Optimizing in vitro culture conditions leads to a significantly shorter production time of human dermo-epidermal skin substitutes

Introduction: Autologous dermo-epidermal skin substitutes (DESS) generated in vitro represent a promising therapeutic means to treat full-thickness skin defects in clinical practice. A serious drawback with regard to acute patients is the relatively long production time of 3-4weeks. With this experimental study we aimed to decrease the production time of DESS without compromising their quality. Methods: Two in vitro steps of DESS construction were varied: the pre-cultivation time of fibroblasts in hydrogels (1, 3, and 6days), and the culture time of keratinocytes (3, 6, and 12days) before transplantation of DESS on nude rats. Additionally, the impact of the air-liquid interface culture during 3days before transplantation was investigated. 3weeks after transplantation, the macroscopic appearance was evaluated and histological sections were produced to analyze structure and thickness of epidermis and dermis, the stratification of the epidermis, and the presence of a basal lamina. Results: Optimal DESS formation was obtained with a fibroblast pre-cultivation time of 6days. The minimal culture time of keratinocytes on hydrogels was also 6days. The air-liquid interface culture did not improve graft quality. Conclusion: By optimizing our in vitro culture conditions, it was possible to very substantially reduce the production time for DESS from 21 to 12days. However, pre-cultivation of fibroblasts in the dermal equivalent and proliferation of keratinocytes before transplantation remain crucial for an equilibrated maturation of the epidermis and cannot be completely skippe

"Trooping the color”: restoring the original donor skin color by addition of melanocytes to bioengineered skin analogs

Purpose: Autologous skin substitutes to cover large skin defects are used since several years. Melanocytes, although essential for solar protection and pigmentation of skin, are not yet systematically added to such substitutes. In this experimental study, we reconstructed melanocyte-containing dermo-epidermal skin substitutes from donor skins of different skin pigmentation types and studied them in an animal model. Features pertinent to skin color were analyzed and compared in both skin substitutes and original donor skin. Methods: Keratinocytes, melanocytes, and fibroblast were isolated, cultured, and expanded from skin biopsies of light- and dark-pigmented patients. For each donor, melanocytes and keratinocytes were seeded in different ratios (1:1, 1:5, 1:10) onto collagen gels previously populated with autologous fibroblasts. Skin substitutes were then transplanted onto full-thickness wounds of immuno-incompetent rats. After 8weeks, macroscopic and microscopic analyses were conducted with regard to skin color and architecture. Results: Chromameter evaluation revealed that skin color of reconstructed light- and dark-pigmented skin was very similar to donor skin, independent of which melanocyte/keratinocyte ratio was added. Histological analyses of the skin analogs confirmed these findings. Conclusion: These data suggest that adding autologous melanocytes to bioengineered dermo-epidermal skin analogs can sustainably restore the patients' native skin colo

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

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 8weeks. 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 3weeks, the skin substitutes of all three epidermal cell variants showed no neuronal ingrowth from the host into the transplant. After 8weeks, 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 8weeks 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 sensitivit

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 15weeks. Results: The skin analogs revealed a significant difference in vascularization patterns between light- and dark-pigmented constructs after 8weeks, 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 15weeks, 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 15weeks, 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-engineered dermo-epidermal skin analogs exhibit de novo formation of a near natural neurovascular link 10weeks 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 8weeks 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: 3weeks after transplantation, blood vessels, but no nerve fibers or lymphatic vessels could be observed. 10weeks 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

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 3weeks) and late (6 and 12weeks) 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 1week post-transplantation. This was followed by monocyte/macrophage recruitment to the graft at 3-12weeks. 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 6-12weeks, 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

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 3weeks 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 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

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