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 21days 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 wa

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 21days 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 engineerin

Matriderm® 1mm versus Integra® Single Layer 1.3mm 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® 1mm and Integra® Single Layer in a one-step procedure with particular focus on neodermis formation. Methods: Matriderm® 1mm and Integra® Dermal Regeneration Template—Single Layer (1.3mm) were compared in a rat model. In three groups of five animals each, a full thickness wound was covered with (a) Matriderm® 1mm and neonatal rat epidermis, (b) Integra® Single Layer and neonatal rat epidermis, or, (c) neonatal rat epidermis only (control). Histological sections 2weeks 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 procedur

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 applie

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 7.5×7.5cm, 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 3weeks. 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 3weeks. 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 envisione

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

Aspects of bio-engineering of human skin : towards clinical application

Skin is the largest organ of the human body and protects it from detrimental effects of the surrounding environment. As skin is directly exposed to the outer environment, it frequently occurs that it is destroyed by accidents. Large full- thickness skin defects may result from burns but also from the surgical excision of congenital giant nevi or scar tissue. The treatment of large (> 40% total body surface area) full-thickness wounds represents a major challenge, as donor sites for autologous skin transplantation often are very limited, and transplantation of autologous split-thickness skin (the present gold standard of treatment) can lead to severe scarring, especially in children. These problems could be significantly reduced applying a bio-engineered autologous dermo-epidermal skin substitute. The Tissue Biology Research Unit (TBRU) has shown that collagen type I hydrogels are a promising scaffold for skin tissue engineering in pre-clinical models. As collagen hydrogels show weak mechanical properties, the first part of my work aimed on engineering human dermo-epidermal skin substitutes based on collagen type I hydrogels which were stabilised by both, plastic compression (established by a postdoctoral fellow in our team) and incorporated biodegradable meshes (established by me). I applied two different meshes of synthetic polymers to mechanically stabilise the collagen hydrogel. I was able to generate skin substitutes which developed into a near normal skin with a dermal compartment and a stratified epidermis in vitro and in vivo using a rat mod. Another aspect of skin tissue engineering and the second part of my work concerns the youngest patients possible, the unborn human fetus. Fetal surgery to treat spina bifida has been convincingly demonstrated to markedly improve the perspectives of the patients. However, closure of skin defects of a fetus is a challenge, and in such cases engineered autologous fetal skin might help. In a first step towards engineering fetal skin, I could successfully apply human amniotic fluid derived cells in the dermal compartment of dermo-epidermal skin analogues, instead of the usually used human dermal fibroblasts, and succeeded in obtaining a well stratified, near normal epidermis. Bio-engineered dermo-epidermal skin substitutes could serve, additionally to their clinical application, for pre-clinical testing of newly designed medicinal products

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

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 x 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