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J. S. Mehta, A. Riau, D. T. Tan, R. W. Beuerman; Analysis of Matrix Proteins, Growth Factors and Membrane Surface in Commercial Available Freeze-Dried Amniotic Membrane. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5745.
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Human amniotic membrane (HAM) has been used with great success in ophthalmology as a surgical patch and as a substrate for stem cell expansion. However, the possibility of transmission of pathogens is an issue and currently its procurement and storage is cumbersome. The aim of this study was to compare a commercially available freeze-dried HAM (FDHAM) with conventional cryopreserved HAM (CHAM) with respect to their cellular components and membrane surface.
The difference in the extra-cellular matrix (ECM) and growth factors (GF) of the two materials was examined by light microscopy and immunohistochemistry using anti-bodies to collagen I, II, IV, VI, VII, elastin, laminin-5, fibronectin and thrombospondin-1, TGFα, TGFβ1, TGFβ2 recep, VEGF, KGF, EGFR, FGF, PDGF-A, PDGF-B, IGF-1. Confirmation of the presence of ECM and GF proteins was made by Western Blot analysis. Examination of the smoothness of the membrane surface was done by scanning electron microscopy (SEM) and by atomic force microscopy (AFM).
Light microscopy with PAS staining showed no evidence of an intact basement membrane on FDHAM. Immuno-recognition of ECM molecules was lost for all basement membrane matrix proteins but was present for collagen I/II/VI and fibronectin. Growth factor expression was similar in both CHAM and FDHAM. Western blot analysis confirmed the presence of the proteins localized by immuno-histochemistry. SEM demonstrated a qualtitavely smooth surface on the FDHAM, confirmed quantitatively by AFM.
Characterization of the components of commercially available FDHAM has shown that the ECM is affected by its preparation. However growth factor expression is not affected and the membrane provides a smooth surface for cellular growth.
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