Effect of dispase denudation on amniotic membrane

[摘要] Purpose: To describe the cellularcomponents, biochemical composition, and membrane surfacecharacteristics of denuded human amniotic membrane (DHAM) treated withDispase II. Methods: DHAM was incubated with DispaseII (1.2 U/ml) for 30 min, 60 min, or 120 min. This was followed bygentle scraping to remove any remaining epithelial cells using a cellscraper. Histology, immunohistochemistry for extracellular matrixmolecules and growth factors, and transmission (TEM) and scanningelectron microscopy (SEM) were performed to assess the effects ofincreasing durations of incubation on DHAM structure. Results: Dispase II treatment wasassociated with the digestion of several ECM molecules, particularlythose in the basement membrane including collagen VI, fibronectin, andlaminin. FGF-2 and PDGF-B expression were unaffected by Dispase II, butTGF-α, TGF-β1, TGF-β2R, PDGF-A, VEGF, and EGFR expression were allreduced by Dispase II incubation. TEM confirmed the disruption of DHAMultrastructure with increasing duration of Dispase II incubation,beginning with disruption of the basal lamina and progressing toloosening of the stromal collagen network as well. Conclusions: The use of Dispase II inthe preparation of DHAM causes significant changes to theultrastructure of the membrane, particularly the BM. Prolongedincubation with dispase may cause significant disruption in DHAMstructure which may affect cell growth in cultured explants.

[学科分类] 生物化学/生物物理