-ribose)polymerase assessed using Western blot. The Ki-67staining showed a significant increase in the kerato-cyte proliferation in cross-linked corneas comparedwith normal and keratoconus. In cross-linkedcorneas CD34-positive keratocytes were regularlydistributed throughout the whole corneal stroma asin the control, and keratoconus was associated withpatchy loss of immunoreactivity. The immunohis-tochemical analysis of collagen type I showed a sig-nificant increase in fibre diameter of cross-linkedcorneas compared with control and keratoconus.

Conclusion: Corneal cross-linking leads to keratocytedamage; after 6 months a repopulation by prolifer-ating cells, a distribution of CD34-positive kerato-cytes as in control and an increase in collagen fibrediameter were observed. These modifications are themorphological correlate of the process leading to anincrease in biomechanical stability.

Key words: collagen fibre, human cornea, keratocyte,riboflavin/UVA corneal cross-linking.

INTRODUCTION

Keratoconus is a non-inflammatory usually bilateralprogressive corneal pathology with frequency of 1 in2.000 in the general population. It is a pathologycharacterized by a progressive thinning and ectasiaof the stroma that results in a cone-shaped corneaand severe visual impairment, making corneal trans-plantation necessary in approximately 20% ofpatients.1 This pathology is characterized by axialthinning, fragmentation of the epithelial basementmembrane, breaks and scarring at the level of Bow-man’s membrane, keratocytes alteration and, ulti-mately, stromal scarring.

Keratocytes, the principal cells of the stroma, playan important role in the preservation of cornealtransparency and mechanical stability. They areresponsible for the synthesis and maintenance of thecollagen component and the extracellular matrix.2

Some studies on keratoconic corneas revealedchanges in keratocyte morphology, decrease in kera-tocyte density and apoptosis increase.3 The causes ofcorneal thinning in keratoconus are still not clear.Takahashi et al. noted that the number of lamellae in

� Correspondence: Dr Rita Mencucci, Department of Oto-Neuro-Ophthalmological Surgical Sciences – Eye Clinic, Viale GB Morgagni 85, 50134

Florence, Italy. Email: [email protected]

Received 27 July 2009; accepted 3 November 2009.

Clinical and Experimental Ophthalmology 2010; 38: 49–56 doi: 10.1111/j.1442-9071.2010.02207.x

© 2010 The AuthorsJournal compilation © 2010 Royal Australian and New Zealand College of Ophthalmologists

the keratoconic cornea is significantly lower than inthe normal cornea but the thickness of lamellae isunaltered.4 Moreover, some studies showed no dif-ference in interfibrillar spacing between keratoconicand control corneas, demonstrating that thinning ofthe corneal stroma in keratoconus is not the result ofcloser packing of the fibrils, but it is due to theprogressive loss of lamellae from the stroma.4–6 It wasalso reported that the orientation of collagen fibrilswithin the lamellae is altered in keratoconus,suggesting that the loss of structural integrity couldplay an important role in the pathogenesis of thedisease.7

Recently, a new method was developed forthe treatment of progressive keratoconus: cornealcollagen cross-linking, that increases the stiffness ofthe cornea using UVA and the photosensitizerriboflavin.8,9 Several studies reported that collagencross-linking can delay or stop keratoconus progres-sion treating some underlying pathophysiologicalmechanisms, improving corneal shape and produc-ing a better quality of vision.10 Extensive experimen-tal studies on rabbit and porcine eyes showed asignificant increase in corneal rigidity of approxi-mately 70% in treated versus untreated corneas afterriboflavin/UVA collagen cross-linking.11–14 It wasdemonstrated that this procedure can strengthen theweak corneal structure by increasing collagen cross-links, which have the role to anchor the collagenfibres preventing the cornea from bulging out andbecoming irregular, as in the keratoconus.14 It hasbeen suggested that a process of keratocytes-inducedapoptosis could be essential for replacement of cellsand new well-structured collagen as confirmed by invivo confocal examinations.15 Wollensak et al. in 2004showed the induction of keratocytes apoptosis in therabbit cornea 24 h after cross-linking.16

Some studies hypothesized that riboflavin/UVAcross-linking increases corneal stroma strength andretards the progression of keratoconus by cross-linking of collagen molecules with mechanisms thatare still largely unknown.17,18 However, recently itwas described in rabbits and spiny dogfish sharksthat the increase in cross-linking of collagen mol-ecules is caused by reactions that require productionof singlet oxygen, whose half-life is extended in thepresence deuterium oxide.19

A previous study reported a statistically signifi-cant increase in corneal collagen fibre diameter afterriboflavin/UVA cross-linking in rabbit corneas, espe-cially in the anterior stroma that underlies theincreased biomechanical stiffness of the cornea afterthe treatment.20 The aggregation of collagen exposedto UVA in the presence of riboflavin was also impu-tated to tyrosine modification.21 A recent study dem-onstrated the presence of high-molecular-weightcollagen type I polymers in the porcine cross-linked

corneas that correlates with an increased collagenfibre diameter after the treatment.22

The clinical importance of a keratocyte loss is notquite clear; at present the effects of cross-linkingtreatment on stromal keratocyte population are notcompletely understood.17,18

The aim of our study was to evaluate the effect ofriboflavin/UVA corneal cross-linking treatment onhuman keratocytes and stromal fibres.

METHODS

Human corneal samples were collected and dividedin three study groups: (i) five cadaver normal corneasfrom Eye-Bank of Lucca as control; (ii) five cornealbuttons with keratoconus without subepithelialscarring removed after penetrating keratoplasty(PKP); and (iii) five corneal buttons with keratoco-nus treated with cross-linking 6 months before,according to Wollensak technique,8 and thenremoved to perform a PKP because of the poorvision. All keratoconus donors gave permission touse their tissue for research. Tissue procurement anduse was carried out in accordance with declaration ofHelsinki and local regulations. In the groups 2 and 3all keratoconus were in Krumeich stage 3.23 Thepatients were informed about the severe stage oftheir keratoconus and gave their permission toperform corneal cross-linking before PKP. In theclinical history of the three groups there was no traceof any other corneal surgical treatment or any ste-roids therapy. Antibiotics drops were used 5 daysafter cross-linking until the new epithelium wasformed completely. The age range of the subjects was30–40 years and was similar in the three groups.

In all groups we analysed central corneal buttonsof similar diameter. Immediately after removal, thecornea buttons were cut and small pieces were pro-cessed for light microscopy and Western blotanalysis.

TUNEL (TdT-mediated dUTP Nick-EndLabeling) assay

Apoptotic cells were identified with Klenow FragelTM DNA Fragmentation Detection Kit (OncogeneResearch Products, San Diego, CA, USA). Briefly,paraffin embedded tissues were deparaffinized,and proteins were digested by applying proteinaseK (20 mg/mL) to specimens for 15 min at roomtemperature. After washing in distilled water,endogenous peroxidase was quenched in 3% hydro-gen peroxide in methanol for 5 min. As described bythe manufacturer, DNA strand breaks were labelledby attaching them to biotin-labelled and unlabelleddeoxynucleotides. Biotinylated nucleotides were

50 Mencucci et al.


detected using a streptavidin-horse radish peroxi-dase conjugate. The immunoreaction product wasvisualized using 3,3′-diaminobenzidine tetrahydro-chloride as chromogen. For counterstaining ofnuclei, 0.3% (w/v) methyl green was used. Theslides were evaluated and photographed using aNikon Microphot-FXA microscope (Nikon, Tokyo,Japan). The number of apoptotic cells was countedin 15 separate fields for each slide.

Western blot analysis

Cornea specimens were incubated with collagenase(0.5 mg/mL) for 20 h at 37°C and centrifuged for15 min at 10 000 g. The pellet was washed inphosphate-buffered saline, homogenized in ice-coldlysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl,1 mM EDTA, 1% Triton, 0.25% SDS) supplementedwith a protease inhibitor cocktail (Sigma-Aldrich,St. Louis, MO, USA) and centrifuged for 15 min at4°C at 10 000 g. The supernatant was collected, andthe protein concentration was measured using a Coo-massie Bio-Rad protein assay kit. Protein aliquots(30 mg) were diluted in 4¥ reducing Laemmli’ssample buffer (250 mM Tris–HCl, pH 6.8, 20% glyc-erol, 8% SDS, 20% 2-mercaptoethanol, 0.008% bro-mophenol blue) and loaded onto 10% SDS-PAGE.After SDS-PAGE, proteins were transferred on poly-vinylidene difluoride membranes. The equal proteinloading in each lane was verified with Ponceau Ssolution staining. Then, membranes were blocked1 h at room temperature in 5% BSA-Tween Tris-buffered saline (TTBS) buffer (0.1% Tween-20,20 mM Tris–HCl, 150 mM NaCl, pH 7.5), washed inTTBS and incubated at 4°C overnight with PARP(poly (ADP-ribose) polymerase) primary policlonalantibody (1 : 1000, H-250, Santa Cruz, CA, USA)diluted in TTBS, followed by peroxidase conjugatedsecondary IgG. The reacted proteins were revealedby the enhanced chemiluminescence system (ECLplus; Amersham Bioscience GE Healthcare, Milan,Italy). Image acquisition and densitometric analysiswere performed with Quantity One software on aChemiDoc XRS instrument (BIO-RAD Laboratories,Hercules, CA, USA).

Immunohistochemical studies

Immunohistochemical studies were performed ondeparaffinized and rehydrated sections, as previ-ously described.24 The slides were exposed to hydro-gen peroxide 3% solution to quench endogenousperoxidase activity. Slides were rinsed in tap water,immersed in ethylenediaminetetraacetic acid (pH 8)and microwaved for 20 min at 350 W to enhanceantigen exposure. The monoclonal anti-Ki-67 (1 : 50

dilution, 7B11; Zymed laboratories San Francisco,CA, USA), anti-CD-34 (1 : 100 dilution, Dakopatts,Carpinteria, CA, USA), anti-collagen type I (1 : 500dilution; COL-1, Sigma-Aldrich), anti-a smoothmuscle actin (1 : 100 diluition; 1A4, Sigma-Aldrich)and polyclonal anti-desmin (1 : 1000; Santa Cruz,CA, USA) antibodies were added to the slides andincubated overnight at 4°C. Sections were rinsedin phosphate-buffered saline, incubated with bio-tinylated secondary antibody and then withstreptavidin-biotin peroxidase complex (Ultravisionlarge volume detection system anti-polyvalent,Laboratory-Vision, Fremont, CA, USA). The reac-tion product was developed with the 3′,3′-diaminobenzidine tetrahydrochloride as chromogen(Sigma-Aldrich). Slides were washed in running tapwater followed by dehydration and coverslipmounting. Controls were performed by processingslides lacking the primary antibody or stained withthe corresponding non-immune serum and counter-stained with haematoxylin. The slides were evalu-ated and photographed using a Nikon Microphot-FXA microscope (Nikon). In the anterior stroma, thecollagen fibres diameter was measured with a com-puterized image analyser programme (Image J1.38¥) on 15 fields for slide. Computer-assistedquantification of CD34 staining has been made usingAdobe Photoshop 6.0 software (Adobe System Incor-porated, San Jose, CA, USA).25

Statistical analysis

Data are expressed as the mean � SD. A one-wayANOVA followed by post-hoc test (Bonferroni correc-tion for multiple comparisons) was performed. Thelevel of P < 0.05 was accepted as statistically signifi-cant. Comparisons of percentages were analysedstatistically after conversion through arcsine transfor-mation from the binomial to the normal distribution.

RESULTS

The anterior stroma was identified considering 250–300 mm from basal membrane of the epithelium.

Keratocytes

The apoptosis was evaluated with TUNEL assay andWestern blotting analysis. The light microscopy inthe TUNEL-stained sections showed apoptotic cellsin the anterior stroma of cross-linked and kerato-conic corneas (Fig. 1a). The number of apoptoticcells both in keratoconus (4.75 � 1.28; n = 4) and incross-linking (4.51 � 1.52; n = 4) was statisticallysignificantly increased in comparison with con-trol (1.25 � 0.91; n = 5; P < 0.01 and P < 0.05,

Effects of cross-linking on human cornea 51


respectively) (Fig. 1b). Western blotting analysiswas performed using PARP antibody. PARP is aprotein involved in the cell protection from apop-totic stimuli, and its cleavage is considered one ofthe classical characteristics of apoptosis. In fact,during the apoptotic process the full-length activeform (116 kDa) is cleaved to an inactive form(85 kDa) that is not able to protect cells from pro-grammed cell death. As shown in Figure 2a, bothisoforms were present and more expressed in cross-linked and keratoconic specimens compared withcontrol. In particular, the densitometric analysis ofthe bands showed a PARP expression increase of,respectively, 338.8% (438.8% � 81.2) in keratoco-nus and 326.4% (426.4% � 83) in cross-linkingcompared with control (100% � 21; P < 0.01) takenas 100 (Fig. 2b).

The keratocyte proliferation was evaluated withKi-67 staining. Almost no Ki-67-positive cells weredetected in the control corneas and only rare positivecells in keratoconus (data not shown). In the corneastreated with cross-linking, there was a statisticallysignificant increase in the number of Ki-67-positivekeratocytes compared with control (2.6 � 0.54 vs.0.2 � 0.11; P < 0.01) and keratoconic corneas(2.6 � 0.54 vs. 0.4 � 0.26; P < 0.01) (Fig. 3).

The stromal keratocytes of the normal cornea werecharacterized by positivity for the CD-34 antigenthroughout the whole thickness of the cornea(Fig. 4a). Some alterations of CD-34 staining wereevident in keratoconus: this pathologic condition is

associated with patchy loss of CD-34 immunoreac-tivity, mainly present in the anterior part of thecornea (Fig. 4a). In cross-linked corneas, the CD-34positivity was regularly distributed throughout thewhole corneal stroma showing distribution similarto the control (Fig. 4a). Accordingly, computer-assisted image analysis indicated that cornea wascharacterized by a significant decrease (P < 0.05) indensity in the keratoconus (79.6% � 18.7) comparedwith normal (taken as 100%) and cross-linkedcorneas (97.9 � 8.3) (Fig. 4b).

The keratocytes were negative for a-smoothmuscle actin and desmin in the three groups (datanot shown) showing that these cells did not exhibita myofibroblast phenotype.

Collagen fibre

Immunohistochemical analysis using anti-collagen(type I) antibody showed a statistically significantdifference (P < 0.05) in collagen fibre diameterbetween the control and keratoconus versus cross-linked corneas (Fig. 5). In particular, an increase incollagen fibre diameter of 22.6% was observed incross-linked corneas compared with control(3.9 � 0.9 vs. 3.2 � 0.8 mm) and an increase of 16.1%compared with keratoconus (3.9 � 0.9 vs. 3.4 �1.1 mm).

The collagen fibre thickness was not statisticallysignificant increased in the posterior stroma in com-parison with normal and keratoconic corneas.

(a)

(b)76 * **

543210T

UN

EL

posi

tive

cells

CTL KER X-LINK

CTL KER X-LINK

Figure 1. (a) Assessment of TUNEL-positive cells in control (CTL), keratoconic (KER) and cross-linked (X-LINK) corneas, as evaluated withimmunocytochemistry. (b) Statistical analysis of data obtained from the different samples. TUNEL-positive cells have been identified bycounting the number of cells showing an intense immunolabelling in at least three slides for each sample and in 15 fields from each slide.Data are expressed as mean � SD of the mean from the separate samples. The images (original magnification ¥20) are representative ofa single experiment. *P < 0.01, **P < 0.05 versus control. Methyl green counterstained.

52 Mencucci et al.


DISCUSSION

Transparency of the cornea is conferred by the highlyorganized extracellular matrix of the corneal stroma.Parallel bundles of collagen fibres of highly uniformdiameter are tightly packed in lamellae lying parallelto the corneal surface. Keratocytes are responsible forthe synthesis and maintenance of the extracellularmatrix components.

This study provided the first evidence that in thehuman cornea, combined riboflavin/UVA cross-linking treatment stimulates both keratocyte apopto-sis and promotes keratocyte repopulation.

One of the first observable changes in the cornealstroma following injury is death of a subpopulationof keratocytes.26 This initial keratocyte cell death is abenign response, thought to have evolved in order toprotect the cornea from further inflammation, andsubsequent loss of transparency. The region and

extent of cell death in the corneal stroma appears tobe dependent on the type of injury induced and thespecies.27 Previous studies on rabbit corneas11,16 havedemonstrated that riboflavin/UVA cross-linkingleads to a dose-dependent damage with keratocyteapoptosis. Wollensak and Redl formed the hypoth-esis that the UVA-induced cellular damage is causedby the production of the so-called reactive oxygenspecies that are bio-toxic but also induce the cross-linking bridges between the collagen proteins.22 Thekeratocyte apoptosis16 was massive when analysed24 h after the treatment. These data were confirmedin humans by in vivo confocal microscopy.15,28

Our results in human specimens demonstratedthat cross-linking treatment is able to stimulate dis-tinct biological responses. In particular, cross-linking treatment induced both apoptosis as wellas cell proliferation. The analysis of the corneas6 months after the treatment showed an increase inapoptotic cells in comparison with controls and thisincrease was similar to the apoptotic shift docu-mented in keratoconic corneas. Furthermore, afterthe riboflavin/UVA cross-linking treatment therewas a small but significant increase in cell prolifera-tion as detected by Ki-67 staining.

The number of proliferating keratocytes in thedeveloping corneal stroma decreased dramaticallyfrom 20% of the total to nearly zero. At the time ofeyelid opening, keratocytes have withdrawn fromthe cell cycle, remaining in G0 rather than undergo-ing complete terminal differentiation.29 Upon injuryto the cornea, keratocytes can transform into diver-gent phenotypes, which are dependent on specificenvironmental signals. Recent research has identi-fied a side population of cells from the human

(a)

PARP

full lenght – 116 kDa

cleaved – 85 kDa

(b)

600*

*

PA

RP

% o

f inc

reas

e

500

400

300

200

100

0

CTL KER X-LINK

CTL KER X-LINK

Figure 2. (a) Western blot detection of poly (ADP-ribose) poly-merase (PARP) in control (CTL), keratoconic (KER) and cross-linked(X-LINK) corneas. Thirty micrograms of proteins for each samplewas separated by 10% sodium dodecyl sulphate polyacrylamidegel electrophoresis (SDS–PAGE), transferred onto polyvinylidenedifluoride membranes and probed for PARP expression withrabbit policlonal anti-PARP human antibody. A single band ofabout 116 kDa is present in all control samples in agreement withthe molecular weight for full-length form of PARP. Western blotdetection of PARP in keratoconus (KER, lane 2) and cross-linking(X-LINK, lane 3) corneal lysates reveals an additional band ofabout 85 kDa, which is in good agreement with the molecularweight for cleaved form of PARP. (b) Percentage of increase inPARP expression over the control value. Quantification of bandswas made directly on the films by image scanning analysis usingPhotoshop software. Data have been reported as mean � SD ofpercentage increase over control taken as 100% (*P < 0.01).

*

Ki-6

7 po

sitiv

e ce

lls

3.2

2.4

1.6

0.8

0CTL KER X-LINK

Figure 3. Statistical analysis of data obtained from differentsample in control (CTL), keratoconic (KER) and cross-linked(X-LINK) corneas. Ki-67-positive cells were identified by countingthe number of cells showing an intense immunolabelling in atleast 15 fields from at least three different slides for each experi-mental point. Data are expressed as mean � SD of the meanfrom the separate samples (*P < 0.01 vs. control).



corneal stroma, which exhibits many aspects of stemcells, including clonal growth in vitro, extendedlifespan and the ability to differentiate into severaldifferent cells types, including keratocytes of thecornea.30 In this study we also showed that aftercross-linking the keratocytes were positively stainedfor CD34, marker for keratocyte phenotype, butnegatively stained for a-smooth muscle actin andfor desmin, markers for myofibroblasts.31,32 Thus,

we suggested that cross-linking treatment is notsufficient to induce keratocyte differentiation tomyofibroblasts.

We also hypothesized that in the human corneathe apoptotic keratocytes may be replaced by precur-sor cells that may be activated under particularstimuli and that can readily respond to reform thenative corneal stroma and restore its transparentproperties.

*

(a)

(b)120

90C

D34

OD

(% o

ver

cont

rol)

60

30

0CTL KER X-LINK

CTL KER X-LINK

Figure 4. (a) CD34 expression in control (CTL), keratoconic (KER) and cross-linked (X-LINK) corneas. Note the high constitutive expres-sion of this protein by keratocytes. In control (CTL) and in cross-linked (X-LINK) corneas CD34 expression was detected in keratocytes ofthe entire corneal stroma and CD34 staining was localized exclusively in the cytoplasmic processes of interlamellar keratocytes. Inkeratoconus (KER) the stained keratocytes are not regularly arranged. Moreover, CD34 expression is lost in small areas most frequentlyfound in the anterior part of the cornea. Haematoxylin counterstained (original magnification ¥20). (b) Computer-assisted quantificationof CD34 staining using Adobe Photoshop 6.0 software in three slides for each sample and 15 fields for each slide. Data are expressed aspercent increase � SD over the control value taken as 100% (*P < 0.05 keratoconus vs. control and cross-linking).

**(b)

5

4

3

μm 2

1

0CTL KER

FIBRE SIZE

X-LINK

(a)

CTL KER X-LINK

Figure 5. (a) Collagen type Istaining of control (CTL), kera-toconic (KER) and cross-linked(X-LINK) corneas (original mag-nification ¥40). (b) Measurementof collagen fibre diameter usingImage J programme in three slidesfor each sample and 15 fieldsfor each slide. Diameters areexpressed in mm � SD. Note thestatistically significant increase incollagen fibre diameter observedin cross-linked corneas comparedwith control and keratoconus(**P < 0.05).

54 Mencucci et al.


Regarding the collagen component, the cause ofcorneal thinning in keratoconus are not still clear,but many authors observed that the thickness oflamellae was unaltered.1,3,4,7 On the other hand, wehave to take into account that the interpretation ofcollagen component diameter may be affected by thehydratation state of the keratoconus buttons andnormal cornea specimens, even if our evaluating pro-cedure was exactly the same for the three groups.

In our study we noticed in treated corneas a sta-tistically significant increase in corneal collagen fibrediameter in the anterior stroma that is due to thephysical cross-linking and underlies the increasedbiomechanical stiffness of the cornea after thetreatment. In particular there is an increase in cross-linked collagen fibre diameter of 22.6% comparedwith control group and an increased of 16.1% com-pared with keratoconus.

An increase in collagen fibre diameter caused bycross-linking induced by aging or diabetes mellitusis a general phenomenon and has been measuredin various other collagenous tissues. According toWollensak et al.,20 the induced cross-links probablypush the collagen molecules apart, resulting in anincreased intermolecular spacing and diameter of thecollagen fibres. To be noticed that the collagen fibrediameter was not statistically significant increased inthe posterior stroma.

Our data confirm in human corneas the observa-tion previously published in rabbit models.16

The presence of an intense polymer band of cross-linked type I protein identified by Wollensak andRedl22 might have a good correlation with this mor-phologic finding of an increased collagen componentdiameter after cross-linking treatment.

In conclusion no satisfactory animal model existsfor the keratoconus, and ex vivo studies are largelylimited to cornea buttons removed from PKP in sub-jects with advanced disease. Our preliminary studyis the first morphological study on human corneasaffected from keratoconus treated with cross-linking.

ACKNOWLEDGEMENT

Authors thank Professor Gianni Virgili, Departmentof Oto-Neuro-Ophthalmological Surgical Sciences –Eye Clinic, University of Florence, Florence, Italy forthe statistic assistance.

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