Evaluation of biological and biomechanical characteristics of decellularized matrix of human glans

Objetivo: Avaliar a integracao de celulas-tronco mesenquimais nao autologas a matrizes descelularizadas de glande humana apos observar se o processo de descelularizacao da glande altera suas caracteristicas biologicas e biomecanicas. Metodos: Amostras de matrizes de glande de penis de doadores de tecidos para transplante foram submetidas a um processo de descelularizacao utilizando solucao de Triton X-100 a 1% com hidroxido de amonio a 0,1% a 4°C, para remocao dos fosfolipidios da membrana plasmatica. Fragmentos da glande descelularizada foram fixados por imersao em formol tamponado a 10% e embebidos em parafina. Foram realizados cortes de 5μm, corados com Hematoxilina-Eosina (HE), e analisados para comprovar a completa acelularidade da matriz descelularizada. A matriz extracelular da glande foi avaliada apos o processo de descelularizacao, analisando a preservacao das fibras colagenas (colageno tipo I) e do arcabouco das fibras musculares, pela coloracao de Tricromico de Masson, e do colageno tipo III e das fibras elasticas, pela coloracao de Verhoff-Van Gieson. As matrizes foram cortadas sob camara de fluxo laminar em fragmentos medindo 0,25 cm2 para semeadura de celulas-tronco mesenquimais, e 0,75 cm2 para realizacao dos testes biomecanicos. Os testes de citotoxicidade foram realizados para detectar o potencial de producao de efeitos letais as celulas 3T3. Utilizouse o teste por contato direto em que uma determinada quantidade do material e colocada diretamente sobre uma monocamada subconfluente de fibroblastos de derme de camundongo 3T3, previamente semeadas em placas de 96 pocos, na densidade de 1x103 celulas por poco e recobertas por meio de cultura. Para a avaliacao da biocompatibilidade das matrizes descelularizadas de glande foram realizados os seguintes testes: Atividade Metabolica da Mitocondria, Atividade Lisossomal, e quantificacao do material genetico. Antes de iniciar os testes, as placas de 96 pocos foram incubadas durante 24 horas com meio de cultura DMEM suplementado a 1% de SBF. Foram realizados testes de biocompatibilidade para avaliacao das interacoes citotoxicas e nao citotoxicas das matrizes com as celulas 3T3 nos intervalos de tempo de 24, 48 e 72 horas. As matrizes de glande foram semeadas com celulas-tronco mesenquimais obtidas de ratos Wistar, e mantidas em cultura durante 7, 14 e 28 dias. Apos os periodos de cultivo as matrizes de glande semeadas foram avaliadas em relacao a integracao e viabilidade das celulas-tronco mesenquimais as matrizes de glande. Testes biomecanicos no tecido nativo, matriz descelularizada e celularizada com celulastronco mesenquimais foram realizados para a caracterizacao de suas propriedades biomecanicas. Resultados: Houve a preservacao da arquitetura tecidual da matriz de glande descelularizada, assim como a manutencao de suas propriedades biologicas e biomecanicas. As analises das matrizes de glande semeadas com celulas-tronco mesenquimais da medula ossea de ratos revelaram a integracao destas celulas as matrizes, e sua viabilidade oin vitroo por duas semanas. Conclusao: Matrizes extracelulares obtidas de glande de cadaveres humanos foram capazes de manter suas propriedades biologicas e biomecanicas, e quando semeadas com celulas-tronco mesenquimais obtidas da medula ossea de ratos mantiveram sua vitalidade oin vitroo durante 14 diasBV UNIFESP: Teses e dissertaçõe

Differentiation of Leydig Cells in the Mongolian Gerbil

Information on postnatal Leydig cell (LC) differentiation in the Mongolian gerbil has been unavailable. Therefore, current investigation was designed to examine LC lineage differentiation in this rodent, from birth to adulthood. Gerbil testes at I day, 1-7 weeks (w), 2 and 3 months of age were conventionally processed by light and transmission electron microscopy. Immunocytochemistry for specific markers of steroidogenic enzymes, namely 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 11 beta-hydroxysteroid steroid dehydrogenase 1 (11 beta-HSD1) and also for androgen receptor (AR) was performed. The establishment of adult Leydig cell populations (ALC) during testis maturation in the gerbil follows the pattern previously described in other mammalian species, with the four progressive stages of differentiation. The LC progenitors were identified at second w by 3 beta-HSD expression; the first newly formed ALC were recognized at fourth w whereas immature ALC appeared at fifth w. The latter were recognized by abundance of cytoplasmic lipid, in addition to expression of 11 beta-HSD1 and intense nuclear AR immunoreaction. Mature ALC in gerbil exhibited irregular eccentric nuclei and a cytoplasmic canaliculus in the perinuclear area. Only one third of mature ALC in adult gerbils showed a high expression of 11 beta-HSD1 and AR expression was highly variable among them. In conclusion, the process of differentiation of ALC population in gerbil follows the pattern previously established for other rodents. However, the resulting mature ALC are strikingly different due their singular asymmetric morphology and presence of a cytoplasmic canaliculus and as well as their functional heterogeneity. Microsc. Res. Tech. 73:119-127, 2010. (C) 2009 Wiley-Liss, Inc.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Differentiation Of Leydig Cells In The Mongolian Gerbil.

Information on postnatal Leydig cell (LC) differentiation in the Mongolian gerbil has been unavailable. Therefore, current investigation was designed to examine LC lineage differentiationin this rodent, from birth to adulthood. Gerbil testes at 1 day, 1-7 weeks (w), 2 and 3 months of age were conventionally processed by light and transmission electron microscopy. Immunocytochemistry for specific markers of steroidogenic enzymes, namely 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 11beta-hydroxysteroid steroid dehydrogenase 1 (11beta-HSD1) and also for androgen receptor (AR) was performed. The establishment of adult Leydig cell populations (ALC) during testis maturation in the gerbil follows the pattern previously described in other mammalian species, with the four progressive stages of differentiation. The LC progenitors were identified at second w by 3beta-HSD expression; the first newly formed ALC were recognized at fourth w whereas immature ALC appeared at fifth w. The latter were recognized by abundance of cytoplasmic lipid, in addition to expression of 11beta-HSD1 and intense nuclear AR immunoreaction. Mature ALC in gerbil exhibited irregular eccentric nuclei and a cytoplasmic canaliculus in the perinuclear area. Only one third of mature ALC in adult gerbils showed a high expression of 11beta-HSD1 and AR expression was highly variable among them. In conclusion, the process of differentiation of ALC population in gerbil follows the pattern previously established for other rodents. However, the resulting mature ALC are strikingly different due their singular asymmetric morphology and presence of a cytoplasmic canaliculus and as well as their functional heterogeneity.73119-2