RESUMO
Bisphosphonates (BPs) inhibit bone resorption and are widely used for the treatment of bone diseases, including osteoporosis. BPs are also being studied for their effects on hydroxyapatite (HAP)-containing biomaterials. There is a growing appreciation that there are hitherto unexpected differences among BPs in their mineral binding affinities that affect their pharmacological and biological properties. To study these differences, we have developed a method based on fast performance liquid chromatography using columns of HAP to which BPs and other phosphate-containing compounds can adsorb and be eluted by using phosphate buffer gradients at pH 6.8. The individual compounds emerge as discrete and reproducible peaks for a range of compounds with different affinities. For example, the peak retention times (min; mean +/- SEM) were 22.0 +/- 0.3 for zoledronate, 16.16 +/- 0.44 for risedronate, and 9.0 +/- 0.28 for its phosphonocarboxylate analog, NE10790. These results suggest that there are substantial differences among BPs in their binding to HAP. These differences may be exploited in the development of biomaterials and may also partly explain the extent of their relative skeletal retention and persistence of biological effects observed in both animal and clinical studies.
Assuntos
Conservadores da Densidade Óssea/química , Difosfonatos/química , Durapatita/química , Ácido Etidrônico/análogos & derivados , Imidazóis/química , Cromatografia Líquida , Ácido Etidrônico/química , Ácido Risedrônico , Espectrofotometria Ultravioleta , Ácido ZoledrônicoAssuntos
Receptores de Ativinas Tipo I/genética , Proteínas de Transporte/genética , Miosite Ossificante/genética , Alelos , Mapeamento Cromossômico , Cromossomos Humanos Par 2/genética , Análise Mutacional de DNA , Triagem de Portadores Genéticos , Humanos , Mutação de Sentido Incorreto , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , Terminologia como AssuntoRESUMO
Human articular cartilage samples were retrieved from the resected material of patients undergoing total knee replacement. Samples underwent automated controlled freezing at various stages of preparation: as intact articular cartilage discs, as minced articular cartilage, and as chondrocytes immediately after enzymatic isolation from fresh articular cartilage. Cell viability was examined using a LIVE/DEAD assay which provided fluorescent staining. Isolated chondrocytes were then cultured and Alamar blue assay was used for estimation of cell proliferation at days zero, four, seven, 14, 21 and 28 after seeding. The mean percentage viabilities of chondrocytes isolated from group A (fresh, intact articular cartilage disc samples), group B (following cryopreservation and then thawing, after initial isolation from articular cartilage), group C (from minced cryopreserved articular cartilage samples), and group D (from cryopreserved intact articular cartilage disc samples) were 74.7% (95% confidence interval (CI) 73.1 to 76.3), 47.0% (95% CI 43 to 51), 32.0% (95% CI 30.3 to 33.7) and 23.3% (95% CI 22.1 to 24.5), respectively. Isolated chondrocytes from all groups were expanded by the following mean proportions after 28 days of culturing: group A ten times, group B 18 times, group C 106 times, and group D 154 times. This experiment demonstrated that it is possible to isolate viable chondrocytes from cryopreserved intact human articular cartilage which can then be successfully cultured.
Assuntos
Cartilagem Articular , Condrócitos , Criopreservação , Idoso , Idoso de 80 Anos ou mais , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Proliferação de Células , Sobrevivência Celular , Condrócitos/citologia , Condrócitos/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
This article reports the mechanical properties and in vitro evaluation of a collagen scaffold fabricated using an indirect 3D printing technique. Collagen scaffolds, featuring predefined internal channels and capillary networks, were manufactured using phase change printing. It was observed that the collagen scaffolds featured internal channels and a hierarchical structure that varied over length scales of 10-400 microm. In vitro evaluation using hMSCs demonstrated that the resultant collagen based scaffolds have the ability to support hMSC cell attachment and proliferation; cells can migrate and survive deep within the structure of the scaffold. The cell numbers increased 2.4 times over 28 days in culture for the lysine treated scaffolds. The cells were spread along the collagen fibers to form a 3D structure and extracellular matrix was detected on the surface of the scaffolds after 4 weeks in culture. The crosslinking treatment enhanced the biostability and dynamic properties of the collagen scaffolds significantly.
Assuntos
Colágeno , Engenharia Tecidual , Animais , Bovinos , Proliferação de Células , Células Cultivadas , Humanos , Teste de Materiais/métodos , Engenharia Tecidual/métodosRESUMO
Autologous chondrocyte implantation (ACI) is used to treat some articular cartilage defects. However, the fate of the cultured chondrocytes after in-vivo transplantation and their role in cartilage regeneration remains unclear. To monitor the survival and fate of such cells in vivo, the chondrocytes were labelled with a lipophilic dye and the resultant regenerated tissue in dogs examined. It was found that, 4 weeks after implantation, the osteochondral defects were filled with regenerative tissue that resembled hyaline cartilage. Fluorescence microscopy of frozen sections of the regenerated tissue revealed that the majority of cells were derived from the DiI-labelled implanted chondrocytes. From these results, it was concluded that a large population of implanted autologous chondrocytes can survive at least 4 weeks after implantation and play a direct role in cartilage regeneration. However, it remains unknown whether other cells, such as periosteal cells or bone marrow stromal stem cells, are involved in the regeneration of cartilage after ACI.
Assuntos
Cartilagem Articular/lesões , Cartilagem Articular/cirurgia , Condrócitos/patologia , Condrócitos/transplante , Fraturas de Cartilagem/patologia , Fraturas de Cartilagem/cirurgia , Regeneração Tecidual Guiada/métodos , Regeneração , Animais , Cartilagem Articular/patologia , Sobrevivência Celular , Células Cultivadas , Cães , Masculino , Resultado do TratamentoRESUMO
In this study, a multiple parallel perfused microbioreactor platform, TissueFlex, was developed which can be used to perform cell and tissue culture under almost uniform and precisely controlled environment in a mid-throughput and parallel manner. These microbioreactors were used to culture human bone marrow cells (hBMCs) in three-dimensional (3D) scaffolds and also in two-dimensional (2D) monolayer for comparison for upto 7 days. Several scaffolding materials were evaluated for this purpose in terms of easiness in handling, ability to support the hBMC growth, and feasibility for non-destructive optical assays. The feasibility and efficacy of using the developed 3D-hBMCs-based model tissue-constructs cultured in TissueFlex microbioreactors for drug evaluation and toxicity testing was then studied. As a demonstration case study, the cultured cells were challenged with two chemicals, trimethoprim and pyrimethamine, both known to be harmful to cellular activities, with different protocols. Cytotoxicity in terms of cell viability and growth was determined using the AlamarBlue assay. The 3D spatial variations in cell morphology and cell survival were also monitored using 3D optical imaging using non-linear multiphoton microscopy. The results show that (i) the data obtained from 3D hBMCs culture and from (2D) monolayer cultures on the effect of the tested chemicals on cell growth are significantly different, and that (ii) the perfused microbioreactor technology could provide a highly controlled and prolonged cell culture environment for testing of various drugs and chemicals. The outcome of this study demonstrated the feasibility and potentials of the using 3D stem cell based model tissues in TissueFlex microbioreactors for drug evaluation and toxicity testing of chemicals as an efficient and standardized alternative testing method.
Assuntos
Técnicas de Cultura de Células , Células-Tronco/metabolismo , Técnicas de Cultura de Tecidos/métodos , Testes de Toxicidade/métodos , Reatores Biológicos , Células da Medula Óssea/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Oxazinas , Pirimetamina/toxicidade , Trimetoprima/toxicidade , XantenosRESUMO
INTRODUCTION: Prostaglandins have an anabolic effect on bone. Possible mediation of this effect is via calcitriol. This study determines in vivo and in vitro effects of PGE(1) on calcitriol synthesis. METHODOLOGY: In vivo: rabbits received intravenous vehicle or prostaglandin E(1) (50 microg/day) for 20 days before measurements of serum total and ionic calcium, magnesium and phosphorus levels, total and bone-specific alkaline phosphatases, 25(OH)D(3), calcitriol, parathyroid hormone and calcitonin. In vitro: rabbit proximal renal tubules were incubated with 25(OH)D(3) (8 microM) together with PGE(1) (2.82 x 10(-6) M) and the prostaglandin receptor inhibitor AH6809 (10(-4) M) in selected samples. After 5 or 30 min incubation, calcitriol production was measured by radioimmunoassay and data analysed statistically. RESULTS: In vivo, in groups receiving PGE(1), levels of total Ca, Mg and calcitriol increased significantly and 25 dihydroxyvitamin D(3), parathyroid hormone and calcitonin remained unchanged. In vitro, PGE(1) increased calcitriol biosynthesis and the prostaglandin inhibitor AH6809 reduced calcitriol levels significantly after prolonged incubation. CONCLUSIONS: In vivo and in vitro results demonstrate that PGE(1) stimulates calcitriol synthesis. This study represent a major advancement in knowledge of bone metabolism.
Assuntos
Alprostadil/farmacologia , Calcitriol/biossíntese , Animais , Biomarcadores , Calcitriol/sangue , Cálcio/sangue , Cinética , Magnésio/sangue , Masculino , Fósforo/sangue , CoelhosRESUMO
Calcium cross-linked sodium alginate hydrogels have several advantageous features making them potentially suitable as tissue engineering scaffolds and this material has been previously used in many biomedical applications. 3D cell culture systems are often very different from 2D petri dish type cultures. in this study the effect of alginate hydrogel architecture was investigated by comparing rat bone marrow cell proliferation and differentiation on calcium cross linked sodium alginate discs and 1mm internal diameter tubes. It was found that bone marrow cell proliferation was diminished as the concentration of alginate in the 2D hydrogel substrates increased, yet proliferation was extensive on tubular alginate constructs with high alginate contents. Alginate gel thickness was found to be an important parameter in determining cell behaviour and the different geometries did not generate significant alterations in BMC differentiation profiles.
Assuntos
Alginatos/química , Materiais Biocompatíveis/química , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Osteoblastos/citologia , Osteoblastos/fisiologia , Osteogênese/fisiologia , Implantes Absorvíveis , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Hidrogéis/química , Masculino , Teste de Materiais , Camundongos , Camundongos Endogâmicos C57BL , RatosRESUMO
Sodium alginate has applications as a material for the encapsulation and immobilisation of a variety of cell types for immunoisolatory and biochemical processing applications. It forms a biodegradable gel when crosslinked with calcium ions and it has been exploited in cartilage tissue engineering since chondrocytes do not dedifferentiate when immobilised in it. Despite its attractive properties of degradability, ease of processing and cell immobilisation, there is little work demonstrating the efficacy of alginate gel as a substrate for cell proliferation, except when RGD is modified. In this study we investigated the ability of rat bone marrow cells to proliferate and differentiate on alginates of differing composition and purity. The mechanical properties of the gels were investigated. It was found that high purity and high G-type alginate retained 27% of its initial strength after 12 days in culture and that comparable levels of proliferation were observed on this material and tissue culture plastic. Depending on composition, calcium crosslinked alginate can act as a substrate for rat marrow cell proliferation and has potential for use as 3D degradable scaffold.
Assuntos
Alginatos , Células da Medula Óssea , Ácido Glucurônico , Ácidos Hexurônicos , Engenharia Tecidual , Animais , Materiais Biocompatíveis , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Cálcio/metabolismo , Células Cultivadas , Hidrogéis , Ratos , Ratos Wistar , Resistência à TraçãoRESUMO
A potential therapy to enhance healing of bone tissue is to deliver isolated mesenchymal stem cells (MSCs) to the site of a lesion to promote bone formation. A key issue within this technology is the development of an injectable system for the delivery of MSCs. Fibrin gel exploits the final stage of the coagulation cascade in which fibrinogen molecules are cleaved by thrombin, convert into fibrin monomers and assembled into fibrils, eventually forming fibers in a three-dimensional network. This gel could have many advantages as a cell delivery vehicle in terms of biocompatibility, biodegradation and hemostasis. The objective of this study was to explore the possibility of using fibrin gel as a delivery system for human MSCs (HMSCs). To this end we have determined the optimal fibrinogen concentrations and thrombin activity for loading HMSCs in vitro into the resultant fibrin gels to obtain cell proliferation. We found that a concentration of 18 mg/ml of fibrinogen and a thrombin activity of 100 IU/ml was optimal for producing fibrin scaffolds that would allow good HMSCs spreading and proliferation. In these conditions, cells were able to proliferate and expressed alkaline phosphatase, a bone marker, in vitro. When implanted in vivo, HMSCs were able to migrate out of the fibrin gel and invade a calcium carbonate based ceramic scaffold suggesting that fibrin gel could serve as a delivery system for HMSCs.
Assuntos
Técnicas de Cultura/métodos , Matriz Extracelular/metabolismo , Fibrina/metabolismo , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cultura/instrumentação , Matriz Extracelular/química , Humanos , Transplante de Células-Tronco Mesenquimais/instrumentação , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Pessoa de Meia-Idade , Trombina/farmacologiaRESUMO
The presence of osteogenic progenitors in human skeletal muscle is suggested by the formation of ectopic bone in clinical and experimental conditions, but their direct identification has not yet been demonstrated. The aims of this study were to identify osteogenic progenitor cells in human skeletal muscle tissue and to expand and characterize them in culture. Specimens of gracilis and semitendinosus muscle were obtained from young adults and digested to separate the connective tissue and satellite cell fractions. The cells were cultured and characterized morphologically and immunohistochemically using antibodies known to be reactive with primitive osteoprogenitor cells, pericytes, intermediate filaments, and endothelial cells. Alkaline phosphatase activity and osteocalcin gene expression were also determined. In the early stages of culture, the connective tissue cells obtained were highly positive for primitive osteoprogenitor cell and for pericyte markers. Alkaline phosphatase activity was detectable at early stages of culture and rose as a function of time, whereas primitive osteoprogenitor cell markers declined and osteocalcin mRNA expression became detectable by reverse transcriptase-polymerase chain reaction (RT-PCR). It is shown that human skeletal muscle connective tissue contains osteogenic progenitor cells. Their identification as pericytes, perivascular cells with established osteogenic potential, suggests a cellular link between angiogenesis and bone formation in muscle tissue. These cells are easily cultured and expanded in vitro by standard techniques, providing an alternative source of osteogenic progenitor cells for possible cell-based therapeutic use in certain conditions.
Assuntos
Osso e Ossos/citologia , Técnicas de Cultura de Células/métodos , Músculo Esquelético/citologia , Células-Tronco/citologia , Actinas/análise , Adulto , Fosfatase Alcalina/metabolismo , Antígenos de Neoplasias , Senescência Celular , Fibroblastos/citologia , Expressão Gênica , Humanos , Antígenos Específicos de Melanoma , Mesoderma/citologia , Proteínas de Neoplasias/análise , Osteocalcina/genética , Pericitos/citologia , RNA Mensageiro/análise , Reprodutibilidade dos Testes , Células-Tronco/química , Células-Tronco/enzimologiaRESUMO
Amplification of multipotential stem cells, with or without ex vivo gene transfer, offers the potential for their use for beneficial repopulation of a host in which there is specific cellular deficiency or functional impairment. The aims of the current study were to immunoselect, genetically mark, and determine the fate of fibroblastic progenitor cells in vivo. A monoclonal antibody, HOP-26, which has high reactivity with a cell surface antigen present on human osteoprogenitors in bone marrow fibroblast populations, was used to select these cells by immunopanning. Following culture in 10% FCS in alphaMEM containing ascorbate-2-phosphate and dexamethasone the amplified cells expressed the osteoblast phenotype as determined by expression of osteocalcin protein determined immunohistochemically, and Type I collagen and osteocalcin mRNA expressions determined by RT-PCR analysis. The selected cells were genetically labeled using a murine leukemia virus (MuLV) encoding a reporter gene (lacZ) with a selective marker gene (neo(r)) using a triple transient transfection protocol. Transfected cells were implanted in CB17 scid/scid mice by local subcutaneous injection over the calvariae. Localization of the genetically marked cells within the calvarial tissues was detected by beta-galactosidase histochemistry and immunocytochemistry. Genetically marked cells were observed within the periosteal layer in close association with the osteoblast layer, covering mineralized bone surfaces and within bone osteoid at 5 and 7 days after injection. This study demonstrates the successful selection, expansion, and retroviral-marking of human osteoprogenitors and their migration and localization within calvariae of SCID mice following in vivo implantation. These basic studies indicate the migration of these cells to skeletal sites and support possibilities for future uses of human osteoprogenitors in therapy of bone deficiency diseases and the potential for development of gene therapy procedures in these conditions.
Assuntos
Células da Medula Óssea/citologia , Transplante de Células , Fibroblastos/citologia , Osteoblastos/citologia , Retroviridae/genética , Crânio , Transplante Heterólogo/fisiologia , Células 3T3 , Animais , Anticorpos Monoclonais , Antígenos de Superfície/imunologia , Técnicas de Cultura de Células/métodos , Divisão Celular , Linhagem Celular , Células Cultivadas , Quimiotaxia , Colágeno/genética , Genes Reporter , Humanos , Rim , Vírus da Leucemia Murina/genética , Camundongos , Camundongos SCID , Osteoblastos/fisiologia , Osteocalcina/genética , TransfecçãoRESUMO
BACKGROUND: Primitive progenitors of bone tissue exist postnatally and exhibit stem-cell characteristics, as shown by extensive renewal potential, and capacity to differentiate into all characteristic connective tissue types, including bone, cartilage, fat, fibrous tissue, muscle and hemopoietic stroma. METHODS: A wide variety of investigative techniques have been applied to characterize and assess differentiation of the normally non-cycling osteogenic stem cells. These include methods to assess in vitro and in vivo differentiation potentials, the production and use of Abs to identify surface markers, the expression of specific genes and, more recently, incorporation of marker genes (beta-galactosidase, green fluorescent protein) to study cell fate after implantation at tissue sites. RESULTS: Some antigenic cell-surface molecules reactive with MAbs generated by a number of laboratories have been identified. For cell-fate studies, retroviral insertion of beta-galactosidase or green fluorescent protein genes into human marrow stromal progenitors has been accomplished with high efficiency. The stromal cell phenotype and cellular functions in vitro are not significantly altered by these genetic modifications. In vivo transplantation in immunodeficient animals demonstrates migration and persistence of marrow stromal cells to skeletal and other tissue sites. DISCUSSION: None of the Abs generated against surface markers of early progenitors are absolutely lineage and cell-stage specific, but the respective Ags appear to participate in cell adhesion and cell-signalling mechanisms. These may be important in stem-cell activation and subsequent early osteogenic development. Studies of cell fate indicate feasibility for future uses in therapy of bone deficiency diseases and the potential for development of gene therapy procedures in these and other conditions.
Assuntos
Osso e Ossos/citologia , Diferenciação Celular , Terapia Baseada em Transplante de Células e Tecidos/métodos , Terapia Baseada em Transplante de Células e Tecidos/tendências , Transplante de Células-Tronco/métodos , Transplante de Células-Tronco/tendências , Células-Tronco/citologia , Antígenos de Superfície/metabolismo , Marcadores Genéticos , Terapia Genética/métodos , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Estromais/citologia , Células Estromais/transplanteRESUMO
Precursor cells, isolated from bone marrow, can develop into various cell types and may contribute to skeletal growth, remodeling, and repair. The D1 cell line was cloned from a multipotent mouse bone marrow stromal precursor and has osteogenic, chondrogenic, and adipogenic properties. The osteogenic phenotype of these precursor cells is relevant to the process of fracture healing and osteointegration of prosthetic implants. The D1 cells were labeled genetically using a replication incompetent retroviral vector encoding beta-galactosidase, an enzyme which is used as a marker. Labeled cells are readily identifiable by staining with 5-bromo-4-chloro-3-indoyl-beta-D-galactoside and by flow cytometry, and retain the desired osteogenic characteristics in vivo as shown by von Kossa staining, alkaline phosphatase assay, an increase in cyclic adenosine monophosphate in response to parathyroid hormone, osteocalcin messenger ribonucleic acid production, and bone formation in diffusion chambers. In addition, the cells cloned from marrow stroma repopulate the marrow of host mice, persist for several weeks, and retain their osteogenic potential ex vivo. The data suggest that such cells may be used to replenish the number of osteoprogenitors in marrow, which appear to decrease with age, thereby leading to recovery from bone loss and improved bone growth and repair. Labeling these cells creates a model in which to study the potential of such cells to participate in fracture repair, ingrowth around prosthetic implants, treatment of osteoporosis, and to explore the possibility of gene delivery to correct mutations or defects in metabolism that are responsible for certain skeletal abnormalities.
Assuntos
Células da Medula Óssea/citologia , Mesoderma/fisiologia , Osteogênese/fisiologia , Células-Tronco/fisiologia , Animais , Divisão Celular , Linhagem Celular , Células Cultivadas , Células Clonais , AMP Cíclico/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Lactente , Injeções , Mesoderma/citologia , Camundongos , Camundongos Endogâmicos BALB C , Retroviridae , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
An experimental model of leg lengthening was used to study the morphology of, the collagenous proteins present, and the collagen genes expressed in the regenerating tissue following 20% lengthening at four different distraction rates. At a distraction rate of 0.3 mm/day (8 weeks distraction), the regenerate consists of intramembranous bone and localized areas of fibrocartilage. At rates of 0.7 (4 weeks) and 1.3 mm/day (2 weeks), the bone that grows from the cut ends of the cortical bone is separated by fibrous tissue and cartilage is present. At 2.7 mm/day (1 week), only fibrous tissue and sparse bone are present. Type I collagen is present in the matrices around the cells expressing its mRNA and similarly, type II collagen is located around the chondrocytes. Type I collagen mRNA is expressed predominantly by the fibroblasts in the fibrous tissue, the bone surface cells and to a reduced extent by the osteocytes. Type II collagen mRNA is expressed by chondrocytes. The results suggest that osteoblasts and chondrocytes within the regenerate originate from the same pool of progenitor cells, and the differentiation of these cells and the expression of types I and II collagen genes are altered by different rates of distraction. These observations suggest that the optimal rate of distraction in the model is 0.7 mm/day.
Assuntos
Colágeno/metabolismo , Osteogênese por Distração , Animais , Regeneração Óssea , Osso e Ossos/citologia , Cartilagem/citologia , Imuno-Histoquímica , Hibridização In Situ , RNA Mensageiro/metabolismo , CoelhosRESUMO
Glucocorticoids have well-documented effects on the skeleton, although their mechanism of action is still poorly understood. The actions of glucocorticoids on bone cells are mediated, in part, directly via specific receptors. The presence of these receptors has been demonstrated in both rodent and human osteoblastic cells in vitro, but their presence in human bone in vivo has not been reported. In this study, we have used specific affinity purified polyclonal antibodies to the functional glucocorticoid receptor alpha (GRalpha) to investigate its expression in both developing and adult human bone using sections of neonatal rib, calvarial, and vertebral bones, tibial growth plates from adolescents, and iliac crest biopsies from adults who were to undergo liver transplantation. In the tibial growth plates, GRalpha was predominantly expressed in the hypertrophic chondrocytes within the cartilage. In the primary spongiosa, the receptor was highly expressed by osteoblasts at sites of bone modeling. Within the bone marrow, receptors were also detected in mononuclear cells and in endothelial cells of blood vessels. In the neonatal rib and vertebrae, GRalpha was widely distributed at sites of endochondral bone formation in resting, proliferating, mature, and hypertrophic chondrocytes. They were also highly expressed in osteoblasts at sites of bone modeling. At sites of intramembranous ossification in neonatal calvarial bone and rib periosteum, GRa was widely expressed in cells within the fibrous tissue and in osteoblasts at both the bone-forming surface and at modeling sites. In the iliac crests from adults, GRalpha was predominantly expressed in osteocytes. The receptors were not detected in osteoclasts. Our results show for the first time the presence of the functional GRalpha in human bone in situ and suggest that the actions of glucocorticoids on bone may be mediated, in part, directly via the GR at different stages of life. The absence of receptor expression in osteoclasts also suggests that the effects of glucocorticoids on bone resorption may be mediated indirectly.
Assuntos
Osso e Ossos/metabolismo , Receptores de Glucocorticoides/metabolismo , Adolescente , Adulto , Remodelação Óssea , Cartilagem/citologia , Cartilagem/metabolismo , Criança , Condrócitos/metabolismo , Feminino , Lâmina de Crescimento/citologia , Lâmina de Crescimento/metabolismo , Humanos , Ílio/metabolismo , Recém-Nascido , Masculino , Osteoblastos/metabolismo , Isoformas de Proteínas/metabolismo , Costelas/metabolismo , Coluna Vertebral/metabolismo , Tíbia/metabolismo , Distribuição TecidualRESUMO
The effect of a hydroxyapatite-tricalcium phosphate (HA) material on collagen synthesis by human osteoblasts was investigated using X-ray photoelectron spectroscopy (XPS). To this aim, thin HA slices were exposed to osteoblasts harvested from three different patients, for 20 days and then analyzed by XPS. Platinum plates were also exposed to the cells for comparison, and control tests were performed on both materials using cell-free media. XPS analysis supported by standard spectra of some polyaminoacids and of collagen deposited on HA suggested that a deposition of collagen occurred on the HA slices in the presence of osteoblasts. On the other hand, only an aspecific deposition of proteins was observed on platinum and when cell-free media were used. These data were confirmed evaluating collagen synthesis by [(3)H]proline incorporation of osteoblasts exposed to HA.
Assuntos
Materiais Biocompatíveis/química , Fosfatos de Cálcio/química , Colágeno/biossíntese , Durapatita/química , Osteoblastos/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Meios de Cultura , Humanos , Peptídeos/farmacologia , Platina , Prolina/metabolismo , Espectrometria por Raios XRESUMO
Adipocytes and osteoblasts have common origins from fibroblastic stem cells. Consequently, modulation of the processes of adipogenesis and osteogenesis has implications for the possible treatment of metabolic bone diseases, such as osteoporosis, in which medullary fat accumulates and trabecular bone volume decreases. It is likely that the balance between these two systems is affected by particular endogenous growth factors which are known to affect bone metabolism. We have therefore investigated the effects of transforming growth factor beta (TGFbeta), basic fibroblast growth factor (bFGF) and dexamethasone (Dex) on cultured human bone marrow (HBM) fibroblastic cells to observe the effects on adipogenesis and osteogenesis. In the absence of fetal calf serum (FCS), TGFbeta caused a dose-dependent increase in cell growth and alkaline phosphatase activity (AP); however, in the presence of FCS growth was inhibited at high concentrations and AP unaffected. TGFbeta increased matrix proteoglycan and collagen synthesis. bFGF inhibited AP and increased colony number and size, while Dex treatment increased AP activity and colony number, and both factors in combination resulted in an additive increase in growth. Dex-induced adipocyte formation was accelerated but not increased by bFGF. A significant inhibition of adipogenesis by TGFbeta was observed within 7 days. These results demonstrate the importance of biological factors known to be involved in bone remodelling in the regulation of osteogenesis and adipogenesis.
Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Células Estromais/citologia , Células Estromais/efeitos dos fármacosRESUMO
Understanding the mechanisms that control the proliferation and commitment of human stem cells into cells of the osteogenic lineage for the preservation of skeletal structure is of basic importance in bone physiology. This study examines some aspects of the differentiation in vitro of human bone marrow fibroblastic cells cultured in the absence (basal media) or presence of 1nM dexamethasone and 50 micrograms/ml ascorbate for 6, 10, 14, and 21 days. Northern blot analysis and in situ hybridisation with digoxygenin-labelled riboprobes for Type I collagen, osteocalcin, bone morphogenetic proteins 2 (BMP-2), and 4 (BMP-4) and the estrogen receptor alpha (ERalpha), together with immunocytochemical analysis of ERalpha expression and histochemical staining of alkaline phosphatase was performed. In basal media, alkaline phosphatase activity and collagen expressions were detected at day 6, ERalpha from day 10 and osteocalcin from day 10. In the presence of dexamethasone and ascorbate, cell proliferation and alkaline phosphatase were markedly stimulated over 10 to 14 days with a dramatic increase in the temporal expression of Type I collagen, ERalpha, and osteocalcin mRNAs in these cultures. Northern blot analysis showed cells cultured in basal media, expressed the highest levels of the mRNA for each marker protein at day 14, whereas in the presence of ascorbate and dexamethasone, the highest levels for alkaline phosphatase, ERalpha, osteocalcin, BMP-2, and BMP-4 were observed at day 21. ERalpha, BMP-2, and BMP-4 expression were found to correlate temporally with induction of the osteoblast phenotype as determined by alkaline phosphatase, collagen, and osteocalcin expression. These results give additional information on the development of the osteoblast phenotype from early fibroblastic stem cells and on the biological factors involved in this process. These studies suggest a role for estrogen and BMP-2 and -4 in the differentiation of osteoprogenitor cells.
Assuntos
Proteínas Morfogenéticas Ósseas/genética , Células-Tronco Hematopoéticas/metabolismo , Osteoblastos/metabolismo , RNA Mensageiro/genética , Receptores de Estrogênio/genética , Fator de Crescimento Transformador beta , Idoso , Idoso de 80 Anos ou mais , Fosfatase Alcalina/metabolismo , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 4 , Diferenciação Celular , Células Cultivadas , Colágeno/genética , Receptor alfa de Estrogênio , Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Humanos , Hibridização In Situ , Osteoblastos/citologia , Osteocalcina/genética , RNA Mensageiro/metabolismoRESUMO
Ideal skeletal reconstruction depends on regeneration of normal tissues that result from initiation of progenitor cell activity. However, knowledge of the origins and phenotypic characteristics of these progenitors and the controlling factors that govern bone formation and remodeling to give a functional skeleton adequate for physiological needs is limited. Practical methods are currently being investigated to amplify in in vitro culture the appropriate autologous cells to aid skeletal healing and reconstruction. Recent advances in the fields of biomaterials, biomimetics, and tissue engineering have focused attention on the potentials for clinical application. Current cell therapy procedures include the use of tissue-cultured skin cells for treatment of burns and ulcers, and in orthopedics, the use of cultured cartilage cells for articular defects. As mimicry of natural tissues is the goal, a fuller understanding of the development, structures, and functions of normal tissues is necessary. Practically all tissues are capable of being repaired by tissue engineering principles. Basic requirements include a scaffold conducive to cell attachment and maintenance of cell function, together with a rich source of progenitor cells. In the latter respect, bone is a special case and there is a vast potential for regeneration from cells with stem cell characteristics. The development of osteoblasts, chondroblasts, adipoblasts, myoblasts, and fibroblasts results from colonies derived from such single cells. They may thus, theoretically, be useful for regeneration of all tissues that this variety of cells comprise: bone, cartilage, fat, muscle, tendons, and ligaments. Also relevant to tissue reconstruction is the field of genetic engineering, which as a principal step in gene therapy would be the introduction of a functional specific human DNA into cells of a patient with a genetic disease that affects mainly a particular tissue or organ. Such a situation is pertinent to osteogenesis imperfecta, for example, where in more severely affected individuals any improvements in long bone quality would be beneficial to the patient. In conclusion, the potentials for using osteogenic stem cells and biomaterials in orthopedics for skeletal healing is immense, and work in this area is likely to expand significantly in the future.