Effect of dispase denudation on amniotic membrane.
OBJECTIVE
To describe the cellular components, biochemical composition, and membrane surface characteristics of denuded human amniotic membrane (DHAM) treated with Dispase II.


METHODS
DHAM was incubated with Dispase II (1.2 U/ml) for 30 min, 60 min, or 120 min. This was followed by gentle scraping to remove any remaining epithelial cells using a cell scraper. Histology, immunohistochemistry for extracellular matrix molecules and growth factors, and transmission (TEM) and scanning electron microscopy (SEM) were performed to assess the effects of increasing durations of incubation on DHAM structure.


RESULTS
Dispase II treatment was associated with the digestion of several ECM molecules, particularly those in the basement membrane including collagen VI, fibronectin, and laminin. FGF-2 and PDGF-B expression were unaffected by Dispase II, but TGF-alpha, TGF-beta1, TGF-beta 2R, PDGF-A, VEGF, and EGFR expression were all reduced by Dispase II incubation. TEM confirmed the disruption of DHAM ultrastructure with increasing duration of Dispase II incubation, beginning with disruption of the basal lamina and progressing to loosening of the stromal collagen network as well.


CONCLUSIONS
The use of Dispase II in the preparation of DHAM causes significant changes to the ultrastructure of the membrane, particularly the BM. Prolonged incubation with dispase may cause significant disruption in DHAM structure which may affect cell growth in cultured explants.
Singapore National Eye Center, Singapore.
Laurence S
LS
Lim
Andri
A
Riau
Rebekah
R
Poh
D T
DT
Tan
R W
RW
Beuerman
J S
JS
Mehta
D000650:Amnion / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure*; D010450:Endopeptidases / Q000378:metabolism* / Q000494:pharmacology; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D017978:Receptors, Growth Factor / Q000378:metabolism
D036341:Intercellular Signaling Peptides and Proteins; D017978:Receptors, Growth Factor; D010450:Endopeptidases; C023139:dispase