Construction of a multi-layer skin substitute: Simultaneous cultivation of keratinocytes and preadipocytes on a dermal template

Abstract

Background After deep excision of burn eschar down to the muscle fascia patients have a non-reversible loss of the skin and underlying subcutaneous tissue. These patients would benefit from the development of a sufficient epidermal, dermal, and hypodermal tissue-engineered replacement provided by new technologies of tissue engineering. The aim of the present study was to determine whether keratinocytes and preadipocytes grow simultaneously on a bovine-derived collagen–elastin matrix under in vitro conditions in order to obtain a multi-layer skin substitute. Methods Human keratinocytes as well as human preadipocytes were seeded onto a collagen–elastin matrix (Matriderm ®). Human preadipocytes were isolated from human subcutaneous adipose tissue and seeded onto the scaffold directly after isolation. Keratinocytes were isolated from fresh human split-thickness skin harvests and seeded onto the surface of the scaffold after 4 days of proliferation. Twenty one days after seeding all scaffolds were histologically evaluated, using hematoxylin eosin, immunohistochemical staining with collagen IV as well as immunofluorescence labeling with anti-Ki67 antibody and DAPI (4′,6-diamidino-2-phenylindole). Results Simultaneous growth of keratinocytes and preadipocytes could be observed on the collagen–elastin matrix. Keratinocytes adhered well to the surface of the matrix and formed a confluent epidermis-like layer. Preadipocytes adhered well and also penetrated into the deeper layers of the matrix. Conclusion In this study, a collagen–elastin matrix served as a suitable scaffold for simultaneous culturing of preadipocytes and keratinocytes. Preadipocytes showed good penetration into deeper layers of the scaffold, whereas keratinocytes attached only to the uppermost surface of the matrix. This approach towards a multi-layered skin substitute might be a useful asset for future reconstructive surgery.