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Fatma Zaguia, Marie Boulze Pankert, Benjamin Goyer, Stephanie Proulx, Isabelle Brunette; Reinnervation of corneal stroma reconstructed by tissue engineering transplanted in the living feline model. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5253.
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© ARVO (1962-2015); The Authors (2016-present)
Tissue-engineering of corneal substitutes opens the way to new solutions to the worldwide growing problem of corneal tissue shortage for transplantation. We have previously demonstrated the feasibility of tissue-engineering a corneal stroma, which when transplanted in a long term living feline model, was shown to be well tolerated and remains transparent. The aim of this study was to assess the capacity of the tissue-engineered (TE)-stroma to reinnervate in vivo.
Eight healthy animals received two intra-stromal TE-stromal transplants produced from keratocytes of allogeneic or xenogeneic origin in one eye, while the controlateral eye was used as a control. During the 4-month follow-up, sensory recovery was assessed on a regular basis (Cochet-Bonnet aesthesiometer). Confocal microscopy (Rostock Cornea Module® of HRT®II) was performed postmortem, focusing on the sub basal nerve plexus (Z1), the sub epithelial nerve plexus (Z2) and the nerves within the grafts (Z3). Images were analyzed using the nerve tracing and analysis plugin NeuronJ in combination with the ImageJ software (NIH) to generate standardized parameters to quantify nerve counts and density. Each graft and control eye was then evaluated by transmission electron microscopy and immunofluorescence using neurofilament and collagen IV antibodies.
The average peripheral corneal sensitivity was 2.61±0.81cm prior to surgery, 0.1±0.17cm on postoperative day 3, it reached a plateau at day 58 (2.2±0.7cm) and ended at 3.1±0.6cm at 4 months, with no differences between the 2 grafted areas. Analysis of confocal microscopy images revealed mean nerve counts and densities of 39/mm2 and 2780 um/mm2 in the xenogeneic TE-grafts, 38/mm2 and 2188 um/mm2 in the sham surgery eye, and 39/mm2 and 2540 um/mm2 in the unoperated control eye. The newly reinnervated zones showed higher nerve densities, but lower degrees of organization. No statistically significant differences were found between the TE-stromas of xenogeneic and allogeneic origin. Immunofluorescence confirmed graft reinnervation.
We have demonstrated that effective sensory reinnervation of the TE-corneal stromas occurs within two months after transplantation in the living feline model.
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