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Differential expression of granulocyte, macrophage, and hypoxia markers during early and late wound healing stages following transplantation of tissue-engineered skin substitutes of human origin

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Abstract

Purpose

Human pigmented tissue-engineered skin substitutes represent an advanced therapeutic option to treat skin defects. The inflammatory response is one of the major factors determining integration and long-term survival of such a graft in vivo. The aim of the present study was to investigate the spatiotemporal distribution of host-derived macrophage and granulocyte graft infiltration as well as hypoxia-inducible factor 1 alpha (HIF-1-alpha) expression in a (nu/nu) rat model.

Methods

Keratinocytes, melanocytes, and fibroblasts derived from human skin biopsies were isolated, cultured, and expanded in vitro. Dermal fibroblasts were seeded into collagen type I hydrogels that were subsequently covered by keratinocytes and melanocytes in 5:1 ratio. These pigmented dermo-epidermal skin substitutes were transplanted onto full-thickness skin wounds on the back of immuno-incompetent rats and analyzed at early (1 and 3 weeks) and late (6 and 12 weeks) stages of wound healing. The expression of distinct inflammatory cell markers specific for granulocytes (HIS48) or macrophages (CD11b, CD68), as well as HIF-1-alpha were analyzed and quantified by immunofluorescence microscopy.

Results

Our data demonstrate that granulocytes infiltrate the entire graft at 1 week post-transplantation. This was followed by monocyte/macrophage recruitment to the graft at 3–12 weeks. The macrophages were initially restricted to the borders of the graft (early stages), and were then found throughout the entire graft (late stages). We observed a time-dependent decrease of macrophages. Only a few graft-infiltrating granulocytes were found between 6–12 weeks, mostly at the graft borders. A heterogeneous expression of HIF-1-alpha was observed at both early and late wound healing stages.

Conclusions

Our findings demonstrate the spatiotemporal distribution of inflammatory cells in our transplants closely resembles the one documented for physiological wound healing.

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Acknowledgments

This work was financially supported by the EU-FP6 project EuroSTEC (soft tissue engineering for congenital birth defects in children: contract: LSHB-CT-2006-037409), by the EU-FP7 project EuroSkinGraft (FP7/2007-2013: grant agreement nr 279024), by the EU-FP7 (MultiTERM, grant agreement nr 238551), and the Clinical Research Priority Programs (CRPP) of the Faculty of Medicine of the University of Zurich. We are particularly grateful to the Gaydoul Foundation and the sponsors of “DonaTissue” (especially Thérèse Meier and Robert Zingg) for their generous financial support and interest in our work.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Tissue Biology Research Unit, University Children’s Hospital Zurich, Zurich, Switzerland

    Agnieszka S. Klar, Sophie Böttcher-Haberzeth, Thomas Biedermann, Katarzyna Michalak & Ernst Reichmann

  2. Department of Surgery, University Children’s Hospital Zurich, Zurich, Switzerland

    Sophie Böttcher-Haberzeth & Martin Meuli

  3. Children’s Research Center, University Children’s Hospital Zurich, Zurich, Switzerland

    Agnieszka S. Klar, Sophie Böttcher-Haberzeth, Thomas Biedermann, Katarzyna Michalak, Ernst Reichmann & Martin Meuli

  4. Surgical Center, Hospital in Enköping, Enköping, Sweden

    Marta Kisiel

Authors
  1. Agnieszka S. Klar
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  2. Sophie Böttcher-Haberzeth
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  3. Thomas Biedermann
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  4. Katarzyna Michalak
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  5. Marta Kisiel
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  6. Ernst Reichmann
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  7. Martin Meuli
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Corresponding author

Correspondence to Martin Meuli.

Additional information

A. S. Klar and S. Böttcher-Haberzeth authors contributed equally to this paper.

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Klar, A.S., Böttcher-Haberzeth, S., Biedermann, T. et al. Differential expression of granulocyte, macrophage, and hypoxia markers during early and late wound healing stages following transplantation of tissue-engineered skin substitutes of human origin. Pediatr Surg Int 30, 1257–1264 (2014). https://doi.org/10.1007/s00383-014-3616-5

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  • DOI: https://doi.org/10.1007/s00383-014-3616-5

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