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Comparison of cryopreserved and air-dried human amniotic membrane for ophthalmologic applications

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Abstract

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-β1 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-β1 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.

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Authors and Affiliations

  1. Department of Ophthalmology, University of Duisburg-Essen, Hufelandstrasse 55, 45122, Essen, Germany

    Henning Thomasen, Mikk Pauklin, Klaus-Peter Steuhl & Daniel Meller

  2. Department of General and Molecular Pathology, University of Tartu, Ravila 19, 50411, Tartu, Estonia

    Mikk Pauklin

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  1. Henning Thomasen
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  4. Daniel Meller
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Correspondence to Daniel Meller.

Additional information

Partially supported by the Grant 5832 from the Estonian Scientific Foundation, Tallinn, Estonia; German Society of Ophthalmology (DOG), Munich, Germany; Grant ME 1623/3-1 from the Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany; Forschungsförderung AG Trockenes Auge, Berlin, Germany.

Conflict of interest

None of the authors has any financial interests to disclose. The authors have full control of all primary data, and agree to allow Graefe’s Archive for Clinical and Experimental Ophthalmology to review our data upon request

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Thomasen, H., Pauklin, M., Steuhl, KP. et al. Comparison of cryopreserved and air-dried human amniotic membrane for ophthalmologic applications. Graefes Arch Clin Exp Ophthalmol 247, 1691–1700 (2009). https://doi.org/10.1007/s00417-009-1162-y

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