| Current treatments for burn, chronic ulcers, pressure ulcers, and reconstructive surgery include tissue transfer from a healthy site in the same or another individual, use of medical devices to support the function of the lost tissue, and pharmacologic supplementation of the metabolic products of the lost tissue. Problems with these treatments include limited number of tissue donors, potential tissue complications such as imperfect matches and dependence on immunosuppressant, and may have a functional impact (or usually an aesthetic impact) on the patient. Currently, due to excellent achievements in a variety of different applications, the performance of tissue engineered skin is very useful in the treatment of burn, chronic ulcers, venous leg ulcers, pressure ulcers, limb amputations and in reconstructive surgery with promising clinical outlooks in the future.Currently, due to excellent achievements in a variety of different biomedical applications, the performance of acellular dermal matrices, which are derived from full-thickness skin treated to remove cells and cellular components but which retain the native dermal structure, has drawn the attention of researchers in many fields. Many recent studies have presented several methods for producing an acellular dermal matrix(ADM) from porcine skin and the submucosal layer of the small intestine. These methods using treatment with trypsin, freeze-thawing, and prolonged incubations with enzymes produced ADMs which were too highly antigenic when implanted into recipients, where they induced immune reactions resulting in poor graft survival. Recently, scaffolds derived from a xenogenic acellular dermal extracellular matrix have been shown to be effective in the repair and reconstruction of several body tissues including the lower urinary tract, dura mater, esophagus, musculotendinous structures, and blood vessels. The characteristic of these scaffolds recognized as important for their effectiveness is their ability to induce a host cellular response that supports constructive remodeling rather than default scar tissue formation. Furthermore, improvement of vascularization and nutrients infiltration could extend the application of xenogenic ADM scaffolds.The object of this study was to prepare a novel xenogenic ADM granule on the base of present preparing methods, on which fibroblasts were seeded on the granules for the characteristics of secreting extracellular matrix and growth factors, to investigate the feasibility of construction of xenogeneic ADM granule skin. Then the in vivo curative effect of xenogeneic ADM granule skin was explored via subcutaneous injection and full-thickness skin wound transplantation. Therefore, was to achieve a convenient and applicable tissue engineering granule skin product and to improve a step in the processing of commercialization of ADM granule skin for biomedical applications.The content of the study was provided as follows:1. Preparation of ADM Granule by Improved Solution Method and its Biocompatibility Tests The xenegeneic ADM was made from the reticular layer of porcine dermis and prepared by solution and freeze thawing methods. Its biocompatibility was tested by cytotoxicity test,cell culture and subdermal implantion. Results: Intact cells were absent from the ADM by the improved method and the tenacity was good;The collagn fibers were a little loose in arrangement but their structure were normal;There were no basement membrane and dermal papillary layer retained,and amounts of xenogeneic were lower. The ADM granule was nontoxic, fibroblasts grew and proliferated well on it and there were no immune rejection in the subdermal implantation. The ADM granules could induce the infiltration of host cells and the vascularization .2. Construction and Characteristic Study of Xenogeneic ADM Granule SkinHuman fibroblasts were cultured in vitro and labeled with PKH26, then combined with ADM granules; the characteristics of tissue engineering granule skin were measured via fluorescent microscope, SEM and MTT assay. Fibroblasts and ADM granules were combined well: cells were adhered at 2h, proliferated at 2d and proliferated to the peak after 10d; cells were layered-adhered and matrix was secreted on the surface of ADM granules.3. Construction and in vivo Study of Xenogeneic ADM Granule SkinHuman fibroblasts were cultured in vitro and labeled with PKH26, then combined with ADM granules to construct tissue engineering granule skin; Restoration of subcutaneous tissue after tissue engineering granule skin injection was measured via fluorescent microscope and HE histological assay; simultaneity restoration of skin on SD rat full-thickness skin wound models after autograft, ADM and tissue engineering granule skin transplantation were measured via macroscopy, fluorescent microscope and HE histological assay. Fibroblasts and ADM granules were combined well: cells were proliferated, layered-adhered and ECM deposited well on ADM;after 2-week subcutaneous injection,cells and vascular tissue were ingrown and inflammatory cells were lessen with time, red fluorescence was observed, 8 weeks later, injected tissues were rebuilt and collagen was regular; there is no statistical differences (P﹥0.05) between autograft and tissue engineering granule skin transplantation on healing time and wound contraction, while tissue engineering granule skin is better than ADM alone(P﹤0.01).In conclusion, The ADM granules prepared by improved solution method possessed very low antigenicity and good biocompatibility and could be a good scaffold for tissue engineering granule skin for further study. The efficient xenogeneic ADM granule skin could be constructed with human fibroblasts and ADM granules well. Xenogeneic ADM granule skin could improve in vivo restoration of subcutaneous tissue and skin wound via injection and full-thickness skin wound transplantation and it might be a promising tool for further medical application. |
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