Optimization of amniotic membrane (AM) denuding for tissue engineering.
BACKGROUND
Amniotic membrane (AM) has gained increasing popularity as a useful carrier for ex vivo-expanded cells for tissue engineering, particularly in ocular surface reconstruction. However, current methods employed for denuding AM are highly variable, and the consequent effects on the structural and molecular composition of the AM basement membrane (BM) are ambiguous. We compare the effects of the main denuding procedures, and propose a highly effective standardized alternative.


METHODS
AMs preserved for transplantation were denuded using published ethylenediaminetetraacetic acid (EDTA)- and dispase-based methodologies and our novel thermolysin-based procedure. Scanning and transmission electron microscopy and immunohistochemistry, for BM components (collagens IV and VII, laminin 5, and integrins alpha6 and beta4), were used to assess effectiveness of denuding epithelium, whilst maintaining the integrity of the BM.


RESULTS
EDTA- and dispase-based denuding techniques resulted in the disaggregation and even destruction of the BM structure and molecular composition. Employing thermolysin effectively denuded epithelium whilst maintaining BM structural and molecular integrity.


CONCLUSIONS
Current procedures for preparing AM are variable and often ineffective, resulting in nonstandard membranes. Our novel thermolysin-based technique effectively denudes the AM whilst preserving an essentially intact and consistent BM. Therefore, we propose that this novel thermolysin procedure may potentially improve overall generation of tissue-engineered constructs using AM.
Hopkinson
Andrew
A
Division of Ophthalmology and Visual Sciences, University of Nottingham, Nottingham, United Kingdom. andy.hopkinson@nottingham.ac.uk
Shanmuganathan
Vijay A
VA
Gray
Trevor
T
Yeung
Aaron M
AM
Lowe
James
J
James
David K
DK
Dua
Harminder S
HS
D000650:Amnion / Q000378:metabolism* / Q000502:physiology* / Q000648:ultrastructure; D001485:Basement Membrane / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004848:Epithelium / Q000378:metabolism; D005123:Eye / Q000378:metabolism; D005260:Female; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D011247:Pregnancy; D013820:Thermolysin / Q000737:chemistry; D023822:Tissue Engineering / Q000379:methods*
D004492:Edetic Acid; D013820:Thermolysin