As time goes by - Overlooking 40 years of inpatient burn treatment at a national pediatric burn center in Switzerland.

BACKGROUND: Despite extensive prevention programs, burns remain a frequent cause of injury in Switzerland with a known age peak in children. Pediatric burns may cause substantial morbidity, a psyochological burden and therapy related high economic costs. To improve preventive measures, precise knowledge of etiology and treatment of pediatric burns in Switzerland as well as their temporal evolution is indispensable. METHODS: The present retrospective analysis included pediatric burn patients admitted for acute treatment to the Pediatric Burn Center of the University Children`s Hospital Zurich over the last four decades. Sociodemographic, injury related, and treatment related data were extracted from medical records. Linear regression analysis was applied to determine temporal changes during the past four decades and chi-square and t-tests were applied wherever applicable. RESULTS: A total of 3425 acute burn patients were included in the study between 1977 and 2020, corresponding to a mean of 89 patients/year. Mean age was 3.60 ± 4.12 years, three quarters of all patients were preschool children (0-5 years) and mean total body surface area (TBSA) burned was 8.01% ± 9.57%, however only around one fifth had severe burns (>10% TBSA). Scald burns (65.31%) and flame burns (32.99%) were most commonly seen. Linear regression analysis showed the total number of thermal injuries treated at our center to have increased significantly as of 2004 (p < 0.001). Separate analysis showed the same for small and medium (<10% TBSA) burns (p < 0.001), whereas the number of severe burns did not increase significantly. Length of stay (LOS) was highly associated with %TBSA burned. The percentage of female patients amongst all patients increased over time (p = 0.012). LOS per TBSA burned decreased significantly (p < 0.001). CONCLUSION: The present data show pediatric burns to remain a major health burden in Switzerland, especially small and medium burns in preschool children. Prevention programs should focus on this age population as well as on scald and flame burns as most common etiologies. The observed decrease in length of stay suggests a major improvement in overall quality of care in pediatric burns and supports centralization of care.

The expression pattern of cytokeratin 6a in epithelial cells of different origin in dermo-epidermal skin substitutes in vivo.

Keratinocytes are the predominant cell type of skin epidermis. Through the programmed process of differentiation, they form a cornified envelope that provides a physical protective barrier against harmful external environment. Keratins are major structural proteins of keratinocytes that together with actin filaments and microtubules form the cytoskeleton of these cells. In this study, we examined the expression pattern and distribution of cytokeratin 6a (CK6a) in healthy human skin samples of different body locations, in fetal and scar skin samples, as well as in dermo-epidermal skin substitutes (DESSs). We observed that CK6a expression is significantly upregulated in fetal skin and scar tissue as well as in skin grafts after short-term transplantation. Importantly, the abundance of CK6a corresponds directly to the expression pattern of wound healing marker CK16. We postulate that CK6a is a useful marker to accurately evaluate the homeostatic state of DESSs.

Surgical treatment of children and youth with congenital melanocytic nevi: self- and proxy-reported opinions.

PURPOSE: The role of surgery in the management of congenital melanocytic nevi (CMN) is controversial. Data on surgical outcomes and predictors of satisfaction remain scarce. METHODS: An online survey was employed following worldwide recruitment of youth aged 14-25 years (n = 44) and parents of children ≤ 18 years (n = 249) with CMN to query patterns of treatment and satisfaction with and opinions about the benefits of surgery. RESULTS: In proxy-reports, 121 of 249 (49%) and in self-reports 30 of 44 (75%) participants underwent CMN excision. The most common reasons for surgery were psychosocial determinants, aesthetic improvement, and melanoma risk reduction. The overall satisfaction with surgical management was good, although no predictors for satisfaction could be identified. Higher current age of the child was found to predict decision regret in proxy-reports. Most participants indicated that having a scar is more socially acceptable than a CMN. Opinions differed on whether surgery should be deferred until the child is old enough to be involved in the decision-making process. CONCLUSIONS: Whether and when to perform surgery in children with CMN is a multifaceted question. Awareness of common concerns as well as risks and benefits of surgery are essential to ensure critical reflection and balanced decision-making.

Multidisciplinary long-term care and modern surgical treatment of congenital melanocytic nevi - recommendations by the CMN surgery network.

In recent years, our knowledge of congenital melanocytic nevi (CMN) has greatly expanded. This has led to a paradigm shift. The present article represents a commentary by an interdisciplinary group of physicians from German-speaking countries with extensive experience in long-term care and surgical treatment of children and adults with CMN (CMN surgery network, "Netzwerk Nävuschirurgie", NNC). The authors address aspects such as the indication for treatment as well as treatment planning and implementation under these new premises. Adequate counseling of parents on conservative and/or surgical management requires an interdisciplinary exchange among physicians and individualized planning of the intervention, which frequently involves a multi-stage procedure. Today, the long-term aesthetic outcome is at the center of any therapeutic endeavor, whereas melanoma prevention plays only a minor role. The premise of "removal at any cost" no longer holds. Potential treatment-related adverse effects (hospitalization, wound healing disorders, and others) must be carefully weighed against the prospects of a beneficial outcome. In this context, the use of dermabrasion in particular must be critically evaluated. At a meeting of the NNC in September 2018, its members agreed on a consensus-based position on dermabrasion, stating that the procedure frequently leads to impaired wound healing and cosmetically unfavorable or hypertrophic scarring. Moreover, dermabrasion is considered to be commonly associated with considerable repigmentation that usually occurs a number of years after the procedure. In addition, the NNC members saw no benefit in terms of melanoma prevention. In the future, physicians should therefore thoroughly caution about the potential risks and often limited cosmetic benefits of dermabrasion.

Bold to do - bald to be? Outcomes decades after harvesting the scalp in burned children.

BACKGROUND: The scalp is the only hidden donor site for split thickness skin grafts. Nevertheless, it is underappreciated due to fear of iatrogenic scarring alopecia. Long-term data showing whether androgenetic hair loss can reveal previously hidden scarring alopecia is unavailable. We aimed to evaluate results and patient satisfaction up to 30years after skin harvest from the scalp. METHODS: Burn patients, hospitalized between 1977 and 1987 at the University Children's Hospital Zurich with scalp skin harvest and currently over 30years old, were studied. Medical records and patient satisfaction were analyzed, and a clinical scalp examination was performed. RESULTS: Thirty-two patients (18 males, 14 females) with a current age of 34.13±3.42years participated. Mean follow-up time was 27.09±3.04years. Fifty-four scalp harvests were performed with 1.69±0.96 sequential harvests. Hair growth was considered normal in 97% patients. Androgenetic alopecia (AGA) type Norwood II-VI was seen in 11 patients. Scalp examination revealed 11 unknown likely harvest-related alopecias with a mean size of 0.7cm2. CONCLUSIONS: Long-term morbidity of scalp skin harvest and the risk of clinically significant alopecia is very low while patient satisfaction is high. AGA is unlikely to reveal harvest damage previously hidden by regrown hair.

Impact of human mesenchymal cells of different body site origins on the maturation of dermo-epidermal skin substitutes.

AIM OF THE STUDY: The use of autologous bio-engineered dermo-epidermal skin substitutes (DESS) yields a pivotal opportunity to cover large skin defects in human patients. These skin grafts consist of both epidermal and dermal compartments necessary for robust and permanent functional wound closure. In this study, we investigated the impact of mesenchymal cells derived from different body site origins on the expression pattern of diverse markers within DESS. METHODS: Human keratinocytes were obtained from interfollicular epidermis, and mesenchymal cells were isolated from foreskin, palmar skin, fat tissue, and tonsils. After expansion, epidermal cells were seeded on collagen I hydrogels containing stromal cells. These human DESS were transplanted on the back of immune-incompetent rats. After 3 weeks, transplants were excised and analyzed using immunohistology techniques. MAIN RESULTS: The macroscopic appearance of skin grafts containing tonsil, fat tissue, or palmar derived mesenchymal cells, was similar to substitutes with foreskin derived dermal fibroblasts. All skin grafts had a strong membrane-localized expression of Lingo-1 in the epidermis. Additionally, we observed an intense expression of transglutaminase 5 in upper epidermal cell layers of the skin grafts confirming a proper keratinocyte differentiation. Tropoelastin was localized throughout the dermal compartments and tightly in contact with the dermo-epidermal junction suggesting an advanced maturation of all skin grafts. CONCLUSIONS: Our data implicate that stromal cells derived from tonsil, fat tissue, and palmar skin can assume fibroblast functions supporting keratinocyte proliferation and differentiation. These findings indicate that distinct types of mesenchymal cells can be clinically used for skin engineering purposes.

UVB exposure of a humanized skin model reveals unexpected dynamic of keratinocyte proliferation and Wnt inhibitor balancing.

We developed human dermo-epidermal skin substitutes that are presently applied in phase I and II clinical trials. Here, we used these very same skin equivalents containing melanocytes, named MelSkin, as an experimental skin model. We investigated the effects of ultraviolet B (UVB) irradiation on the skin grafts transplanted on immune-compromised rats. The irradiation induces a strong wound healing response going along with massive proliferation of basal keratinocytes, basically quiescent under nonirradiated, homeostatic conditions. As a consequence of UVB irradiation, the initially clearly defined basal keratinocyte (mono)layer expands into about 3 layers of keratinocytes, all of which still express the basal keratinocyte marker keratin 15. In contrast, epidermal melanocytes remain quiescent under these circumstances. Moreover, the Wnt inhibitors Dickkopf 3 and Wif1 are downregulated upon UVB irradiation in basal keratinocytes, whereas melanocytes continue to express Wnt inhibitors. These findings suggest that there is (a) a suprabasal population, proliferating in the homeostatic state, hence maintaining the integrity of the epidermis, and (b) a basal, usually quiescent keratinocyte population that is induced to massively proliferate upon irradiation. Importantly, the finding that MelSkin responds in a physiological fashion to UVB is of paramount importance in light of the planned clinical application.

The expression pattern of keratin 24 in tissue-engineered dermo-epidermal human skin substitutes in an in vivo model.

AIMS AND OBJECTIVES: The use of autologous tissue-engineered skin substitutes is a promising approach to cover large skin defects in patients. Preclinical investigation is pivotal to test and improve the quality of these bio-engineered substitutes. In the skin, the epidermis, formed mainly by keratinocytes, provides the first physical barrier protecting from the environment. Proper keratinocyte differentiation and, thus, formation of a stratified epidermis is essential for this function. Keratins, the main structural support of keratinocytes, play a vital role regarding differentiation of keratinocytes. Here, we examined the expression pattern of a recently described keratinocyte differentiation marker, namely Keratin 24, in our skin substitutes. MATERIALS AND METHODS: Human epidermal keratinocytes, melanocytes, dermal fibroblasts, palmar fibroblasts or sweat gland cells were used to prepare skin substitutes. Fibroblast-containing collagen hydrogels were prepared, and keratinocytes or sweat gland cells and melanocytes were seeded onto the hydrogels. The generated tissue-engineered dermo-epidermal skin analogs were transplanted onto full-thickness skin wounds created on the back of immuno-incompetent rats. The skin substitutes were excised at different time points and histologically examined with regard to Keratin 24 expression. RESULTS: We observed the expression of Keratin 24 in keratinocytes of the upper stratum spinosum of the epidermis. In particular, we observed an intensified expression of Keratin 24 13 weeks after transplantation compared to 4 weeks after transplantation. Importantly, we noticed a markedly higher presence of Keratin 24 in more spinous layers if we used palmar fibroblasts or sweat gland cells in our skin substitutes compared non-palmar fibroblasts or epidermal keratinocytes. CONCLUSION: Our observations prove that the keratinocyte differentiation marker Keratin 24 is expressed in our dermo-epidermal skin substitutes in a normal pattern. This highlights that our bio-engineered skin analogs mature and reach homeostasis in an in vivo assay. These findings harbor favorable implications regarding future clinical application.

The Effect of Wound Dressings on a Bio-Engineered Human Dermo-Epidermal Skin Substitute in a Rat Model.

Autologous bio-engineered dermo-epidermal skin substitutes are a promising treatment for large skin defects such as burns. For their successful clinical application, the graft dressing must protect and support the keratinocyte layer and, in many cases, possess antimicrobial properties. However, silver in many antimicrobial dressings may inhibit keratinocyte growth and differentiation. The purpose of our study was to evaluate the effect of various wound dressings on the healing of a human hydrogel-based dermo-epidermal skin substitute in preparation for the first-in-human clinical trials. Human dermo-epidermal skin substitutes approved for clinical trials were produced under good manufacturing practice conditions, transplanted onto immuno-incompetent rats, and dressed with either Vaseline Gauze™ (Kendall Medtronic, Minneapolis, USA), Suprathel (PolyMedics Innovations GmbH, Denkendorf, Germany), Urgotul SSD (Urgo Medical, Shepshed, United Kingdom), Mepilex AG (Mölnlycke Health Care, Gothenburg, Sweden), or Acticoat™ (Smith&Nephew, Baar, Switzerland). Grafts were assessed clinically for take, epithelialization, and infection at 10 and 21 days post-transplantation, and histologically at 21 days. There were three subjects each in the Vaseline Gauze™ and Suprathel groups, and four subjects each in the Urgotul SSD, Mepilex AG, and Acticoat™ groups. For all samples, the take rate was 100% and the expected keratinocyte number, epithelialization and epidermal stratification were observed. All of the dressings in the current study were well tolerated by our human dermo-epidermal skin substitute. The tolerance of the silver-based dressings is particularly relevant given the high risk of bacterial contamination with large skin defects, and provides pivotal information as we embark on clinical trials for this novel skin substitute.

Characterization of vasculogenic potential of human adipose-derived endothelial cells in a three-dimensional vascularized skin substitute.

PURPOSE: The need for clinically applicable skin substitutes continues to be a matter of fact. Hypothetically, a laboratory grown autologous skin analog with near normal architecture might be a suitable approach to yield both satisfactory functional and cosmetic long-term results. In this study, we explored the use of human endothelial cells derived from freshly isolated adipose stromal vascular fraction (SVF) in a three-dimensional (3D) co-culture model of vascularized bio-engineered skin substitute. METHODS: The SVF was isolated from human white adipose tissue samples and keratinocytes from human skin biopsies. The SVF, in particular endothelial cells, were characterized using flow cytometry and immuofluorescence analysis. Endothelial and mesenchymal progenitors from the SVF formed blood capillaries after seeding into a 3D collagen type I hydrogel in vitro. Subsequently, human keratinocytes were seeded on the top of those hydrogels to develop a vascularized dermo-epidermal skin substitute. RESULTS: Flow cytometric analysis of surface markers of the freshly isolated SVF showed the expression of endothelial markers (CD31, CD34, CD146), mesenchymal/stromal cell-associated markers (CD44, CD73, CD90, CD105), stem cell markers (CD49f, CD117, CD133), and additionally hematopoietic markers (CD14, CD15, CD45). Further analysis of white adipose-derived endothelial cells (watECs) revealed the co-expression of CD31, CD34, CD90, CD105, and partially CD146 on these cells. WatECs were separated from adipose-stromal cells (watASCs) using FACS sorting. WatASCs and watECs cultured separately in a 3D hydrogel for 3 weeks did not form any vascular structures. Only if co-cultured, both cell types aligned to develop a ramified vascular network in vitro with continuous endothelial lumen formation. Transplantation of those 3D-hydrogels onto immuno-incompetent rats resulted in a rapid connection of human capillaries with the host vessels and formation of functional, blood-perfused mosaic human-rat vessels within only 3-4 days. CONCLUSIONS: Adipose tissue represents an attractive cell source due to the ease of isolation and abundance of endothelial as well as mesenchymal cell lineages. Adipose-derived SVF cells exhibit the ability to form microvascular structures in vitro and support the accelerated blood perfusion in skin substitutes in vivo when transplanted.

Collagen hydrogels strengthened by biodegradable meshes are a basis for dermo-epidermal skin grafts intended to reconstitute human skin in a one-step surgical intervention.

Extensive full-thickness skin loss, associated with deep burns or other traumata, represents a major clinical problem that is far from being solved. A promising approach to treat large skin defects is the use of tissue-engineered full-thickness skin analogues with nearly normal anatomy and function. In addition to excellent biological properties, such skin substitutes should exhibit optimal structural and mechanical features. This study aimed to test novel dermo-epidermal skin substitutes based on collagen type I hydrogels, physically strengthened by two types of polymeric net-like meshes. One mesh has already been used in clinical trials for treating inguinal hernia; the second one is new but consists of a FDA-approved polymer. Both meshes were integrated into collagen type I hydrogels and dermo-epidermal skin substitutes were generated. Skin substitutes were transplanted onto immuno-incompetent rats and analyzed after distinct time periods. The skin substitutes homogeneously developed into a well-stratified epidermis over the entire surface of the grafts. The epidermis deposited a continuous basement membrane and dermo-epidermal junction, displayed a well-defined basal cell layer, about 10 suprabasal strata and a stratum corneum. Additionally, the dermal component of the grafts was well vascularized.

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. In this study, we investigated in vivo the influence of mesenchymal-epithelial interactions in human tissue-engineered skin substitutes reconstructed from palmar- and nonpalmoplantar-derived fibroblasts. Tissue-engineered dermo-epidermal analogs based on collagen type I hydrogels were populated with either human palmar or nonpalmoplantar fibroblasts and seeded with human nonpalmoplantar-derived melanocytes and keratinocytes. These skin substitutes were transplanted onto full-thickness skin wounds of immunoincompetent 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 nonpalmoplantar-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.

Long-term expression pattern of melanocyte markers in light- and dark-pigmented dermo-epidermal cultured human skin substitutes.

PURPOSE: Transplantation of pigmented tissue-engineered human autologous skin substitutes represents a promising procedure to cover skin defects. We have already demonstrated that we can restore the patient's native light or dark skin color by adding melanocytes to our dermo-epidermal skin analogs. In this long-term study, we investigated if melanocytes in our skin substitutes continue to express markers as BCL2, SOX9, and MITF, known to be involved in survival, differentiation, and function of melanocytes. METHODS: Human epidermal melanocytes and keratinocytes, as well as dermal fibroblasts from light- and dark-pigmented skin biopsies were isolated and cultured. Bovine collagen hydrogels containing fibroblasts were prepared, and melanocytes and keratinocytes were seeded in a 1:5 ratio onto the gels. Pigmented dermo-epidermal skin substitutes were transplanted onto full-thickness wounds of immuno-incompetent rats and analyzed for the expression of melanocyte markers after 15 weeks. RESULTS: Employing immunofluorescence staining techniques, we observed that our light and dark dermo-epidermal skin substitutes expressed the same typical melanocyte markers including BCL2, SOX9, and MITF 15 weeks after transplantation as normal human light and dark skin. CONCLUSIONS: These data suggest that, even in the long run, our light and dark dermo-epidermal tissue-engineered skin substitutes contain melanocytes that display a characteristic expression pattern as seen in normal pigmented human skin. These findings have crucial clinical implications as such grafts transplanted onto patients should warrant physiological numbers, distribution, and function of melanocytes.

Characterization of pigmented dermo-epidermal skin substitutes in a long-term in vivo assay.

In our laboratory, we have been using human pigmented dermo-epidermal skin substitutes for short-term experiments since several years. Little is known, however, about the long-term biology of such constructs after transplantation. We constructed human, melanocyte-containing dermo-epidermal skin substitutes of different (light and dark) pigmentation types and studied them in a long-term animal experiment. Developmental and maturational stages of the epidermal and dermal compartment as well as signs of homoeostasis were analysed 15 weeks after transplantation. Keratinocytes, melanocytes and fibroblasts from human skin biopsies were isolated and assembled into dermo-epidermal skin substitutes. These were transplanted onto immuno-incompetent rats and investigated 15 weeks after transplantation. Chromameter evaluation showed a consistent skin colour between 3 and 4 months after transplantation. Melanocytes resided in the epidermal basal layer in physiological numbers and melanin accumulated in keratinocytes in a supranuclear position. Skin substitutes showed a mature epidermis in a homoeostatic state and the presence of dermal components such as Fibrillin and Tropoelastin suggested advanced maturation. Overall, pigmented dermo-epidermal skin substitutes show a promising development towards achieving near-normal skin characteristics and epidermal and dermal tissue homoeostasis. In particular, melanocytes function correctly over several months whilst remaining in a physiological, epidermal position and yield a pigmentation resembling original donor skin colour.

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.

Tissue-engineered dermo-epidermal skin grafts prevascularized with adipose-derived cells.

The major problem in skin grafting is that tissue-engineered skin grafts after their transplantation are initially entirely dependent on diffusion. Since this process is slow and inefficient, nutrients, growth factors, and oxygen will insufficiently be supplied and the regenerating graft will undergo a physiological crisis, resulting in scar-like dermal structures and shrinkage. The tissue-engineering of a vascular network in human dermo-epidermal skin substitutes (DESS) is a promising approach to overcome this limitation. Here we report, for the first time, on the use of the adipose stromal vascular fraction (SVF)-derived endothelial cell population to tissue-engineer DESS containing a highly efficient capillary plexus. To develop vascular networks in vitro, we employed optimized 3D fibrin or collagen type I hydrogel systems. Upon transplantation onto immune-deficient rats, these pre-formed vascular networks anastomosed to the recipient's vasculature within only four days. As a consequence, the neo-epidermis efficiently established tissue homeostasis, the dermis underwent almost no contraction, and showed sustained epidermal coverage in vivo. Overall, the here described rapid and efficient perfusion of SVF-based skin grafts opens new perspectives for the treatment of hitherto unmet clinical needs in burn/plastic surgery and dermatology.

De novo epidermal regeneration using human eccrine sweat gland cells: higher competence of secretory over absorptive cells.

In our previous work, we showed that human sweat gland-derived epithelial cells represent an alternative source of keratinocytes to grow a near normal autologous epidermis. The role of subtypes of sweat gland cells in epidermal regeneration and maintenance remained unclear. In this study, we compare the regenerative potential of both secretory and absorptive sweat gland cell subpopulations. We demonstrate the superiority of secretory over absorptive cells in forming a new epidermis on two levels: first, the proliferative and colony-forming efficiencies in vitro are significantly higher for secretory cells (SCs), and second, SCs show a higher frequency of successful epidermis formation as well as an increase in the thickness of the formed epidermis in the in vitro and in vivo functional analyses using a 3D dermo-epidermal skin model. However, the ability of forming functional skin substitutes is not limited to SCs, which supports the hypothesis that multiple subtypes of sweat gland epithelial cells hold regenerative properties, while the existence and exact localization of a keratinocyte stem cell population in the human eccrine sweat gland remain elusive.

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.

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.