| Background and ObjectiveLarge areas of skin defect by severe trauma, burns and other causes is a common clinical condition. The traditional ways of wound closure are skin graft or skin flap transfer on granulation tissue of the wound through dressing after wound debridement. Granulation tissues by traditional dressing grow slowly which lead to a long time to achieve healing. These will cause great pain to the patient and increase the workload of doctors. However, the split-thickness skin graft is lack of a complete leather structure, so it is difficult to complete reconstruction of dermal tissue. Patient is often difficult to accept full thickness skin, thick skin or skin flap grafting because they will cause new wounds and scars. In addition, the large skin defect will lead to supply shortages. Skin tissue engineering offers new ideas and methods for skin defect repair, and dermal substitutes become a hot research of skin tissue engineering. Dermal substitute plays an important role in the reconstruction process of the skin, which can enhance wound healing in skin elasticity, flexibility and mechanical wear, reducing scar hyperplasia, control wound contraction.More and more studies have found that tissue-engineered skin needs the support of the dermal scaffold which will improve the function of the epidermis and dermis reconstruction. On the one hand, dermal template can guide fibroblasts and vascular endothelial cell ingrowth and proliferation which promote the formation of new dermal tissue and dermal remodeling. On the other hand, dermal provides nutrients to the epidermis through the interaction between epidermis and dermis to ensure the survival of the skin. Dermal scaffold also provides a good signal for the epidermis to promote their maturation and differentiation. Foreign researchers have successfully developed a variety of dermal substitutes. So far, one of the ideal dermal substitutes is Integra which is initially used in the repair severe trauma and the depth of burn wounds, and has made certain clinical effect. But these tissue-engineered skins are expensive and limited. Ordinary people are difficult to pay for it. In this experiment, bilayer artificial skin developed by Shenzhen Qi Kang Medical Devices Co., Ltd. is similar with the Integra. The upper layer is made of silicone and the under layer is comprised of a porous matrix of cross-linked bovine tendon collagen and glycosaminoglycan. Whether this artificial skin can be quickly vascularization and provide a good bed for autologous skin graft transplant which are the foundation of the late wounds repair?To establish the animal model of full-thickness skin wounds of Tibetan pig for bilayer artificial skin transplants in this experiment. We carried out a preliminary experiment about bilayer artificial skin vascularization impact. The results showed that the artificial skin had not been separated from the double structures and did not close adhesion with the wound substrate at 3d,7d post operation after transplantation. If we tried to unfold the upper silica film which would not only damage the vascular tissue, but also made the destruction of dermal integrity. Artificial skin survived well and its double-layer structure had been isolated at 14d,21 d post operation after transplantation. We could saw granulation tissue when we peeled off the upper silicone membrane. Artificial skin and wound tissue substrate are of adhesion very closely at these times. On this basis, we further observe double artificial skin vascularization after implantation and observe wound healing at 14d,21d post operation with autologous skin grafting. Our aims are to suggest the best time point for autologous skin grafting and observe the efficacy of the bilayer artificial skin with autologous skin grafts in repairing full-thickness skin wounds in order to provide theoretical and experimental basis for future clinical application.Materials and methodsAnimals and groups:24 Tibetan pig (provided by the Southern Medical University Animal Center), weight 30-35Kg, male or female. Before surgery, animals were fasted for 12 h and 4 h water deprivation. Anesthesia was using SU-MIAN-XIN Ⅱ 0.1 ml/kg intramuscularly and 3% sodium pentobarbital 1 ml/kg intraperitoneal injection. After the animals were anesthetized, the animals were fixed on the operating table. Each animal produced full-thickness skin defect wounds on the back of each side, length of 5 cm, deep into muscle fascia. Each side of back had three wounds from the spine at least 3cm.The distance between the ipsilateral wounds up more than 5cm. After stanch bleeding and debridement of the wounds, we repeatedly washed the wounds with saline containing gentamicin. The wounds were covered with saline gauze. This study used self-controlled. The right side wounds were covered by bilayer artificial skin (experimental group), while the left side by Vaseline gauze (control group). Experimental group putted the bilayer artificial skin, which silicone membrane side up, into the right side of the wounds. Bilayer artificial skin was fixed to the edge of normal skin, and set aside 10cm long suture in order to package. Vaseline gauze directly covered the wounds of control group, the rest steps of the operation referred to the experimental group. Sterile gauze and sterile single coverage the wounds again after all wounds completion of packaged, and bandage wrapped. Eight animals were used for observing wound vascularization which were respectively sacrificed two at 3d,7d,14d,21 d post operation after transplantation. Observation indexes included the general situation of wounds, HE staining, Masson staining and CD31 immunohistochemical staining. Another 16 animals were used for observing wound healing at 2w,4w,8w,12w post operation of 14d,21d post operation with autologous skin grafting. Observation indexes included the general situation of wounds, HE staining, Masson staining and CD31 immunohistochemical staining.Results1. Wound general observationAfter I period surgery, overall, wounds in the experimental group were dryer than the control group, with lower incidence of infection, better granulation tissue formation and growth. There were not obvious red and swollen at the margin of wounds. At the 3d post operation, there were not granulation tissues in the wounds of the experimental group. Granulation tissue gradually increased at the 7d post operation, dermal scaffold form kept intact, but not closely adhered to the wound base structure. At the 14d post operation, artificial skin double layer structure showed partial separation. Peeling off the silicone membrane, the entire frame was almost filled of bright red granulation tissue which surface was flat. Dermal scaffold closely adhered to the wound base structure. At 21d post operation, artificial skin double layer structure was completely separated. Dermal scaffold did not be visible. Granulation tissues were still flat, but not easy bleeding.At 2w post operation of 14d post operation with autologous skin grafting, almost skin survived well in the experimental group and control group. The rate of skin survival was higher in the experimental group than control group, but there were no significant difference between groups. At 2w post operation of 21 d post operation with autologous skin grafting, skin survived less than ideal in the experimental group. The rate of skin survival was higher in the control group than experimental group and had significant difference between groups. Compared with the experimental group of 14d post operation with autologous skin grafting, the rate of skin survival was lower in the experimental group and control group of 21d post operation with autologous skin grafting and had significant difference between groups. Skin with good elasticity and flexibility, was smooth and soft, and shrank relatively insignificant in the experimental group of 14d post operation with autologous skin grafting. While skin with poor elasticity and flexibility, shrank significantly in the control group of 14d and the experimental group and control group of 21d post operation with autologous skin grafting.2. Histological examinationAfter I period surgery, overall, HE staining demonstrated that the granulation tissues in the study group increased faster than in control group, and less inflammation and more regular arrangement of collagen in the study group than in control. Most of the capillaries growth pattern was parallel to the wound, which was closer to normal skin. The bottom of dermal scaffold occurred fibroblast at the 3d post operation. At the 7d post operation, scaffold porosity had many fibroblasts which produced extracellular matrix components. The newborn capillaries could be observed at the bottom of dermal scaffold. At the 14d post operation, dermal scaffolds were filled of a large number of fibroblasts, inflammatory cell infiltration, newly formed capillaries which were closed to the surface of the scaffold. At the 21 d post operation, dermal scaffolds were surrounded by the upper granulation tissue and underlying muscle tissue. Original scaffold porosity were filled with fibroblasts and extracellular matrix with less inflammatory cells. Fibroblasts and collagen were orderly arranged. Dermal regeneration scaffolds were gradually of degradation with time.After Ⅱ period skin graft surgery, compared with other groups, the experimental group of 14d post operation with autologous skin grafting showed more well-differentiated epidermis spikes structures. The epidermis was more closely connected to the dermis. Collagen fibers arranged in relatively coarse and orderly. With time, collagen deposition and distribution, in the experimental group was more similar to normal tissue. But all groups had no hair follicles, sweat glands, sebaceous glands and so on. At 2w post operation of 14d post operation with autologous skin grafting in the experimental group, HE staining showed that epidermal became alive with more spikes structure. Dermal scaffold existed degradation. At 4w post operation, the epidermis was continuously connected to the dermis. Spikes structures were well-differentiated and collagen fibers deposited obviously. We observed that the dermal scaffold continued degradation. At 8w post operation, collagen fibers arranged orderly. Inflammatory cells, fibroblasts and capillaries gradually reduced in the experimental group. Dermal scaffold almost completely degraded. At 12w post operation, a large number of epidermal cells grow into the dermis. Epidermis closely connected with dermal. Dermal collagen fibers arranged in order, and there were no nodules of scar tissue structures in the dermal layer.3. CD31 immunohistochemistry and counted the number of new blood vesselsAfter Ⅰ period surgery, two groups had no significant expression of CD31 at the 3d post operation,. CD31 expression significantly increased at the 7d post operation in two groups. The CD31 expression,in study group, reached a peak at the 14d, while control group at the 21 d. The CD31 expression was higher in study group at the 7d and 14d than in control group and had significant difference between groups. However, it was significantly lower in study group at the 21d when compared with control group.The number of CD31 expression positive neovascularization in two groups after 14d,21d post operation with autologous skin grafting was decreasing with time. The number of CD31 expression in the study group of 14d post operation with autologous skin grafting reduced the fastest, while the control group of 21d post operation with autologous skin grafting reduced the slowest. Each group in different time points had significant difference. Compared with the same time point of the study group of 14d post operation with autologous skin grafting, there were significant differences in different groups at different time points, except for 2w post operation of 21d post operation with autologous skin grafting in control group.Conclusion1. Compared with conventional gauze dressing, bilayer artificial skin has good biocompatibility and biodegradability, which can induce repair cells ingrowth and adequate and obtain rapid and sufficient vascularization rate in the early time.2.14d after transplantation of artificial skin is a better time for combining with skin grafting which can improve the quality of wound healing. |
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