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N. Farooqui, D. Myung, M. Masek, R. Dalal, W. Koh, S. Gupta, J. Noolandi, C. Frank, C.N. Ta; Histological Evaluation of Poly(Ethylene Glycol)–Poly(Acrylic Acid) (PEG–PAA) Double Network Hydrogel Corneal Implant . Invest. Ophthalmol. Vis. Sci. 2005;46(13):873.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Due to their unique composition, physiologic properties, and high water content, many hydrogel scaffolds are unable to be successfully processed for histological evaluation using routine tissue processing methods. Our goal is to adapt novel methods for hydrogel processing to successfully prepare a PEG–PAA double network (DN) hydrogel for histological analysis. Methods:Three methods were assessed for their ability to produce accurate light microscopy analysis. A modified protocol for glycol methacrylate (GMA) resin was used. The specimen, consisting of a PEG–PAA hydrogel (water content 80%) implanted within the stroma of a bovine cornea, was fixed overnight using 2.5% glutaraldehyde–2% paraformaldehyde. Pre–infiltration was achieved with increasing concentrations of GMA every hour under vacuum pressure. The Technovit 7100 kit recommendations call for dilution of the GMA monomer with ethanol. Due to the high water content of the PEG–PAA hydrogel and to preserve the morphology, ethanol was substituted with deionized water. Infiltration was performed overnight under vacuum with 100ml 100% GMA monomer and 1g Hardener I (dibenzoylperoxide). The sample was embedded in gelatin capsules and polymerized at room temperature, using 15:1 of pre–infiltration solution and Hardener II (dimethylsulfoxide). Thin sections (2 microns) were taken using a glass knife. Sections were taken off of a water bath and placed directly onto a dry slide and stained with toluidine blue. Bovine corneas with PEG–PAA implants were also processed by using frozen section and the standard TEM epoxy methods as bases for comparison. Results:Slides processed using the GMA method were evaluated under a light microscope, and the implant was clearly visible and intact within the stroma, with excellent preservation of both tissue and hydrogel morphology. Frozen sectioning of the implanted tissue resulted in loss of the hydrogel due to ice crystal formation of the water in the hydrogel. Hydrogels embedded in epoxy resin showed gross distortion of morphology due to the high ethanol concentrations required in the process. Conclusions: The GMA method is successful in yielding light microscopy sections for the histological evaluation of a PEG–PAA corneal implant. It proves to be a promising method for further evaluation of our hydrogel implants in their various applications.
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