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Alvaro Meana, Jose L Cenis, Salvador D Aznar-Cervantes, Manuel Chacón, Natalia Vázquez, David Cereijo, Jose Antonio Rodríguez-Cortés, David Álvarez, Jesus Merayo-Lloves; Silk Fibroin Membranes as a Carrier for the Treatment of Corneal Lesions.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1450.
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© ARVO (1962-2015); The Authors (2016-present)
We analyze the viability of silk fibroin (SF) membranes as a carrier of corneal cells and their potential use as a treatment of corneal lesions.
Cocoons of Bombyx mori were degummed (boiling in 0.02N Na2CO3) and SF was dissolved in 9.3M LiBr and dialyzed against distilled water into dialysis bags (3,500Da MWCO) for 3 days. Then, 580µL of the resultant 5% w/v SF solution was cast upon plastic Petri dish 5,8cm in diameter to give a 10 microns thickness film. Once dried at room temperature, the water-annealing was performed by placing the SF films in a water-filled desiccator in vacuum conditions for 24h. Cells were obtained from normal New Zealand white rabbits and normal human corneoscleral rims obtained during surgery after 8mm trephination of the graft. Limbal stem cells were obtained from explants of 2-3mm in diameter of the limbal region. Stromal and endothelial cells were obtained by digesting the stromal tissue and the dissected endothelium with Tripsin/EDTA and Collagenase I respectively. Cells were cultured in different culture media onto SF membranes using a handling device designed by Prodintec Foundation for our laboratory. Corneal cells growing on the SF membranes were examined by phase contrast microscopy, scanning electron microscopy (SEM) and immunocytochemistry for antibodies: Cytokeratin High Molecular Weight and p.63, Vimentin and ZO-1. Limbal stem cell deficiency (LSCD) and Descemet Membrane and Endothelial Keratoplasty (DMEK) are currently being performed in order to test the capabilities of SF membranes in vivo.
Stromal and limbal stem cells showed a high proliferation capacity in human and rabbit primary cell cultures and were able to attach and grow onto SF membranes while maintaining their morphology and cellular markers (Vimentin and Cytokeratin) while showing a highly population of p.63 positive cells in the limbal stem cell cultures. Rabbit endothelial cells were effectively cultured on SF membrane while maintaining a positive stain of ZO-1, however, human endothelial cells were unable to attach and proliferate in vitro on this type of membrane.
SF membranes could be improved and studied for their use as a carrier for the treatment of corneal diseases.
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