RESUMO
Loss of skeletal muscle profoundly affects the health and well-being of patients, and there currently is no way to replace lost muscle. We believe that a key step in the development of a prosthesis for reconstruction of dysfunctional muscular tissue is the ability to reconstitute the in vivo-like 3-dimensional (3D) organization of skeletal muscle in vitro with isolated satellite cells. In our present proof of principle studies, we have successfully constructed a multilayered culture of skeletal muscle cells, derived from neonatal satellite cells, that are distributed in a 3D pattern of organization that mimics many of the features of intact tissue. These multilayered cultures are composed of elongated multinucleated myotubes that are MyoD positive. Histological studies indicate that the multiple layers of myotubes can be distinguished. Expression of muscle-specific markers such as myosin heavy chain, dystrophin, integrin alpha-7, alpha-enolase, and beta-enolase was detected using real-time reverse transcriptase polymerase chain reaction at levels near adult values. Physiological measurements of the engineered skeletal muscle showed that they tetanize and display physiologic force length behavior, although developed force per cross-sectional area was below that of native rat skeletal muscle.
Assuntos
Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Engenharia Tecidual , Animais , Animais Recém-Nascidos , Técnicas de Cultura de Células , Separação Celular , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Meios de Cultura , Fluoresceína-5-Isotiocianato/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Corantes Fluorescentes/metabolismo , Géis , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Contração Isométrica , Microscopia Confocal , Contração Muscular , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Proteína MyoD/metabolismo , Faloidina/metabolismo , RNA Mensageiro/análise , Ratos , Células Satélites de Músculo Esquelético/fisiologia , Especificidade por Substrato , Temperatura , Fatores de Tempo , Azul Tripano/metabolismo , Azul Tripano/farmacologiaRESUMO
In our studies to develop a simple and reliable method for the generation of recombinant adenoviruses (Fotadar et al . 2003), we noted that the E. coli (BJ5183 and DH5alpha) survived a heat-shock of 45 degrees C for 5 minutes in 0.1 M calcium chloride. As a result, we investigated the growth of E. coli (DH5alpha) at elevated temperatures. We hereby demonstrate that contrary to previous observations (Cooper et al . 2001 and Bronikowski et al . 2001) E. coli can grow consistently at a temperature as high as 49 degrees C, in spite of the fact that growth beyond 40 degrees C can generally be prohibitive. Hence, it is quite likely that these E. coli (DH5alpha) may have acquired mutations which permit them to grow reproducibly at 49 degrees C. Growth of E. coli above 49 degrees C (up to 53 degrees C) was also observed, but this was sporadic and not reproducible. This result could extend the utility of these organisms for cloning and manipulations requiring high temperature.
