Phenotypic analysis of migratory dendritic cell (DC) subsets from skin and gingiva.

<p>Chemotaxis, maturation and differentiation-associated marker expression on DC subsets 1–5 from skin vs. gingiva, shown per indicated subset (*P<0.05, n = 4–9 for skin and gingiva). Explants (6mm diameter) from skin and gingiva were taken and cultured floating in medium for 48h, after which they were discarded and migrated DC harvested, stained and analysed by flowcytometry.</p

Inflammatory cytokine release profile of skin vs. gingival explants.

<p>Shown in pg/ml and measured in supernatants of skin and gingiva explants (6 mm diameter, 3mm depth) after 48 h of culture in 1 ml volume. IL-8, IL-6, IL-1β, IL-10 and TNFα were all significantly higher in the gingiva-conditioned cultures, whereas IL-12p70 levels were below the detection limit for both skin and gingiva (*P<0.05, **P<0.05; n = 3 skin, n = 3 gingiva).</p

Dendritic cell (DC) subset definition and distribution.

<p>Dendritic cell (DC) subset definition and distribution according to CD1a and CD14 expression in CD11c^hi DC migrated from human skin or gingiva. Explants (6mm diameter) from skin and gingiva were taken and cultured floating in medium for 48h, after which they were discarded and migrated DC harvested, stained and analysed by flowcytometry. (A) Flow cytometry dot plots with gates denoting five migrated DC subsets (numbered 1 to 5) in skin and gingiva. (B) Frequency distribution of the five subsets among migrated DC (n = 9).</p

Comparison of cell density between full-thickness skin and gingiva (per 100μm tissue cross-section).

<p>Comparison of cell density between full-thickness skin and gingiva (per 100μm tissue cross-section).</p

Dendritic cell (DC) marker expression, density and distribution over full-thickness human gingiva or skin.

<p>(A) CD1a staining of representative full-thickness skin (left panel) and gingiva (right panel) biopsies. Red dotted lines denote full-area epithelial surface (left panel, used for quantitation as shown in Fig 1B upper panel) and 100 μm wide full-thickness cross-section (right panel, used for quantitation as shown in Fig 1B lower panel, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180333#sec002" target="_blank">Materials and Methods</a>). (B) Quantitation of CD1a⁺ DC according to total epithelium area (upper panel) or epithelium area in 100 μm wide full-thickness cross-section (lower panel), see areas denoted by red dotted lines in Fig 1A for the respective definitions (n = 10). The number of positively stained cells in the epidermis, dermis, mucosal epithelium and subjacent lamina propria, were assessed for each sample per 100 μm² tissue. * P<0.05, ***P<0.001. (C) Representative staining of indicated additional DC maturation/differentiation markers shows distribution between epithelial and underlying connective tissue layers in skin and gingival biopsies (n = 10).</p

Monoclonal antibodies used for immunohistochemical staining.

<p>Monoclonal antibodies used for immunohistochemical staining.</p

Secretion of angiogenic factors, cytokines and chemokines by endothelial cells.

<p>A) Secretion of angiogenic factors 24 h after a 4 h exposure to 0, 2 and 10 ng/ml TNF-α. B) Secretion of cytokines and chemokines 24 h after a 4 h exposure to 0, 2 and 10 ng/ml TNF-α. Significance of the dose response curve was calculated using a one-way ANOVA followed by a Dunn’s multiple comparison test. Data is shown for 4 donors as mean ± SEM. Grey bars represent A-EC and black bars D-EC.</p

Proliferation of endothelial cells in response to bFGF or VEGF.

<p>A) Proliferation during normal culture conditions: lines of individual donors are shown, grey squares represent A-EC and black circles D-EC. B) 3H incorporation during 16h of proliferation of A-EC and D-EC in nutrient poor medium. C-D) Relative proliferation of A-EC and D-EC in response to bFGF or VEGF in nutrient poor medium. The dose-response curve to VEGF of dermal-EC shows more proliferation compared to adipose-EC (Cell type: *; two-way ANOVA followed by a Sidak’s multiple comparison test). *P<0.05, **P < 0.01, ***P<0.001. Data is shown for 4 donors as mean ± SEM. Grey bars represent A-EC and black bars D-EC. cpm = counts per minute.</p

Endothelial cell migration in response to bFGF in a wound healing scratch assay.

<p>A) Basal migration of A-EC and D-EC after 16h. B) Relative migration in response to bFGF after 16h. Significance of stimulation was determined using a one-way ANOVA test. *P<0.05, **P < 0.01. Data is shown for 3–5 donors as mean ± SEM. Grey bars represent A-EC and black bars D-EC.</p

<i>In vitro</i> sprouting of adipose- and dermal-EC in a fibrin matrix.

<p>A) Surface view of A-EC or D-EC sprouting in a fibrin matrix after stimulation with HMEC medium supplemented with 2 ng/ml TNF-α alone or in combination with 10 ng/ml bFGF or 25 ng/ml VEGF. B) H&E staining of fibrin matrices with A-EC or D-EC upon stimulation with HMEC medium supplemented with 2 ng/ml TNF-α alone (Control) or in combination with 10 ng/ml bFGF or 25 ng/ml VEGF. Quantification of sprouting in response to bFGF (C) or VEGF (D). Significance of the dose response effect was determined using a repeated measures one-way ANOVA. **P < 0.01, ***P<0.001. Data is shown for 5 donors as mean ± SEM. Grey bars represent A-EC and black bars D-EC. The scale bars represent 50 μm.</p