| Background: One of the most important treatment in severely burned patients is appropriate burn wound management for rapid skin repair and regeneration. Timely and effective coverage on burn wound is vital for the patients suffering from burn injury. The classic therapies for skin loss are auto-transplantations, but these methods of dermoplasty more or less lack the component of dermis, would seriously affect the appearance and function of reconstructive skin. The research of ideal dermal graft and dermis transplantation is the chief and important responsibility for the skin tissue engineering researchers and burn surgeons. Xenogenic acellular dermal matrix (ADM) which is usually derived from swine is attracting widespread attention because it is easily available and has some similarities with the structure and function of human dermis. However, the following deficiencies still exist in clinical applications: (1) the strong immunogenicity, long-term presence of local inflammatory and immune response; (2) the current acellular dermal matrix is very dense and has poor nutrition permeability with slow vascularization, subsequently leads to low survival rate after transplantation. This research is focused on the problems in xenogenic ADM. We firstly compared the histological differences of dermis among three species including human, swine and rat, and obtained the morphological evidences for selecting the animal species on xenogenic acellular dermal matrix. Then the methods of preparing acellular dermal matrix were compared between their effects of decellularization. The xenogenic acellular dermal matrix was implanted underneath the skin of rabbits for evaluating the inflammatory response of ADM. Subsequently, the effect of decellularization on the heparin level of ADM was investigated, and the methods of restoring the heparin level of ADM to normal dermis were explored.Objectives1. To perform a comparative study of dermal tissue structure, content of difference types of collagen and biomechanical properties between Bama miniature pig, Sprague-Dawley(SD) rat and human skin, obtain experimental information of resource selection on xenogenic acellular dermal matrix.2. To improve the preparation of acellular dermal matrix (ADM), and evaluate the decellular effects by HE staining, then assess the in vivo biocompatibility by subcutaneous implantation.3. To find a simple method of combining the heparin on the ADM, establish the conditions for further clinical application.Methods1. The dorsal full-thickness skin samples were collected from Bama miniature pig, SD rat and human, and observed grossly. The sections of these samples were stained by hematoxylin and eosin (HE) staining, Masson's trichrome staining, sirius red staining and Verhoeff's Van Gieson (VVG) staining, and were examined under light microscopy. These samples were also observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The relevant data of histological images were measured with image analysis software. In the meantime, these samples were detected by material testing machine for biomechanical properties.2. ADM was prepared by using various concentration of enzymatic digestive solution in various reaction times.The decellular effect was evaluated by HE staining. The decellularization of two methods (with or without hypotonic solution) was also compared. The relevant data of histological images were measured with image analysis software.3. New-Zealand rabbits were divided into four groups randomly. They were cellular dermal matrix (DM) with basement membrane (group A); ADM with basement membrane (group B); ADM without basement membrane (group C); sham operation (group D). Each group had six rabbits. A 2 cm midline incision was made on the dorsum of rabbit, prepared of subcutaneous capsule on both sides of the incision. The size of the subcutaneous capsule was 4 cm×2 cm. The space between two capsules was 2 cm. There were four capsules on each side. The ADMs were cut into 2 cm×2 cm in each piece. Each subcutaneous capsule implanted a piece of ADM. At 1, 2, 4, 8 and 12 weeks after implantation operation, the implants were taken for evaluating the inflammatory reaction. The implant samples were fixed by 4% paraformaldehyde, stained by HE staining, then were examined under light microscopy for observing the inflammatory cells.4. The ADMs were soaked in heparin solution at different times, then measured the content of heparin by toluidin blue spectrophotometry and compared with that of DM.Results1. Compared with human dermis by light microscope, Bama miniature pig had much thicker dermal collagen bundles [(39.29±10.17) m vs (17.21±5.20) m] (P=0.0003), and had significant decrease in the gap rate between these bundles [(19.01±1.55)% vs (32.36±1.28)%] (P=0.029), and SD rats had slight thicker dermal collagen bundles [(19.27±0.36) m] (P=0.049), and was similar in the gap rate between these bundles [(30.54±3.50)%,P=0.837]. The distribution of elastic fiber, the contents of type I and III collagen and their ratio also had significant differences between human and porcine dermis (P<0.05), but they were similar between human and SD rat dermis.2. The results from SEM showed that the dermal collagen bundles of Bama miniature pig were densely arranged and had smaller pores. The dermal collagen bundles of human and SD rat were porous arranged and had bigger pores. The collagenous fibrils of Bama miniature pig were thinner and had longer cyclical transverse striation than human by TEM. The collagenous fibrils of SD rat were thicker but had similar cyclical transverse striation than human by TEM.3. The results of biomechanical testing showed that the maximum strain of Bama miniature pig dermis [(0.52±0.05) mm/mm] was smaller than that of human dermis [(0.80±0.06) mm/mm] (P=0.003) while the elastic modulus [(69.65±12.16) MPa] and maximum stress [(20.72±1.49) MPa] of Bama miniature pig dermis were stronger than that of human dermis [(44.23±4.63) MPa, (11.85±0.19) MPa] (P=0.028, P=0.001). The maximum strain of SD rat dermis [(0.63±0.08) mm/mm] (P=0.042) was also smaller. But the elastic modulus [(35.60±7.29) MPa] (P=0.158) and maximum stress [(8.60±2.16) MPa] (P=0.120) of SD rat dermis were similar with that of human dermis.4. The ADM made by using 2 ml enzymatic digestive solution per cm2 flap of reticular layer of SD rat dermis with 2 h digestion had less residual cell components without over-digestion. This was the best protocol for preparating ADM by enzymatic digestion. The method of preparing ADM with hypotonic solution could reach thoroughly decellularization and maintain the three-dimensional construction of collagen fibers. 5. Compared with the rabbits implanted cellular DM, the rabbits implanted ADM had no skin necrosis, The histological changes showed that the main infiltrating cells were mononuclear cells in the early period after implantation, the fibroblasts and capillaries could be found inside of the implants after 2 or 3 months. There was no apparent inflammatory reaction and immunologic rejection in rabbits implanted with ADM.6. The content of heparin in the rat cellular dermis was (4.34±0.83) g/cm2, that in ADM was (2.36±0.13) g/cm2 (P=0.000). The decellularized process obviously reduced the content of heparin in the dermis. The content [(4.25±0.77) g/cm2 ] of heparin in the ADM after soaking in heparin solutions for 15 min could reach the level of the content of heparin in cellular DM, the content [(8.43±1.43) g/cm2] of heparin in the ADM reached to peak after soaking in heparin solutions for 12 h.Conclusion1. The morphology characteristics of pig and human dermis are quite different, while that of SD rat and human are more similar, therefore using SD rat as the animal resource of xenogenic ADM has reliable and solid histological fundament.2. We design the physical method to remove the basement membrane according to the distribution of the antigenicity of dermis, and apparently decreased immunogenicity of xenogenic ADM could be achieved by improved enzymatic digestion method following the treatment with hypotonic solution. This xenogenic ADM might cause slight inflammation after implantation.3. The decellularized process could significantly reduce the content of heparin in the dermis. The content of heparin in soaking the ADM in heparin solutions for short time (15 -30 min) could reach the level of heparin content in cellular DM. This simple method with the combination of the heparin on the ADM could provide the conditions for further clinical application.
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