214 Chapter 7 Figure 2. Monocytes after 7 days of culture in (burn-injured) FSEs. (A) Immunohistochemical CD68 staining of an injured FSE. (B) Number of CD68+ cells (macrophages) per FSE after isolation based on flow cytometry; dashed line indicates the number of monocytes added to the dermal side of the FSE. Percentage of CD68+ cells (macrophages) that were (C) CD14+; (D) CD11b+; (E) HLA-DR+; (F) CD163+. Experiments were performed in duplicate using keratinocytes and fibroblasts from 6 different donors and monocytes from 4 different donors. Only comparisons between monocytes in matrix, in uninjured FSEs and in burn-injured FSEs are shown. Statistically significant differences were calculated using Wilcoxon signed-rank test. Significant differences are indicated by asterisks: *: p < 0.05. Production of inflammatory cytokines seem to be increased upon administration of monocytes into full skin equivalents, regardless of burn injury Next, cytokine levels in the culture media of the FSEs were determined at day 3 (when medium was changed) and at day 7 (when FSEs were terminated) to study the effect of administering monocytes into the FSE (Figure 3). FSEs without monocytes produced high levels of IL-6, IL-8 (CXCL8) and MCP-1 (CCL2) (Figure 3 and Figure 5). Burn injury increased the levels of IL-4, IL-6, IL-8 and IL-12p70 and decreased the level of IP-10 (CXCL10) (Supplementary Figure 2) at both day 3 and day 7. The inclusion of monocytes in the FSEs seemed to further increase the levels of IL-4, IL-6, IL-8 and IL-12p70, and burn injury led to a further increase of IL-1β, IL-6, IL-8 and IL-12p70 at both time points. No significant differences between the different conditions were observed for the levels of IL-2, MCP-1 and TNF-α (Supplementary Figure 2).
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