Claire L. Allen, Gerry Clare, Elizabeth A. Stewart, Matthew J. Branch, Owen D. McIntosh, Megha Dadhwal, Harminder S. Dua, Andrew Hopkinson

Abstract:

Purpose: Dried amniotic membrane (AM) can be a useful therapeutic adjunct in ophthalmic surgery and possesses logistical
advantages over cryopreserved AM. Differences in preservation techniques can significantly influence the biochemical
composition and physical properties of AM, potentially affecting clinical efficacy. This study was established to investigate
the biochemical and structural effects of drying AM in the absence and presence of saccharide lyoprotectants and its
biocompatibility compared to cryopreserved material.

Methods: AM was cryopreserved or dried with and without pre-treatment with trehalose or raffinose and the antioxidant
epigallocatechin (EGCG). Structural and visual comparisons were assessed using electron microscopy. Localisation,
expression and release of AM biological factors were determined using immunoassays and immunofluorescence. The
biocompatibility of the AM preparations co-cultured with corneal epithelial cell (CEC) or keratocyte monolayers were
assessed using cell proliferation, cytotoxicity, apoptosis and migration assays.

Results: Drying devitalised AM epithelium, but less than cryopreservation and cellular damage was reduced in dried AM
pre-treated with trehalose or raffinose. Dried AM alone, and with trehalose or raffinose showed greater factor retention
efficiencies and bioavailability compared to cryopreserved AM and demonstrated a more sustained biochemical factor time
release in vitro. Cellular health assays showed that dried AM with trehalose or raffinose are compatible and superior
substrates compared to cryopreserved AM for primary CEC expansion, with increased proliferation and reduced LDH and
caspase-3 levels. This concept was supported by improved wound healing in an immortalised human CEC line (hiCEC) cocultured
with dried and trehalose or raffinose membranes, compared to cryopreserved and fresh AM.

Conclusions: Our modified preservation process and our resultant optimised dried AM has enhanced structural properties
and biochemical stability and is a superior substrate to conventional cryopreserved AM. In addition this product is stable
and easily transportable allowing it to be globally wide reaching for use in clinical and military sectors.

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Donald E. Fetterolf, MD; and Robert J. Snyder, DPM, MSc

Abstract:

Amniotic membrane has been employed in the treatment of wounds for almost 100 years, beginning with early application of natural amniotic membrane obtained from labor and delivery to various types of burns and wounds. Amniotic membrane is rich in collagen and various growth factors that support the healing process to both improve wound closure and reduce scar formation. Unique properties of the material include the lack of immunologic markers, conferring an “immune privileged” status on the allografts; antibacterial properties; and the ability to reduce pain on application. The resurgence of interest in the use of amniotic membrane in a number of applications, including wound treatment, has occurred following improved techniques for preserving the natural membrane. Recently, techniques have been developed to dehydrate the material while preserving many of these wound-healing attributes, to produce a temperature-stable allograft. Future research will continue to yield more information on the unique properties of the amniotic membrane allografts.

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