Re-epithelialization and immune cell behaviour in an ex vivo human skin model methods
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A multi-layered epidermis expressing epidermal differentiation markers forms after wound closure
We next tested whether keratinocytes upon wound closure can also differentiate and form multiple epidermal layers upon prolonged culture. Indeed, several K14 + keratinocyte layers were observed upon 8 days of culture above the initial wound bed (Fig. ). During the continued culture period of 12 days, multiple K14 + layers have formed (Fig. ). In normal, unwounded skin, K10 is exclusively localized in suprabasal keratinocytes (Rakita, unpublished observation). In wounded skin, a few K10 + basal keratinocytes, in particular at the wound edge, were observed in addition to the conventional staining pattern in suprabasal keratinocytes at day 8 of culture (Fig. ). K10 expression appeared in all layers except the basal layer at later time points (Fig. ). Next, expression kinetics of particular epidermal differentiation markers in unwounded skin and blister-induced wou...
Increased frequency of matrix metalloproteinase 9 (MMP-9) expressing cells in wounded skin upon prolonged culture
MMPs become activated during wound repair. In particular, MMP-9 influences the detachment of basal keratinocytes from the basement membrane thus improving their migration,. We noticed scarce MMP-9 + cells directly under the wound bed and in unwounded skin areas (Fig. ). In contrast, strong MMP-9 expression was observed at the leading wound edges at day 4 and already decreased 6 days after wounding (Fig., inserts). MMP-9 + cells greatly accumulated in the dermis at day 10 of culture (Fig., arrowheads). Of note, different MMP-9 expression patterns were observed in dermal cells implying that distinct cell types upregulate this enzyme. MMP-9 appeared in dots around the nucleus (Fig., right insert), on the cell surface (Fig. arrowhead) or interconnecting cells forming a "vessel-like" shape (Fig., upper insert). Inspired by the MMP-9 ...
Methods
Healthy skin was obtained from routine reduction surgeries from anonymous donors and experiments on ex vivo skin were typically performed within 1-2 hours (h) after surgery. The study was approved by the ethics committee of the Medical University of Vienna and conducted according to the Declaration of Helsinki principles. Written informed consent from all participants was obtained.
Suction blister-induced wound model
A negative pressure instrument (Electronic Diversities, Finksburg, MD, USA) constructed to produce standard suction blisters upon application of negative pressure, was used on healthy skin ( ex vivo: abdominal skin; in vivo: lower forearm). Subcutaneous fat was partially removed from ex vivo skin using a scissor. Subsequently, skin (10 × 10 cm 2 ) was placed (not fixed, not kept in medium) on a styrofoam lid that was covered with aluminium foil to provide (at least partial) backpressure. Suction chambers with 5 openings (Ø = 5 mm) on the orifice plate were attached to skin, topped with a styrofoam lid and pressed with 1 kg weight in order to avoid movement of the plate. A pressure of 200-250 millimeter (mm) mercury (Hg) ( ex vivo ) or 150-200 mm Hg ( in vivo ) caused the skin to be drawn through the openings creatin...
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We next tested whether keratinocytes upon wound closure can also differentiate and form multiple epidermal layers upon prolonged culture. Indeed, several K14 + keratinocyte layers were observed upon 8 days of culture above the initial wound bed (Fig. ). During the continued culture period of 12 days, multiple K14 + layers have formed (Fig. ). In normal, unwounded skin, K10 is exclusively localized in suprabasal keratinocytes (Rakita, unpublished observation). In wounded skin, a few K10 + basal keratinocytes, in particular at the wound edge, were observed in addition to the conventional staining pattern in suprabasal keratinocytes at day 8 of culture (Fig. ). K10 expression appeared in all layers except the basal layer at later time points (Fig. ). Next, expression kinetics of particular epidermal differentiation markers in unwounded skin and blister-induced wounds were assessed. Desmoglein-1 (DSG-1), a cell adhesion structure playing a role in maintaining the structure of the epidermis through its adhesive function, is located above the basal layer in normal skin. Corneodesmosin (CDSN) is synthesized in lower granular keratinocytes and represents an epid...
MMPs become activated during wound repair. In particular, MMP-9 influences the detachment of basal keratinocytes from the basement membrane thus improving their migration,. We noticed scarce MMP-9 + cells directly under the wound bed and in unwounded skin areas (Fig. ). In contrast, strong MMP-9 expression was observed at the leading wound edges at day 4 and already decreased 6 days after wounding (Fig., inserts). MMP-9 + cells greatly accumulated in the dermis at day 10 of culture (Fig., arrowheads). Of note, different MMP-9 expression patterns were observed in dermal cells implying that distinct cell types upregulate this enzyme. MMP-9 appeared in dots around the nucleus (Fig., right insert), on the cell surface (Fig. arrowhead) or interconnecting cells forming a "vessel-like" shape (Fig., upper insert). Inspired by the MMP-9 "vessel-like" staining pattern, we tested whether they co-express CD31 which in normal human skin is expressed primarily in endothelial cells lining the interior surface of blood and lymphatic vessels. Surprisingly, we found that dermal cells expressing MMP-9 and CD31 are mutually exclusiv...
Healthy skin was obtained from routine reduction surgeries from anonymous donors and experiments on ex vivo skin were typically performed within 1-2 hours (h) after surgery. The study was approved by the ethics committee of the Medical University of Vienna and conducted according to the Declaration of Helsinki principles. Written informed consent from all participants was obtained.
A negative pressure instrument (Electronic Diversities, Finksburg, MD, USA) constructed to produce standard suction blisters upon application of negative pressure, was used on healthy skin ( ex vivo: abdominal skin; in vivo: lower forearm). Subcutaneous fat was partially removed from ex vivo skin using a scissor. Subsequently, skin (10 × 10 cm 2 ) was placed (not fixed, not kept in medium) on a styrofoam lid that was covered with aluminium foil to provide (at least partial) backpressure. Suction chambers with 5 openings (Ø = 5 mm) on the orifice plate were attached to skin, topped with a styrofoam lid and pressed with 1 kg weight in order to avoid movement of the plate. A pressure of 200-250 millimeter (mm) mercury (Hg) ( ex vivo ) or 150-200 mm Hg ( in vivo ) caused the skin to be drawn through the openings creating typical suction blisters of different size within 6-8 h ( ex vivo ) and 1-2 h ( in vivo ). Suction blister fluid (~110 µl/5 blisters) was collected using a syringe with a needle. Cells within the fluid were counted and placed on adhesion slides for staining and a...
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