The requirement for basement membrane antigens in the production of human epidermal/dermal composites in vitro

Abstract

The importance of a dermal element when providing permanent wound cover for skin loss has become evident as the shortcomings of pure epidermal grafts are recognized. We are developing a skin composite formed from sterilized human de-epidermized acellular dermis, keratinocytes and fibroblasts with the ultimate aim of using this composite to cover full-thickness excised burn wounds. These composites can be prepared with or without basement membrane (BM) antigens initially present on the dermis. This study investigates the importance of retaining BM antigens on the dermis to the production and appearance of these composites in vitro. Skin composites prepared from dermis with BM antigens either present or absent initially were studied throughout 3 weeks. Composites with BM antigens present initially were significantly better than those initially lacking BM antigens in: (i) the degree of epithelial cell attachment to the underlying dermis (hemidesmosomes were seen only in the former); (ii) the morphology of the epithelial layer; (iii) the consistent presence of collagen IV and laminin and the increasing expression of tenascin; and (iv) the amount of soluble collagen IV and fibronectin detected in the conditioned media. We conclude that an initial BM antigen template is vital in this skin composite model for the attachment and differentiation of the epithelial layer and for the subsequent remodelling of the BM in vitro.

Epithelialization of hydrogels achieved by amine functionalization and co-culture with stromal cells

“…In our own work we have shown extensive fibronectin production by fibroblasts in monoculture which is attenuated when epithelial cells are co-cultured with them [31] . Fibronectin acts as an adhesive protein in binding cells to both natural and synthetic substrates.…”

Development of a Basement Membrane Substitute Incorporated Into an Electrospun Scaffold for 3D Skin Tissue Engineering

“…22 However in our experience based on looking at the formation of hemidesmosomes in reconstructed skin based on decellularised dermis they failed to achieve any structural organization even after 3 weeks in vitro unless there was some basement membrane already present in which case they were able to form hemi-desmosomes which help anchor the keratinocytes to the underlying dermal collagen. 23 In the current study we have provided cells with a physically dense nano-fibrous mesh as a basement membrane substitute. Alternative approaches of introducing natural ECM proteins may well work but are rarely going to be suitable for scale -up for clinical use for reasons of cost and more importantly regulatory concerns about safety will mean that such ECM proteins must be sourced so they are no risk to the patient-implying recombinant proteins or synthesised peptide sequences from these proteins.…”

The Development and Characterization of an Organotypic Tissue-Engineered Human Esophageal Mucosal Model

“…However there is little published characterisation of this model itself and the approach does not include a basement membrane (BM). Experiments on human skin have shown the importance of the presence of a basement membrane in the degree of epithelial cell attachment, the differentiation of the epithelium and the subsequent remodelling of the BM in vitro 17 . A rat-derived de-epithelialised acellular oesophagus with an intact BM has been used for the culture of oesophageal cells 18 , however in these experiments the cells used were also derived from the rat.…”