RESUMO
Human bone marrow-derived mesenchymal cells (hBMMCs) originate from cell populations in the bone marrow and are capable of differentiating along multiple mesenchymal lineages. To differentiate hBMMCs into osteoblasts, adipocytes and chondrocytes, dexamethasone has been used as a differentiation reagent. We hypothesized that dexamethasone would augment the responsiveness of BMMCs to other differentiation reagents and not define the lineage. This study investigated the effect of continuous treatment with 100 nM dexamethasone on the differentiation of BMMCs into three different lineages. hBMMCs cultured with continuous dexamethasone treatment (100 nM) exhibited higher mRNA expression levels of osteogenic markers and higher positive rates of colony forming unit assays for osteogenesis compared to hBMMCs treated with dexamethasone only during the differentiation culture. Furthermore, continuous dexamethasone treatment augmented bone formation capability of monkey-derived BMMCs in a bone induction experimental model at an extra skeletal site. In addition, continuously dexamethasone-treated hBMMCs formed larger chondrogenic pellets and expressed SOX9 at higher level than the control BMMCs. Likewise, continuous dexamethasone treatment facilitated adipogenic differentiation based on mRNA level and colony forming unit analysis. To investigate the mechanism of the augmentation of differentiation, further studies on apoptosis were conducted. The studies indicated that dexamethasone selectively induced apoptosis of some populations of hBMMCs which were thought to have poor differentiation capability.
Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Dexametasona/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Biomarcadores , Células da Medula Óssea/metabolismo , Caspase 3/metabolismo , Células Cultivadas , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Citofotometria , Feminino , Humanos , Macaca , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacosRESUMO
In this study, the effects of gamma-ray irradiation on the mechanical properties, absorbability, and osteoconductivity of porous hydroxyapatite/collagen (HAp/Col) were investigated. Porous HAp/Col was exposed to 16, 25, 35, or 50 kGy of gamma-ray irradiation. The compressive elastic modulus showed irradiation dose-dependence, with a particularly pronounced decrease in the 50-kGy treatment group. An in vitro enzymatic digestion test showed that gamma-ray irradiation of porous HAp/Col resulted in accelerated degradation by collagenase. For in vivo studies, porous HAp/Col was transplanted into the back muscles or bone defects in the femoral condyle of rats. Specimens were obtained at 2, 4, and 8 weeks postoperatively. Absorption of the implants in the muscle was time- and irradiation dose-dependent, with notable absorption for the 35- and 50-kGy groups at 2 weeks. At the skeletal sites, porous HAp/Col demonstrated high osteoconductivity in all irradiation treatment groups. Interestingly, not only implant absorption but also bone formation was irradiation dose-dependent at early time points.
Assuntos
Substitutos Ósseos , Osso e Ossos/química , Colágeno/química , Durapatita/química , Raios gama , Absorção , Animais , Osso e Ossos/efeitos da radiação , Microscopia Eletrônica de Varredura , RatosRESUMO
Trabecular bone fragments can be percutaneously harvested from the ilium using methods that are similar in invasiveness to aspiration of bone marrow. In this study, we investigated the use of the trabecular bone as a cell source for bone tissue engineering. Trabecular bone-derived progenitor cells (TB cells) were isolated with a simple method in which trabecular fragments were first cultured as explants, and then the cells were released by trypsin digestion and advanced to a monolayer culture. The properties of TB cells prepared in this procedure were compared with bone marrow-derived progenitor cells (BM cells). A large number of TB cells could be obtained with less variation among donors, compared with BM cells. In multiple harvests of donor tissue through the same aspiration hole at the cortex, TB cells could be more consistently obtained in primary culture. The proliferative potential of BM and TB cells was similar in serial subculture. TB cells showed a higher alkaline phosphatase expression in the surface marker analysis and greater in vitro osteogenic abilities than BM cells after the initial 14 days of culture. In in vivo bone formation studies, TB cells also showed a higher osteogenic potential than BM cells. The results of this study suggest that TB cells can be considered an attractive source for clinical bone regeneration.
Assuntos
Osso e Ossos/citologia , Osteogênese , Células-Tronco/citologia , Engenharia Tecidual/métodos , Animais , Biomarcadores/metabolismo , Células da Medula Óssea/citologia , Contagem de Células , Diferenciação Celular , Proliferação de Células , Separação Celular , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Propriedades de SuperfícieRESUMO
Articular cartilage has a limited capacity for self-renewal. This article reports the development of a porous hydroxyapatite/collagen (HAp/Col) scaffold as a bone void filler and a vehicle for drug administration. The scaffold consists of HAp nanocrystals and type I atelocollagen. The purpose of this study was to investigate the efficacy of porous HAp/Col impregnated with FGF-2 to repair large osteochondral defects in a rabbit model. Ninety-six cylindrical osteochondral defects 5 mm in diameter and 5 mm in depth were created in the femoral trochlear groove of the right knee. Animals were assigned to one of four treatment groups: porous HAp/Col impregnated with 50 microl of FGF-2 at a concentration of 10 or 100 microg/ml (FGF10 or FGF100 group); porous HAp/Col with 50 microl of PBS (HAp/Col group); and no implantation (defect group). The defect areas were examined grossly and histologically. Subchondral bone regeneration was quantified 3, 6, 12, and 24 weeks after surgery. Abundant bone formation was observed in the HAp/Col implanted groups as compared to the defect group. The FGF10 group displayed not only the most abundant bone regeneration but also the most satisfactory cartilage regeneration, with cartilage presenting a hyaline-like appearance. These findings suggest that porous HAp/Col with FGF-2 augments the cartilage repair process.
Assuntos
Doenças Ósseas/tratamento farmacológico , Doenças das Cartilagens/tratamento farmacológico , Colágeno/farmacologia , Durapatita/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Engenharia Tecidual/métodos , Animais , Doenças Ósseas/diagnóstico por imagem , Doenças Ósseas/cirurgia , Doenças das Cartilagens/diagnóstico por imagem , Doenças das Cartilagens/cirurgia , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/cirurgia , Terapia Combinada , Sistemas de Liberação de Medicamentos/métodos , Fêmur/diagnóstico por imagem , Fêmur/cirurgia , Masculino , Nanofibras , Complicações Pós-Operatórias , Coelhos , Regeneração/efeitos dos fármacos , Regeneração/fisiologia , Microtomografia por Raio-XRESUMO
To potentiate the bone formation capability of bone marrow stromal cell (BMSC)/beta-tricalcium phosphate (beta-TCP) constructs, we devised an autologous plasma-based construct. We tested its effectiveness and investigated the effects of its components on a monkey ectopic bone formation model. The autologous plasma (platelet-rich plasma, PRP, or platelet-poor plasma, PPP)/BMSC/beta-TCP construct (R group or P group) showed significantly more bone formation at 3 and 6 weeks after implantation than a conventional BMSC/beta-TCP construct using a culture medium (M group). There was no significant difference between the P and R groups. Moreover, the P group constructs with a 10-fold lower cell concentration yielded equivalent bone formation to the M group at 5 weeks after implantation. To elucidate the effect of fibrin and serum contained in the plasma, five constructs were prepared using the following cell vehicles: autologous serum + fibrinogen (0, 1, 4, or 16 mg/mL) or phosphate-buffered saline + fibrinogen (4 mg/mL). The serum + fibrinogen (4 mg/mL, physiological concentration of monkeys) construct showed the most abundant bone formation at 3 weeks after implantation, though at 5 weeks no statistical difference existed among the groups. Autologous plasma efficiently promoted osteogenesis of BMSCs/porous beta-TCP constructs, and both fibrin and serum proved to play significant roles in the mechanism.