Gesa Maria Grobe1, Stephan Reichl. 1. Institut für Pharmazeutische Technologie, Technische Universität Carolo-Wilhelmina zu Braunschweig, Braunschweig, Germany.
Abstract
PURPOSE: To improve the mechanical stability of a tissue-engineered human cornea construct, which is used as an in vitro model for drug absorption studies, the collagen matrix of this construct is to be strengthened by collagen cross-linking. A suitable method to induce photooxidative cross-linking of collagen fibrils is UVA irradiation combined with riboflavin as a photosensitizer. MATERIALS AND METHODS: After riboflavin/UVA treatment, the viscoelastic properties of the collagen matrix and the molecular weight of its proteins, as well as cell viability of the human corneal keratocytes (HCK) incorporated in the stromal matrix, were analyzed depending on the dose of irradiation. In addition, the cell damage to the HCKs after riboflavin/UVA treatment was also analyzed in monolayer cultures. Various luminescent cell assays were performed to clarify whether the decrease of cell viability was a consequence of apoptosis or necrosis. Furthermore, fluorescent double staining was carried out using an apoptotic/necrotic cells detection kit. RESULTS: The improvement of mechanical properties was low, whereas resultant cell damage was considerable and enduring. When lower doses of irradiation were used, the reduction of cell viability was triggered by apoptosis while necrosis supervened for increased doses of irradiation. CONCLUSION: We conclude that in contrast to clinical applications, the riboflavin/UVA treatment does not seem to be a suitable method to obtain a sufficiently firm stromal matrix including vital keratocytes to build a tissue-engineered human cornea construct to be used as an in vitro model for drug absorption studies.
PURPOSE: To improve the mechanical stability of a tissue-engineered human cornea construct, which is used as an in vitro model for drug absorption studies, the collagen matrix of this construct is to be strengthened by collagen cross-linking. A suitable method to induce photooxidative cross-linking of collagen fibrils is UVA irradiation combined with riboflavin as a photosensitizer. MATERIALS AND METHODS: After riboflavin/UVA treatment, the viscoelastic properties of the collagen matrix and the molecular weight of its proteins, as well as cell viability of the human corneal keratocytes (HCK) incorporated in the stromal matrix, were analyzed depending on the dose of irradiation. In addition, the cell damage to the HCKs after riboflavin/UVA treatment was also analyzed in monolayer cultures. Various luminescent cell assays were performed to clarify whether the decrease of cell viability was a consequence of apoptosis or necrosis. Furthermore, fluorescent double staining was carried out using an apoptotic/necrotic cells detection kit. RESULTS: The improvement of mechanical properties was low, whereas resultant cell damage was considerable and enduring. When lower doses of irradiation were used, the reduction of cell viability was triggered by apoptosis while necrosis supervened for increased doses of irradiation. CONCLUSION: We conclude that in contrast to clinical applications, the riboflavin/UVA treatment does not seem to be a suitable method to obtain a sufficiently firm stromal matrix including vital keratocytes to build a tissue-engineered human cornea construct to be used as an in vitro model for drug absorption studies.