Bio-engineering of fetal cartilage for in utero spina bifida repair.

PURPOSE: During in utero surgical spina bifida repair, a multi-layer closure is used to cover the defect. These soft tissues, however, might be not sufficient to protect the spinal cord during the future life. Our goal is to develop a more rigid protective tissue construct consisting of bioengineered cartilage and skin. METHODS: Ovine fetal chondrocytes were tested for their in vitro chondrogenic potential in three-dimensional cultures. Scaffolds based on natural biopolymers (collagen I, fibrin glue) were loaded with varying amounts of fetal chondrocytes and assessed for their ability to support cartilage formation in vitro. The bioengineered constructs were analyzed using cartilage-specific histology stainings and compared to native fetal cartilage. RESULTS: Fetal chondrocytes actively produced cartilage extracellular matrix in three-dimensional cultures, even at high passages. Among all bioengineered scaffolds, only the collagen I-based hydrogels loaded with high densities of fetal chondrocytes showed cartilage-like structure in vitro but also extensive shrinking. CONCLUSION: Fetal chondrocytes represent a good cell source for cartilage bioengineering. Collagen I scaffolds support cartilage formation in vitro, but the construct shrinking constitutes a major limitation. Future steps include the identification of suitable bioprintable materials which maintain their shape and size, as well as the analysis of the interphase between bioengineered cartilage and skin.

Bioengineering of Fetal Skin: Differentiation of Amniotic Fluid Stem Cells into Keratinocytes.

PURPOSE: Open fetal spina bifida repair has become a novel clinical standard of care. In very large spina bifida lesions, the skin defect cannot be covered primarily, asking for alternative solutions. We hypothesize that amniotic fluid stem cells (AFSC) could be differentiated into keratinocytes that could then be used to bioengineer autologous skin usable for in utero back coverage. METHODS: To obtain human AFSC, amniotic fluid samples obtained from fetal surgeries were subjected to immunoselection for c-kit. C-kit-positive samples and controls were cultured with the additives morphogenetic protein 4 and vitamin C to induce differentiation towards keratinocytes. This process was monitored by measuring the expression of K8 and K14 via immunohistochemical staining, flow cytometry, and polymerase chain reaction. RESULTS: After immunoselection and expansion, most cells were positive for K8, but none for K14. After completion of the differentiation protocol, cell colonies with keratinocyte-like appearance could be observed, but cells remained positive for K8 and negative for K14, indicating failed differentiation into keratinocytes. CONCLUSIONS: Culturing of keratinocyte-like cells from AFSC, harvested intraoperatively, was not feasible in this setting. The reasons for failure must be investigated and eliminated, as bioengineering of fetal skin for clinical use during fetal surgery for spina bifida remains an attractive goal.

Epithelial proliferation in inflammatory skin disease is regulated by tetratricopeptide repeat domain 7 (Ttc7) in fibroblasts and lymphocytes.

BACKGROUND: Mutations in tetratricopeptide repeat domain 7A (TTC7A) and its mouse orthologue, Ttc7, result in a multisystemic disease, mostly affecting the epithelial barriers and immune system. Despite successful hematopoietic stem cell transplantation, ongoing progression of gastrointestinal manifestations can be life-threatening in TTC7A-deficient patients. OBJECTIVE: We sought to identify whether TTC7A mutations dysregulate epithelial cells only or whether a cell-intrinsic defect in lymphocytes or other cells contributes to disease manifestations. METHODS: Ttc7-mutated (Ttc7fsn/fsn) mice were crossed to generate double-mutant (Rag2-/-Ttc7fsn/fsn) and triple-mutant (Rag2-/-IL2rg-/-Ttc7fsn/fsn) mice. These models, together with bone marrow chimeras, were used to explore the role of adaptive and innate lymphocytes in the flaky skin phenotype. The effect of the Ttc7fsn/fsn mutation on stromal cells was tested in a xenograft model in conjunction with transcriptomic analysis of Ttc7fsn/fsn fibroblasts. RESULTS: We observed that the severity of epithelial hyperproliferation was accentuated by lymphocytes, whereas the phenotype was not induced by transfer of Ttc7-mutated hematopoietic cells. Furthermore, mice completely lacking the lymphocytic compartment were not protected from epithelial hyperproliferation. Ttc7-mutated mouse fibroblasts expressed increased transcript levels of insulin-like growth factor 1 (Igf1) and the antimicrobial protein regenerating islet-derived protein 3γ (Reg3γ). In a xenograft model Ttc7-mutated fibroblasts markedly increased epithelial proliferation of keratinocytes. Thus Ttc7-mutated fibroblasts were identified as potent instigators of epithelial hyperproliferation. CONCLUSION: Our results reveal a previously unsuspected fundamental cell-extrinsic role of Ttc7. We have identified potential candidates for molecularly targeted treatment strategies that will need to be evaluated in future preclinical studies.

Induction of angiogenic and inflammation-associated dermal biomarkers following acute UVB exposure on bio-engineered pigmented dermo-epidermal skin substitutes in vivo.

PURPOSE: Ultraviolet (UV) radiation adversely affects skin health at cellular and molecular levels. Hence, UV radiation can directly induce inflammatory responses in the dermis by inducing erythema, edema, inflammation, dermal fibroblasts alterations, and extracellular matrix modifications. METHODS: Human keratinocytes, melanocytes, and fibroblasts were isolated from skin biopsies, cultured, and expanded in vitro. Fibroblasts were seeded into collagen type I hydrogels that were subsequently covered by keratinocytes and melanocytes. These pigmented dermo-epidermal skin substitutes (pigmDESS) were transplanted for 5 weeks onto full-thickness skin wounds on the back of immuno-incompetent rats, exposed to a single UVB dose of 250 mJ/cm2 or unexposed and excised after 1 week. The effects onto the dermis were assessed regarding cell number, cell phenotype, and cell proliferation. Local inflammation by granulocytes (HIS48) or macrophages (CD11b, iNOS) was analyzed by immunohistochemistry staining. RESULTS: We observed a significantly enhanced ingrowth rate of blood capillaries, but not of lymphatic capillaries at 1 week post-irradiation. Moreover, the enhanced vascularization of pigmDESS after UVB exposure was concomitant with a high infiltration of granulocytes and monocytes/macrophages to the dermal part of grafts. In addition, a heterogeneous expression of HIF-1α and TNFα was detected at this early phase after UVB exposure. In local cellular response examination, results only show a moderate cell proliferation in the dermis. CONCLUSIONS: We were able to define early markers of UVB-induced effects in the dermis of pigmDESS. Overall, a single UVB dose induces temporary acute angiogenic and immune responses during the early post-irradiation phase in vivo.

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.

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.

Characterization of M1 and M2 polarization of macrophages in vascularized human dermo-epidermal skin substitutes in vivo.

AIMS AND OBJECTIVES: Vascularized bio-engineered human dermo-epidermal skin substitutes (vascDESS) hold promise for treating burn patients, including those with severe full-thickness wounds. We have previously shown that vascDESS promote wound healing by enhanced influx of macrophages and granulocytes. Immediately following transplantation, macrophages infiltrate the graft and differentiate into a pro-inflammatory (M1) or a pro-healing M2 phenotype. The aim of this study was to characterize the activation state of macrophages infiltrating skin transplants at distinct time points following transplantation. METHODS: Keratinocytes and the stromal vascular fraction (SVF) were derived from human skin or adipose tissue, respectively. Human SVF containing both endothelial and mesenchymal/stromal cells was used to generate vascularized dermal component in vitro, which was subsequently covered with human keratinocytes. Finally, vascDESS were transplanted on the back of immuno-incompetent rats, excised, and analyzed after 1 and 3 weeks using immunohistological techniques. RESULTS: A panel of markers of macrophage M1 (nitric oxide synthase: iNOS) and M2 (CD206) subclass was used. All skin grafts were infiltrated by both M1 and M2 rat macrophages between 1-3 weeks post-transplantation. CD68 (PG-M1) was used as a pan-macrophage marker. The number of CD68+CD206+ M2-polarized macrophages was higher in 3-week transplants as compared to early-stage transplants (1 week). In contrast, the number of CD68+iNOS+ M1 cells was markedly decreased in later stages in vivo. CONCLUSIONS: Macrophages exhibit a heterogeneous and temporally regulated polarization during skin wound healing. Our results suggest that the phenotype of macrophages changes during healing from a more pro-inflammatory (M1) profile in early stages after injury, to a less inflammatory, pro-healing (M2) phenotype in later phases in vivo.

Comparison of in vivo immune responses following transplantation of vascularized and non-vascularized human dermo-epidermal skin substitutes.

PURPOSE: Autologous bio-engineered dermo-epidermal skin substitutes (DESS) represent an alternative therapeutic option for a definitive treatment of skin defects in human patients. Largely, the interaction of host immune cells with transplanted DESS is considered to be essential for the granulation tissue formation, graft take, and its functionality. The aim of this study was to compare the spatiotemporal distribution and density of host-derived monocytes/macrophages and granulocytes in vascularized (vascDESS) versus non-vascularized DESS (non-vascDESS) in a rat model. METHODS: Keratinocytes and the stromal vascular fraction (SVF) were derived from human skin or human adipose tissue, respectively. Human SVF containing both endothelial and mesenchymal/stromal progenitors was used to develop a vascularized collagen type I-based dermal component in vitro. The donor-matched, monolayer-expanded adipose stromal cells lacking endothelial cells were used as a negative control. Subsequently, human keratinocytes were seeded on top of hydrogels to build dermo-epidermal skin grafts. After transplantation onto full-thickness skin wounds on the back of immuno-incompetent rats, grafts were excised and analyzed after 1 and 3 weeks. The expression of distinct inflammatory cell markers specific for host-derived monocytes/macrophages (CD11b, CD68) or granulocytes (HIS48) was analyzed by immunofluorescence microscopy. RESULTS: All skin grafts were infiltrated by host-derived monocytes/macrophages (CD11b+, CD68+) and granulocytes (HIS48+) between 1-3 week post-transplantation. When compared to non-vascDESS, the vascDESS showed an increased granulocyte infiltration at all time points analyzed with the majority of cells scattered throughout the whole dermal part. Whereas a moderate number of rat monocytes/macrophages (CD11b+, CD68+) were found in vascDESS at 1 week, only a few cells were detected in non-vascDESS. We observed a time-dependent decrease of monocytes/macrophages in all transplants at 3 weeks. CONCLUSIONS: These results demonstrate a distinct spatiotemporal distribution of monocytes/macrophages as well as granulocytes in our transplants that closely resemble the one observed during physiological wound healing. The differences identified between vascDESS and non-vascDESS may indicate that human endothelial cells lining blood capillaries of vascDESS accelerate infiltration of monocytes and leukocytes.

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.

About ATMPs, SOPs and GMP: The Hurdles to Produce Novel Skin Grafts for Clinical Use.

BACKGROUND: The treatment of severe full-thickness skin defects represents a significant and common clinical problem worldwide. A bio-engineered autologous skin substitute would significantly reduce the problems observed with today's gold standard. METHODS: Within 15 years of research, the Tissue Biology Research Unit of the University Children's Hospital Zurich has developed autologous tissue-engineered skin grafts based on collagen type I hydrogels. Those products are considered as advanced therapy medicinal products (ATMPs) and are routinely produced for clinical trials in a clean room facility following the guidelines for good manufacturing practice (GMP). This article focuses on hurdles observed for the translation of ATMPs from research into the GMP environment and clinical application. RESULTS AND CONCLUSION: Personalized medicine in the field of rare diseases has great potential. However, ATMPs are mainly developed and promoted by academia, hospitals, and small companies, which face many obstacles such as high financial burdens.

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.

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.

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 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.

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.

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.

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 3-4 weeks. 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 6 days), and the culture time of keratinocytes (3, 6, and 12 days) before transplantation of DESS on nude rats. Additionally, the impact of the air-liquid interface culture during 3 days before transplantation was investigated. 3 weeks 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 6 days. The minimal culture time of keratinocytes on hydrogels was also 6 days. 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 12 days. 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 skipped.

Human amniotic fluid derived cells can competently substitute dermal fibroblasts in a tissue-engineered dermo-epidermal skin analog.

PURPOSE: Human amniotic fluid comprises cells with high differentiation capacity, thus representing a potential cell source for skin tissue engineering. In this experimental study, we investigated the ability of human amniotic fluid derived cells to substitute dermal fibroblasts and support epidermis formation and stratification in a humanized animal model. METHODS: Dermo-epidermal skin grafts with either amniocytes or with fibroblasts in the dermis were compared in a rat model. Full-thickness skin wounds on the back of immuno-incompetent rats were covered with skin grafts with (1) amniocytes in the dermis, (2) fibroblasts in the dermis, or, (3) acellular dermis. Grafts were excised 7 and 21 days post transplantation. Histology and immunofluorescence were performed to investigate epidermis formation, stratification, and expression of established skin markers. RESULTS: The epidermis of skin grafts engineered with amniocytes showed near-normal anatomy, a continuous basal lamina, and a stratum corneum. Expression patterns for keratin 15, keratin 16, and Ki67 were similar to grafts with fibroblasts; keratin 1 expression was not yet fully established in all suprabasal cell layers, expression of keratin 19 was increased and not only restricted to the basal cell layer as seen in grafts with fibroblasts. In grafts with acellular dermis, keratinocytes did not survive. CONCLUSION: Dermo-epidermal skin grafts with amniocytes show near-normal physiological behavior suggesting that amniocytes substitute fibroblast function to support the essential cross-talk between mesenchyme and epithelia needed for epidermal stratification. This novel finding has considerable implications regarding tissue engineering.

"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 8 weeks, 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 color.