Ösophagotracheale Fistel: Hervorgerufen durch eine im Ösophagus stecken gebliebene Knopfbatterie

Zusammenfassung: Verschluckte, im Ösophagus stecken gebliebene, Knopfbatterien müssen notfallmäßig entfernt werden, da sie trotz rascher Entfernung in kürzester Zeit durch verschiedene Mechanismen zu tiefen Verletzungen der Mukosa führen können. Dies wird anhand eines Fallbeispiels eines 2-jährigen Mädchens demonstriert, das nach rascher, unproblematischer endoskopischer Entfernung einer Knopfbatterie aus dem Ösophagus und einem zunächst guten Verlauf eine ösophagotracheale Fistel entwickelt

Differential expression of granulocyte, macrophage, and hypoxia markers during early and late wound healing stages following transplantation of tissue-engineered skin substitutes of human origin

PURPOSE Human pigmented tissue-engineered skin substitutes represent an advanced therapeutic option to treat skin defects. The inflammatory response is one of the major factors determining integration and long-term survival of such a graft in vivo. The aim of the present study was to investigate the spatiotemporal distribution of host-derived macrophage and granulocyte graft infiltration as well as hypoxia-inducible factor 1 alpha (HIF-1-alpha) expression in a (nu/nu) rat model. METHODS Keratinocytes, melanocytes, and fibroblasts derived from human skin biopsies were isolated, cultured, and expanded in vitro. Dermal fibroblasts were seeded into collagen type I hydrogels that were subsequently covered by keratinocytes and melanocytes in 5:1 ratio. These pigmented dermo-epidermal skin substitutes were transplanted onto full-thickness skin wounds on the back of immuno-incompetent rats and analyzed at early (1 and 3 weeks) and late (6 and 12 weeks) stages of wound healing. The expression of distinct inflammatory cell markers specific for granulocytes (HIS48) or macrophages (CD11b, CD68), as well as HIF-1-alpha were analyzed and quantified by immunofluorescence microscopy. RESULTS Our data demonstrate that granulocytes infiltrate the entire graft at 1 week post-transplantation. This was followed by monocyte/macrophage recruitment to the graft at 3-12 weeks. The macrophages were initially restricted to the borders of the graft (early stages), and were then found throughout the entire graft (late stages). We observed a time-dependent decrease of macrophages. Only a few graft-infiltrating granulocytes were found between 6-12 weeks, mostly at the graft borders. A heterogeneous expression of HIF-1-alpha was observed at both early and late wound healing stages. CONCLUSIONS Our findings demonstrate the spatiotemporal distribution of inflammatory cells in our transplants closely resembles the one documented for physiological wound healing

Analysis of blood and lymph vascularization patterns in tissue-engineered human dermo-epidermal skin analogs of different pigmentation

PURPOSE: Bioengineered dermo-epidermal skin analogs containing melanocytes represent a promising approach to cover large skin defects including restoration of the patient's own skin color. So far, little is known about the development of blood and lymphatic vessels in pigmented skin analogs after transplantation. In this experimental study, we analyzed the advancement and differences of host blood and lymphatic vessel ingrowth into light- and dark-pigmented human tissue-engineered skin analogs in a rat model. METHODS: Keratinocytes, melanocytes, and fibroblasts from light- and dark-pigmented skin biopsies were isolated, cultured, and expanded. For each donor, melanocytes and keratinocytes were seeded in ratios of 1:1, 1:5, and 1:10 onto fibroblast-containing collagen gels. The skin analogs were subsequently transplanted onto full-thickness wounds of immuno-incompetent rats and quantitatively analyzed for vascular and lymphatic vessel density after 8 and 15 weeks. RESULTS: The skin analogs revealed a significant difference in vascularization patterns between light- and dark-pigmented constructs after 8 weeks, with a higher amount of blood vessels in light compared to dark skin. In contrast, no obvious difference could be detected within the light- and dark-pigmented group when varying melanocyte/keratinocyte ratios were used. However, after 15 weeks, the aforementioned difference in blood vessel density between light and dark constructs could no longer be detected. Regarding lymphatic vessels, light and dark analogs showed similar vessel density after 8 and 15 weeks, while there were generally less lymphatic than blood vessels. CONCLUSION: These data suggest that, at least during early skin maturation, keratinocytes, melanocytes, and fibroblasts from different skin color types used to construct pigmented dermo-epidermal skin analogs have distinct influences on the host tissue after transplantation. We speculate that different VEGF expression patterns might be involved in this disparate revascularization pattern observed

Tissue engineering of skin: human tonsil-derived mesenchymal cells can function as dermal fibroblasts

PURPOSE: It is unclear whether dermal fibroblasts are indispensable key players for tissue engineering of dermo-epidermal skin analogs. In this experimental study, we wanted to test the hypothesis that tonsil-derived mesenchymal cells can assume the role of dermal fibroblasts when culturing pigmented skin analogs for transplantation. METHODS: Mesenchymal cells from excised tonsils and keratinocytes, melanocytes, and fibroblasts from skin biopsies were isolated, cultured, and expanded. Melanocytes and keratinocytes were seeded in a ratio of 1:5 onto collagen gels previously populated either with tonsil-derived mesenchymal cells or with autologous dermal fibroblasts. These laboratory engineered skin analogs were then transplanted onto full-thickness wounds of immuno-incompetent rats and analyzed after 3 weeks with regard to macroscopic and microscopic epidermal characteristics. RESULTS: The skin analogs containing tonsil-derived mesenchymal cells showed the same macroscopic appearance as the ones containing dermal fibroblasts. Histologically, features of epidermal stratification, pigmentation, and cornification were identical to those of the controls assembled with autologous dermal fibroblasts. Transmission electron microscopy confirmed these findings. CONCLUSION: These data suggest that human tonsil-derived mesenchymal cells can assume dermal fibroblast functions, indicating that possibly various types of mesenchymal cells can successfully be employed for "skingineering" purposes. This aspect may have clinical implications when sources for dermal fibroblasts are scarce

Tissue-engineered dermo-epidermal skin analogs exhibit de novo formation of a near natural neurovascular link 10 weeks after transplantation

PURPOSE: Human autologous tissue-engineered skin grafts are a promising way to cover skin defects. Clearly, it is mandatory to study essential biological dynamics after transplantation, including reinnervation. Previously, we have already shown that human tissue-engineered skin analogs are reinnervated by host nerve fibers as early as 8 weeks after transplantation. In this study, we tested the hypothesis that there is a de novo formation of a "classical" neurovascular link in tissue-engineered and then transplanted skin substitutes. METHODS: Keratinocytes, melanocytes, and fibroblasts were isolated from human skin biopsies. After expansion in culture, keratinocytes and melanocytes were seeded on dermal fibroblast-containing collagen type I hydrogels. These human tissue-engineered dermo-epidermal skin analogs were transplanted onto full-thickness skin wounds on the back of immuno-incompetent rats. Grafts were analyzed after 3 and 10 weeks. Histological sections were examined with regard to the ingrowth pattern of myelinated and unmyelinated nerve fibers into the skin analogs using markers such as PGP9.5, NF-200, and NF-160. Blood vessels were identified with CD31, lymphatic vessels with Lyve1. In particular, we focused on alignment patterns between nerve fibers and either blood and/or lymphatic vessels with regard to neurovascular link formation. RESULTS: 3 weeks after transplantation, blood vessels, but no nerve fibers or lymphatic vessels could be observed. 10 weeks after transplantation, we could detect an ingrowth of myelinated and unmyelinated nerve fibers into the skin analogs. Nerve fibers were found in close proximity to CD31-positive blood vessels, but not alongside Lyve1-positive lymphatic vessels. CONCLUSION: These data suggest that host-derived innervation of tissue-engineered dermo-epidermal skin analogs is initiated by and guided alongside blood vessels present early post-transplantation. This observation is consistent with the concept of a cross talk between neurovascular structures, known as the neurovascular link

The influence of stromal cells on the pigmentation of tissue-engineered dermo-epidermal skin grafts

It has been shown in vitro that melanocyte proliferation and function in palmoplantar skin is regulated by mesenchymal factors derived from fibroblasts. Here, we investigated in vivo the influence of mesenchymal-epithelial interactions in human tissue-engineered skin substitutes reconstructed from palmar- and non-palmoplantar-derived fibroblasts. Tissue-engineered dermo-epidermal analogs based on collagen type I hydrogels were populated with either human palmar or non-palmoplantar fibroblasts and seeded with human non-palmoplantar-derived melanocytes and keratinocytes. These skin substitutes were transplanted onto full-thickness skin wounds of immuno-incompetent rats. Four weeks after transplantation the development of skin color was measured and grafts were excised and analyzed with regard to epidermal characteristics, in particular melanocyte number and function. Skin substitutes containing palmar-derived fibroblasts in comparison to non-palmoplantar derived fibroblasts showed a) a significantly lighter pigmentation; b) a reduced amount of epidermal melanin granules; and c) a distinct melanosome expression. However, the number of melanocytes in the basal layer remained similar in both transplantation groups. These findings demonstrate that human palmar fibroblasts regulate the function of melanocytes in human pigmented dermo-epidermal skin substitutes after transplantation, whereas the number of melanocytes remains constant. This underscores the influence of site-specific stromal cells and their importance when constructing skin substitutes for clinical application

Rebuild, restore, reinnervate: do human tissue engineered dermo-epidermal skin analogs attract host nerve fibers for innervation?

PURPOSE: Tissue engineered skin substitutes are a promising tool to cover large skin defects, but little is known about reinnervation of transplants. In this experimental study, we analyzed the ingrowth of host peripheral nerve fibers into human tissue engineered dermo-epidermal skin substitutes in a rat model. Using varying cell types in the epidermal compartment, we wanted to assess the influence of epidermal cell types on reinnervation of the substitute. METHODS: We isolated keratinocytes, melanocytes, fibroblasts, and eccrine sweat gland cells from human skin biopsies. After expansion, epidermal cells were seeded on human dermal fibroblast-containing collagen type I hydrogels as follows: (1) keratinocytes only, (2) keratinocytes with melanocytes, (3) sweat gland cells. These substitutes were transplanted into full-thickness skin wounds on the back of immuno-incompetent rats and were analyzed after 3 and 8 weeks. Histological sections were examined with regard to myelinated and unmyelinated nerve fiber ingrowth using markers such as PGP9.5, NF-200, and NF-145. RESULTS: After 3 weeks, the skin substitutes of all three epidermal cell variants showed no neuronal ingrowth from the host into the transplant. After 8 weeks, we could detect an innervation of all three types of skin substitutes. However, the nerve fibers were restricted to the dermal compartment and we could not find any unmyelinated fibers in the epidermis. Furthermore, there was no distinct difference between the constructs resulting from the different cell types used to generate an epidermis. CONCLUSION: Our human tissue engineered dermo-epidermal skin substitutes demonstrate a host-derived innervation of the dermal compartment as early as 8 weeks after transplantation. Thus, our substitutes apparently have the capacity to attract nerve fibers from adjacent host tissues, which also grow into grafts and thereby potentially restore skin sensitivity

A new model for preclinical testing of dermal substitutes for human skin reconstruction

BACKGROUND: Currently, acellular dermal substitutes used for skin reconstruction are usually covered with split-thickness skin grafts. The goal of this study was to develop an animal model in which such dermal substitutes can be tested under standardized conditions using a bioengineered dermo-epidermal skin graft for coverage. METHODS: Bioengineered grafts consisting of collagen type I hydrogels with incorporated human fibroblasts and human keratinocytes seeded on these gels were produced. Two different dermal substitutes, namely Matriderm(®), and an acellular collagen type I hydrogel, were applied onto full-thickness skin wounds created on the back of immuno-incompetent rats. As control, no dermal substitute was used. As coverage for the dermal substitutes either the bioengineered grafts were used, or, as controls, human split-thickness skin or neonatal rat epidermis were used. Grafts were excised 21 days post-transplantation. Histology and immunofluorescence was performed to investigate survival, epidermis formation, and vascularization of the grafts. RESULTS: The bioengineered grafts survived on all tested dermal substitutes. Epidermis formation and vascularization were comparable to the controls. CONCLUSION: We could successfully use human bioengineered grafts to test different dermal substitutes. This novel model can be used to investigate newly designed dermal substitutes in detail and in a standardized way

Glycerol treatment as recovery procedure for cryopreserved human skin allografts positive for bacteria and fungi

Human donor skin allografts are suitable and much used temporary biological (burn) wound dressings. They prepare the excised wound bed for final autografting and form an excellent substrate for revascularisation and for the formation of granulation tissue. Two preservation methods, glycerol preservation and cryopreservation, are commonly used by tissue banks for the long-term storage of skin grafts. The burn surgeons of the Queen Astrid Military Hospital preferentially use partly viable cryopreserved skin allografts. After mandatory 14-day bacterial and mycological culture, however, approximately 15% of the cryopreserved skin allografts cannot be released from quarantine because of positive culture. To maximize the use of our scarce and precious donor skin, we developed a glycerolisation-based recovery method for these culture positive cryopreserved allografts. The inactivation and preservation method, described in this paper, allowed for an efficient inactivation of the colonising bacteria and fungi, with the exception of spore-formers, and did not influence the structural and functional aspects of the skin allografts