| Purpose:To establish Bama minipig skin hypertrophic scar model by taking the half-thickness skin graft and preparing the half dermal-thickness burn. Detect the expression of TGF-β1/TGF-β3 in wound tissues during the process of wound healing. Evaluate the stimulation of skin hypertrophic scar of animal model and human.Methods:Six healthy 4 month-old female Bama minipigs were selected, which weighed about 13±0.6 kg. Wounds were prepared on animal back after anesthesia. Half full-thickness skin grafts were incised on left side and half dermal-thickness burn wounds were created on the right side. Each wound area was 3*3 cm2 and interval measured 2cm. Animals were feeded separately after the wound created to insure the wounds healed naturally, and the wounds healing status was observed.24 hours,7,14,21,28,56 days after the wound created, tissue specimens were taken from the wound randomly. Observe the epidermis and the dermis histologic changes in the wound healing process and measure the scar thickness by H&E staining. Observe the form and arrangement of corium layer collagen fiber in the wound healing process and scar formation and measure the AA by Masson staining. Detect the expression of TGF-β1/TGF-β3 in the process of wound healing and scar formation by immunohistochemical staining. And detect the expression of TGF-β1/TGF-β3 mRNA in the process of wound healing and scar formation by RT-qPCR. Observe the fibroblast and the structure of collagen fiber between normal skin and day 56 scar tissue by transmission electron microscope.Results:1. After the wounds created, errhysiscould be seen on the surface of incised wounds. There were clear boundaries between burn wound and surrounding tissues and blisters dispersed visibly. Around 28 days after wound created, the wound healing rate of incised wound and burn wound is respectively 99.8% and 99.0%. Wounds contracted with red color; on day 56 after wounds created, tough hyperplastic tissue could be palpated in the center of wound, with no obvious hair growth.2. H&E staining showed that in the wound healing process, gradually increased inflammatory cells and fibroblasts chemotactic in dermal papilla of wound tissue which compound and secret abundant collagen fibers could be seen 7 days after the wounds were made. Comparing with normal skins, there were more fibroblasts and collagen fibers in the scar tissue on day 56 after wound created. The thickness of normal skin is 1.298±0.305 mm, and the thickness of incised wound and burn wound is respectively 3.039±0.169 mm and 4.023±0.176 mm. According to Masson staining observation, in the wound healing process, dermal papilla showed abundant slim collagen fibers which encircled fibroblasts, and collagen fibers in the lamina reticular layerwere thick with tight and disorder arrangement.3. Transmission electron microscope revealed that in scar tissues, the nucleolus of fibroblasts was enlarged and fibroblasts cytoplasm was rich. A large number of rough endoplasmic reticulums were expanded and enlarged spindle cells could be easily observed.The intensity of collagen fiber bundle was increased.4. The immunehistochemical staining:there were significant statistical differences in the expression of TGF-β1/TGF-β3 between tissues from different period of wound healing and normal skin (P< 0.05). More over, TGF-β1 had the highest positive expression on day 7 after wound created (P< 0.05), TGF-β3 had the highest positive expression on day 21 after wound created (P<0.05)5. RT-qPCR:there were significant statistical differences in the expression of TGF-β1/TGF-β3 mRNA between tissues from different period of wound healing and normal skin (P<0.05). The relative expression level of TGF-β1 mRNA reached the summit at 7th day after wound created (P< 0.05). Scar tissue TGF-β1 level still exceeded normal skins’even on day 56 after wound created (P <0.05). TGF-β3 was slowly up-regulated and reached the highest level on day 21 (P<0.05). And the expression of TGF-β3 in scar tissue still exceeded normal skin on day 56 after wound created (P<0.05)Conclusion:1. The half full-thickness skin graft incised wound can lead to skin hypertrophic scar. It can cause half dermal-thickness burn by the equipment reached 100℃ contacting skin 20s, which also can form hypertrophic scar on Bama minipig’s back. Histology characteristics of hypertrophic scar are similar to human beings.2. The expression of TGF-(3i/TGF-β3 in hypertrophic scar tissues are similar to human beings, which validates the reliability of the hypertrophic scar model. |
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