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 collagencrosslinking 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.
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.
Department of Ophthalmology University of Dresden
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.
Fetscherstr. 74 01309 Dresden
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Germany
flap flap from deeper layers
Contact: Thomas Schilde
[email protected] Fig.1: experimental equipment
Fig.2: riboflavin/UVA application
Fig.3: cutting flaps with a microkeratom
Fig.4: dissected flaps (200µm)
Fig.5: half-life of the surfaces 80 70
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Fig.6: solvent behaviour: treated superficial flaps after 3 hours
Commercial Relationship: T. Schilde, None; E. Spoerl, None; M. Kohlhaas, None; L.E. Pillunat, None
Fig.7: solvent behaviour: treated superficial flaps after 171 hours
Fig.8: digitally measurement calibrate the computerprogram
Fig.9: digitally measurement of the solved surfaces
Fig.10: results
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.
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 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. Department of Ophthalmology University of Dresden Fetscherstr. 74 01309 Dresden Germany
Contact: Gabriele Unger
[email protected]
Fig.1: UVA-radiation
Fig. 2: rotormicrokeratom
Fig. 3: cornea stripe
Fig. 4: stress-strain measuring
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±62x10³ 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. 1200
1200
porcine cornea
human cornea (65 years)
Fig. 6:
1000
Stress in 10³ N/m²
Stress in 10³ N/m²
first treated flaps
Commercial Relationship: G. Unger, none T. Schilde, none E. Spoerl, none M. Kohlhaas, none L.E. Pillunat, none
Fig. 5: biomechanical surveyors table
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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.
