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123. Kongress der Deutschen Gesellschaft für Chirurgie

Deutsche Gesellschaft für Chirurgie

02. bis 05.05.2006, Berlin

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Construction of tissue-engineered skin by genetically modified human keratinocytes and fibroblasts in a collagen-GAG matrix

Meeting Abstract

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  • corresponding author T. Egana - Department of Plastic Surgery and Hand Surgery, Burn Care Center
  • F. Liu - Department of Plastic Surgery and Hand Surgery, Burn Care Center
  • S. Krengel - Department of Dermatology
  • S. Krüger
  • W. Lindenmaier
  • H.-G. Machens - Department of Plastic Surgery and Hand Surgery, Burn Care Center

Deutsche Gesellschaft für Chirurgie. 123. Kongress der Deutschen Gesellschaft für Chirurgie. Berlin, 02.-05.05.2006. Düsseldorf, Köln: German Medical Science; 2006. Doc06dgch4691

The electronic version of this article is the complete one and can be found online at: http://www.egms.de/en/meetings/dgch2006/06dgch583.shtml

Published: May 2, 2006

© 2006 Egana et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en). You are free: to Share – to copy, distribute and transmit the work, provided the original author and source are credited.

Outline

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Einleitung: To evaluate the biological character of PDGF-BB gene modified human keratinocytes in tissue-engineered skin templates including fibroblasts and collagen-GAG matrix for full-thickness skin defects.

Material und Methoden: We isolated human keratinocytes from human foreskin, cultured them in vitro and introduced a gene encoding human PDGF-BB into human keratinocytes using an adenovirus-derived vector (pAdcos bicistronic). Similarily, human fibroblasts were modified to express a combination of VEGF165 and bFGF as angiogenic stimuli. Growth curve, cloning efficiency and target gene expression were measured in vitro. The gene modified keratinocytes and fibroblasts were seeded into the collagen-GAG matrix to reconstruct the tissue-engineered skin and to observe the growth of seeded cells in vitro for two weeks. Two types of skin substitutes were prepared with keratinocytes and fibroblasts: group A, containing both modified keratinocytes and fibroblasts and group B, containing both unmodified keratinocytes and fibroblasts. Matrices were transplanted into full-thickness wounds on athymic nu/nu mice. Wound healing was evaluated, tissue samples were harvested and examined by means of histology, immunohistochemistry and microangiography. The results were compared among the groups.

Ergebnisse: The in vitro results showed successful gene transfection of both keratinocytes and fibroblasts with the adenoviral vector by fluorescent microscope inspection of the cells and ELISA measurement of the culture medium. Protein expression persisted for at least 1 week with a peak at about 3 to 7 days after transfection. Keratinocytes and fibroblasts of group A maintained significantly higher proliferative capacity in vitro after seeding in collagen-GAG matrix both in vitro and in vivo, compared to group B (P<0.001). Angiogenesis were also significantly improved in grafts of group A compared to the control grafts in group B (P<0.05).

Schlussfolgerung: : PDGF-BB gene transfection can promote the proliferation of human keratinocytes in our model. Combined expression of VEGF165 and bFGF has a strong angiogenic impact on the accelerated vascularisation of bioactivated matrices. Our model may become a feasible tool for constructing bioengineered skin substitutes using cell-based gene therapy.