Comparison of cryopreserved and air-dried human amniotic membrane for ophthalmologic applications.
BACKGROUND
Cryopreserved amniotic membrane (Cryo-AM) is widely used in ocular surface surgery because of its positive effect on wound healing and its anti-inflammatory properties. A new peracetic acid/ethanol sterilized air-dried amniotic membrane (AD-AM) recently became available which might be an alternative to Cryo-AM. Our aim was to compare AM preserved with both methods with regard to the release of wound-healing modulating proteins, the preservation of basement membrane components, and the ability to serve as a substrate for the cultivation of human limbal epithelial cells (HLECs).


METHODS
Pieces of Cryo-AM and AD-AM from three different donors were incubated in DMEM for five days. The culture supernatant was collected after an incubation period of 0.1, 24, 48, 72 and 120 h; in the case of AD-AM, this period was extended up to 14 days. TIMP-1, IL-1ra, CTGF and TGF-beta1 were detected in the culture supernatant using Western blotting. Twenty human limbal epithelial cultures were initiated on both AD- and Cryo-AM. The cultures were analyzed morphologically, and the outgrowth area was measured in 3-day intervals. Cryosections of Cryo- and AD-AM from three different donors were analyzed histochemically to detect the basement membrane components collagen IV, collagen VII, laminin, laminin 5 and fibronectin.


RESULTS
The release of TIMP-1, IL-1ra and TGF-beta1 from Cryo-AM was constant for the studied period. CTGF showed a stronger signal after 120 h. None of the analyzed proteins, except for a small amount of IL-1ra, could be detected in the supernatant of AD-AM. An outgrowth of HLEC was observed in all cultures on Cryo-AM, but in only 30% of cultures on AD-AM. The outgrowth area on Cryo-AM was at all time points significantly higher than on AD-AM (p < 0.0001). Collagen IV, -VII, laminins and fibronectin were detectable in the basement membrane of Cryo-AM, but only collagen IV and fibronectin in AD-AM.


CONCLUSIONS
Cryo-AM is a more suitable substrate for the cultivation of HLECs than AD-AM. The higher outgrowth rate of cultured limbal epithelium, release of intact soluble wound-healing modulating factors and a better preservation of basement membrane components suggest the superiority of Cryo-AM for use in ophthalmology in comparison to AD-AM.
Thomasen
Henning
H
Department of Ophthalmology, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany.
Pauklin
Mikk
M
Steuhl
Klaus-Peter
KP
Meller
Daniel
D
D000650:Amnion / Q000378:metabolism*; D001485:Basement Membrane / Q000378:metabolism; D001684:Biological Dressings; D015153:Blotting, Western; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor / Q000378:metabolism; D015925:Cryopreservation; D003890:Desiccation; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology; D009926:Organ Preservation; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism; D014945:Wound Healing
C526317:CCN2 protein, human; D016326:Extracellular Matrix Proteins; D053590:Interleukin 1 Receptor Antagonist Protein; D019715:Tissue Inhibitor of Metalloproteinase-1; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor
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