Depth dependence of stiffening on riboflain/UVA treated corneas

<strong>Depth</strong> <strong>dependence</strong> <strong>of</strong> <strong>stiffening</strong> on rib<strong>of</strong>lavin/UVA treated corneasSchilde, T.; Spoerl, E.; Kohlhaas, M.; Pillunat, L. E.Department <strong>of</strong> Ophthalmology, University <strong>of</strong> DresdenFetscherstr. 74, Germany-01307 DresdenPurpose:Treatment <strong>of</strong> keratoconus with rib<strong>of</strong>lavin/UVA is based on a significant <strong>stiffening</strong> <strong>of</strong> the cornea. This second studyshould show until which depth the mechanical stabilization after collagen-crosslinking would be provablebiochemically.Method:10 out <strong>of</strong> 20 enucleated porcine eyes were treated with rib<strong>of</strong>lavin as photosensitizer and UVA (370nm, 3mW/cm²,30min), the other 10 eyes served as controls. With a microkeratom two flaps <strong>of</strong> 200µm thickness were cut fromeach eye and afterwards laid in collagenase solution(NaCl + collagenase; 1:1). The surfaces <strong>of</strong> the flaps weremeasured digitally and compared daily to characterize the solvent behaviour.Results:The resistance (regarding corneal collagen against enzymatic digestion) <strong>of</strong> the treated flaps, that were cut firstlywas considerably (p=0,001) higher compared to those which were cut secondly and to the control flaps. But eventhe flaps that were cut secondly showed a significant increase <strong>of</strong> resistance (p=0,02) in comparison with theuntreated flaps. The half-life <strong>of</strong> the surfaces <strong>of</strong> the treated flaps which were cut firstly was 220 hours, <strong>of</strong> those cutsecondly 80 hours. The both untreated flaps had a half-life <strong>of</strong> 50 hours.Conclusions:The biochemical study showed, that treatment <strong>of</strong> the cornea with rib<strong>of</strong>lavin/UVA leads to a significant collagencrosslinkingnot only in the anterior slice <strong>of</strong> 200µm but also in the following 200µm. This locally limitedcrosslinking-effect may be explained by the absorption behaviour for UVA <strong>of</strong> the rib<strong>of</strong>lavin-treated cornea. 65% <strong>of</strong>UVA-irradiation is absorbed in the first 200µm and only 25-30% in the next 200µm. Therefore, deeper layingstructures and especially the endothelium are not influenced.

<strong>Depth</strong> Dependence <strong>of</strong> Stiffeningon Rib<strong>of</strong>lavin/UVA Treated CorneasT. Schilde; E. Spoerl; M. Kohlhaas; L.E. PillunatDepartment <strong>of</strong> Ophthalmology, University <strong>of</strong> Dresden, Dresden, GermanyPurpose: It was shown that treatment <strong>of</strong> keratoconus with rib<strong>of</strong>lavin/UVA is based on a significant <strong>stiffening</strong> <strong>of</strong> the cornea (ARVO 2004abstr.nr.2888). Aim <strong>of</strong> this study was to evaluate how deep the mechanical stabilization after collagen-crosslinking could be shownbiochemically.Methods: 10 out <strong>of</strong> 20 enucleated porcine eyes were treated with rib<strong>of</strong>lavin as photosensitizer and UVA (370nm; 3mW/cm²; 30 min), theother 10 eyes served as controls. With a microkeratom two flaps <strong>of</strong> 200µm thickness were cut from each eye and afterwards were put incollagenase solution (NaCl + collagenase A). The surfaces <strong>of</strong> the flaps were measured digitally and compared daily to characterize thesolvent behavior.Department <strong>of</strong> OphthalmologyUniversity <strong>of</strong> DresdenResults: The resistance (regarding corneal collagen against enzymatic digestion) <strong>of</strong> 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 significantincrease <strong>of</strong> resistance (P=0.02) in comparison with the untreated flaps. The half-life <strong>of</strong> the surfaces <strong>of</strong> the treated superficial flaps was 220hours, <strong>of</strong> those cut secondly 80 hours. The both untreated flaps had a half-life <strong>of</strong> 50 hours.Fetscherstr. 7401309 DresdentreateduntreatedGermanysuperficialflap22050P=0.001flap from8050P=0.02Contact:Thomas SchildeThomas.Schilde@web.dedeeperlayersP=0.01P=0.3Fig.1: experimental equipment Fig.2: rib<strong>of</strong>lavin/UVA application Fig.3: cutting flaps with a Fig.4: dissected flaps (200µm) Fig.5: half-life <strong>of</strong> themicrokeratomsurfaces807060surface in mm²504030k<strong>of</strong>lap1k<strong>of</strong>lap2uvflap1uvflap2201001 21 28 46 68 73 127 146 194 240 315time in hFig.6: solvent behaviour: Fig.7: solvent behaviour: Fig.8: digitally measurement Fig.9: digitally measurement Fig.10: resultstreated superficial flaps treated superficial flaps calibrate the computer- <strong>of</strong> the solved surfacesafter 3 hours after 171 hours programCommercial Relationship:T. Schilde, None;E. Spoerl, None;M. Kohlhaas, None;L.E. Pillunat, NoneConclusion: This biochemical study showed that treatment <strong>of</strong> cornea with rib<strong>of</strong>lavin/UVA leads to a significant collagen-crosslinking notonly in the anterior slice <strong>of</strong> 200µm but also in the following 200µm. This locally limited crosslinking-effect may be explained by theabsorption behaviour for UVA <strong>of</strong> the rib<strong>of</strong>lavin-treated cornea. 65% <strong>of</strong> UVA- irradiation is absorbed in the first 200µm and only 25 – 30% inthe next 200µm. Therefore, deeper laying structures and especially the endothelium seem to be not influenced.

Evaluation <strong>of</strong> the <strong>stiffening</strong> effect <strong>of</strong> rib<strong>of</strong>lavin/UVA treatment indifferent depth <strong>of</strong> the corneaG.Unger, T. Schilde, E. Spoerl, M. Kohlhaas, L.E. PillunatDepartment <strong>of</strong> Ophthalmology, University <strong>of</strong> Dresden, Dresden, GermanyPurpose: Treatment <strong>of</strong> keratoconus with rib<strong>of</strong>lavin/UVA is based on a significant <strong>stiffening</strong> <strong>of</strong> the cornea. Aim <strong>of</strong> the study was to examine to which depth <strong>of</strong> thecornea the <strong>stiffening</strong> effect is present biomechanically.Methods: 20 enucleated porcine eyes were treated with rib<strong>of</strong>lavin as photosensitizer and UVA (370 nm, 3 mW/cm², 30 min) and 20 eyes served as controls. Twoconsecutive flaps <strong>of</strong> 200 μm thickness were cut with a microkeratom from each. Each flap was divided into stripes <strong>of</strong> 5 mm width and 7 mm length. Stress-strain curveswere measured with a material tester to characterize the <strong>stiffening</strong> effect. For statictics ANOVA was used. Additionally, the same procedure was realized at a pair <strong>of</strong>human corneas.Department <strong>of</strong> OphthalmologyUniversity <strong>of</strong> DresdenFetscherstr. 7401309 DresdenGermanyContact:Gabriele Ungergabriele.unger@uniklinikumdresden.deFig.1: UVA-radiation Fig. 2: rotormicrokeratom Fig. 3: cornea stripe Fig. 4: stress-strain measuring Fig. 5: biomechanical surveyors tableResults: 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 differencewas statistically significant (P=0.0001). The deep treated flaps showed a stress <strong>of</strong> 105±56x10³ N/m² compared to the corresponding control flaps with 103±62x10³N/m². This difference was not statistically significant (P=0.94). The stiffness <strong>of</strong> the superficial flap was statistically significant increased in comparison to the deeperflaps (P = 0.001). The stiffness between the superficial and deep control flaps was not statistically significant different (P=0.9).The same <strong>stiffening</strong> effect was also observed in the human eye. Contrary to the porcine cornea the superficial flap <strong>of</strong> the human cornea is stiffer than the second flap.Commercial Relationship:G. Unger, noneT. Schilde, noneE. Spoerl, noneM. Kohlhaas, noneL.E. Pillunat, noneStress in 10³ N/m²1200porcine cornea10008006004002000Fig. 6: Fig. 70 2 4 6 8 10Strain in %first treated flapsfirst untreated flapssecond untreated flapssecond treated flaps1200human cornea (65 years)100080060040020000 2 4 6 8 10Strain in %Conclusions: Treatment <strong>of</strong> the cornea with rib<strong>of</strong>lavin/UVA leads to a significant <strong>stiffening</strong> only in the anterior 200 μm <strong>of</strong> the cornea. This locally limited <strong>stiffening</strong>effect might be explained by the absorption behaviour <strong>of</strong> the rib<strong>of</strong>lavin-treated cornea for UVA. 65 - 70 % <strong>of</strong> UVA-irradiation is absorbed in the first 200 μm and only 20% in the next 200 μm.Stress in 10³ N/m²first treated flapfirst untreated flapsecond untreated flapsecond treated flap