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

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.

Comparison of in vivo immune responses following transplantation of vascularized and non-vascularized human dermo-epidermal skin substitutes.

PURPOSE: Autologous bio-engineered dermo-epidermal skin substitutes (DESS) represent an alternative therapeutic option for a definitive treatment of skin defects in human patients. Largely, the interaction of host immune cells with transplanted DESS is considered to be essential for the granulation tissue formation, graft take, and its functionality. The aim of this study was to compare the spatiotemporal distribution and density of host-derived monocytes/macrophages and granulocytes in vascularized (vascDESS) versus non-vascularized DESS (non-vascDESS) in a rat model. METHODS: Keratinocytes and the stromal vascular fraction (SVF) were derived from human skin or human adipose tissue, respectively. Human SVF containing both endothelial and mesenchymal/stromal progenitors was used to develop a vascularized collagen type I-based dermal component in vitro. The donor-matched, monolayer-expanded adipose stromal cells lacking endothelial cells were used as a negative control. Subsequently, human keratinocytes were seeded on top of hydrogels to build dermo-epidermal skin grafts. After transplantation onto full-thickness skin wounds on the back of immuno-incompetent rats, grafts were excised and analyzed after 1 and 3 weeks. The expression of distinct inflammatory cell markers specific for host-derived monocytes/macrophages (CD11b, CD68) or granulocytes (HIS48) was analyzed by immunofluorescence microscopy. RESULTS: All skin grafts were infiltrated by host-derived monocytes/macrophages (CD11b+, CD68+) and granulocytes (HIS48+) between 1-3 week post-transplantation. When compared to non-vascDESS, the vascDESS showed an increased granulocyte infiltration at all time points analyzed with the majority of cells scattered throughout the whole dermal part. Whereas a moderate number of rat monocytes/macrophages (CD11b+, CD68+) were found in vascDESS at 1 week, only a few cells were detected in non-vascDESS. We observed a time-dependent decrease of monocytes/macrophages in all transplants at 3 weeks. CONCLUSIONS: These results demonstrate a distinct spatiotemporal distribution of monocytes/macrophages as well as granulocytes in our transplants that closely resemble the one observed during physiological wound healing. The differences identified between vascDESS and non-vascDESS may indicate that human endothelial cells lining blood capillaries of vascDESS accelerate infiltration of monocytes and leukocytes.