Elsevier

Burns

Volume 37, Issue 4, June 2011, Pages 626-630
Burns

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

https://doi.org/10.1016/j.burns.2010.07.016Get rights and content

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.

Section snippets

Background

Treatment of severely burned patients has significantly improved over the past decades. Thus, the focus has shifted from survival to an improved functional as well as aesthetic outcome. Patients after deep excision of burn eschar down to the muscle fascia suffer from an irreversible loss of the skin and underlying subcutaneous tissue. Especially these patients would benefit from the development of sufficient epidermal, dermal, and hypodermal replacement. Great efforts have been made to develop

Isolation, culturing, and seeding of preadipocytes

Connective tissue was removed from excised human adipose tissue. Isolation of preadipocytes was performed as described in earlier studies [13]. Afterwards, the cells were filtrated over a 70-mM nylon filter and after that seeded onto a transwell plate (Transwell 24 mm Inserts Costar 3450). A collagen–elastin matrix (Matriderm®, Dr. Otto Suwelack Skin and Health Care GmbH, Billerbeck, Germany) was positioned on top of the cell suspension (Fig. 1), so that preadipocytes could enter the scaffold

Results

In all five experiments, the keratinocytes, which were isolated from human tissue adhered well to the surface of the matrix, and formed a confluent epidermis-like sheet in one to three layers, as shown with nuclear staining using DAPI (Fig. 2). Preadipocytes showed good adherence and good penetration into the matrix (Fig. 3). Keratinocytes did not penetrate into deeper layers of the scaffold. The average penetration depth was 200 μm.

Immunohistochemical staining with basal membrane collagen IV

Discussion

In burn patients deep excision to the muscle fascia is one of the most frequent forms of debridement, and full-scale substitution of the excised tissue seems so far to be an elusive reconstructive goal.

Some new therapeutic options have offered alternatives to treat massive skin loss: cultured autologous and allogenic keratinocytes grafts, autologous or allogenic composites, acellular biological matrices, and cellular matrices with fibrin sealant or various types of collagen and hyaluronic acid

Conflict of interest

Authors declare that there is no conflict of interest.

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