title: Augmented dried versus cryopreserved amniotic membrane as an ocular surface dressing.
abstract: OBJECTIVE
  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) co-cultured 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.
authors.affiliation: Ophthalmology, Division of Clinical Neuroscience, University of Nottingham, Nottingham, United Kingdom.
authors.forename: Claire L
authors.initials: CL
authors.lastname: Allen
authors.forename: Gerry
authors.initials: G
authors.lastname: Clare
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Stewart
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Branch
authors.forename: Owen D
authors.initials: OD
authors.lastname: McIntosh
authors.forename: Megha
authors.initials: M
authors.lastname: Dadhwal
authors.forename: Harminder S
authors.initials: HS
authors.lastname: Dua
authors.forename: Andrew
authors.initials: A
authors.lastname: Hopkinson
mesh_terms: D000328:Adult; D000650:Amnion / Q000502:physiology*; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001684:Biological Dressings; D053148:Caspase 3 / Q000378:metabolism; D002392:Catechin / Q000031:analogs & derivatives / Q000494:pharmacology; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D018920:Coculture Techniques; D003315:Cornea / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D060527:Corneal Keratocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D015925:Cryopreservation / Q000379:methods; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D005612:Freeze Drying / Q000379:methods; D006801:Humans; D011887:Raffinose / Q000494:pharmacology; D014199:Trehalose / Q000494:pharmacology
chemical_list: D000975:Antioxidants; D002392:Catechin; D014199:Trehalose; D053148:Caspase 3; C057580:gallocatechol; D011887:Raffinose