Characterization of M1 and M2 polarization of macrophages in vascularized human dermo-epidermal skin substitutes in vivo

Pediatr Surg Int. 2018 Feb;34(2):129-135. doi: 10.1007/s00383-017-4179-z. Epub 2017 Nov 9.


Aims and objectives: Vascularized bio-engineered human dermo-epidermal skin substitutes (vascDESS) hold promise for treating burn patients, including those with severe full-thickness wounds. We have previously shown that vascDESS promote wound healing by enhanced influx of macrophages and granulocytes. Immediately following transplantation, macrophages infiltrate the graft and differentiate into a pro-inflammatory (M1) or a pro-healing M2 phenotype. The aim of this study was to characterize the activation state of macrophages infiltrating skin transplants at distinct time points following transplantation.

Methods: Keratinocytes and the stromal vascular fraction (SVF) were derived from human skin or adipose tissue, respectively. Human SVF containing both endothelial and mesenchymal/stromal cells was used to generate vascularized dermal component in vitro, which was subsequently covered with human keratinocytes. Finally, vascDESS were transplanted on the back of immuno-incompetent rats, excised, and analyzed after 1 and 3 weeks using immunohistological techniques.

Results: A panel of markers of macrophage M1 (nitric oxide synthase: iNOS) and M2 (CD206) subclass was used. All skin grafts were infiltrated by both M1 and M2 rat macrophages between 1-3 weeks post-transplantation. CD68 (PG-M1) was used as a pan-macrophage marker. The number of CD68+CD206+ M2-polarized macrophages was higher in 3-week transplants as compared to early-stage transplants (1 week). In contrast, the number of CD68+iNOS+ M1 cells was markedly decreased in later stages in vivo.

Conclusions: Macrophages exhibit a heterogeneous and temporally regulated polarization during skin wound healing. Our results suggest that the phenotype of macrophages changes during healing from a more pro-inflammatory (M1) profile in early stages after injury, to a less inflammatory, pro-healing (M2) phenotype in later phases in vivo.

Keywords: Adipose stem cells; Macrophages; Skin tissue engineering.

MeSH terms

  • Adipose Tissue / cytology*
  • Adolescent
  • Animals
  • Biomarkers / metabolism
  • Cells, Cultured
  • Child
  • Child, Preschool
  • Dermis / cytology*
  • Dermis / metabolism
  • Epidermal Cells*
  • Epidermis / metabolism
  • Humans
  • Infant
  • Keratinocytes / cytology*
  • Keratinocytes / metabolism
  • Lectins, C-Type / metabolism
  • Macrophages / cytology*
  • Macrophages / metabolism
  • Male
  • Mannose Receptor
  • Mannose-Binding Lectins / metabolism
  • Mesenchymal Stem Cells / cytology
  • Models, Animal
  • Nitric Oxide Synthase Type II / metabolism
  • Phenotype
  • Rats
  • Receptors, Cell Surface / metabolism
  • Skin Transplantation / methods*
  • Skin, Artificial
  • Tissue Engineering / methods*
  • Wound Healing


  • Biomarkers
  • Lectins, C-Type
  • Mannose Receptor
  • Mannose-Binding Lectins
  • Receptors, Cell Surface
  • Nitric Oxide Synthase Type II