A Cultured Autologous Dermo-epidermal Skin Substitute for Full-Thickness Skin Defects: A Phase I, Open, Prospective Clinical Trial in Children.

BACKGROUND: The management of deep partial-thickness and full-thickness skin defects remains a significant challenge. Particularly with massive defects, the current standard treatment, split-thickness skin grafting, is fraught with donor-site limitations and unsatisfactory long-term outcomes. A novel, autologous, bioengineered skin substitute was developed to address this problem. METHODS: To determine whether this skin substitute could safely provide permanent defect coverage, a phase I clinical trial was performed at the University Children's Hospital Zurich. Ten pediatric patients with acute or elective deep partial- or full-thickness skin defects were included. Skin grafts of 49 cm were bioengineered using autologous keratinocytes and fibroblasts isolated from a patient's small skin biopsy specimen (4 cm), incorporated in a collagen hydrogel. RESULTS: Graft take, epithelialization, infection, adverse events, skin quality, and histology were analyzed. Median graft take at 21 days postoperatively was 78 percent (range, 0 to 100 percent). Healed skin substitutes were stable and skin quality was nearly normal. There were four cases of hematoma leading to partial graft loss. Histology at 3 months revealed a well-stratified epidermis and a dermal compartment comparable to native skin. Mean follow-up duration was 15 months. CONCLUSIONS: In the first clinical application of this novel skin substitute, safe coverage of skin defects was achieved. Safety and efficacy phase II trials comparing the novel skin substitute to split-thickness skin grafts are ongoing. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

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.

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.

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.

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.

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.

The genome of Chelonid herpesvirus 5 harbors atypical genes.

The Chelonid fibropapilloma-associated herpesvirus (CFPHV; ChHV5) is believed to be the causative agent of fibropapillomatosis (FP), a neoplastic disease of marine turtles. While clinical signs and pathology of FP are well known, research on ChHV5 has been impeded because no cell culture system for its propagation exists. We have cloned a BAC containing ChHV5 in pTARBAC2.1 and determined its nucleotide sequence. Accordingly, ChHV5 has a type D genome and its predominant gene order is typical for the varicellovirus genus within the alphaherpesvirinae. However, at least four genes that are atypical for an alphaherpesvirus genome were also detected, i.e. two members of the C-type lectin-like domain superfamily (F-lec1, F-lec2), an orthologue to the mouse cytomegalovirus M04 (F-M04) and a viral sialyltransferase (F-sial). Four lines of evidence suggest that these atypical genes are truly part of the ChHV5 genome: (1) the pTARBAC insertion interrupted the UL52 ORF, leaving parts of the gene to either side of the insertion and suggesting that an intact molecule had been cloned. (2) Using FP-associated UL52 (F-UL52) as an anchor and the BAC-derived sequences as a means to generate primers, overlapping PCR was performed with tumor-derived DNA as template, which confirmed the presence of the same stretch of "atypical" DNA in independent FP cases. (3) Pyrosequencing of DNA from independent tumors did not reveal previously undetected viral sequences, suggesting that no apparent loss of viral sequence had happened due to the cloning strategy. (4) The simultaneous presence of previously known ChHV5 sequences and F-sial as well as F-M04 sequences was also confirmed in geographically distinct Australian cases of FP. Finally, transcripts of F-sial and F-M04 but not transcripts of lytic viral genes were detected in tumors from Hawaiian FP-cases. Therefore, we suggest that F-sial and F-M04 may play a role in FP pathogenesis.

Matriderm® 1 mm versus Integra® Single Layer 1.3 mm for one-step closure of full thickness skin defects: a comparative experimental study in rats.

PURPOSE: Dermal templates, such as Matriderm® and Integra®, are widely used in plastic and reconstructive surgery, often as two-step procedures. A recent development is the application of thin dermal templates covered with split thickness skin grafts in one-step procedures. In this experimental study, we compare the two thin matrices Matriderm® 1 mm and Integra® Single Layer in a one-step procedure with particular focus on neodermis formation. METHODS: Matriderm® 1 mm and Integra® Dermal Regeneration Template-Single Layer (1.3 mm) were compared in a rat model. In three groups of five animals each, a full thickness wound was covered with (a) Matriderm® 1 mm and neonatal rat epidermis, (b) Integra® Single Layer and neonatal rat epidermis, or, (c) neonatal rat epidermis only (control). Histological sections 2 weeks post transplantation were analyzed with regard to take of template and epidermis, neodermal thickness, collagen deposition, vascularization, and inflammatory response. RESULTS: Take of both templates was complete in all animals. The Matriderm®-based neodermis was thinner but showed a higher cell density than the Integra®-based neodermis. The other parameters were similar in both matrices. CONCLUSION: The two templates demonstrate a comparable biological behavior early after transplantation. The only difference was found regarding neodermal thickness, probably resulting from faster degradation of Matriderm®. These preliminary data suggest that both dermal templates appear similarly suitable for transplantation in a one-step procedure.

Modified plastic compression of collagen hydrogels provides an ideal matrix for clinically applicable skin substitutes.

Tissue engineering of clinically applicable dermo-epidermal skin substitutes is crucially dependent on the three-dimensional extracellular matrix, supporting the biological function of epidermal and dermal cells. This matrix essentially determines the mechanical stability of these substitutes to allow for safe and convenient surgical handling. Collagen type I hydrogels yield excellent biological functionality, but their mechanical weakness and their tendency to contract and degrade does not allow producing clinically applicable transplants of larger sizes. We show here that plastically compressed collagen type I hydrogels can be produced in clinically relevant sizes (7×7 cm), and can be safely and conveniently handled by the surgeon. Most importantly, these dermo-epidermal skin substitutes mature into a near normal skin that can successfully reconstitute full-thickness skin defects in an animal model.

Skingineering I: engineering porcine dermo-epidermal skin analogues for autologous transplantation in a large animal model.

BACKGROUND: Extended full thickness skin defects still represent a considerable therapeutic challenge as ideal strategies for definitive autologous coverage are still not available. Tissue engineering of whole skin represents an equally attractive and ambitious novel approach. We have recently shown that laboratory-grown human skin analogues with near normal skin anatomy can be successfully transplanted on immuno-incompetent rats. The goal of the present study was to engineer autologous porcine skin grafts for transplantation in a large animal model (pig study = intended preclinical study). MATERIALS AND METHODS: Skin biopsies were taken from the pig's abdomen. Epidermal keratinocytes and dermal fibroblasts were isolated and then expanded on culture dishes. Subsequently, highly concentrated collagen hydrogels and collagen/fibrin hydrogels respectively, both containing dermal fibroblasts, were prepared. Fibroblast survival, proliferation, and morphology were monitored using fluorescent labelling and laser scanning confocal microscopy. Finally, keratinocytes were seeded onto this dermal construct and allowed to proliferate. The resulting in vitro generated porcine skin substitutes were analysed by H&E staining and immunofluorescence. RESULTS: Dermal fibroblast proliferation and survival in pure collagen hydrogels was poor. Also, the cells were mainly round-shaped and they did not develop 3D-networks. In collagen/fibrin hydrogels, dermal fibroblast survival was significantly higher. The cells proliferated well, were spindle-shaped, and formed 3D-networks. When these latter dermal constructs were seeded with keratinocytes, a multilayered and partly stratified epidermis readily developed. CONCLUSION: This study provides compelling evidence that pig cell-derived skin analogues with near normal skin anatomy can be engineered in vitro. These tissue-engineered skin substitutes are needed to develop a large animal model to establish standardized autologous transplantation procedures for those studies that must be conducted before "skingineering" can eventually be clinically applied.

Skingineering II: transplantation of large-scale laboratory-grown skin analogues in a new pig model.

BACKGROUND: Tissue engineering of skin with near-normal anatomy is an intriguing novel strategy to attack the still unsolved problem of how to ideally cover massive full-thickness skin defects. After successful production of large, pig cell-derived skin analogues, we now aim at developing an appropriate large animal model for transplantation studies. MATERIALS AND METHODS: In four adult Swiss pigs, full-thickness skin defects, measuring 7.5 × 7.5 cm, were surgically created and then shielded against the surrounding skin by a new, self-designed silicone chamber. In two animals each, Integra dermal regeneration templates or cultured autologous skin analogues, respectively, were applied onto the wound bed. A sophisticated shock-absorbing dressing was applied for the ensuing 3 weeks. Results were documented photographically and histologically. RESULTS: All animals survived uneventfully. Integra healed in perfectly, while the dermo-epidermal skin analogues showed complete take of the dermal compartment but spots of missing epidermis. The chamber proved effective in precluding ("false positive") healing from the wound edges and the special dressing efficiently kept the operation site intact and clean for the planned 3 weeks. CONCLUSION: We present a novel and valid pig model permitting both transplantation of large autologous, laboratory-engineered skin analogues and also keeping the site of intervention undisturbed for at least three postoperative weeks. Hence, the model will be used for experiments testing whether such large skin analogues can restore near-normal skin, particularly in the long term. If so, clinical application can be envisioned.

Oxidoreductase activity of oligosaccharyltransferase subunits Ost3p and Ost6p defines site-specific glycosylation efficiency.

Asparagine-linked glycosylation is a common posttranslational modification of diverse secretory and membrane proteins in eukaryotes, where it is catalyzed by the multiprotein complex oligosaccharyltransferase. The functions of the protein subunits of oligoasccharyltransferase, apart from the catalytic Stt3p, are ill defined. Here we describe functional and structural investigations of the Ost3/6p components of the yeast enzyme. Genetic, biochemical and structural analyses of the lumenal domain of Ost6p revealed oxidoreductase activity mediated by a thioredoxin-like fold with a distinctive active-site loop that changed conformation with redox state. We found that mutation of the active-site cysteine residues of Ost6p and its paralogue Ost3p affected the glycosylation efficiency of a subset of glycosylation sites. Our results show that eukaryotic oligosaccharyltransferase is a multifunctional enzyme that acts at the crossroads of protein modification and protein folding.