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P.A. Steinfeld, F. Versen–Höynck, J. Becker, M. Hermel, P. Walter, U. Hesselbarth; Development of a Standardized Method to Sterilize and Preserve Amniotic Membrane Transplants . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3935.
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© ARVO (1962-2015); The Authors (2016-present)
Amniotic membrane transplants (AM) are used to treat patients with limbal deficiency. They may provide a scaffold for corneal epithelial stem cell transplantation. We examined the biophysical and histological characteristics of AM sterilized and preserved using different techniques to find a method that complies with international standards (Council of Europe, 2000; EATB Standards 2002a,b).
Amniotic membranes were prepared using 3 different methods. Group A: sterilization with peracetic acid (PA) and preservation with glycerol (GLY), group B: sterilisation with PA and drying in air (AIR), group C: preservation at –80°C without sterilization. After thawing, tensile strength (TS) (Zwick–Universal–Testmachine 1455, nominal load 200N, 10mm/min) was determined. Sulphur content in the AM was measured by atomic EDX–Analysis (Falcon–EDAXTM). Immunohistochemistry was performed to determine the presence of fibrillar collagen type V (Chemicon MAB 3393) and VII (Sigma C6805) in the basement membrane. Statistical analysis was performed using the Mann–Whitney U–Test.
The mean TS was 18,4+8,23 N/cm², 9,9+4,14 N/cm² and 4,75+2,02 N/cm² in groups B, A and C, resp (p<0.05 for B vs. A and B vs. C). The mean concentration of sulphur 35,64+11,1 EDX–Units (EDXU), 24,9+1,5 EDXU and 13+2.7 EDXU in groups B, C and A, resp. (p<0.05 for B vs. C and B vs. A). The typical layered structure of the AM was unchanged in all groups. Collagen type VII was clearly detectable in C, scarcely in A and not traceable in B. Collagen type V was scarcely detectable in A and C, and not detectable in B.
Sulphur content is a parameter for the amount of disulfide bonds in the tissue matrix. The AM preserved with GLY and –80°C showed a significantly less sulphur content and tensile strength than AIR. Consistent with our previous results (Versen et. al., Cell a Tissue Banking 2004: 5(1):57), sterilization with PA and drying with air appears to be superior to the others preservation methods in terms of their biophysical properties. However, freezing leads to better preservation of AM tissue structures. The influence of both methods on the ability of AM to provide a scaffold for limbal stem cell culture will be the subject of future studies.
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