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Fusion of epithelial sheets as seen in formation of the chick amnion.

Fusion of the amniotic folds over the chick embryo provides an example of epithelial fusion in which one group of ectodermal cells appears to contribute to traction and a second group appears to respond by moving to the midline, becoming internalized and degenerating. Neutral red staining of living embryos and transmission electron microscopy indicate that a patch of dying cells is present in the fusion region. Scanning electron microscopy shows that ectoderm cells at the rim of the amniotic folds are cylindrical in shape with long axes oriented parallel to the rim. These cells have a smooth surface, but show increasing amounts of blebbing toward the midline. Characteristically, a globular region of rounded cells showing extreme blebbing is present at the midline. Just anterior to the globular region, flattened cells are present on the surface with many taut cellular processes. Orientation of ectodermal cells around the region of fusion suggests that traction is exerted from these flattened cells. When treated with cytochalasin D, posterior progression of the zone of fusion is blocked. Also, the flattened cells anterior to the globular region round up, revealing many elongated, anteriorly-directed cytoplasmic processes presumably leading to sites of original attachment.

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Affinity chromatography of primary human amnion interferon.

The chromatographic behavior of human amniotic interferon on various affinity chromatography ligands was studied. Most of this interferon bound strongly to bovine plasma albumin-agarose, cibacron blue F3GA-agarose, concanavalin A-agarose and L-tryptophyl-L-tyrosine-omega-carboxyl-pentyl-agarose. After binding most of the interferon activity was eluted only with 50 percent ethylene glycol, showing the high hydrophobicity of this interferon. Smaller quantities could be recovered after phosphate-buffered saline elution or with increased salt concentration. On BPA-omega-carboxy-pentyl-agarose and omega-amino-hexyl-agarose, the majority of the biological activity was found in the break-through fraction (eluted with phosphate buffered saline) while some interferon was displaced with high salt or ethylene glycol. Increasing the salt concentration and lowering the pH was necessary to elute interferon from zinc chelate-agarose. These patterns indicate that human amniotic interferon is similar to human fibroblast (beta) interferon but different from human leukocyte (alpha) interferon. However, the heterogeneity displayed by amniotic interferon on bovine plasma albumin-agarose requires further investigation.

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New skin-equivalent model from de-epithelialized amnion membrane.

The presence of pre-existing basement membrane (BM) components improves the morphogenesis of epidermis and BM in constructing a human living skin-equivalent (LSE). De-epithelialized amniotic membrane (AM) retains key BM components. We have therefore investigated the usefulness of AM for constructing LSE. De-epithelialized AM was overlaid on type I collagen gel embedded with fibroblasts. Normal human keratinocytes (NHKs) were then seeded onto the epithelial side of the AM to construct an AM-LSE. A conventional LSE was constructed by seeding NHKs on a fibroblast-populated type I collagen gel. When the keratinocytes reached confluence, the LSE was lifted to the air-liquid interface and cultured for up to 3 weeks. Samples were harvested at various times and investigated morphologically, immunohistochemically, and ultrastructurally. In AM-LSE, the epidermis was better stratified, with more compact, polarized, columnar basal cells, and the expression of differentiation and proliferation markers was more similar to that of normal human skin than was that of LSE without AM. A more continuous BM and better-developed hemidesmosomes were found in AM-LSE. The epidermis of AM-LSE outgrew much faster than that of LSE without AM. When transplanted onto nude mice, both LSEs took well; however, the AM-LSE graft showed better morphogenesis of the epidermis, BM, and hemidesmosomes. The better epidermal morphology and better-developed BM in AM-LSE in vitro and in vivo indicates its superiority over LSE without AM for clinical applications.

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