title: Assessment of the use of cryopreserved x freeze-dried amniotic membrane (AM) for reconstruction of ocular surface in rabbit model.
abstract: OBJECTIVE
  To determine the efficacy of freeze-dried amniotic membrane (AM) for reconstruction of the ocular surface in rabbit eyes.
  
  
  METHODS
  The sterilized, freeze-dried amniotic membrane (lyophilized or FD-AM) is a preservative method that uses the drying by freezing process to maintain the AM well preserved for a long time even at room temperature. This paper is an experimental animal interventional study. One eye of each of 15 male New Zealand rabbits (1.5 - 3.0 kg) had the central cornea marked with a 6.0 mm trephine. The marked area was deepithelialized with a No.15 blade. The denuded corneal surface was covered as follows: Group 1: cryopreserved AM (n=6); Group 2: freeze-dried AM (n=6); and Group 3: not covered (control group, n=3). The AM in group 1 and 2 and the periphery of the denuded area in group 3 were secured with continuous 10-0 nylon sutures. The clinical evaluation was made by a blinded observer and graded on a four-point scale (1= minimal, 4= marked) for conjunctival and ciliary hyperemia, eyelid edema, corneal neovascularization, corneal opacity and reepithelialization on postoperative (PO) days 1, 7 and 30 . After PO day 30, the rabbits were euthanized and their corneas were sent for histopathological and ultrastructural analysis to evaluate tissue inflammation, reepithelialization, and basement membrane integrity.
  
  
  RESULTS
  Two eyes in group 2 had a corneal infection and were excluded from the analysis. No statistically significant differences among the three groups were found (p>0.05) regarding the clinical evaluation on 1st, 7th and 30th PO days. On transmission electron microscopy, the basement membrane in lyophilized and control groups was more continuous and homogeneous than in the glycerol group.
  
  
  CONCLUSIONS
  The freeze-drying method seems to be a good option to preserve human amniotic membrane to be used in ocular surface reconstruction. This preservative method reduces the preservation costs and may enhance the use of AM, facilitating its storage and transport.
authors.affiliation: Department of Ophthalmology, Federal University of São Paulo, São Paulo, Brazil. rodrigo.doyle@superig.com.br
authors.forename: Rodrigo Doyle
authors.initials: RD
authors.lastname: Libera
authors.forename: Gustavo Barreto de
authors.initials: GB
authors.lastname: Melo
authors.forename: Acácio de Souza
authors.initials: Ade S
authors.lastname: Lima
authors.forename: Edna Freymüller
authors.initials: EF
authors.lastname: Haapalainen
authors.forename: Priscila
authors.initials: P
authors.lastname: Cristovam
authors.forename: Jose Alvaro Pereira
authors.initials: JA
authors.lastname: Gomes
mesh_terms: D000650:Amnion / Q000637:transplantation* / Q000648:ultrastructure; D000818:Animals; D003315:Cornea / Q000601:surgery* / Q000648:ultrastructure; D015925:Cryopreservation / Q000379:methods*; D005612:Freeze Drying; D008297:Male; D011817:Rabbits