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A. d. Frota, A. B. T. Dias, S. Maloney, C. Martins, M. N. Burnier, Jr.; Freeze-Drying as an Alternative Method for Human Sclera Preservation. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3455.
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Ninety-six samples of formalin-fixed and paraffin-embedded human sclera were studied. Half of them were submitted to the freeze-drying process and the other half preserved in 95% ethanol. Preservation periods of 18, 45, 90 and 174 days were studied. Automated immunostaining was carried out in the Ventana BenchMarkR LT platform using Collagen (COL-1) and Fibronectin (FIB) monoclonal antibodies. Histological analysis was also performed with Hematoxilin-Eosin to evaluate collagen fiber parallelism, and Mason-Trichrome to evaluate its integrity. Samples were classified according to the degree of collagen fibers parallelism (0-2), intensity of Mason staining (0-2), and the expression of both COL-1 and FIB monoclonal antibodies (0-3). Friedman and Wilcoxon tests were applied to compare preservation methods through statistical analysis of the mean scores of each sclera sample studied. Inherent to the methods used, p-values below 0.05 were considered to ensure statistical significance
Relevant results were found in four situations: 1) freeze-dried scleras showed higher level of collagen fiber integrity in the ones re-hydrated after 174 day preservation as compared to the 90-day preservation ones; 2) the freeze-dried group showed better collagen fiber integrity in the samples re-hydrated after 18 and 174 days-preservation as compared to the ethanol group with the same preservation period; 3) the freeze-dried group disclosed higher immunohistochemical expression for COL-1 antibody in the sclera samples re-hydrated after 45, 90 and 174 days of preservation period as compared to the ones re-hydrated after 18 days; 4) freeze-dried samples re-hydrated after 174 day preservation period showed lesser immunohistochemical expression for FIB antibody as compared to samples preserved in 95% ethanol.
Histopathological and immunohistochemical analysis showed freeze-drying to be a superior method for sclera graft preservation as compared to 95% ethanol. This technique provides an easy to manipulate tissue, with long shelf life, and storage at room temperature.
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