New skin-equivalent model from de-epithelialized amnion membrane

Abstract

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.

The basement membrane microenvironment directs the normalization and survival of bioengineered human skin equivalents

“…Since epithelial BMs are composed of an intricate network of proteins at the epithelial-stromal interface (Yurchenko and O'Rear, 1994;Christiano and Uitto 1996;Li et al 2001), BM assembly needs to be studied in biological systems in which a high degree of tissue complexity can be achieved, such as human skin equivalents (HSEs) (Andriani et al 2003;Berking and Herlyn 2001). However, HSEs have shown limited success forming structured BM (Black et al 2005;Yang et al 2006) due to MMP-mediated degradation of BM components after their synthesis and secretion (Amano et al 2001;Amano et al 2005). These studies have shown that Type IV Collagen can suppress this MMP-9 expression and stabilize the BM interface during the early stages of BM assembly.…”

“…Thus, it appears that the balance between degradation and stabilization of newly synthesized BM components is essential for BM assembly and maturation (Amano et al 2001) It has been shown that pre-existing BM proteins serve as a template for the deposition and integration of newly synthesized proteins at the dermal-epidermal junction (Vailly et al, 1995). HSEs constructed by seeding keratinocytes on an interface containing pre-existing BM components, such as de-epithelialized skin (Andriani et al 2003) or amniotic membrane (Yang et al 2006) promote rapid assembly of well-structured BM. This is thought to occur due to interactions between Type IV Collagen and β1 integrins (Fleischmajer et al 1998) that has been shown to provide an early scaffold for BM organization.…”

Properties of the amniotic membrane for potential use in tissue engineering

“…These experiments suggest a promising new approach for the repair of a prematurely ruptured foetal membrane (Portmann-Lanz et al, 2007). Cultivation and seeding of epithelial cells on an amnion scaffold is a frequently used method for ocular surface and skin reconstruction (Fatima et al, 2006;Yang et al, 2006;Capeans et al, 2003). And lastly, cultivation of endothelial cells on an AM scaffold has also been reported as a potential approach for vascular TE (Ishino et al, 2004;Tsai et al, 2007).…”

Production of an acellular matrix from amniotic membrane for the synthesis of a human skin equivalent

“…The HAM is the innermost layer of the placenta and is composed of: (1) a single layer of columnar or cuboid epithelial cells; (2) a basal membrane, which resembles that of skin both morphologically and ultrastructurally, consisting of laminin 5 and types IV, VII, XVII collagen (Wilshaw et al 2008); (3) an acellular compact layer; and (4) underlying fibroblast and spongy layers (Yang et al 2006). The HAM contains no blood vessels or nerves (Niknejad et al 2008).…”