RESUMO
PURPOSE: The aim of this study was to construct functional tissue-engineered bone in dogs using cell sheet engineering, a new technique to gain and transfer seed cells. MATERIALS AND METHODS: Demineralized bone matrixes, prepared from homologous bone, were coated with recombinant human bone morphogenetic protein-2. Bone marrow stromal cells (BMSCs) were isolated and subcultured. Osteogenic-induced BMSCs were incubated in a temperature-responsive culture dish to form the BMSC sheet. The complex of demineralized bone matrix, recombinant human bone morphogenetic protein-2, and BMSCs wrapped with BMSC sheets was implanted around the blood vessels of the latissimus dorsi muscle in the experimental side, and the same complex without BMSC sheets was implanted around the blood vessels of the latissimus dorsi muscle on the other side as a control. At 4, 8, 12, and 16 weeks after implantation, the implants were removed for radiographic evaluation, descriptive histologic observation, and histologic quantitative analysis. RESULTS: Radiographic analysis showed that the optical density of the tissue-engineered bone on the 2 sides increased with time. However, the optical density of the experimental side was significantly greater than that of the control side at the same points. Sixteen weeks after implantation, mature lamellar bone was formed in the experimental side, with red bone marrow in the bone marrow cavity. In contrast, the control side exhibited significantly less lamellar bone. Histologic quantitative analysis showed that the experimental side exhibited significantly more bone per area compared with the control side. CONCLUSION: BMSC sheet engineering may be useful to construct functional tissue-engineered bone.
Assuntos
Células da Medula Óssea/fisiologia , Osso e Ossos/fisiologia , Células-Tronco Mesenquimais/fisiologia , Osteogênese/fisiologia , Engenharia Tecidual/métodos , Animais , Técnica de Desmineralização Óssea , Matriz Óssea , Proteína Morfogenética Óssea 2/química , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Capilares/patologia , Técnicas de Cultura de Células , Separação Celular , Meios de Cultura , Cães , Fáscia/irrigação sanguínea , Fasciotomia , Feminino , Ósteon/patologia , Humanos , Umidade , Processamento de Imagem Assistida por Computador/métodos , Masculino , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/cirurgia , Osteoblastos/fisiologia , Radiografia , Distribuição Aleatória , Proteínas Recombinantes/química , Temperatura , Engenharia Tecidual/instrumentação , Alicerces Teciduais/química , Fator de Crescimento Transformador beta/químicaRESUMO
OBJECTIVE: To analyze and compare the ITS sequences of Aconitum vilmorinianum and its medicinal adulterant Aconitum austroyunnanense. METHODS: Total genomic DNA were extracted from sample materials by improved CTAB method, ITS sequences of samples were amplified using PCR systems, directly sequenced and analyzed using software DNAStar, ClustalX1.81 and MEGA 4.0. RESULTS: 299 consistent sites, 19 variable sites and 13 informative sites were found in ITS1 sequences, 162 consistent sites, 2 variable sites and 1 informative sites were found in 5.8S sequences, 217 consistent sites, 3 variable sites and 1 informative site were found in ITS2 sequences. Base transition and transversion was not found only in 5.8S sequences, 2 sites transition and 1 site transversion were found in ITS1 sequences, only 1 site transversion was found in ITS2 sequences comparting the ITS sequences data matrix. By analyzing the ITS sequences data matrix from 2 population of Aconitum vilmorinianum and 3 population of Aconitum austroyunnanense, we found a stable informative site at the 596th base in ITS2 sequences, in all the samples of Aconitum vilmorinianum the base was C, and in all the samples of Aconitum austroyunnanense the base was A. CONCLUSION: Aconitum vilmorinianum and Aconitum austroyunnanense can be identified by their characters of ITS sequences, and the variable sites in ITS1 sequences are more than in ITS2 sequences.