RESUMEN
This study has examined the osteogenic and chondrogenic differentiation of human foetal femur-derived cells in 3-dimensional pellet cultures. After culture for 21-28 days in osteogenic media, the pellets acquired a unique configuration that consisted of an outer fibrous layer, an osteoid-like shell surrounding a cellular and cartilaginous region. This configuration is typical to the cross section of the foetal femurs at the same age and was not observed in pellets derived from adult human bone marrow stromal cells. Time course study showed that after 7-14 days, the cells of the inner cellular region were viable, proliferated rapidly, and were immuno-positive for c-myc, as well as for bone sialoprotein and type I collagen. After 21-28 days, the cells accumulated at the inner edge of the osteoid shell. The direction of osteoid formation thus differed from that of periosteal bone formation. Following micro-dissection of the human foetal femurs into epiphyses, bone cylinder and hypertrophic cartilage, epiphyseal chondrocytes and osteoblasts both gave rise to osteoid-shell forming cells. These studies demonstrate the developmental plasticity of human foetal skeletal and epiphyseal chondrocytes and suggest that the microenvironment modulates lineage commitment and matrix formation. Furthermore, this ex vivo model offers a new approach to delineate human bone development as well as a model with potential application for evaluation of therapeutic compounds for bone formation.
Asunto(s)
Diferenciación Celular , Condrogénesis , Fémur/citología , Osteogénesis , Calcificación Fisiológica , Técnicas de Cultivo de Célula , Proliferación Celular , Células Cultivadas , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Medios de Cultivo , Feto , Humanos , Sialoproteína de Unión a Integrina/metabolismo , Osteonectina/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismoRESUMEN
Osteoarthritis, a degenerative joint disease, is the most disabling condition of the Western world. It affects first and foremost the articular cartilages and leads to a molecular and supramolecular destruction of the extracellular cartilage matrix. In addition, the cells, the chondrocytes, show severe alterations of their phenotype: they get anabolically and catabolically activated, change accordingly their gene expression pattern, lose their differentiated phenotype, and undergo focally cell death and cell degeneration. All these processes represent potential targets for therapeutic intervention and drug development. Apart from the cartilage itself, however, other joint tissues are also involved in the disease: thus, the synovial capsule and membrane as well as the subchondral bone account not only for most of the symptoms of the disease, but are also presumably involved in the progression of the degenerative process. Both, inflammation and stiffening within the joint capsule accelerate joint destruction. Stiffening of the subchondral bone increases the mechanical stress over the overlying cartilage during physiological movement. Altogether, there is a plethora of tissues, disease processes and targets for treating osteoarthritic joint degeneration, which will need to be followed up systematically in the future.
Asunto(s)
Osteoartritis/tratamiento farmacológico , Antiinflamatorios/uso terapéutico , Cartílago Articular/patología , Diseño de Fármacos , Matriz Extracelular/patología , Humanos , Osteoartritis/patologíaRESUMEN
OBJECTIVE: To review the current knowledge of the mechanism of DNA methylation, its association with transcriptional silencing, possible mechanisms of hyper- and hypomethylation and how epigenetic changes may relate to the pathogenesis of osteoarthritis (OA). METHODS: Journal literature was searched using Pubmed. Since there are very few publications directly on epigenetic phenomena in OA, the search was extended to give an overview of epigenetic mechanisms as they relate to the molecular mechanisms of the disease. RESULTS: While the epigenetics of cancer cells have been intensively investigated, little attention has so far been paid as to whether epigenetic changes contribute to the pathology of non-neoplastic diseases such as OA. This review explains the mechanisms of DNA methylation, its role in transcriptional regulation, and possible demethylation mechanisms that may be applicable to OA. Preliminary evidence suggests that changes in DNA methylation, together with cytokines, growth factors and changes in matrix composition, are likely to be important in determining the complex gene expression patterns that are observed in osteoarthritic chondrocytes. CONCLUSION: Early evidence points to a role of epigenetics in the pathogenesis of OA. Since epigenetic changes, although heritable at the cellular level, are potentially reversible, epigenetics could be a new molecular target for therapeutic intervention, especially early in the disease.
Asunto(s)
Metilación de ADN , Osteoartritis/genética , Condrocitos/metabolismo , Epigénesis Genética/genética , Humanos , Osteoartritis/metabolismoRESUMEN
OBJECTIVE: Collagen fibril degeneration involves initially the cleavage within the triple helix by the collagenases 1 (MMP-1) and 3 (MMP-13), but then mainly involves also the gelatinases A (MMP-2) and B (MMP-9). The objective of this study was to determine the quantitative expression levels as well as the distribution in normal and osteoarthritic cartilage of gelatinase B and in cultured articular chondrocytes with and without stimulation by Il-1Beta. METHODS: Conventional and real-time quantitative PCR technology and immunohistochemistry were used to determine gelatinase B expression on the mRNA and protein level. RESULTS: Conventional PCR analysis could demonstrate the presence of gelatinase B mRNA only in osteoarthritic chondrocytes. Real-time quantitative PCR confirmed the increased expression of gelatinase B mRNA expression in osteoarthritic chondrocytes. No significant up-regulation of gelatinase B was observed by Il-1Beta. Immunostaining for gelatinase B showed the presence of gelatinase B in a subset of normal and in a large portion of osteoarthritic chondrocytes with a more extended distribution in the latter. CONCLUSION: In osteoarthritic cartilage destruction, gelatinase B is involved in collagen destruction though still at a very much lower level than gelatinase A. Only a very small subset of normal adult articular chondrocytes express gelatinase B in vivo suggesting that gelatinase B unlike gelatinase A is hardly or only very focally involved in physiological collagen turnover.
Asunto(s)
Cartílago Articular/metabolismo , Condrocitos/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Osteoartritis de la Rodilla/metabolismo , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/patología , Relación Dosis-Respuesta a Droga , Cabeza Femoral/citología , Expresión Génica , Humanos , Inmunohistoquímica , Interleucina-1/farmacología , Metaloproteinasa 9 de la Matriz/genética , Persona de Mediana Edad , Osteoartritis de la Rodilla/patología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: To investigate the immunolocalisation of beta-dystroglycan (beta-DG) and specific matrix metalloproteinases (MMPs)-3, -9, -13 and a disintegrin like and metalloproteinase thrombospondin type 1 motif 4 (ADAMTS-4) within the joint tissues of patients with osteoarthritis (OA) and unaffected controls. DESIGN: Cartilage, synovium and synovial fluid were obtained from the hip joints of five osteoarthritic (patients undergoing total hip replacement) and five control hip joints (patients undergoing hemiarthroplasty for femoral neck fracture). The samples were analysed for beta-DG protein using Western blot technique and by immunohistochemistry for tissue distribution of beta-DG, MMP-3, -9, -13, and ADAMTS-4. RESULTS: beta-DG was detected in the smooth muscle of both normal and osteoarthritic synovial blood vessels. Importantly, beta-DG was detected in endothelium of blood vessels of OA synovium, but not in the control endothelium. In the endothelium of osteoarthritic synovial blood vessels, beta-DG co-localised with MMP-3 and -9. MMP-13 and ADAMTS-4 showed no endothelial staining, and only weak staining of the vascular smooth muscle was found. In contrast, we did not detect beta-DG protein in cartilage or synovial fluid. CONCLUSIONS: beta-DG has been shown to have a role in angiogenesis, and our results demonstrate for the first time that there are clear differences in beta-DG staining between OA and control synovial blood vessels. The specific immunolocalisation of beta-DG within endothelium of inflamed OA blood vessels and its co-localisation with MMP-3 and -9, reported to have pro-angiogenic roles and believed to be involved in beta-DG cleavage, may also suggest that beta-DG plays a role in angiogenesis accompanying OA.
Asunto(s)
Proteínas ADAM/análisis , Distroglicanos/análisis , Metaloproteinasas de la Matriz/análisis , Osteoartritis de la Cadera/metabolismo , Procolágeno N-Endopeptidasa/análisis , Proteína ADAMTS4 , Western Blotting/métodos , Cartílago Articular/enzimología , Cartílago Articular/metabolismo , Endotelio Vascular/enzimología , Endotelio Vascular/metabolismo , Articulación de la Cadera/enzimología , Articulación de la Cadera/metabolismo , Humanos , Inmunohistoquímica/métodos , Metaloproteinasa 13 de la Matriz/análisis , Metaloproteinasa 3 de la Matriz/análisis , Metaloproteinasa 9 de la Matriz/análisis , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/metabolismo , Osteoartritis de la Cadera/enzimología , Líquido Sinovial/enzimología , Líquido Sinovial/metabolismo , Membrana Sinovial/enzimología , Membrana Sinovial/metabolismoRESUMEN
Osteoarthritis is first and foremost the ongoing destruction of the articular cartilages of joints. Therefore, the extracellular matrix and the cells of the articular cartilages are the primary targets of osteoarthritis therapy. This tries to inhibit enzymatic destruction of the extracellular cartilage matrix as well as the modification of the cellular phenotype of the chondrocytes: cell degeneration and cell death are alongside anabolic activation and stabilization of the cellular phenotype of major interest. However, apart from the cartilage and its cells, other tissues of the joints are also important for the symptoms of the disease, which basically all originate outside the articular cartilage. In addition, changes in the subchondral bone as well as the synovial capsule and membrane are important at least for the progression of the disease process. All the named tissues offer different directions and ways for therapeutic intervention.
Asunto(s)
Antirreumáticos/administración & dosificación , Antirreumáticos/uso terapéutico , Sistemas de Liberación de Medicamentos/tendencias , Artropatías/tratamiento farmacológico , Humanos , Osteoartritis/tratamiento farmacológicoRESUMEN
Evidence has accumulated in recent years that programmed cell death (PCD) is not necessarily synonymous with the classical apoptosis, as defined by Kerr and Wyllie, but that cells use a variety of pathways to undergo cell death, which are reflected by different morphologies. Although chondrocytes with the hallmark features of classical apoptosis have been demonstrated in culture, such cells are extremely rare in vivo. The present review focuses on the morphological differences between dying chondrocytes and classical apoptotic cells. We propose the term 'chondroptosis' to reflect the fact that such cells are undergoing apoptosis in a non-classical manner that appears to be typical of programmed chondrocyte death in vivo. Unlike classical apoptosis, chondroptosis involves an initial increase in the endoplasmic reticulum and Golgi apparatus, reflecting an increase in protein synthesis. The increased ER membranes also segment the cytoplasm and provide compartments within which cytoplasm and organelles are digested. In addition, destruction occurs within autophagic vacuoles and cell remnants are blebbed into the lacunae. Together these processes lead to complete self-destruction of the chondrocyte as evidenced by the presence of empty lacunae. It is speculated that the endoplasmic reticulum pathway of apoptosis plays a greater role in chondroptosis than receptor-mediated or mitochondrial pathways and that lysosomal proteases are at least as important as caspases. Because chondroptosis does not depend on phagocytosis, it may be more advantageous in vivo, where chondrocytes are isolated within their lacunae. At present the initiation factors or the molecular pathways involved in chondroptosis remain unclear.
Asunto(s)
Apoptosis , Condrocitos/patología , Animales , Autofagia , Compartimento Celular , Condrocitos/ultraestructura , Retículo Endoplásmico/ultraestructura , Aparato de Golgi/ultraestructura , Placa de Crecimiento/patología , Placa de Crecimiento/ultraestructura , Humanos , Modelos Biológicos , Osteoartritis/patología , Proteínas/metabolismo , Vacuolas/ultraestructuraRESUMEN
The development of new bone formation strategies offers tremendous therapeutic implications in a variety of musculoskeletal diseases. One approach involves harnessing the regenerative capacity of osteoprogenitor bone cells in combination with biomimetic scaffolds generated from appropriate scaffold matrices and osteoinductive factors. The aims of our study were to test the efficacy of two innovative osteoinductive agents: the osteoblast stimulating factor-1 (osf-1), an extracellular matrix-associated protein, and osteoinductive extracts of Saos-2 cells on human osteoprogenitor cells. Saos-2 extracted osteoinductive factors significantly stimulated alkaline phosphatase specific activity in basal and osteogenic conditions. Osf-1 significantly stimulated chemotaxis, total colony formation, alkaline phosphatase-positive colony formation, and alkaline phosphatase specific activity at concentrations as low as 10 pg/ml compared with control cultures. Osteoinductive factors present in Saos-2 cell extracts and osf-1 promoted adhesion, migration, expansion, and differentiation of human osteoprogenitor cells on 3-D scaffolds. The successful generation of 3-D biomimetic structures incorporating osf-1 or osteoinductive factors from Saos-2 cells indicates their potential for de novo bone formation that exploits cell-matrix interactions.
Asunto(s)
Materiales Biocompatibles , Proteínas Portadoras/farmacología , Medios de Cultivo Condicionados/farmacología , Citocinas/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Anciano , Anciano de 80 o más Años , Fosfatasa Alcalina/metabolismo , Células de la Médula Ósea , Calcificación Fisiológica/efectos de los fármacos , Calcificación Fisiológica/fisiología , División Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inmunohistoquímica , Ácido Láctico , Masculino , Células Madre Mesenquimatosas/enzimología , Persona de Mediana Edad , Osteogénesis/fisiología , Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Polímeros , Ingeniería de TejidosRESUMEN
Epidemiological studies suggest that poor growth during fetal life and infancy is associated with decreased bone mass in adulthood. However, theses observations have not, to date, been corroborated in animal models. To address this issue we evaluated the influence of maternal protein restriction on bone mass and growth plate morphology among the adult offspring, using a rat model. Maternal protein restriction resulted in a reduction in bone area and BMC, but not BMD, among the offspring in late adulthood. The widened epiphyseal growth plate in the protein-restricted offspring is compatible with the programming of cartilage and bone growth by maternal nutrition in early life.
Asunto(s)
Desarrollo Óseo/fisiología , Huesos/fisiología , Dieta con Restricción de Proteínas/efectos adversos , Placa de Crecimiento/anomalías , Efectos Tardíos de la Exposición Prenatal , Absorciometría de Fotón , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Densidad Ósea/fisiología , Huesos/diagnóstico por imagen , Femenino , Fémur/patología , Masculino , Embarazo , Ratas , Ratas Wistar , Tibia/patologíaRESUMEN
The ability to generate new bone for skeletal use is a major clinical need. Biomimetic scaffolds that interact and promote osteoblast differentiation and osteogenesis offer a promising approach to the generation of skeletal tissue to resolve this major health-care issue. In this study we examine the ability of surface-modified poly(lactic acid) (PLA) films and poly(lactic-co-/glycolic acid) (PLGA) (75:25) porous structures to promote human osteoprogenitor adhesion, spreading, growth, and differentiation. Cell spreading and adhesion were examined using Cell Tracker green fluorescence and confocal microscopy. Osteogenic differentiation was confirmed with alkaline phosphatase activity as well as immunocytochemistry for type I collagen, core binding factor-1 (Cbfa-1), and osteocalcin. Poor cell growth was observed on nonmodified PLA films and PLGA scaffolds. The polymers were then coupled with RGD peptides [using poly(L-lysine), or PLL] and physical adsorption as well as PLA films presenting adsorbed fibronectin (FN). Both modifications enhanced cell attachment and spreading. On PLA-FN and PLA-PLL-GRGDS films, the osteoblast response was dose dependent (20 pmol/L to 0.2 micromol/L FN and 30 nmol/L to 30 micromol/L PLL-GRGDS) and significant at concentrations as low as 2 nmol/L FN and 30 nmol/L PLL-GRGDS. With optimal concentrations of FN or RGD, adhesion and cell spreading were comparable to tissue culture plastic serum controls. In PLGA (75:25) biodegradable porous scaffolds, coated with FN, PLL-GRGDS, or fetal calf serum for 24 h in alpha MEM alone, prior to growth in dexamethasone and ascorbate-2-phosphate for 4-6 weeks, extensive osteoblast impregnation was observed by confocal and fluorescence microscopy. Cell viability in extended culture was maintained as analyzed by expression of Cell Tracker green and negligible ethidium homodimer-1 (a marker of cell necrosis) staining. Alkaline phosphatase activity, type I collagen, Cbfa-1, and osteocalcin expression were observed by immunocytochemistry. Mineralization of collagenous matrix took place after 4 weeks, which confirmed the expression of the mature osteogenic phenotype. These observations demonstrate successful adhesion and growth of human osteoprogenitors on protein- and peptide-coupled polymer films as well as migration, expansion, and differentiation on three-dimensional biodegradable PLGA scaffolds. The use of peptides/proteins and three-dimensional structures that provide positional and environmental information indicate the potential for biomimetic structures coupled with appropriate factors in the development of protocols for de novo bone formation.
Asunto(s)
Materiales Biocompatibles , Células de la Médula Ósea/citología , Diferenciación Celular , División Celular , Células Madre/citología , Anciano , Anciano de 80 o más Años , Células Cultivadas , Femenino , Humanos , Inmunohistoquímica , Masculino , Microscopía Fluorescente , Persona de Mediana Edad , Propiedades de SuperficieRESUMEN
Six calcaneal fragments from patients aged 2, 3, 4, and 5 years with relapsed talipes, and two normal feet from a 40-week-old stillborn fetus were studied. All tissue was sectioned in the sagittal or coronal plane and stained using alcian blue and sirius red to distinguish cartilage and bone. Immunocytochemistry was performed to illustrate collagen types I and II. Within the clubfoot calcaneum, there were fewer chondrocytes and a diminished number of cartilage canals. Although a growth plate was present, the zones of differentiated chondrocytes were not apparent and the chondrocytes were smaller and flatter. The alcian blue staining within the spherical physis was paler than normal, suggesting that the amount of extracellular proteoglycans was reduced. Overall, the growth plate region of the talipes calcaneum resembled that of a permanent cartilage, like articular cartilage. Abnormalities were also seen in the ossification center. Cartilage spicules were rare, and developing bone frequently abutted directly onto the growth plate cartilage. The relative absence of a primary spongiosa suggested that the physis was virtually inactive and endochondral bone formation was retarded. These findings are consistent with the hypothesis that an intrinsic primary growth disorder causes the formation of a small hypoplastic bone and, subsequently, a smaller foot.
Asunto(s)
Calcáneo/patología , Pie Equinovaro/patología , Calcáneo/química , Preescolar , Pie Equinovaro/metabolismo , Colágeno/análisis , Feto/patología , Placa de Crecimiento/patología , Humanos , Inmunohistoquímica , Lactante , Osificación Heterotópica/patologíaRESUMEN
Chondrocytes at the lower zone of the growth plate must be eliminated to facilitate longitudinal growth; this is generally assumed to involve apoptosis. We attempted to provide definitive electron-microscopic evidence of apoptosis in chondrocytes of physes and chondroepiphyses in the rabbit. We were, however, unable to find a single chondrocyte with the ultrastructure of 'classical' apoptosis in vivo, although such a cell was found in vitro. Instead, condensed chondrocytes had a convoluted nucleus with patchy chromatin condensations while the cytoplasm was dark with excessive amounts of endoplasmic reticulum. These cells were termed 'dark chondrocytes'. A detailed study of their ultrastructure combined with localisation methods in situ suggested a different mechanism of programmed cell death. In addition, another type of death was identified among the immature chondrocytes of the chondroepiphysis. These cells had the same nucleus as dark chondrocytes, but the lumen of the endoplasmic reticulum had expanded to fill the entire non-nuclear space, and all cytoplasm and organelles had been reduced to dark, worm-like inclusions. Since these cells appeared to be 'in limbo', they were termed 'paralysed' cells. It is proposed that 'dark chondrocytes' and 'paralysed cells' are examples of physiological cell death which does not involve apoptosis. It is possible that the confinement of chondrocytes within their lacunae, which would prevent phagocytosis of apoptotic bodies, necessitates different mechanisms of elimination.
Asunto(s)
Apoptosis , Condrocitos/ultraestructura , Placa de Crecimiento/ultraestructura , Fosfatasa Ácida/metabolismo , Animales , Apoptosis/fisiología , Condrocitos/enzimología , Cabeza Femoral/enzimología , Cabeza Femoral/ultraestructura , Colorantes Fluorescentes , Placa de Crecimiento/enzimología , Histocitoquímica , Húmero/enzimología , Húmero/ultraestructura , Etiquetado Corte-Fin in Situ , Microscopía Electrónica , ConejosRESUMEN
The exits from metaphase arrest and anatomy of mitotic catastrophe were studied in two human osteosarcoma cell lines, nontumorigenic HOS TE85 and its chemically transformed strain MNNG-HOS, applying mild genotoxic damage by heat shock at 41.8 degrees C for 24 h. Under these conditions, both cell lines doubled or tripled their mitotic index entering arrest in metaphase. On return to 37 degrees C, the arrest was either released or ended in apoptosis. The transformed strain showed a greater capacity to arrest in metaphase as well as a greater probability of developing the third pathway: to restitute this arrest in polyploid interphase. This, in turn, either entered an 'endocycle' or, following a delay, apoptosis. Thus, arrest in metaphase was a cross-point of the mitotic cycle, apoptosis, and endocycle. Mitotic catastrophe can morphologically manifest combinations of elements of these three processes.
Asunto(s)
Calor , Metafase , Mitosis , Osteosarcoma/metabolismo , Anafase , Apoptosis , ADN/metabolismo , Células Gigantes/fisiología , Humanos , Modelos Biológicos , Telofase , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Growth plates taken from five- to 20-week-old Japanese white rabbits were immunostained for c-Myc protein. This was localised both in the proliferating zone and upper hypertrophic zone at five weeks, whereas after ten weeks it was found mostly in the lower hypertrophic zone. The proliferating chondrocytes tended to show nuclear staining and the hypertrophic cells cytoplasmic staining, although the terminal hypertrophic chondrocytes sometimes expressed the protein in their nuclei. In the younger rabbits, c-Myc co-localised with proliferating cell nuclear antigen, whereas in the hypertrophic zone of older rabbits, it was present in some chondrocytes the nuclei of which also contained DNA breaks. Our study suggests that, in the rabbit growth plate, c-Myc is associated with different cellular processes, depending on the age and the developmental stage of the chondrocytes.
Asunto(s)
Envejecimiento/fisiología , Apoptosis/fisiología , Placa de Crecimiento/metabolismo , Proteínas Proto-Oncogénicas c-myc/análisis , Animales , Núcleo Celular/química , Condrocitos/química , Condrocitos/patología , Citoplasma/química , Fragmentación del ADN , Cabeza Femoral/patología , Placa de Crecimiento/química , Placa de Crecimiento/patología , Hipertrofia , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Antígeno Nuclear de Célula en Proliferación/análisis , ConejosRESUMEN
The efficient elimination of apoptotic cells depends on heterophagocytosis by other cells, which is difficult or impossible when the dying cells are embedded in an extracellular matrix. This situation is exemplified by the epiphyseal chondrocytes during the development of the chondroepiphyses of long bones. A detailed ultrastructural study identified an unusual type of epiphyseal chondrocyte which contained a very dark nucleus with irregular patches of condensed chromatin and a crenated nuclear membrane. The cytosol consisted of excessively expanded endoplasmic reticulum lumen, containing "islands" of cytoplasm and organelles. Since these cells appeared to be "in limbo," neither viable nor dead, they are referred to as "paralyzed" cells. By studying cells of intermediate morphologies, we were able to demonstrate the sequence of events leading to cell paralysis. It is proposed that the paralysis represents an intermediate state in the physiological cell death of epiphyseal chondrocytes in which destruction is orderly and avoids a inflammatory, potentially locally destructive, reaction. The cell is rendered paralyzed in terms of function but impotent in respect of damaging consequences. Paralysis is compared and contrasted with apoptosis, autophagocytosis, and necrosis and may represent another mode of programmed cell death in situations where cells are immature and/or where phagocytosis by neighboring cells is difficult.
Asunto(s)
Apoptosis/fisiología , Condrocitos/patología , Placa de Crecimiento/patología , Animales , Animales Recién Nacidos , Núcleo Celular/ultraestructura , Supervivencia Celular/fisiología , Desarrollo Embrionario y Fetal/fisiología , Fémur/embriología , Fémur/crecimiento & desarrollo , Fémur/patología , Placa de Crecimiento/embriología , Placa de Crecimiento/crecimiento & desarrollo , Húmero/embriología , Húmero/crecimiento & desarrollo , Húmero/patología , Cuerpos de Inclusión/ultraestructura , Mitocondrias/ultraestructura , ConejosRESUMEN
The activities of acid and alkaline phosphatases were localized by enzyme histochemistry in the chondroepiphyses of 5 week old rabbits. Using paraformaldehyde-lysine-periodate as fixative, the activity of acid phosphatase was particularly well preserved and could be demonstrated not only in osteoclasts, but also in chondrocytes as well as in the cartilage and early endochondral matrices. The acid phosphatase in the chondrocytes and the matrix was tartrate-resistant, but inhibited by 2 mM sodium fluoride, whereas for osteoclasts 50-100 mM sodium fluoride were required for inhibition. Simultaneous localisation of both acid and alkaline phosphatase activities was possible in tissue that had been fixed in 85% ethanol and processed immediately. In the growth plates of the secondary ossification centre and the physis, there was a sequential localisation of the two phosphatases associated with chondrocyte maturation. The matrix surrounding immature epiphyseal chondrocytes or resting/proliferating growth plate chondrocytes contained weak acid phosphatase activity. Maturing chondrocytes were positive for alkaline phosphatase which spread to the matrix in the pre-mineralizing zone, in a pattern that was consistent with the known location of matrix vesicles. The region of strong alkaline phosphatase activity was the precise region where acid phosphatase activity was reduced. With the onset of cartilage calcification, alkaline phosphatase activity disappeared, but strong acid phosphatase activity was found in close association with the early mineral deposition. Acid phosphatase activity was also present in the matrix of the endochondral bone, but was only found in early spicules which had recently mineralised. The results suggest that alkaline phosphatase activity is required in preparation of mineralization, whereas acid phosphatase activity might have a contributory role during the early progression of mineral formation.
Asunto(s)
Fosfatasa Ácida/metabolismo , Fosfatasa Alcalina/metabolismo , Matriz Ósea/enzimología , Huesos/fisiología , Calcificación Fisiológica , Cartílago/fisiología , Animales , Desarrollo Óseo , Huesos/enzimología , Cartílago/enzimología , Cartílago/crecimiento & desarrollo , Condrocitos/citología , Condrocitos/efectos de los fármacos , Condrocitos/enzimología , Fijadores/farmacología , Formaldehído/farmacología , Placa de Crecimiento/efectos de los fármacos , Placa de Crecimiento/enzimología , Isoenzimas/metabolismo , Lisina/farmacología , Ácido Peryódico/farmacología , Polímeros/farmacología , Conejos , Fosfatasa Ácida Tartratorresistente , Tartratos/farmacología , Fijación del TejidoRESUMEN
The epiphyses of femurs from 7.5-15 day chicken embryos were studied by electron microscopy. Several forms of aberrant cell cycles were present: (1) in the perichondrium, polyploid metaphases, segmentating large (giant) cells, and mitotic catastrophe (midway between mitosis and apoptosis) were observed; (2) in the resting zone, premature chromosome condensation was found; (3) in the proliferative zone, approximately 5% of divisions were aberrant, representing most often mitosis restitution from metaphase and more seldom from the anaphase; (4) in all layers, 'dark chondrocytes' representing a premortal form of hypersecretory cells undergoing often a-mitotic nuclear segmentation were present. Many of the aberrations of cell cycle were combined with cell death. These deviations omitting or adapting the cell cycle check-points represent evidently the normal epigenetic mechanisms of development and repair. At the same time, by origin and appearances they seem very close to the loss of the growth control displayed by malignant tumours. This connection is briefly analysed in view of some current concepts of carcinogenesis.
Asunto(s)
Ciclo Celular , Muerte Celular , Placa de Crecimiento/embriología , Animales , Apoptosis/fisiología , División Celular , Embrión de Pollo , Fémur/citología , Fémur/embriología , Fémur/ultraestructura , Placa de Crecimiento/citología , Placa de Crecimiento/ultraestructura , Microscopía ElectrónicaRESUMEN
Chondrocytes of the growth plate are generally assumed to undergo apoptosis, but the mechanisms which induce this cell death are not known. The Fas receptor is a mediator of the apoptotic signal in some systems. We studied its expression in situ in growth plates of rabbits aged from five to 20 weeks. In addition, we investigated the immunolocalisation in the growth plates of the bone proteins, osteonectin and osteocalcin, and the changes in their expression with age. The Fas-positive chondrocytes were found mostly in the hypertrophic zone, as were the osteonectin-positive and osteocalcin-positive cells. The percentage of Fas-positive cells increased with age whereas little change was found in the number of osteonectin-positive and osteocalcin-positive chondrocytes. Many of the Fas-positive chondrocytes were also TUNEL-positive. This strongly suggests that apoptosis in the growth plate is mediated through the Fas system. Double immunostaining for osteocalcin and Fas showed that not all hypertrophic chondrocytes were of the same cell type. Some chondrocytes stained for osteocalcin only, others for Fas only, while some were positive for both.
Asunto(s)
Envejecimiento/metabolismo , Placa de Crecimiento/metabolismo , Osteocalcina/metabolismo , Osteonectina/metabolismo , Receptor fas/metabolismo , Animales , Condrocitos/metabolismo , Inmunohistoquímica , ConejosRESUMEN
Many events occur concurrently during the initiation of the secondary ossification center in the cartilaginous epiphyses of long bones. We have investigated the chronology of interactions between the vascular system and epiphyseal chondrocytes by culturing explanted heads of femurs and humeri from pre- and neonatal rabbits on the chorioallantoic membrane (CAM) of growing chick embryos. We confirmed that, on the whole, the epiphyseal cartilage was resistant to vascular invasion, whereas the physeal growth plate was resorbed. However, new CAM-derived cartilage canals occasionally penetrated through the articular surface. This caused death of those chondrocytes in the immediate vicinity of the canal but no further reaction. If explants already contained a bony epiphysis and were halved prior to culture, CAM-derived vessels were attracted to the spongiosa. From there they pushed into the uncalcified cartilage, indicating that calcification was not a prerequisite for vascular invasion. Where at least two vessels were in apposition, a new pseudo-ossification center was initiated: chondrocytes became hypertrophic and the matrix calcified. This suggests that cumulative release of diffusible factors from more than one vessel was the trigger for chondrocyte hypertrophy, which, in turn, led to the initiation of the bony epiphysis. CAM cultures thus provide an experimental model for both the quiescent angiogenesis of cartilage canal formation and the reactionary angiogenesis associated with chondrocyte hypertrophy. By exploiting the different anatomy of CAM-derived vascularity, events that occur concurrently in vivo can be specially separated in CAM culture.
Asunto(s)
Condrocitos/fisiología , Epífisis/embriología , Cabeza Femoral/embriología , Húmero/embriología , Neovascularización Fisiológica/fisiología , Alantoína , Animales , Apoptosis/fisiología , Cartílago Articular/irrigación sanguínea , Cartílago Articular/embriología , Células Cultivadas , Vellosidades Coriónicas/irrigación sanguínea , Epífisis/ultraestructura , Cabeza Femoral/irrigación sanguínea , Húmero/irrigación sanguínea , ConejosRESUMEN
Growth plate chondrocytes of embryonic chick femurs were examined by electron microscopy, cytophotometry and autoradiography. Apart from the well-described 'light' chondrocyte, a different 'dark' type of chondrocyte was present, comprising 10 - 35% of the cell population. They were found at all stages of chondrocyte differentiation and in all ages of the femurs studied. Well developed rough endoplasmatic reticulum and Golgi complex, many secretory vesicles, energetically active mitochondria and a lot of glycogen, indicating high activity of the cytoplasm, were combined with low RNA synthesis, gentle margination and scattered compaction of the chromatin. DNA cytometry revealed that most of dark cells were diploid, but 15 - 30% were tetraploid, with the absence of an S-phase. Substantial loss of DNA was found in about 10% of dark chondrocytes. The TUNEL reaction demonstrated a limited number of DNA strand breaks. Advanced dark cells possessed the nuclear features of both apoptosis and necrosis. Besides chromomeric-chromonemic compaction, a chromatin arrangement similar to that of prometaphase and metaphase, as well as amitotic nuclear segregation, all of them degenerative, were found. Our interpretation is that the dark chondrocytes undergo an aberrant type of cell death which may be combined with aberrant cell cycle. Cell death of dark chondrocytes is preceded by a pre-mortal burst of secretion.