Keratinocyte‐driven Contraction of Reconstructed Human Skin

Abstract

We have previously reported that reconstructed human skin, using deepidermized acellular sterilized dermis and allogeneic keratinocytes and fibroblasts, significantly contracts in vitro. Contracture of split skin grafts in burns injuries remains a serious problem and this in vitro model provides an opportunity to study keratinocyte/mesenchymal cell interactions and cell interactions with extracted normal human dermis. The aim of this study was to investigate the nature of this in vitro contraction and explore several approaches to prevent or reduce contraction. Three different methodologies for sterilization of the dermal matrix were examined: glycerol, ethylene oxide and a combination of glycerol and ethylene oxide. While the nature of the sterilization technique influenced the extent of contraction and thinner dermal matrices contracted proportionately more than thicker matrices, in all cases contraction was driven by the keratinocytes with relatively little influence from the fibroblasts. The contraction of the underlying dermis did not represent any change in tissue mass but rather a reorganization of the dermis which was rapidly reversed (within minutes) when the epidermal layer was removed. Pharmacological approaches to block contraction showed forskolin and mannose-6-phosphate to be ineffective and ascorbic acid-2-phosphate to exacerbate contraction. However, Galardin, a matrix metalloproteinase inhibitor and keratinocyte conditioned media, both inhibited contraction.

Development of biodegradable electrospun scaffolds for dermal replacement

“…Again this is further evidence of an epithelial/mesenchymal dialogue and suggests that contraction of these scaffolds by skin cells may not prove to be a major problem in vivo. We have found contraction of tissue engineered skin based on a natural dermal scaffold -sterilised allodermis -can be a considerable problem [31][32][33][34]. Contraction of conventional splitthickness skin grafts is also a major concern as recently discussed from this laboratory [35], accordingly this will definitely be one area for further investigation prior to progressing this to clinical application but these preliminary results in this area look very promising.…”

Fluorescence imaging of reepithelialization from skin explant cultures on acellular dermis

“…Thus fibroblast‐driven contraction of the substrate did not interfere with neo‐epidermal area measurements. Similarly, keratinocyte‐induced contraction of the dermis, which is known to occur with thin (0.2–0.6 mm) dermal matrices repopulated with keratinocytes, 30 was not a problem in our model utilizing thicker DEDs. The outgrowth characteristics of neo‐epidermis formed on alternative substrates, such as synthetic matrices, dermal equivalents, or DEDs with abolished basement membrane structures, were not investigated in this study.…”

“…The model therefore concentrates directly on epidermal cells, abolishing the mixed cell–type interactions and distant effects acting in vivo, e.g., during cutaneous tissue repair. Although primarily aimed for studies of pure keratinocyte‐populated epithelium, the model may be modified to allow investigations of epithelial–mesenchymal interactions, e.g., by coculturing the DEDs with dermal fibroblasts 29,30 …”

Organotypic culture of normal, dysplastic and squamous cell carcinoma‐derived oral cell lines reveals loss of spatial regulation of CD44 and p75NTR in malignancy

“…HOME culture of oral epithelial cells at the air/liquid interface uses techniques that have been optimised for other epithelial cells by several research groups over many years (30, 33, 34). Key features of HOME culture using intact acellular dermis include retention of an intact basement membrane, promotion of hierarchical epithelial tissue formation and good compatibility with established immunohistochemical techniques.…”