Amniotic membrane for ocular surface reconstruction: donor variations and the effect of handling on TGF-beta content.
OBJECTIVE
Amniotic membrane (AM) transplantation is an accepted procedure in ocular surgery. However, little is known of the interdonor and intradonor variability within the membrane. In addition, the effects of the methods of processing, storage, and preoperative preparation on the membrane are not fully elucidated. The purpose of this study was to use TGF-beta as an example to investigate interdonor and intradonor variability and to determine the effect of "handling " on TGF-beta1 within fresh, processed and stored, and transplantation-ready AM (TRAM).


METHODS
Seventeen human AMs, both fresh and handled, were analyzed for TGF-beta1 by real-time polymerase chain reaction, immunohistochemistry, SDS-PAGE, and Western blotting.


RESULTS
TGF-beta1 was the highest normalized expressed isoform of TGF-beta in all samples, but it varied between membranes of different donors and at different sites within the same membrane. The highest concentration was noted in the spongy layer. Removal of the spongy layer successfully removed the bulk of TGF-beta1 from TRAM. Latency-associated protein (LAP) and a latent TGF-beta-binding protein (LTBP) were also detected.


CONCLUSIONS
TGF-beta1 is present in various regulatory forms in the AM. A degree of intermembrane and intramembrane variation is modified by handling. Unless a standardized protocol is adopted that delivers a membrane with consistent constituents, clinical outcomes may vary and comparisons may be invalid.
Hopkinson
Andrew
A
Division of Ophthalmology and Visual Sciences and.
McIntosh
Richard S
RS
Tighe
Patrick J
PJ
James
David K
DK
Dua
Harminder S
HS
D000650:Amnion / Q000378:metabolism* / Q000637:transplantation; D000956:Antigens, Viral / Q000378:metabolism; D015153:Blotting, Western; D003316:Corneal Diseases / Q000601:surgery; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D053779:Latent TGF-beta Binding Proteins; D009687:Nuclear Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013048:Specimen Handling; D014019:Tissue Donors; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D053773:Transforming Growth Factor beta1
D000956:Antigens, Viral; D047908:Intracellular Signaling Peptides and Proteins; D053779:Latent TGF-beta Binding Proteins; D009687:Nuclear Proteins; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C109540:latency-associated nuclear antigen