Epidermal growth factor variations in amniotic membrane used for ex vivo tissue constructs.
BACKGROUND
The amniotic membrane (AM) is used for engineering ex vivo tissue constructs used in ocular surface reconstruction. Epidermal growth factor (EGF) content of the AM is believed to play a key role in supporting corneal epithelial cell expansion on AM. This study investigated EGF content in AM in relation to intra- and inter-donor variations and the effect of processing and preservation (handling).


METHODS
Fifteen human AM, both fresh and handled, were analyzed for EGF gene and protein expression by real-time polymerase chain reaction and ELISA, respectively.


RESULTS
EGF gene expression was predominantly seen in the AM epithelium (p<0.01). Similarly, EGF protein too was predominantly seen in the epithelial layer (p<0.01) for fresh and handled samples. EGF protein content varied between membranes (inter-donor) and at different sites within the same membrane (intra-donor). The highest EGF protein concentration was noted in the AM apical and mid-region epithelium. Significant EGF protein loss (p<0.01) was observed after handling.


CONCLUSIONS
There is a considerable variation in EGF content between and within donors. This is further affected by handling of the AM. Such variations could affect the clinical efficacy of tissue constructs. Current use of AM for ex vivo expansion of epithelial cells is not standardized and remains an area of concern.
Gicquel
Jean-Jacques
JJ
Division of Ophthalmology and Visual Sciences, Hospital Nottingham, Queen's Medical Centre, Nottingham, United Kingdom.
Dua
Harminder S
HS
Brodie
Andrew
A
Mohammed
Imran
I
Suleman
Hanif
H
Lazutina
Elena
E
James
David K
DK
Hopkinson
Andrew
A
D000650:Amnion / Q000378:metabolism* / Q000648:ultrastructure; D001485:Basement Membrane / Q000378:metabolism / Q000648:ultrastructure; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000235:genetics* / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013048:Specimen Handling; D013820:Thermolysin / Q000378:metabolism; D023822:Tissue Engineering
D012333:RNA, Messenger; D004815:Epidermal Growth Factor; D013820:Thermolysin