RESUMO
Embryonic stem cells can provide an unlimited supply of pluripotent cells for tissue engineering applications. Bone tissue engineering by directly differentiating ES cells (ESCs) into osteoblasts has been unsuccessful so far. Therefore, we investigated an alternative approach, based on the process of endochondral ossification. A cartilage matrix was formed in vitro by mouse ESCs seeded on a scaffold. When these cartilage tissue-engineered constructs (CTECs) were implanted s.c., the cartilage matured, became hypertrophic, calcified, and was ultimately replaced by bone tissue in the course of 21 days. Bone aligning hypertrophic cartilage was observed frequently. Using various chondrogenic differentiation periods in vitro, we demonstrated that a cartilage matrix is required for bone formation by ESCs. Chondrogenic differentiation of mesenchymal stem cells and articular chondrocytes showed that a cartilage matrix alone was not sufficient to drive endochondral bone formation. Moreover, when CTECs were implanted orthotopically into critical-size cranial defects in rats, efficient bone formation was observed. We report previously undescribed ESC-based bone tissue engineering under controlled reproducible conditions. Furthermore, our data indicate that ESCs can also be used as a model system to study endochondral bone formation.
Assuntos
Osso e Ossos/citologia , Condrogênese , Células-Tronco Embrionárias/citologia , Engenharia Tecidual/métodos , Animais , Encéfalo/patologia , Cartilagem/citologia , Diferenciação Celular , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/citologia , Osteogênese , Fatores de TempoRESUMO
In this study, the development is described of a tissue-engineered construct mimicking the structure of a natural blood vessel. Smooth muscle cells (SMC) were cultured under pulsatile flow conditions in porous tubular scaffolds composed of crosslinked type I insoluble collagen and insoluble elastin. Under these dynamic culture conditions, average wall shear rate, systolic and diastolic pressures and pressure wave-forms comparable to conditions in the human carotid artery were obtained. Culturing of SMC in tubular scaffolds under dynamic conditions resulted in enhanced tissue formation compared to static conditions. Higher SMC numbers, a more homogeneous distribution of SMC throughout the scaffolds and higher collagen mRNA expression levels were found when cells were cultured under dynamic compared to static conditions. mRNA expression levels of markers of proliferation and apoptosis showed that the higher cell numbers in the scaffolds cultured under dynamic conditions can be explained by increased cell proliferation but not by decreased apoptosis. Glucose consumption and lactate formation by the cells showed that cell metabolism was more aerobic under dynamic compared to static conditions. Lining of the dynamically cultured constructs with a luminal monolayer of endothelial cells might result in vessels suitable for in vivo applications.
Assuntos
Reatores Biológicos , Prótese Vascular , Engenharia Tecidual , Materiais Biocompatíveis , Fenômenos Biomecânicos , Artérias Carótidas/anatomia & histologia , Artérias Carótidas/fisiologia , Células Cultivadas , Colágeno Tipo I/genética , Reagentes de Ligações Cruzadas , Ciclina E/genética , Elastina/genética , Proteínas de Ligação ao GTP , Expressão Gênica , Humanos , Teste de Materiais , Microscopia Eletrônica de Varredura , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Proteína 2 Glutamina gama-Glutamiltransferase , Fluxo Pulsátil , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos , Transglutaminases/genéticaRESUMO
Tissue engineering offers the opportunity to develop vascular scaffolds that mimic the morphology of natural arteries. We have developed a porous three-dimensional scaffold consisting of fibres of collagen and elastin interspersed together. Scaffolds were obtained by freeze-drying a suspension of insoluble type I collagen and insoluble elastin. In order to improve the stability of the obtained matrices, they were cross-linked by two different methods. A water-soluble carbodi-imide, alone or in combination with a diamine, was used for this purpose: zero- or non-zero-length cross-links were obtained. The occurrence of cross-linking was verified by monitoring the thermal behaviour and the free-amino-group contents of the scaffolds before and after cross-linking. Smooth-muscle cells (SMCs) were cultured for different periods of time and their ability to grow and proliferate was investigated. SMCs were isolated from human umbilical and saphenous veins, and the purity of the cultures obtained was verified by staining with a specific monoclonal antibody (mAb). Cultured cells were also identified by mAbs against muscle actin and vimentin. After 14 days, a confluent layer of SMCs was obtained on non-cross-linked scaffolds. As for the cross-linked samples, no differences in cell attachment and proliferation were observed between scaffolds cross-linked using the two different methods. Cells cultured on the scaffolds were identified with an anti-(alpha-smooth-muscle actin) mAb. The orientation of SMCs resembled that of the fibres of collagen and elastin. In this way, it may be possible to develop tubular porous scaffolds resembling the morphological characteristics of native blood vessels.