Construction of a multi-layer skin substitute: Simultaneous cultivation of keratinocytes and preadipocytes on a dermal template Maike Keck a, *, Daniela Haluza b,c , David B. Lumenta a , Sonja Burjak a , Bettina Eisenbock a , Lars-Peter Kamolz a , Manfred Frey a a Vienna Burn Centre, Division of Plastic and Reconstructive Surgery, Department of Surgery, Medical UniversityVienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria b Institute of Environmental Health, Center for Public Health, Medical University Vienna, Austria c Department of Pathology, Medical University Vienna, Austria burns 37 (2011) 626–630 article info Article history: Accepted 31 July 2010 Keywords: Preadipocyte Keratinocytes Tissue engineering Collagen scaffold Collagen–elastin matrix Multi-layered skin substitute 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 colla- gen–elastin matrix (Matriderm 1 ). Human preadipocytes were isolated from human subcu- taneous 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 hema- toxylin eosin, immunohistochemical staining with collagen IV as well as immunofluores- cence labeling with anti-Ki67 antibody and DAPI (4 0 ,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 simul- taneous culturing of preadipocytes and keratinocytes. Preadipocytes showed good penetra- tion 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. # 2010 Elsevier Ltd and ISBI. All rights reserved. * Corresponding author. Tel.: +43 1 40400 6860; fax: +43 1 40400 6862. E-mail address: maike.keck@meduniwien.ac.at (M. Keck). available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/burns 0305-4179/$36.00 # 2010 Elsevier Ltd and ISBI. All rights reserved. doi:10.1016/j.burns.2010.07.016