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Enzymatic evidence of the depth dependence of stiffening on riboflavin/UVA treated corneas [in German]

Authors:

Abstract

It has been shown that the treatment of keratoconus with riboflavin/ultraviolet A (UVA) causes significant stiffening of the cornea due to cross-linking. The aim of this study was to evaluate how deep the mechanical stabilization after collagen cross-linking could be shown biochemically. Ten out of 20 enucleated porcine eyes were treated with riboflavin as a photosensitizer and UVA (370 nm, 3 mW/cm2, 30 min). The other 10 eyes served as controls. With a Microkeratom device, two flaps with a thickness of 200 microm and a diameter of 8 mm were cut off from each eye and put in a collagenase solution (NaCl plus collagenase A, 1:1). The surfaces of the flaps were measured digitally every day to characterize the dissolving behavior. The resistance (regarding corneal collagen against enzymatic digestion) of the treated superficial flaps was considerably higher (p=0.001) compared to those that were cut secondarily and to the control flaps. But even the flaps from deeper layers showed a significant increase in resistance (p=0.02) compared with the untreated flaps. The half-life of the surfaces of the treated superficial flaps was 220 h; of those cut secondarily, it was 80 h. Both untreated flaps had a half-life of 50 h. The biochemical study showed that the treatment of the cornea with riboflavin/UVA leads to significant collagen cross-linking not only in the anterior slice of 200 microm but also in the following 200 microm. This locally limited cross-linking effect may be explained by the absorption behavior for UVA of the riboflavin-treated cornea; 65% of UVA irradiation is absorbed in the first 200 microm and only 25-30% in the next 200 microm. Therefore, deeper-lying structures and especially the endothelium are not affected.
Depth dependence of stiffening on riboflavin/UVA treated corneas
Schilde, T.; Spoerl, E.; Kohlhaas, M.; Pillunat, L. E.
Department of Ophthalmology, University of Dresden
Fetscherstr. 74, Germany-01307 Dresden
Purpose:
Treatment of keratoconus with riboflavin/UVA is based on a significant stiffening of the cornea. This second study
should show until which depth the mechanical stabilization after collagen-crosslinking would be provable
biochemically.
Method:
10 out of 20 enucleated porcine eyes were treated with riboflavin as photosensitizer and UVA (370nm, 3mW/cm²,
30min), the other 10 eyes served as controls. With a microkeratom two flaps of 200µm thickness were cut from
each eye and afterwards laid in collagenase solution (NaCl + collagenase; 1:1). The surfaces of the flaps were
measured digitally and compared daily to characterize the solvent behaviour.
Results:
The resistance (regarding corneal collagen against enzymatic digestion) of the treated flaps, that were cut firstly
was considerably (p=0,001) higher compared to those which were cut secondly and to the control flaps. But even
the flaps that were cut secondly showed a significant increase of resistance (p=0,02) in comparison with the
untreated flaps. The half-life of the surfaces of the treated flaps which were cut firstly was 220 hours, of those cut
secondly 80 hours. The both untreated flaps had a half-life of 50 hours.
Conclusions:
The biochemical study showed, that treatment of the cornea with riboflavin/UVA leads to a significant collagen-
crosslinking not only in the anterior slice of 200µm but also in the following 200µm. This locally limited
crosslinking-effect may be explained by the absorption behaviour for UVA of the riboflavin-treated cornea. 65% of
UVA-irradiation is absorbed in the first 200µm and only 25-30% in the next 200µm. Therefore, deeper laying
structures and especially the endothelium are not influenced.
Department of Ophthalmology
University of Dresden
Fetscherstr. 74
01309 Dresden
Germany
Contact:
Thomas Schilde
Thomas.Schilde@web.de
Commercial Relationship:
T. Schilde, None;
E. Spoerl, None;
M. Kohlhaas, None;
L.E. Pillunat, None
Depth Dependence of Stiffening
on Riboflavin/UVA Treated Corneas
T. Schilde; E. Spoerl; M. Kohlhaas; L.E. Pillunat
Department of Ophthalmology, University of Dresden, Dresden, Germany
Purpose: It was shown that treatment of keratoconus with riboflavin/UVA is based on a significant stiffening of the cornea (ARVO 2004
abstr.nr.2888). Aim of this study was to evaluate how deep the mechanical stabilization after collagen-crosslinking could be shown
biochemically.
Methods: 10 out of 20 enucleated porcine eyes were treated with riboflavin as photosensitizer and UVA (370nm; 3mW/cm²; 30 min), the
other 10 eyes served as controls. With a microkeratom two flaps of 200µm thickness were cut from each eye and afterwards were put in
collagenase solution (NaCl + collagenase A). The surfaces of the flaps were measured digitally and compared daily to characterize the
solvent behavior.
Results: The resistance (regarding corneal collagen against enzymatic digestion) of the treated superficial flaps was considerably higher
(P=0.001) compared to those which were cut secondly and to the control flaps. But even the flaps from deeper layers showed a significant
increase of resistance (P=0.02) in comparison with the untreated flaps. The half-life of the surfaces of the treated superficial flaps was 220
hours, of those cut secondly 80 hours. The both untreated flaps had a half-life of 50 hours.
Fig.1: experimental equipment Fig.2: riboflavin/UVA application Fig.3: cutting flaps with a Fig.4: dissected flaps (200µm) Fig.5: half-life of the
microkeratom surfaces
Fig.6: solvent behaviour: Fig.7: solvent behaviour: Fig.8: digitally measurement Fig.9: digitally measurement Fig.10: results
treated superficial flaps treated superficial flaps calibrate the computer- of the solved surfaces
after 3 hours after 171 hours program
Conclusion: This biochemical study showed that treatment of cornea with riboflavin/UVA leads to a significant collagen-crosslinking not
only in the anterior slice of 200µm but also in the following 200µm. This locally limited crosslinking-effect may be explained by the
absorption behaviour for UVA of the riboflavin-treated cornea. 65% of UVA- irradiation is absorbed in the first 200µm and only 25 30% in
the next 200µm. Therefore, deeper laying structures and especially the endothelium seem to be not influenced.
0
10
20
30
40
50
60
70
80
1 21 28 46 68 73 127 146 194 240 315
time in h
surface in mm²
koflap1
koflap2
uvflap1
uvflap2
P=0.3P=0.01
deeper
layers
P=0.025080flap from
flap
P=0.00150220superficial
untreatedtreated
Purpose: Treatment of keratoconus with riboflavin/UVA is based on a significant stiffening of the cornea. Aim of the study was to examine to which depth of the
cornea the stiffening effect is present biomechanically.
Methods: 20 enucleated porcine eyes were treated with riboflavin as photosensitizer and UVA (370 nm, 3 mW/cm², 30 min) and 20 eyes served as controls. Two
consecutive flaps of 200 μm thickness were cut with a microkeratom from each. Each flap was divided into stripes of 5 mm width and 7 mm length. Stress-strain curves
were measured with a material tester to characterize the stiffening effect. For statictics ANOVA was used. Additionally, the same procedure was realized at a pair of
human corneas.
Fig.1: UVA-radiation Fig. 2: rotormicrokeratom Fig. 3: cornea stripe Fig. 4: stress-strain measuring Fig. 5: biomechanical surveyors table
Results: Stress at 5 % strain was 261±133x10³ N/m² for the superficial treated flaps in comparison to 104±52x10³ N/m² corresponding control flaps. This difference
was statistically significant (P=0.0001). The deep treated flaps showed a stress of 105±56x10³ N/m² compared to the corresponding control flaps with 103±62x1
N/m². This difference was not statistically significant (P=0.94). The stiffness of the superficial flap was statistically significant increased in comparison to the deeper
flaps (P = 0.001). The stiffness between the superficial and deep control flaps was not statistically significant different (P=0.9).
The same stiffening effect was also observed in the human eye. Contrary to the porcine cornea the superficial flap of the human cornea is stiffer than the second flap.
Fig. 6: Fig. 7
Conclusions: Treatment of the cornea with riboflavin/UVA leads to a significant stiffening only in the anterior 200 μm of the cornea. This locally limited stiffening
effect might be explained by the absorption behaviour of the riboflavin-treated cornea for UVA. 65 - 70 % of UVA-irradiation is absorbed in the first 200 μm and only 20
% in the next 200 μm.
Evaluation of the stiffening effect of riboflavin/UVA treatment in
different depth of the cornea
G.Unger, T. Schilde, E. Spoerl, M. Kohlhaas, L.E. Pillunat
Department of Ophthalmology, University of Dresden, Dresden, Germany
Department of Ophthalmology
University of Dresden
Fetscherstr. 74
01309 Dresden
Germany
Contact:
Gabriele Unger
gabriele.unger@uniklinikum-
dresden.de
Commercial Relationship:
G. Unger, none
T. Schilde, none
E. Spoerl, none
M. Kohlhaas, none
L.E. Pillunat, none
0
200
400
600
800
1000
1200
0 2 4 6 8 10
Strain in %
first treated flaps
first untreated flaps
second untreated flaps
second treated flaps
porcine cornea
0
200
400
600
800
1000
1200
0 2 4 6 8 10
Strain in %
Stress in 10³ N/m²
first untreated flap
first treated flap
second untreated flap
second treated flap
human cornea (65 years)
... This melting processes can be halted by two mechanisms: by decreasing the production of degrading enzymes or by increasing the strength of the cornea to the enzymatic digestion. The later effect is achieved by combined Riboflavin/UVA CXL of the corneal stroma [10][11][12][13]. Thus, the corneal stroma becomes more resistant to digestive enzymes from microbial and inflammatory origin [14]. ...
... Thus, the corneal stroma becomes more resistant to digestive enzymes from microbial and inflammatory origin [14]. The structural and biomechanical effects of Riboflavin/UVA CXL in the corneal stroma have been evaluated both clinically and experimentally [10][11][12][13]15,16]. For in vitro evaluations light, electron and confocal microscopy, x-ray scattering and second harmonic generation imaging, 2-dimensional fast Fouriertransform analysis stress-strain biomaterial tester and enzymatic digestion have been used [17]. ...
Article
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Objective: Crosslinking (CXL) of the cornea by using riboflavin (RBF)/ultraviolet-A (UVA) has been developed for stiffening the collagenous matrix of the cornea. Collagenases and other metalloproteinases are known to play an important role in the pathogenesis of corneal ulceration. Our aim in this study was to show and compare the morphological and histopathological changes of the CXL human corneas against enzymatic digestion by collagenase, trypsin and pepsin solutions.Materials and Methods: Eighteen corneas from nine fresh frozen cadavers were included in this study. The crosslinked group (n=12) was treated with the combined riboflavin (RBF) and ultraviolet-A (UVA) and the control group (n=6) was only treated with RBF. After treatment corneas were exposed to collagenase, trypsin and pepsin enzymes. For each enzyme group one cornea was evaluated morphologically and the other one was evaluated microscopically. Results: The collagenase treated control group, digestion of the cornea was noted in the first days and completed on the 5th day. The control cornea treated with pepsin was fully digested after 14 days. In the trypsin treated CXL cornea complete digestion was noted after 18 days. In both groups, enzyme activity was observed to be parallel to each other morphologically, but CXL cornea was evaluated to be more resistant to enzymatic digestion. Conclusion: In conclusion, CXL increases resistance of the cornea against enzymatic digestion. In addition to the biomechanical support, this study was also showing the histopathological changes of CXL procedure and the results of enzymatic digestion, supporting new treatment options in the corneal diseases.
... Ultraviolet A light was utilized to ina-ctivate microorganisms in drinking water and to disinfect blood products [6]. Riboflavin prompts a change in properties of the collagen and affects the corneal stroma, which stabilizes it and increases its stiffness and resistance to enzymatic bacterial degradation avoiding the progression of corneal melting [7]. In practice, the standard riboflavin/ultraviolet A crosslink protocol seems to produce only short-term improvement in pain, irritation, and discomfort and sometimes reducing the corneal thickness [8]. ...
... 12 It induces a change in the property of the collagen and has a stiffening effect on the corneal stroma which stabilizes it and increases its resistance to enzymatic degradation avoiding the progression of corneal melting. 16,17 This is also utilized to prevent melting of the corneal graft used as a carrier in Boston keratoprosthesis. 18 The photoactivation of riboflavin damages the RNA and DNA of microorganisms by oxidation processes causing lesions in their chromosomal strands. ...
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Collagen crosslinking (CXL), used as a treatment modality in infectious keratitis, can be further studied for the prevention of recurrent infection in cases undergoing therapeutic penetrating keratoplasty (TPK). To study the role of collagen crosslinking of the donor corneal tissue and the graft host junction during TPK in preventing recurrence of infection. This was a prospective, clinical, interventional pilot case study done from May 2017 to July 2018. Fifteen cases and 15 controls who had infectious keratitis and needed TPK were included. Pediatric patients, one eyed patients, perforated ulcers and those with any other significant co-morbidity were excluded. In patients who were enrolled as cases, the donor corneal graft and the graft host junction were subjected to CXL using modified Dresden protocol just after the TPK. TPK was done as per standard protocol. The patients were followed up for at least four months duration and the recurrences in both the groups were recorded. The Odds Ratio of the cases not developing a recurrent infection vis-a-vis the controls has been calculated along with the Confidence Interval using the SPSS Statistics software. Recurrence of infection was found in one case and in four controls within one month of TPK. CXL done on donor corneal tissue and the graft host junction intra operatively, has a role in reducing recurrence of infection after TPK.
... 8,9 A decade ago, Wollensak and associated have introduced a novel minimally invasive technique using riboflavin\UVA corneal collagen cross-linking (CXL), which has been shown to enhance corneal biomechanical stiffening, stabilize and even arrest progression of keratoconus in adult eyes, with no permanent deleterious effects, thus rendering corneal transplantation unnecessary in some patients. [10][11][12][13][14][15][16][17][18] It seems that keratoconus has a tendency to progress faster in children than in adults, and therefore the consequence of unsatisfactory visual acuity or contact lens intolerance may be encountered sooner. Vinciguerra and associates have recently reported their results of a 2 years follow-up after corneal CXL in patients younger than 18 years with documented progressive keratoconus. ...
Article
Purpose To report refractive, topographic and safety outcomes of corneal cross-linking (CXL) in patients younger than 18 years of age with progressive keratoconus. Materials and methods In this retrospective study, we enrolled 31 eyes of 21 children aged 11 to 17 years that underwent corneal riboflavin-ultraviolet A induced CXL due to progressive keratoconus at three different ophthalmology departments in Israel. They were followed for 3 to 48 months (average 23 ± 13.6 months). Evaluated parameters were uncorrected visual acuity (UCVA), best spectacle corrected visual acuity (BSCVA), manifest refraction, pachymetry, slit- lamp examination and corneal topography at baseline and at 1,3,6,12,24 and 48 months. Results We found a nonsignificant improvement in UCVA and BSCVA with a small reduction of manifest cylinder and no significant change in spherical equivalent or K-values. Following CXL, stability of UCVA and BSCVA at the last follow-up examination was found in 71 and 77% of treated eyes, respectively. No permanent adverse events have been recorded throughout the study period. Conclusion In our series, CXL was a safe procedure in the pediatric population. Stabilization of progressive keratoconus was achieved in visual acuity, refractive and topography parameters with no improvement in corneal indices in contrary to adult CXL treatment. How to cite this article Bakshi E, Barequet IS, Aizenman I, Levinger S, Avni I, Zadok D. Corneal Corss-linking in Patients Younger than 18 Years: Long-term Follow-up in Three Israeli Medical Centers. Int J Kerat Ect Cor Dis 2014;3(2):84-87.
... 4 It induces a change in the property of collagen and has a stiffening effect on corneal stroma which stabilizes it and increases its resistance to enzymatic degradation avoiding progression of corneal melting. 15,16 This is also utilized to prevent melting of corneal graft used as a carrier in Boston Keratoprosthesis. 9 The photoactivation of riboflavin damages the RNA and DNA of micro-organisms by oxidation processes causing lesions in their chromosomal strands. ...
Thesis
Ziel der Studie: Evaluation der Effektivität und Sicherheit des Kollagen-Crosslinkings der Hornhaut mit Riboflavin und UV-A (CXL) bei progressivem Keratokonus über einen Nachbeobachtungszeitraum von bis zu 10 Jahren. Design: Retrospektive klinische Längsschnittstudie Methoden: 131 Augen von 131 Patienten (männlich:weiblich = 95:36) erhielten an der Universitäts-Augenklinik Würzburg zwischen 2006 und 2016 ein Kollagen-Crosslinking (CXL) nach dem Dresdner Standardprotokoll, bestehend aus einer Abrasio, Applikation von iso-osmolaren Riboflavin/Dextran-Augentropfen für 30 min und anschließender UV-A-Bestrahlung mit 3 mW/cm2 für 30 min. Die retrospektive Nachbeobachtung betrug 1 (n=103 Augen) bis maximal 10 Jahre (n=44 Augen). Einschlusskriterien waren eine Zunahme des maximalen Hornhautkrümmungsradius (Kmax, gemessen mittels Pentacam HR) >1 dpt und eine Hornhautdicke >400 µm nach Abrasio. Für parametrische bzw. nichtparametrische Daten wurde der T-Test bzw. der Wilcoxon-Vorzeichen-Rangsummentest durchgeführt. Ergebnisse: 1 bis 3 Jahre präoperativ nahm der Median von Kmax und K2 signifikant um 1,5 dpt (p=0,001) und 1,1 dpt (p<0,001) bis zur Behandlung zu. Die apikale Hornhautdicke nahm nach 1-3 Jahren präoperativ um 12 µm ab (p=0,003). Postoperativ stieg der Median von K2 zunächst bis nach 1 Jahr um 0,1 dpt, nahm dann aber über den weiteren Nachbeobachtungszeitraum kontinuierlich ab, nach 10 Jahren lag er um 0,85 dpt (p=0,021) unter dem Ausgangswert. Die mittlere apikale Hornhautdicke nahm nach 3, 7 bzw. 10 Jahren um 11 µm (p<0,001), 9 µm (p=0,014) und 3 µm (p=0,358) ab. Der mediane Kmax zeigte Schwankungen ohne signifikante Veränderung. Der mittlere bestkorrigierte Visus (logMAR) nahm nach 5 Jahren signifikant um 0,13 und nach 10 Jahren um 0,08 ab (p=0,013 und p=0,010). Der Anteil der Non-Responder, definiert durch einen postoperativen Anstieg von Kmax>2 dpt, nahm von 16% nach 5 auf 33% nach 10 Jahren zu. Risikofaktoren waren ein junges Alter, hoher Astigmatismus, eine dünne Hornhaut und atopische Dermatitis. 4 Augen erhielten im Verlauf komplikationslos eine Revernetzung, woraufhin sich keine weitere Krankheitsprogression zeigte. Fazit: Die CXL-Behandlung kann die Progression des Keratokonus verlangsamen oder stoppen. Allerdings war ab 5 Jahren nach dem Eingriff eine Abnahme des Anteils der Responder zu beobachten. Regelmäßige Nachkontrollen sollten daher besonders auch im Langzeitverlauf durchgeführt werden, um eine erneute Progression frühzeitig erkennen und behandeln zu können.
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Collagen-crosslinking using combined riboflavin/ UVA treatment has been developed by us as a new treatment for keratoconus by stiffening the collagenous matrix. Recently, we have started to use the same method for the treatment of corneal ulcers. The aim of the present study was to evaluate the influence of the crosslinking treatment on the resistance of the cornea against enzymatic degradation. 60 enucleated porcine eyes were treated with the photosensitizer riboflavin and UVA-irradiation (370 nm; irradiance of 1, 2 or 3 mW/cm2) for 30 minutes and compared with 20 untreated control eyes. After crosslinking treatment, the corneal buttons were trephined and exposed to pepsin, trypsin and collagenase solutions. The extent of the corneal digestion was monitored daily. Selected cases were examined by light microscopy. The corneal buttons crosslinked with riboflavin/ UVA at 3 mW/cm2 were dissolved only by day 13 following pepsin digestion and by day 14 following collagenase treatment versus 6 days in the untreated control corneas. Digestion by trypsin was observed on day 5 in buttons crosslinked at 3 mW/cm2 compared to day 2 in the control corneas. Microscopically, a prolonged preservation especially of the anterior portion of the crosslinked corneas could be demonstrated. Photochemical crosslinking of the cornea using riboflavin and UVA results in a markedly increased resistance versus collagen digesting enzymes. The findings support the use of the new method in the treatment of corneal ulcers.
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