A new model for in vitro testing of vitreous substitute candidates
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To describe a new model for in vitro assessment of novel vitreous substitute candidates.
The biological impact of three vitreous substitute candidates was explored in a retinal explant culture model; a polyalkylimide hydrogel (Bio-Alcamid®), a two component hydrogel of 20 wt.% poly (ethylene glycol) in phosphate buffered saline (PEG) and a cross-linked sodium hyaluronic acid hydrogel (Healaflow®). The gels where applied to explanted adult rat retinas and then kept in culture for 2, 5 and 10 days. Gel-exposed explants were compared with explants incubated under standard tissue culture conditions. Cryosections of the specimens were stained with hematoxylin and eosin, immunohistochemical markers (GFAP, Vimentin, Neurofilament 160, PKC, Rhodopsin) and TUNEL.
Explants kept under standard conditions as well as PEG-exposed explants displayed disruption of retinal layers with moderate pyknosis of all neurons. They also displayed moderate labeling of apoptotic cells. Bio-Alcamid®-exposed explants displayed severe thinning and disruption of retinal layers with massive cell death. Healaflow®-treated explants displayed normal retinal lamination with significantly better preservation of retinal neurons compared with control specimens, and almost no signs of apoptosis. Retinas exposed to Healaflow® and retinas kept under standard conditions showed variable labeling of GFAP with generally low expression and some areas of upregulation. PEG-exposed retinas showed increased GFAP labeling and Bio-Alcamid®-exposed retinas showed sparse labeling of GFAP.
Research into novel vitreous substitutes has important implications for both medical and surgical vitreoretinal disease. The in vitro model presented here provides a method of biocompatibility testing prior to more costly and cumbersome in vivo experiments. The explant culture system imposes reactions within the retina including disruption of layers, cell death and gliosis, and the progression of these reactions can be used for comparison of vitreous substitute candidates. Bio-Alcamid® had strong adverse effects on the retina which is consistent with results of prior in vivo trials. PEG gel elicits reactions similar to the control retinas whereas Healaflow® shows protection from culture-induced trauma indicating favorable biocompatibility.
KeywordsVitreous substitute Immunohistochemistry Retinal culture Vitreoretinal surgery Hyaluronic acid Polyethylene oxide Polyal kylimide
This work was supported by: The Faculty of Medicine, University of Lund, The Swedish Research Council, The Princess Margareta’s Foundation for Blind Children, The Wallenberg Foundation. T.M.O. was supported by a Sir General John Monash Scholarship. Some of this work was sponsored by a gift to MIT by the In Vivo Therapeutics Corporation.
Thanks to Karin Arnér for excellent technical support and Linnéa Taylor for valuable input on the manuscript.
The authors have full control of all primary data, available for review by Graefe's Archive for Clinical and Experimental Ophthalmology upon request. The “Principles of laboratory animal care” (NIH publication No. 85–23, revised 1985), the OPRR Public Health Service Policy on the Humane Care and Use of Laboratory Animals (revised 1986) and the U.S. Animal Welfare Act, as amended, were followed, as well as the current version of the Swedish Law on the Protection of Animals, where applicable.
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