title: The effect of amniotic membrane de-epithelialization method on its biological properties and ability to promote limbal epithelial cell culture.
abstract: OBJECTIVE
  We characterized the de-epithelialized human amniotic membrane (HAM), and compared cell attachment and proliferation efficiencies.
  
  
  METHODS
  HAM was de-epithelialized by 20% ethanol (AHAM), 1.2 U/mL Dispase (DHAM), 0.02% EDTA (EHAM), 0.25% trypsin-EDTA (THAM), and 5 M urea (UHAM), respectively, followed by gentle scraping with a #15 blade. Surface topology, extracellular matrix (ECM), and growth factor content were characterized and compared to intact HAM by electron microscopies (EM), atomic force microscopy (AFM), immunohistochemistry, and Western blotting. Primary human limbal epithelial cells (LEC) attachment and proliferation efficiencies were assayed. Statistical significance was calculated by SPSS and Fisher's least significant difference test.
  
  
  RESULTS
  EHAM, THAM, and UHAM had intact basal lamina and smooth basement membrane surface shown under transmission and scanning EM, and AFM. Cell remnants stayed on AHAM. Disrupted basement membrane and stroma was found in DHAM. Immunostaining intensity quantification and hierarchical clustering revealed that ECM composition of EHAM and UHAM resembled intact HAM. In contrast, DHAM and THAM had drastic loss of ECM and growth factor content. LEC attachment efficiency at 24 hours after seeding was the highest in EHAM (51% as on conventional culture surface), followed by UHAM and AHAM. However, cell proliferation indices at day 10 of culture were similar among different HAM substrates, suggesting repair of ECM and basement membrane by growing epithelial cells.
  
  
  CONCLUSIONS
  Urea denudation preserved the basement membrane integrity, ECM, and growth factor composition, and had higher cell attachment and proliferation efficiencies. With its short processing time, urea treatment offers a novel alternative for HAM de-epithelialization.
authors.affiliation: Duke-NUS Graduate Medical School, Singapore.
authors.forename: Ting
authors.initials: T
authors.lastname: Zhang
authors.forename: Gary Hin-Fai
authors.initials: GH
authors.lastname: Yam
authors.forename: Andri K
authors.initials: AK
authors.lastname: Riau
authors.forename: Rebekah
authors.initials: R
authors.lastname: Poh
authors.forename: John C
authors.initials: JC
authors.lastname: Allen
authors.forename: Gary S
authors.initials: GS
authors.lastname: Peh
authors.forename: Roger W
authors.initials: RW
authors.lastname: Beuerman
authors.forename: Donald T
authors.initials: DT
authors.lastname: Tan
authors.forename: Jodhbir S
authors.initials: JS
authors.lastname: Mehta
mesh_terms: D000293:Adolescent; D000328:Adult; D000650:Amnion / Q000166:cytology*; D001485:Basement Membrane / Q000648:ultrastructure; D018929:Cell Culture Techniques / Q000379:methods; D049109:Cell Proliferation; D002675:Child, Preschool; D004492:Edetic Acid / Q000494:pharmacology; D010450:Endopeptidases / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000166:cytology*; D000431:Ethanol / Q000494:pharmacology; D005260:Female; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D008297:Male; D018625:Microscopy, Atomic Force; D008855:Microscopy, Electron, Scanning; D008875:Middle Aged; D012997:Solvents / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology; D055815:Young Adult
chemical_list: D012997:Solvents; D000431:Ethanol; D014508:Urea; D004492:Edetic Acid; D010450:Endopeptidases; D014357:Trypsin; C023139:dispase