RESUMO
In this study, the mutual fusion of chondrocyte pellets was promoted in order to produce large-sized tissue-engineered cartilage with a three-dimensional (3D) shape. Five pellets of human auricular chondrocytes were first prepared, which were then incubated in an agarose mold. After 3 weeks of culture in matrix production-promoting medium under 5.78g/cm(2) compression, the tissue-engineered cartilage showed a sufficient mechanical strength. To confirm the usefulness of these methods, a transplantation experiment was performed using beagles. Tissue-engineered cartilage prepared with 50 pellets of beagle chondrocytes was transplanted subcutaneously into the cell-donor dog for 2 months. The tissue-engineered cartilage of the beagles maintained a rod-like shape, even after harvest. Histology showed fair cartilage regeneration. Furthermore, 20 pellets were made and placed on a beta-tricalcium phosphate prism, and this was then incubated within the agarose mold for 3 weeks. The construct was transplanted into a bone/cartilage defect in the cell-donor beagle. After 2 months, bone and cartilage regeneration was identified on micro-computed tomography and magnetic resonance imaging. This approach involving the fusion of small pellets into a large structure enabled the production of 3D tissue-engineered cartilage that was close to physiological cartilage tissue in property, without conventional polyper scaffolds.
Assuntos
Cartilagem/citologia , Fusão Celular/métodos , Condrócitos , Engenharia Tecidual/métodos , Animais , Cartilagem/fisiologia , Células Cultivadas , Cães , Humanos , Regeneração , Microtomografia por Raio-XRESUMO
To enrich the subpopulation that preserves self-renewal and multipotentiality from conventionally prepared bone marrow stromal cells (MSCs), we attempted to use 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer-coated plates that selected the MSCs with strong adhesion ability and evaluated the proliferation ability or osteogenic/chondrogenic potential of the MPC polymer-selected MSCs. The number of MSCs that were attached to the MPC polymer-coated plates decreased with an increase in the density of MPC unit (0-10%), whereas no significant difference in the proliferation ability was seen among these cells. The surface epitopes of CD29, CD44, CD105, and CD166, and not CD34 or CD45, were detectable in the cells of all MPC polymer-coated plates, implying that they belong to the MSC category. In the osteogenic and chondrogenic induction, the MSCs selected by the 2-5% MPC unit composition showed higher expression levels of osteoblastic and chondrocytic markers (COL1A1/ALP, or COL2A1/COL10A1/Sox9) at passage 2, compared with those of 0-1% or even 10% MPC unit composition, while the enhanced effects continued by passage 5. The selection based on the adequate cell adhesiveness by the MPC polymer-coated plates could improve the osteogenic and chondrogenic potential of MSCs, which would provide cell sources that can be used to treat the more severe and various bone/cartilage diseases.
Assuntos
Células da Medula Óssea/fisiologia , Técnicas de Cultura de Células/instrumentação , Condrogênese/fisiologia , Metacrilatos/metabolismo , Osteogênese/fisiologia , Fosforilcolina/análogos & derivados , Células Estromais/fisiologia , Fosfatase Alcalina/metabolismo , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Células da Medula Óssea/citologia , Adesão Celular/fisiologia , Técnicas de Cultura de Células/métodos , Proliferação de Células , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Epitopos , Humanos , Teste de Materiais , Metacrilatos/química , Fosforilcolina/química , Fosforilcolina/metabolismo , Polímeros/química , Polímeros/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Células Estromais/citologia , Propriedades de SuperfícieRESUMO
Recently we reported that mouse germ cells in the testis contain active P450 aromatase (P450arom), the enzyme that converts androgens to estrogens. This finding suggested that germ cells have the ability to produce estrogen. Further studies have shown that germ cells in the testis of several species contain P450arom. The goal of this study was to determine if epididymal sperm contain P450arom and if P450arom activity in sperm changes during traversion of the epididymis in the adult mouse. P450arom was localized in sperm present in the efferent ductules and epididymis by immunocytochemistry using an antiserum generated against purified human placental cytochrome P450arom. P450arom immunostaining in sperm was most prominent in sperm located in the proximal caput epididymis, decreased as sperm traversed the corpus epididymis, and was only slightly apparent in sperm in the cauda epididymis. The immunolocalization of P450arom in epididymal sperm was supported by the measurement of P450arom activity in sperm by the 3H2O assay. We found that P450arom activity in sperm significantly decreases as sperm traverse the epididymis. Based upon these observations, we conclude that sperm can synthesize estrogen and that the synthesis of estrogen by sperm present in the efferent ductules and caput epididymis could be important in the process of sperm maturation.