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    Aplicações da bioengenharia tecidual e do potencial anti-inflamatório e pró-angiogênico da proteína anexina A1 em heteroenxertos de pele acelular
    (Universidade Federal de São Paulo (UNIFESP), 2016-03-30) Mimura, Kallyne Kioko Oliveira [UNIFESP]; Oliani, Sonia Maria [UNIFESP]; http://lattes.cnpq.br/5102737730539655; http://lattes.cnpq.br/8017096776183586; Universidade Federal de São Paulo (UNIFESP)
    The development of skin substitutes is essential to overcome the shortage at organs transplantation. Furthermore, the use of drugs to modulate the inflammation and angiogenesis processes is important to the successful tissue regeneration. Objectives: The aims of our investigations were to standardize techniques of porcine skin decellularization, to develop acellular skins matrix (scaffolds) without cross-linking, to evaluate its biocompatibility and the anti-inflammatory / pro-angiogenic potential of the AnxA12-26 peptide of the annexin A1 protein in heterologous transplants. Materials and methods: Scaffolds (1 and 2), produced without cross-linking by two types of decellularization protocols (osmotic shock and enzymatic digestion), were evaluated by histological, molecular and biomechanical methods. For the biocompatibility analysis, the scaffolds and Permacol? as control (commercially available porcine dermal scaffold with crosslinking) were implanted in rats (Rattus norvegicus) for 3, 14, 21 and 90 days. The peripheral blood was collected for leukocytes quantification and plasma was isolated for the pro-inflammatory cytokines IL-6, TNF-? and IL-1? measurement. The implants and the surrounding tissues were processed for histological, immuno-histochemical (blood vessels, myofibroblasts, IL-6 positive cells and ED-1 / CCR7 / CD163 positive macrophages) and molecular (IL-1?, VEGF-A, TGF-?1, COL3A1, COL1A1 and COL1A2) analysis. In the following experiments, the scaffold 1, that presented the best biological properties, was used for transplantation in Balb/c mice, treated or not i.p. with 4 mg/kg AnxA12-26 peptide, for 3, 10, 15 and 60 days. The leukocytes were quantified on peripheral blood. The transplanted scaffolds and adjacent tissue were processed for histological, biochemical (cytokines and growth factors measurement) and molecular (VEGF-A, FGF-b, TGF-?1 and ?-SMA) analysis. Results: The decellularization methods were effective in both scaffolds (1 and 2), with preservation of extracellular matrix components (collagen and glycosaminoglycans) and adequate tensile strength at biomechanical testing. Initially, on 3rd day post-implantation, polymorphonuclear cells were observed inside scaffolds and were gradually replaced by IL-6 positive cells and M1 macrophages, at the 14th day. After 21 days, M2 macrophages, myofibroblasts and blood vessels were observed inside the scaffold. The cellular events observed in heterograft were consistent with the gene expression of investigated trophic factors, which were involved in tissue remodeling (IL-1?, TGF- ?1, VEGF-A, COL1A1, COL1A2 and COL3A1). The peptide AnxA12-26 administration in heterologous transplants was effective, promoting the reduction of proinflammatory mediators (IL-1?, IL-6, TNF-?, IL-17 and IFN-?) and increasing of cellular infiltration, pro-angiogenic factors expression (VEGF-A, b-FGF and TGF-?1) and myofibroblasts recruitment. Conclusion: Together, our results show the efficiency of the two decellularization protocols, indicating that both scaffolds are biologically compatible, allowing the cells recruitment and tissue remodeling with the gradual replacement of the acellular matrix. Moreover, our data reveals an important role of the AnxA1 protein in angiogenic and inflammatory processes on the experimental model of acellular skin heterologous transplants.