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J. S. Mehta, R. Beuerman, Z. M. Thein, D. T. Tan; Modification of Human Amniotic Membrane as a Carrier for Stem Cell Transplantation. Invest. Ophthalmol. Vis. Sci. 2007;48(13):453.
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Tissue engineered stem cell transplantation has revolutionized ocular surface surgery in the last decade. Human amniotic membrane (HAM) has most of the attributes necessary for a substrate for engineered cells from corneal limbal, conjunctival or oral sources. Growth factors and extra-cellular matrix molecules that are native to HAM are thought to enhance the adhesion and phenotype of the epithelial cells for transplantation. The aim of this study was to examine the effect of preparative procedures to remove the HAM epithelium on these critical biomolecules.
Cryopreserved amniotic membrane was subjected to different treatments to allow subsequent denudation i.e. 1.2UDispase for 2hours, 0.02%EDTA, 5M urea and 20% ethanol. The effect on the extracellular matrix and growth factors 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, TGFß1, TGFß2 recep, VEGF, KGF, EGFR, FGF, PDGF-A, PDGF-B. Confirmation of the presence of extra-cellular matrix and growth factor proteins was made Western Blot analysis. Growth rates were compared on different treated membranes by measuring areas of cellular outgrowth from primary conjunctival explants.
After treatment with Dispase, immuno-recognition of extra-cellular matrix molecules was lost except for collagen I/II. Incubation with EDTA, urea and alcohol preserved immuno-recognition of all extra-cellular matrix components as and an intact basement membrane was seen on light microscopy with PAS staining. Growth factor expression was robust to the various preparative techniques. Western blot analysis confirmed the presence of the proteins localized by immuno-histochemistry. Growth rates were significantly faster on EDTA, urea and alcohol treated membranes compared to Dispase treated HAM.
Dispase denudation for 2 hrs has been a standard treatment for HAM; however, less damage to extra-cellular matrix molecules responsible for cell growth and epithelial organization and a considerable savings in time can be realized with novel alternative agents e.g. urea and alcohol that allow rapid denudation and are preferable to maintain HAM integrity as a structural matrix for epithelial cell expansion.
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