Figure 1.
Scheme of the utilised dorsal skin fold chambers in mice.
The chambers are attached to the back skin of the mice. The printed skin construct consisting of 20 layers of fibroblasts and 20 layers of keratinocytes on top of Matriderm® is placed into a round full-thickness wound in the mouse skin, while the opposite side remains intact. To close the chamber a cover glass is used.
Figure 2.
Tissue engineered skin construct in the dorsal skin fold chamber in nude mice.
The pictures show a skin construct inserted into the wound directly after the implantation (left) and on day 11 (right). The implanted constructs were created via LaBP, consisting of 20 layers of fibroblasts and 20 layers of keratinocytes on top of Matriderm®. They fill the full-thickness wound completely.
Figure 3.
Histological sections of the tissue engineered skin constructs in vivo.
Skin constructs were implanted in dorsal skin fold chambers in mice for 11 days. Sections were stained with Masson’s trichrome (A–D) or analyzed with fluorescence microscopy (E), respectively. (A) illustrates an overview with the junction between the inserted skin construct (m = Matriderm®) and native mouse skin (n) at the wound edge after 11 days in the dorsal skin fold chamber in mice. The intact mouse skin opposite of the skin construct can be seen in the lower part of the picture (n) (see also Figure 1). The skin construct and the intact skin part in the sandwiched skin are separated by the panniculus carnosus (pc). Both in native mouse skin (B) and the printed skin construct (C) a dense epidermis (empty asterisks) and a corneal layer can be observed. In case of the skin construct, the epidermis is formed by the printed keratinocytes (E). This can clearly be seen by the green fluorescence emitted by the used HaCaT-eGFP cells. The fibroblasts (NIH3T3-mCherry) partly migrated into the Matriderm® (yellowish fibres). The fibroblasts, which stayed on top of the Matriderm®, display an outstretched morphology (C), being accompanied by collagen deposition (filled asterisks). Blood vessels (arrows) can be detected in the skin constructs (D). Scale bars depict 200 µm (A, D, E) and 100 µm (B, C).
Figure 4.
Fluorescent pictures of the tissue engineered skin constructs in vivo.
Skin constructs were implanted in dorsal skin fold chambers in mice for 11. The sections show an overview of the junction between the inserted skin construct (m = Matriderm®) and native mouse skin (n) with either fluorescence microscopy (A, C) or transmitted light microscopy (B). Two different situations concerning the epidermis were observed during analysis of the junction zones: In some cases, as depicted in (A), the normal mouse epidermis (ne) started to grow on top of the Matriderm®, where it connected to the epidermis formed by the printed keratinocytes (pk). The latter were labelled in green by stable transduction (HaCaT-eGFP). In other cases, as depicted in (B) and (C), the epidermis formed by the printed keratinocytes ended at the border of the Matriderm®, synchronous to the presence of the printed fibroblasts (pf) labelled in red (NIH3T3-mCherry). As can be seen by comparing (B) and (C) - which depict the same location - the keratinocytes even partly grew on top of the normal mouse epidermis. In both cases, the printed fibroblasts formed a multi-layer tissue underneath the printed keratinocytes. Partly, they also migrated into the Matriderm®. All scale bars depict 200 µm.
Figure 5.
Blood vessel detection via immunohistochemistry in skin constructs cultivated in vivo for 11 days.
Skin constructs were cultivated in vivo for 11 days in the dorsal skin fold chamber in mice. Collagen IV expression (brown) – indicating blood vessels/capillaries – can be detected in the Matriderm® as small tubes reaching from the wound bed in the direction of the cells (A). Small and large blood vessels are present in the normal mouse skin (B). Matriderm® (C) and normal mouse skin (D) without first antibody serve as the respective negative controls. Scale bars depict 200 µm each.
Figure 6.
E-cadherin detection via immunohistochemistry in skin constructs cultivated in vivo for 11 days.
Skin constructs were cultivated in vivo for 11 days in the dorsal skin fold chamber in mice. E-cadherin expression (dark brown) can be found throughout the epidermis in both normal mouse skin (A) and the skin constructs (B). Normal mouse skin without first antibody serves as a negative control (C). All scale bars depict 100 µm.
Figure 7.
Detection of cytokeratin 14 and proliferation via immunohistochemistry in skin constructs cultivated in vivo.
Skin constructs were cultivated in vivo in the dorsal skin fold chamber in mice for 11 days. The left column shows normal mouse skin, the middle column the skin construct and the right column the respective negative controls (normal mouse skin without first antibody) of the immunohistochemistry stainings. Cytokeratin 14 expression is limited to the suprabasal layers of the epidermis in mouse skin (A) but present in the whole epidermis in the skin constructs (B). Proliferation via Ki67 can be detected in the suprabasal layers of the epidermis and in the dermis in both normal mouse skin and skin constructs (D, E). Scale bars depict 200 µm (A–C) and 100 µm (D–F).
Figure 8.
Histological sections of the tissue engineered skin constructs in vitro.
Skin constructs were cultivated at the air-liquid-interface with differentiation medium for 11 days. Sections show cells using fluorescent microscopy and Masson’s trichrome staining, respectively. The time points indicated in A–C are valid for the whole respective columns. The skin constructs were cultivated at the air-liquid-interface. The keratinocytes (HaCaT-mCherry) exhibit red fluorescence while the fibroblasts (NIH3T3-eGFP) appear in green (A–C). Masson’s trichrome staining reveals the connective tissue containing collagen (green) and the cells (reddish) (D–I). The fibroblasts already start to grow into the Matriderm® underneath one day after printing (A, D, G). The keratinocytes, which still are rounded and are not connected to each other on day 0 (A, D, G), already form a dense tissue on day 5 (B, E, H). The thereby formed epidermis increases in height until day 11 (C, F, I). Scale bars depict 200 µm (A–F) and 100 µm (G–I).
Figure 9.
Sections of immunohistochemically stained skin constructs cultivated in vitro.
Skin constructs were cultivated in vitro at the air-liquid-interface with differentiation medium for 11 days. The indicated time points in A–C are valid for the whole respective column. E-cadherin expression is absent on day 0 but can be detected on days 5 and 11 (A–C) while cytokeratin 14 expression is clearly visible at all time points in the whole epidermis (D–F). While nearly all cells exhibit Ki67 staining on day 0, only few cells do so at days 5 and 11 (G–I). The corresponding negative controls of the stainings (skin constructs without first antibody) are shown below (K – e-cadherin, L – cytokeratin 14, M – Ki67). All scale bars depict 100 µm.