| In daily life, skin defects caused by diseases and incidents such as burns and traffic accidents are common. It is reported, there are 4 million patients needed skin graft annually in China. The traditional methods of skin transplatation are autograft, allograft and xenograft. All of them have their disadvantages, the emergence of tissue engineered skin offers us a better method to treat skin wounds.In 1987 the word of tissue engineering was first put forword in Wastington science fund committee. In the following decades, it had made great progress, its study aims kept expanding and its meaning changed rapidly, so it is hard to make a clear define for it. Recently, the most well acceptable define of tissue engineering is a cross science which utilitize the basic principle of engineering and life sciences to research and develop biological artifical substitutes to maintain, recover or enhance the function of injured body tissue which expressed by R. Langer, a professor of massachusetts institute of technology and J.P. Vacanti, a Harvard medical school professor in their article published in Science 1993.The fabrication of tissue engineered skin involves three parts: seed cells, scaffolds with good biocompatibility and biodegradability and the interaction of key cells and scaffolds.Among those the fabrication of ECM analogue-tissue engineering scaffold is one of key processes. In addition, growth factors are important for the fabrication of tissue engineered skin.The artifical skins were studied earlier than the concept of tissue engineering was put forward. 1981 for the first time.Yannas and Berk successfully constructed a skin analogue made of collagen/6-sulphate-chondroitin which could induce wound tissue regeneration. Today there are many engineered skin products to be chosed, such as Integra~? which not including cells component, Dermagraf and Artigraf which including cells component. But those products are hard to get in China, and their prices are too expenseive for Chinese. So it is urgent for us to design and construct our tissue engineered skin products.Researchers in Department of Polymer Science and Engineering of Zhejiang University have worked in this field many years. They have constructed a collagen-chitosan/ silicone bilayer artifical skin as skin analogues, collagen/chitosan porous scaffolds as derml substitutes is made of collagen and chitosan, both of them are nature biological polymolecular, have good biocompatibility and can be biodegraded, can induce cells adherence and to excrete ECM, which would accelerate the healing of skin woud. It was observed in our pilot experiment, collagen-chitosan scaffolds combined with angiogenin could promote grafts angiogenesis when embedded subcutaneously on the New Zealand rabbit ears. The silicone film, functions as a temporary epidermis, which can play a role in controlling water loss and inhibiting bacterial entry and so on.ObjectivesEstablish a minipig skin defect wound model for artifical skin transplantation and observe its biological activities after grafted, which is the foundation for further laboratorial study and clinical application.Method and materialsBama minpigs supplied by zhe Zhejiang University Animal Unit were used in this study. Animals were anaesthetised by 3% pentobarbital sodium and SU MIAN XIN. After anaesthesiacircular full-thickness wounds of 3.3 cm diameter down to muscle fascia were made by lancet on the flank of each animal. Wounds were isolated by percutaneou ploypropylene chambers and grafted with biolayer Artificial Skin supplied by Department of Polymer Science and Engineering of Zhejiang Univrsity. This chamber was sutured interruptedly with wound edge skin. To achieve close approximation of the bilayer Artificial Skins to the wound bed the disk perimeter was sutured to the wound bed using a circumferential over and over suture. After suture the vaseline gauze, 0.25% aqueous silver nitrate soaked gauze and PVP soaked gauze were inserted in the chamber. The wound then firmly packed with PVP soaked gauze.At 3,7,14,21 days after surgery the dressings were changed and antibiotic prophylaxis was provided by a single intra-muscular injection of penicillin. The wounds were inspected and histological biopsies were taken at 7,14,21,28 days. Tissue section were stained with HE and immunohistochemical methods. The blood vessels were calculated.Result1. Animal modelWe have successfully established an animal model for artifical skin transplantation. The take rate for the artifical skin was 94%.2. General inspection and wound inspectionThe collagen-chitosan/silicone bilayer artifical skins were firmly adherent to the wound beds and no subcutaneous liquid. There were slight inflammatory reactions in wounds.There were granulation tissues between the chamber and the artifical skin at 7 days.At 14 days the granulation tissues growed more.At 1 week the silicone memberanes was sticked with collagen-chitosan scaffolds, but till 2 weeks it was partially separate from the scaffolds. At 3 weeks or later, it was totally apart. To observe the wounds after removing the silicone memberanes, we found the scaffolds were whrite and remained integrity before 2 weeks. At 3 weeks or later, there were only granulation tissues in wounds. The spirit and the appetite of the minipigs after surgery were similar to which of pre-operation. The body temperatures ofminipigs in 3 days after surgery were normal. 3. Histological observation3.1 Inflammatory reactions1 week after grafted there were many inflammation cells infiltrated in the lower part of scaffolds.Among them lymphocytes, neutrophilic cells were prominent. At 2 weeks the number of inflammation cells increased and the infiltrated area was expanded. The macrophage cells could be found in the scaffolds. At 3 weeks the whole scaffolds were infiltrated with inflammation cells, the number of macrophage cells significantly improved. In some areas we could see foreign body granuloma. The inflammation reactions of the 4 weeks were similar to 3 weeks.3.2 Scaffolds and fibroblasts1 week after grafted there were lots of fibroblasts infiltrated in the lower part of scaffolds which excreted a great deal of ECM at the same time. At the conjunct areas between dermal substitutes and muscle fascias we could see the Fb ingrowth to the scaffolds.2 weeks after grafted fibroblasts infiltrated to the upper part. 3 weeks and 4 weeks even on the top of scaffolds there were granulation tissues. The granulation tissues, the scaffolds and the muscles formed the Sandwich structure.3.3 Vascularization of scaffoldsAt 1 week after grafted there were a few blood vessels in the scaffolds. The blood vessels hardly were seen in the middle of scaffolds. At 2 weeks the number of blood vessels increased, most of the blood vessels were in the conjunct areas. 3 weeks and 4 weeks after transplantation there were many blood vessels in the scaffolds, we also could find some blood vessels in the middle part of scaffolds. Compared with 3 weeks the number of blood vessels added and the density of it enhanced at 4 weeks.3.4 Biodegradation of scaffoldFrom 1 week to 4 weeks after surgery, there were biodegradation of scaffolds at different levels. At 1 week the biodegradation was slight. With the time went by, the biodegradationbecome severity. At 4 weeks the scaffold were half-baked, the scaffold partial disappeared and replaceded by new born dermal tissue.Conclusion1. The minipig model could be well used in artifical skins transplatation and to observe and investigate the biological activities of artifical skins after grafted on big animals.2. Initial study shows the collagen-chitosan/silicone bilayer artifical skin has good biocompatibility and can induce fibroblasts and vessels ingrowth.3. The complete biodegradation of scaffolds needs more time than 4 weeks.4. The intensity of biocompatible glue is not strong enough for the study, it should be improved.
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