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H. Thomasen, M. Pauklin, K.-P. Steuhl, D. Meller; Comparison of Cryo-Preserved and Freeze-Dried Amniotic Membrane for Ophthalmologic Applications. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1792.
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The cryopreserved amniotic membrane (Cryo-AM) is widely used for the treatment of ocular surface conditions because of its’ positive effect on wound healing and inflammation. Recently a new peracetic acid/ethanol sterilized air-dried amniotic membrane (AD-AM) was introduced which might be an alternative to Cryo-AM. Our aim was to analyze Cryo- and AD-AM regarding the release of wound-healing modulating proteins by and the suitability to serve as a substrate for the cultivation of human limbal epithelial cells (HLEC).
Slices (9cm2) of Cryo-AM and AD-AM (n=3) were incubated at 37°C in DMEM medium for five days. The medium was collected at 0.1 h, 24 h, 48 h, 72 h and 120 h, in case of AD-AM this period was extended up to 14 days. Proteins were isolated by TCA-precipitation and separated by SDS-PAGE. The proteins TIMP-1, IL-1ra, CTGF and TGF-β1 were detected by Western Blotting.Limbal tissue was obtained from four enucleated eyes. Each sample was divided into ten pieces (1x1mm), treated with Dispase (1.2 U/ml), randomly divided into two groups and placed on the epithelial side of either AD- (n=5) or Cryo-AM (n=5). Cultures were incubated for 18 days at 37°C under 5% carbon dioxide and 95% air in SHEM medium. The outgrowth area was monitored by mean of phase contrast microscopy. The outgrowth area of each culture was documented at day 9, 11, 14, 16 and 18 and measured using ImagJ (NIH). Two-sample t-test was used for statistical analysis of the outgrowth.
The examined proteins were detectable in Cryo-AM after 24 h of incubation. The amount of TIMP-1, IL-1ra and TGF-β1 was constant for the studied period. In contrast CTGF showed a weak constant signal for the first 72 h, but a stronger signal was obtained after 120 h. In AD-AM only IL-1ra could be detected after 7-days of incubation.An outgrowth was observed in all HLEC cultures on Cryo-AM but in only 30% of cultures on AD-AM. The cells showed a significantly higher outgrowth area on Cryo-AM than on AD-AM. The differences were statistically significant (p< 0.0001) at all analyzed time points.
Cryo-AM releases intact soluble proteins while in AD-AM these were barely detectable. The release of proteins seems to be time dependent for some factors.These results indicate that the cryopreservation method preserves soluble proteins while the sterilization and preservation process of AD-AM denaturates them. Cryo-AM is a more suitable substrate for HLEC than AD-AM.
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