Burn eschar stimulates fibroblast and adipose mesenchymal stromal cell proliferation and migration but inhibits endothelial cell sprouting

The majority of full-thickness burn wounds heal with hypertrophic scar formation. Burn eschar most probably influences early burn wound healing, since granulation tissue only forms after escharotomy. In order to investigate the effect of burn eschar on delayed granulation tissue formation, burn wound extract (BWE) was isolated from the interface between non-viable eschar and viable tissue. The influence of BWE on the activity of endothelial cells derived from dermis and adipose tissue, dermal fibroblasts and adipose tissue-derived mesenchymal stromal cells (ASC) was determined. It was found that BWE stimulated endothelial cell inflammatory cytokine (CXCL8, IL-6 and CCL2) secretion and migration. However, BWE had no effect on endothelial cell proliferation or angiogenic sprouting. Indeed, BWE inhibited basic Fibroblast Growth Factor (bFGF) induced endothelial cell proliferation and sprouting. In contrast, BWE stimulated fibroblast and ASC proliferation and migration. No difference was observed between cells isolated from dermis or adipose tissue. The inhibitory effect of BWE on bFGF-induced endothelial proliferation and sprouting would explain why excessive granulation tissue formation is prevented in full-thickness burn wounds as long as the eschar is still present. Identifying the eschar factors responsible for this might give indications for therapeutic targets aimed at reducing hypertrophic scar formation which is initiated by excessive granulation tissue formation once eschar is removed

Reconstructed human skin shows epidermal invagination towards integrated neopapillae indicating early hair follicle formation in vitro

Application of reconstructed human Skin (RhS) is a promising approach for the treatment of extensive wounds and for drug efficacy and safety testing. However, incorporating appendages, such as hair follicles, into RhS still remains a challenge. The hair follicle plays a critical role in thermal regulation, dispersion of sweat and sebum, sensory and tactile functions, skin regeneration, and repigmentation. The aim of this study was to determine whether human neopapilla could be incorporated into RhS (differentiated epidermis on fibroblast and endothelial cell populated dermis) and whether the neopapillae maintain their inductive follicular properties in vitro. Neopapillae spheroids, constructed from expanded and self-aggregating dermal papilla cells, synthesized extracellular matrix typically found in follicular papillae. Compared with dermal fibroblasts, neopapillae showed increased expression of multiple genes (Wnt5a, Wnt10b, and LEF1) known to regulate hair development and also increased secretion of CXCL1, which is a strong keratinocyte chemoattractant. When neopapillae were incorporated into the dermis of RhS, they stimulated epidermal down-growth resulting in engulfment of the neopapillae sphere. Similar to the native hair follicle, the differentiated invaginating epidermis inner side was keratin 10 positive and the undifferentiated outer side keratin 10 negative. The outer side was keratin 15 positive confirming the undifferentiated nature of these keratinocytes aligning a newly formed collagen IV, laminin V positive basement membrane within the hydrogel. In conclusion, we describe a RhS model containing neopapillae with hair follicle-inductive properties. Importantly, epidermal invagination occurred to engulf the neopapillae, thus demonstrating in vitro the first steps towards hair follicle morphogenesis in RhS

Surface marker expression in cultured endothelial cells derived from human adipose tissue or dermis

<p>Surface marker expression in cultured endothelial cells derived from human adipose tissue or dermis</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

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

IgG Antibodies in Food Allergy Influence Allergen-Antibody Complex Formation and Binding to B Cells: A Role for Complement Receptors

Allergen-IgE complexes are more efficiently internalized and presented by B cells than allergens alone. It has been suggested that IgG Abs induced by immunotherapy inhibit these processes. Food-allergic patients have high allergen-specific IgG levels. However, the role of these Abs in complex formation and binding to B cells is unknown. To investigate this, we incubated sera of peanut-or cow's milk-allergic patients with their major allergens to form complexes and added them to EBV-transformed or peripheral blood B cells (PBBCs). Samples of birch pollen-allergic patients were used as control. Complex binding to B cells in presence or absence of blocking Abs to CD23, CD32, complement receptor 1 (CR1, CD35), and/or CR2 (CD21) was determined by flow cytometry. Furthermore, intact and IgG-depleted sera were compared. These experiments showed that allergen-Ab complexes formed in birch pollen, as well as food allergy, contained IgE, IgG1, and IgG4 Abs and bound to B cells. Binding of these complexes to EBV-transformed B cells was completely mediated by CD23, whereas binding to PBBCs was dependent on both CD23 and CR2. This reflected differential receptor expression. Upon IgG depletion, allergen-Ab complexes bound to PBBCs exclusively via CD23. These data indicated that IgG Abs are involved in complex formation. The presence of IgG in allergen-IgE complexes results in binding to B cells via CR2 in addition to CD23. The binding to both CR2 and CD23 may affect Ag processing and presentation, and (may) thereby influence the allergic respons

Methods to study differences in cell mobility during skin wound healing in vitro

Wound healing events which occur in humans are difficult to study in animals due to differences in skin physiology. Furthermore there are increasing restrictions in Europe for using animals for testing the therapeutic properties of new compounds. Therefore, in line with the 3Rs (reduction, refinement and replacement of test animals), a number of human in vitro models of different levels of complexity have been developed to investigate cell mobility during wound healing. Keratinocyte, melanocyte, fibroblast and endothelial cell mobility are described, since these are the residential cells which are responsible for restoring the main structural features of the skin. A monolayer scratch assay is used to study random fibroblast and endothelial cell migration in response to EGF and bFGF respectively and a chemotactic assay is used to study directional fibroblast migration towards CCL5. In order to study endothelial sprouting in response to bFGF or VEGF, which involves continuous degradation and resynthesis of a 3D matrix, a fibrin gel is used. Human physiologically relevant tissue-engineered skin models are used to investigate expansion of the stratified, differentiated epidermis (keratinocytes and melanocytes) over a fibroblast populated dermis and also to study migration and distribution of fibroblasts into the dermis. Together these skin models provide a platform for testing the mode of action of novel compounds for enhanced and scar free wound healing