June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
The Effects of Amniotic Membrane Extract on Primary Human Corneal Epithelial Cells
Author Affiliations & Notes
  • David Dudok
    Ophthalmology, Western University Canada, London, ON, Canada
  • Kevin Cheung
    Ophthalmology, Western University Canada, London, ON, Canada
  • Hong Liu
    Ophthalmology, Western University Canada, London, ON, Canada
  • Luca Vedovelli
    Division of Nephrology, Cell Biology, Human Biology and Physiology, Massachusetts General Hospital, Boston, MA
  • Emiliano Ghinelli
    Ophthalmology, Massachusetts Eye & Ear Infirmary, Boston, MA
  • Ken Kenyon
    Ophthalmology, Massachusetts Eye & Ear Infirmary, Boston, MA
  • Sunil Parapuram
    Ophthalmology, Western University Canada, London, ON, Canada
  • Cindy Hutnik
    Ophthalmology, Western University Canada, London, ON, Canada
  • Footnotes
    Commercial Relationships David Dudok, None; Kevin Cheung, None; Hong Liu, None; Luca Vedovelli, None; Emiliano Ghinelli, None; Ken Kenyon, None; Sunil Parapuram, None; Cindy Hutnik, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 5236. doi:
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      David Dudok, Kevin Cheung, Hong Liu, Luca Vedovelli, Emiliano Ghinelli, Ken Kenyon, Sunil Parapuram, Cindy Hutnik, ; The Effects of Amniotic Membrane Extract on Primary Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5236.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract
 
Purpose
 

1) To assess the effects of amniotic membrane extract (AMX) on the cellular activity of primary human corneal epithelial (HCE) cells. 2) To assess the effects of AMX on HCE activity under mechanical and oxidative stress conditions.

 
Methods
 

A lyophilized form of AMX was obtained from Keera Inc; later reconstituted in balanced salt solution. Primary human corneal epithelial cells were cultured. MTT assay for cellular viability over 48 hours using various AMX concentrations was carried out with 10% fetal bovine serum and serum-free media controls. The second phase involved mechanical “scratch” stress of HCE culture simulating corneal injury. 0.1% AMX was incubated for 48 and 72 hours in culture and comparison of scratch healing was made versus serum-free control. The oxidative phase involved use of 0.5 mM tertiary-butylhydroperoxide (t-BOOH) addition to HCE culture for 1 hour, washout of cells, then addition of 0.1% AMX versus control for 48 hours. Further oxidative study was undertaken using 12 hour pre-treatment with 0.1% AMX.

 
Results
 

MTT assay cellular viability was 100% for control, 101% for 0.01% AMX, 95.5% for 0.05% AMX, 95.3% for 0.1% AMX, 78.5% for 0.5% AMX, and 91% for 10% fetal bovine serum. 0.1% AMX was further studied due to highest viability for highest concentration. Mechanical “scratch” test on the HCE cultures revealed a significant healing distance ratio at 48 and 72 hours in favor of 0.1% AMX treated cultures (p = 0.021, 0.035). Oxidative stress with 0.5 mM t-BOOH 1 hour administration did not reveal significant difference in MTT assay of AMX treated versus control cultures (71% vs. 69%). Pre-treatment with 0.1% AMX for 12 hours before repeat of t-BOOH protocol revealed significant difference at 24 hours (73% AMX vs. 66% control, p=0.04) but not at 48 hours (81% AMX vs. 71% control, p=0.38).

 
Conclusions
 

The concentration of AMX with the best cellular viability in relation to highest concentration was 0.1%. The 0.1% AMX treated cells healed faster with a mechanical insult compared to controls, suggesting benefit in an acute corneal injury with increased HCE proliferation. The oxidative impact on cellular viability of t-BOOH did not improve with 0.1% AMX administration vs. control unless the cells were pre-treated with AMX. In more chronic corneal stress, AMX pre-treatment may provide some benefit.

 
 
Photographic comparison of mechanical "scratch" healing of AMX-treated HCE cells vs. control
 
Photographic comparison of mechanical "scratch" healing of AMX-treated HCE cells vs. control
 
Keywords: 480 cornea: basic science • 482 cornea: epithelium  
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