Construction of a multi-layer skin substitute: Simultaneous cultivation of keratinocytes and preadipocytes on a dermal template
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.
References (32)
- et al.
New research in breast reconstruction: adipose tissue engineering
Clin Plast Surg
(2002) Experimental implantation of adipose tissue fragments
Br J Plast Surg
(1989)- et al.
Human clinical experience with adipose precursor cells seeded on hyaluronic acid-based spongy scaffolds
Biomaterials
(2008) - et al.
Generation of mature fat pads in vitro and in vivo utilizing 3-D long-term culture of 3T3-L1 preadipocytes
Exp Cell Res
(2004) - et al.
Tissue engineering of white adipose tissue using hyaluronic acid-based scaffolds. I.: In vitro differentiation of human adipocyte precursor cells on scaffolds. Biomaterials
(2003) - et al.
First experiences with the collagen–elastin matrix Matriderm as a dermal substitute in severe burn injuries of the hand
Burns
(2007) - et al.
Matriderm® versus Integra®: a comparative experimental study
Burns
(2009) - et al.
Introduction to soft tissue augmentation: a historical perspective
- et al.
Skin tissue engineering for tissue repair, regeneration
Tissue Eng B Rev
(2008) Design principles for composition and performance of cultured skin substitutes
Burns
(2001)