Use of A Collagen/Elastin Matrix As Transport Carrier System to Transfer Proliferating Epidermal Cells to Human Dermis in Vitro

This in vitro study describes a novel cell culture, transport, and transfer protocol that may be highly suitable for delivering cultured proliferating keratinocytes and melanocytes to large open skin wounds (e.g., burns). We have taken into account previous limitations identified using other keratinocyte transfer techniques, such as regulatory issues, stability of keratinocytes during transport (single cell suspensions undergo terminal differentiation), ease of handling during application, and the degree of epidermal blistering resulting after transplantation (both related to transplanting keratinocyte sheets). Large numbers of proliferating epidermal cells (EC) (keratinocytes and melanocytes) were generated within 10-14 days and seeded onto a three-dimensional matrix composed of elastin and collagen types I, III, and V (Matriderm®), which enabled easy and stable transport of the EC for up to 24 h under ambient conditions. All culture conditions were in accordance with the regulations set by the Dutch Central Committee on Research Involving Human Subjects (CCMO). As an in vitro model system for clinical in vivo transfer, the EC were then transferred from Matriderm onto human acellular dermis during a period of 3 days. After transfer the EC maintained the ability to regenerate into a fully differentiated epidermis containing melanocytes on the human dermis. Proliferating keratinocytes were located in the basal layer and keratin-10 expression was located in differentiating suprabasal layers similar to that found in human epidermis. No blistering was observed (separation of the epidermis from the basement membrane). Keratin-6 expression was strongly upregulated in the regenerating epidermis similar to normal wound healing. In summary, we show that EC-Matriderm contains viable, metabolically active keratinocytes and melanocytes cultured in a manner that permits easy transportation and contains epidermal cells with the potential to form a pigmented reconstructed epidermis. This in vitro study has produced a robust protocol that is ready for clinical studies in the future

A cytotoxic analysis of antiseptic medication on skin substitutes and autograft

Background: There is an increasing demand for the clinical application of human skin substitutes (HSSs) for treating ulcers, burns and surgical wounds. Due to this increasing demand and due to the simultaneous requirement for the administration of topical antiseptic medications, there is a need to determine potential cytotoxic effects of these medications on HSSs compared with autograft skin. Objectives: To perform such an evaluation. Methods: Two different HSSs were used (autologous reconstructed epidermis on fibroblast-populated human dermis and allogeneic reconstructed epidermis on a fibroblast-populated rat collagen gel) and were compared with conventional full-thickness autograft. Twelve different antiseptics were applied topically to the stratum corneum in vitro for 24 h. The degree of cytotoxicity was analysed as detrimental changes in histology, metabolic activity (MTT assay) and RNA staining of tissue sections. Results: The antiseptic medications tested showed different degrees of cytotoxicity. Acticoat®, Aquacel Ag®, Dermacyn®, Fucidin®, 0.5% silver nitrate solution and chlorhexidine digluconate were not cytotoxic for either HSS or autograft, and can therefore be used as required. Flamazine® and zinc oxide cream resulted in moderate cytotoxicity. However, application of Betadine®, cerium-silver sulfadiazine cream, silver sulfadiazine cream with 1% acetic acid and Furacine® resulted in a substantial decrease in cell viability and a detrimental effect on tissue histology when applied to autograft and especially to HSS. Conclusions: Due to the potential cytotoxic effect of some antiseptics on HSS, it is advised that clinicians balance the cytotoxicity of the medication, its antiseptic properties and the severity of colonization in choosing which one to apply

Methods to study differences in cell mobility during skin wound healing in vitro

Wound healing events which occur in humans are difficult to study in animals due to differences in skin physiology. Furthermore there are increasing restrictions in Europe for using animals for testing the therapeutic properties of new compounds. Therefore, in line with the 3Rs (reduction, refinement and replacement of test animals), a number of human in vitro models of different levels of complexity have been developed to investigate cell mobility during wound healing. Keratinocyte, melanocyte, fibroblast and endothelial cell mobility are described, since these are the residential cells which are responsible for restoring the main structural features of the skin. A monolayer scratch assay is used to study random fibroblast and endothelial cell migration in response to EGF and bFGF respectively and a chemotactic assay is used to study directional fibroblast migration towards CCL5. In order to study endothelial sprouting in response to bFGF or VEGF, which involves continuous degradation and resynthesis of a 3D matrix, a fibrin gel is used. Human physiologically relevant tissue-engineered skin models are used to investigate expansion of the stratified, differentiated epidermis (keratinocytes and melanocytes) over a fibroblast populated dermis and also to study migration and distribution of fibroblasts into the dermis. Together these skin models provide a platform for testing the mode of action of novel compounds for enhanced and scar free wound healing