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Alvaro Meana, Iriana Zambrano, Manuel Chacon, Natalia Vazquez, Juan Jose Fernandez, Adolfo Fernandez, Pablo Fanjul, Clara Blanco, Zoraida Gonzalez, Rosa Menendez, Jesus Merayo-Lloves; Graphene oxide membranes for ocular tissue engineering.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):897.
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© ARVO (1962-2015); The Authors (2016-present)
We analyze the viability of graphene oxide (GO) membranes as a carrier of ocular cells and their potential use as a treatment of ocular lesions.
1-Scaffold development: Three different GO suspensions were used for obtaining GO membranes by casting into a silicon mold and air-drying at room temperature. GO membranes were treated using a reducing agent to improve its mechanical strength.2-Donors: Fresh human eyes and corneal tissue were attained from our local tissue bank after informed consent.3-Cellular culture: Explants, 2-3 mm in diameter, of the limbal region were cultured onto different GO membranes. Retinal pigment epithelial (RPE) cells and stromal keratocytes were obtained by digesting RPE and corneal tissue respectively with trypsin/EDTA. Once confluent, RPE cells and stromal keratocytes were detached and subcultured on different GO membranes.4-Cellular analysis: Human limbal, RPE and corneal keratocytes cells growing on the GO membranes were fixed and analyzed by scanning electron microscopy (SEM) and immunocytochemistry against Cytokeratin High Molecular Weight (CK) and p63 for limbal cells, against CK18 and ZO-1 for RPE cells and vimentin for corneal keratocytes.5-Cytotoxicity and inflammation assays: DNA damage to cells growing on GO membranes was evaluated using the single cell gel electrophoresis assay. Moreover, in vivo biocompatibility was evaluated by grafting the different GO membranes into the sub-dermal tissue of healthy Winstar rats.
Human limbal, RPE cells and stromal keratocytes were able to attach and grow onto GO membranes. All the cells maintained their morphology and cellular markers. Limbal cells exhibited a positive stain to CK and p63, RPE cells to CK18 and ZO1 and stromal keratocytes to vimentin.No sing of inflammatory response was detected in the tissue surrounding the GO membranes implanted in rats. Moreover, no significantly DNA damage was detected when compared to a control group.
GO membranes have shown to be a good scaffold for culturing limbal, stromal keratocytes and human RPE cells; therefore, future applications of graphene oxide membranes may be developed for the reconstruction of the eye.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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