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G. M. Grobe, S. Reichl; Examining the Suitability of Riboflavin/UVA-Treatment for Tissue Engineering Applications. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4977.
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To improve the mechanical stability of a tissue-engineered cornea construct which is used as an in-vitro-model for drug absorption studies, the collagen matrix of this construct was to be strengthened by crosslinking using the riboflavin/UVA-method. Afterwards cell viability of keratocytes incorporated in the stromal matrix was analyzed depending on the dose of irradiation. The pathway of cell death induced by the treatment was studied in monolayer culture.
SV40-immortalized human corneal keratocytes (HCK-Ca) were dispersed in a collagen gel. After 7 days of cultivation the stromal bioequivalents were treated with the riboflavin/UVA-method. Oscillation rheology was used to determine the improvement of viscoelastic properties of the collagen gels. Cell viability was measured 24 hours after the treatment using the MTT-Assay.In order to clarify whether the decrease of cell viability was consequence of an apoptotic process or necrosis different luminescent assays were performed in monolayer culture. On the one hand the activities of caspases 3, 7, 8 and 9 were measured. On the other hand the activity of a protease associated with cytotoxicity was determined.
As expected, increasing the dose of irradiation led to a considerable decrease of cell viability of the HCK-Ca incorporated in the collagen matrix as well as in monolayer culture. Luminescent assays showed that lower doses of irradiation up to 1.5 J/cm^2 induced an apoptotic cell death whereas a cytotoxic effect supervened for higher doses of irradiation.The improvement of the elastic components of the stromal bioequivalents was weak but statistically significant. Ongoing studies are carried out to clarify if plastic compression of collagen gels in combination with riboflavin/UVA-treatment is able to enforce the gain of mechanical strength.
Riboflavin/UVA-treatment led to dose-dependent cell death via apoptosis or necrosis. In contrast to clinical applications, the improvement of mechanical stability was weak. This might be ascribed to the lower collagen content of the tissue engineered stromal bioequivalent compared to human cornea.
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