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KA Kobuch, B Wild, E Eckert, C Fischbach, VP Gabel; Expression of Collagen Type II by Hyalocytes In Vitro - First Steps Towards a Biological Vitreous Substitute . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3495.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Current standard therapies in vitreoretinal surgery replace the vitreous body by synthetic vitreous substitutes like silicon oil or perfluorocarbon liquids. To overcome the negative effects of these substances in the long term therapy of vitreoretinal diseases, we evaluated the potential of hyalocytes in culture to form a biological vitreous-tranplant in vitro, characterized by the extracellular expression of collagen type II. Methods: Hyalocytes were isolated from porcine eyes within three hours after death of the animals, dissociated mechanically through a syringe and grown to confluence in culture-flasks. In order to obtain a sufficient number of hyalocytes, the cells were proliferated and subcultured three times. Then they were grown to redifferentiate in 12-well culture-dishes or in three-dimensional biodegradable porous scaffolds respectively. After 1, 2, 3, 8 weeks, the degree of redifferentiation and the formation of extracellular matrix was assessed by light- and electron microscopy and immunohistochemical staining with a monoclonal collagen type II-antibody. Results: A sufficient number of vital hyalocytes still with the potential to redifferentiate could be obtained by proliferating the cells up to three passages. Under modified conditions in the 12-well plates or in the three-dimensional scaffolds, as signs of redifferentiation the rate of cell division decreased and the synthesizing activity of the cells increased, evidenced by a large number of intracytoplasmatic vacuoles and granules. Finally, after three to eight weeks, extracellular matrix was expressed increasingly, forming granular and network- or fiber-like structures around the cells and a coherent mass in the culture dish. The extracellular material stained positive for collagen type II, which is the main matrix component of the vitreous. Conclusion: Hyalocytes in tissue culture did effectively proliferate to sufficient numbers, redifferentiate and express extracellular collagen type II. Although culture conditions have to be optimized, this may be a first step towards a tissue-engineered biological, potentially autologous vitreous-transplant and contribute to improve the treatment schemes for vitreoretinal diseases.
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