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Updated: Jun 20, 2026
Lumican Extraction from Amniotic Membrane and Determination of its Storage Temperature
Published on: October 14, 2022
Effect of dispase denudation on amniotic membrane
Laurence S Lim1, Andri Riau, Rebekah Poh
1Singapore National Eye Center, Singapore.
Purpose:
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.
Insights
Dispase II treatment alters denuded human amniotic membrane (DHAM) structure, affecting basement membrane components and growth factor expression. Prolonged incubation may disrupt DHAM, impacting cell growth in explants.
Area of Science:
- Regenerative Medicine
- Tissue Engineering
- Biomaterials Science
Background:
- Human amniotic membrane is a promising biomaterial for tissue regeneration.
- Enzymatic treatment is often used to prepare amniotic membrane for therapeutic applications.
- Understanding the effects of enzymes on membrane structure is crucial for optimizing its use.
Purpose of the Study:
- To characterize the cellular and biochemical changes in denuded human amniotic membrane (DHAM) after Dispase II treatment.
- To evaluate the impact of varying Dispase II incubation durations on DHAM ultrastructure and composition.
Main Methods:
- DHAM was incubated with Dispase II (1.2 U/ml) for 30, 60, or 120 minutes.
- Histology, immunohistochemistry, transmission electron microscopy (TEM), and scanning electron microscopy (SEM) were employed.
- Analysis focused on extracellular matrix molecules, growth factors, and ultrastructural integrity.
Main Results:
- Dispase II digested key basement membrane components like collagen VI, fibronectin, and laminin.
- Expression of several growth factors (e.g., TGF-alpha, VEGF) and receptors (e.g., EGFR) was reduced.
- TEM revealed progressive disruption of the basal lamina and stromal collagen network with increased incubation time.
Conclusions:
- Dispase II significantly alters DHAM ultrastructure, particularly the basement membrane.
- Extended Dispase II incubation can lead to substantial structural disruption.
- These structural changes may negatively influence cell growth in cultured amniotic membrane explants.
