Reconstrução do corpo cavernoso de coelhos utilizando técnicas de engenharia de tecidos
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Data
2013
Autores
Laks, Marcus [UNIFESP]
Orientadores
Freitas Filho, Luiz Gonzaga de [UNIFESP]
Tipo
Tese de doutorado
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Objetivo: Comparar a reconstrucao de segmentos do corpo cavernoso com matrizes de origem homologa, quando semeadas com celulas-tronco mesenquimais nao autologas, e quando estas celulas-tronco mesenquimais sao injetadas por via intravenosa.. Metodos: Foram utilizados 16 coelhos da linhagem Nova Zelandia, com aproximadamente 3 Kg, idade aproximada de 6 meses, divididos em 3 grupos Grupo A (n=4) Reconstrucao do corpo cavernoso com matriz homologa descelularizada Grupo B (n=6) - Reconstrucao do corpo cavernoso com matriz semeada com celulas-tronco mesenquimais em matriz homologa descelularizada; Grupo C (n=6)- Reconstrucao do corpo cavernoso com matriz homologa descelularizada seguido de infusao venosa de celulas-tronco mesenquimais. A matriz homologa foi obtida em abatedouro comercial e descelularizada com triton X100 e hidroxido de amonio. A matriz foi interposta entre os dois cotos do corpo cavernoso seccionado em sua porcao central e suturada com pontos separados. As celulas-tronco mesenquimais foram obtidas por puncao e aspiracao de 5 mL do liquido da medula ossea da crista iliaca de coelhos, e cultivadas oin vitroo. Todos os grupos foram avaliados na decima segunda semana de pos-operatorio. Quanto ao aspecto macroscopico avaliou-se o enchimento venoso distal dos corpos cavernosos. Para a avaliacao do processo de reparacao dos implantes as pecas cirurgicas foram submetidas a coloracao com hematoxilina e eosina e imunoistoquimica com os anticorpos primarios anti - actina de musculo liso e anti fator VIII para avaliacao de celulas musculares lisas e endotelio, respectivamente. Um animal de cada grupo foi submetido a cavernosometria Resultados: Tres animais do grupo B e tres do grupo C apresentaram enchimento do corpo cavernoso a jusante do local de interposicao da matriz no momento da eutanasia, o que nao ocorreu em nenhum coelho do grupo A. O animal do grupo B atingiu a cavernosometria pico pressorico de 50 cm de H2O, o animal do grupo C pico pressorico de 110 cm de H2O, e o do grupo A pico de 20 cm de H2O. Na avaliacao histologica, em todos os grupos houve grande deposicao de colageno, sendo que nos animais dos grupos B e C haviam areas semelhantes a trabeculas de tecido cavernoso, com feixes musculares lisos organizados, formando seios venosos com neovascularizacao evidente. No animais do grupo A as areas de musculo liso estavam desorganizadas e sobrepostas as areas em que ocorreu grande deposicao de colageno, formando apenas poucos seios venosos. Nao houve diferenca estatistica entre os tres grupos estudados quanto a quantidade de musculo liso e endotelio por avaliacao imunoistoquimica. Conclusao: A reconstrucao do corpo cavernoso utilizando matrizes descelularizadas e celulas tronco mesenquimais injetadas por via intravenosa ou semeadas diretamente e possivel, havendo crescimento de tecido semelhante ao corpo cavernoso nativo. Nao foram observadas diferencas significantes quando as celulas-tronco foram semeadas diretamente na matriz ou infundidas por via intravenosa
Objective: To compare the reconstruction of entire cross-sectional segments of corpus cavernosum when they are seeded with non-autologous mesenchymal stem cells and when this stem cells are infused by intravenous injection. Methods: Sixteen New Zealand white rabbits, weighting 3 kg, and aged 6 months were distributed in three groups: Group A (n=4)- reconstruction of the corpus cavernosum using homologous decellularized matrices, Group B (n=6) - reconstruction of the corpus cavernosum using homologous decellularized matrices seeded with mesenchymal stem cells Group C (n=6) - reconstruction of the corpus cavernosum using homologous decellularized matrices submitted to intravenous infusion of mesenchymal stem cells. The homologous matrices were obtained in commercial slaughterhouses and decellularized with triton X100 and ammonium hydroxide. The matrices were interposed between the two stumps of the sectioned corpus cavernosum of the animal host on its central portion and sutured with interrupted stitches. The mesenchymal stem cells were obtained by bone marrow aspiration of the iliac crest and cultivated “in vitro”. All groups were evaluated at the twelfth postoperative week. The venous filling aspect of the distal end of the corpus cavernosum was evaluated. To assess the reparatory process surgical specimens were submitted to hematoxylin and eosin staining protocol in addition to immunohistochemistry assessment with anti a-smooth muscle actin and anti factor VIII primary antibodies for evaluation of both smooth muscle cells and endothelium. Cavernosometry was done in one animal of each group. Results: Three animals on group B and three animals on group C presented full filling of distal end of at time of euthanasia. No animals in group A presented filling of the corpus cavernosum. At cavernosometry the animal on group B attained 50 cmH2O, on group C 110 cmH2O and the animal on group A 20 cmH2O. The histologic evaluation showed in all groups great collagen deposition. Animals from groups B and C presented trabecular areas “cavernous-like” tissue with organized muscular bundles and venous sinus with neovascularization. Animals from group A presented disorganized smooth muscle areas with collagen and few sinuses. There were no statistical difference between the three groups with regard to the amount of smooth muscle and endothelium on immunohistochemical evaluation. Conclusion: The reconstruction of corpus cavernosum using descellularized matrices and mesenchymal stem cells, either by intravenous injetion or directly seeded is possible, with growth of “corpus cavernosum-like tissue”. There were no significant differences when the stem cells were directly seeded in the matrices or administred by intravenous injection.
Objective: To compare the reconstruction of entire cross-sectional segments of corpus cavernosum when they are seeded with non-autologous mesenchymal stem cells and when this stem cells are infused by intravenous injection. Methods: Sixteen New Zealand white rabbits, weighting 3 kg, and aged 6 months were distributed in three groups: Group A (n=4)- reconstruction of the corpus cavernosum using homologous decellularized matrices, Group B (n=6) - reconstruction of the corpus cavernosum using homologous decellularized matrices seeded with mesenchymal stem cells Group C (n=6) - reconstruction of the corpus cavernosum using homologous decellularized matrices submitted to intravenous infusion of mesenchymal stem cells. The homologous matrices were obtained in commercial slaughterhouses and decellularized with triton X100 and ammonium hydroxide. The matrices were interposed between the two stumps of the sectioned corpus cavernosum of the animal host on its central portion and sutured with interrupted stitches. The mesenchymal stem cells were obtained by bone marrow aspiration of the iliac crest and cultivated “in vitro”. All groups were evaluated at the twelfth postoperative week. The venous filling aspect of the distal end of the corpus cavernosum was evaluated. To assess the reparatory process surgical specimens were submitted to hematoxylin and eosin staining protocol in addition to immunohistochemistry assessment with anti a-smooth muscle actin and anti factor VIII primary antibodies for evaluation of both smooth muscle cells and endothelium. Cavernosometry was done in one animal of each group. Results: Three animals on group B and three animals on group C presented full filling of distal end of at time of euthanasia. No animals in group A presented filling of the corpus cavernosum. At cavernosometry the animal on group B attained 50 cmH2O, on group C 110 cmH2O and the animal on group A 20 cmH2O. The histologic evaluation showed in all groups great collagen deposition. Animals from groups B and C presented trabecular areas “cavernous-like” tissue with organized muscular bundles and venous sinus with neovascularization. Animals from group A presented disorganized smooth muscle areas with collagen and few sinuses. There were no statistical difference between the three groups with regard to the amount of smooth muscle and endothelium on immunohistochemical evaluation. Conclusion: The reconstruction of corpus cavernosum using descellularized matrices and mesenchymal stem cells, either by intravenous injetion or directly seeded is possible, with growth of “corpus cavernosum-like tissue”. There were no significant differences when the stem cells were directly seeded in the matrices or administred by intravenous injection.
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Citação
LAKS, Marcus. Reconstrução do corpo cavernoso de coelhos utilizando técnicas de engenharia de tecidos. 2013. 61 f. Tese (Doutorado em Ciências) – Escola Paulista de Medicina, Universidade Federal de São Paulo. São Paulo, 2013.