RESUMEN
OBJECTIVE: Articular cartilage harbors chondrogenic progenitor cells (CPCs), a population that responds chemotactically to cell death. Because this behavior is reminiscent of macrophages, we hypothesized that CPCs have macrophage-like capabilities for scavenging cell and tissue debris through phagocytosis. DESIGN: CPCs, chondrocytes, synoviocytes, and macrophages were cultured with fluorophore-labeled chondrocyte debris for 3, 6, 12, or 24 h. Debris internalization was quantified by confocal microscopy and flow cytometry. Confocal microscopy was also used to test CPCs and chondrocytes for uptake of fluorophore-labeled fibronectin fragments (Fn-fs), a form of extracellular matrix debris. Lysosome activity and mass in CPCs and chondrocytes were measured using fluorescent probes. The relative expression of phagocytosis-related genes and proteins was evaluated by polymerase chain reaction (PCR) and immunoblotting, respectively. Pulse-chase experiments were performed to determine if the debris internalized by CPCs and chondrocytes was cleared, and if clearance was affected by a cathepsin B inhibitor. RESULTS: More macrophages, synoviocytes, and CPCs internalized cell debris than chondrocytes at all time points. While uptake remained flat in chondrocytes at â¼10%, in the other cell types it peaked at more than 60% after 12-24 h. Relative to chondrocytes, CPCs showed significantly higher rates of Fn-fs engulfment, greater lysosome activity and mass, and over-expressed phagocytosis-related genes and proteins. Pulse-chase experiments revealed time- and cathepsin B-dependent clearance of cell debris in CPCs, but not in chondrocytes. CONCLUSIONS: CPCs phagocytized cell and matrix debris much more efficiently than chondrocytes, supporting the hypothesis that they play a macrophage-like role in injured cartilage.
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Cartílago Articular/lesiones , Condrogénesis , Células Cultivadas , Condrocitos , Humanos , Células Madre Multipotentes , Fagocitos , Células MadreRESUMEN
OBJECTIVE: To date, no approved clinical intervention successfully prevents the progressive degradation of injured articular cartilage that leads to osteoarthritis (OA). Stem/progenitor cell populations within tissues of diarthrodial joint have shown their therapeutic potential in treating OA. However, this potential has not been fully realized due in part to the heterogeneity of these subpopulations. Characterization of clonal populations derived from a single cell may help identify more homogenous stem/progenitor populations within articular cartilage. Moreover, chondrogenic potential of clonal populations from different zones could be further examined to elucidate their differential roles in maintaining articular cartilage homeostasis. METHOD: We combined Fluorescence-activated cell sorting (FACS) and clonogenicity screening to identify stem/progenitor cells cloned from single cells. High-efficiency colony-forming cells (HCCs) were isolated, and evaluated for stem/progenitor cell characteristics. HCCs were also isolated from different zones of articular cartilage. Their function was compared by lineage-specific gene expression, and differentiation potential. RESULTS: A difference in colony-forming efficiency was observed in terms of colony sizes. HCCs were highly clonogenic and multipotent, and overexpressed stem/progenitor cell markers. Also, proliferation and migration associated genes were over-expressed in HCCs. HCCs showed zonal differences with deep HCCs more chondrogenic and osteogenic than superficial HCCs. CONCLUSION: Our approach is a simple yet practical way to identify homogeneous stem/progenitor cell populations with clonal origin. The discovery of progenitor cells demonstrates the intrinsic self-repairing potential of articular cartilage. Differences in differentiation potential may represent the distinct roles of superficial and deep zone stem/progenitor cells in the maintenance of articular cartilage homeostasis.
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Cartílago Articular/citología , Células Madre/citología , Animales , Cartílago Articular/metabolismo , Bovinos , Diferenciación Celular/fisiología , Proliferación Celular/genética , Separación Celular/métodos , Células Cultivadas , Quimiotaxis/genética , Quimiotaxis/fisiología , Condrocitos/citología , Condrocitos/metabolismo , Condrogénesis/genética , Condrogénesis/fisiología , Ensayo de Unidades Formadoras de Colonias , Citometría de Flujo/métodos , Expresión Génica , Células Madre Multipotentes/citología , Células Madre/metabolismoRESUMEN
OBJECTIVE: Although the majority of the adenosine triphosphate (ATP) in chondrocytes is made by glycolysis rather than by oxidative phosphorylation in mitochondria there is evidence to suggest that reactive oxygen species produced by mitochondrial electron transport (ET) help to maintain cellular redox balance in favor of glycolysis. The objective of this study was to test this hypothesis by determining if rotenone, which inhibits ET and blocks oxidant production inhibits glycolytic ATP synthesis. DESIGN: Bovine osteochondral explants were treated with rotenone, an ET inhibitor; or oligomycin an ATP synthase inhibitor; or 2-fluoro-2-deoxy-D-glucose, a glycolysis inhibiter; or peroxide, an exogenous oxidant; or mitoquinone (MitoQ), a mitochondria-targeted anti-oxidant. Cartilage extracts were assayed for ATP, nicotine adenine dinucleotide (NAD+/H), and culture medium was assayed for pyruvate and lactate after 24 h of treatment. Imaging studies were used to measure superoxide production in cartilage. RESULTS: Rotenone and 2-FG caused a significant decline in cartilage ATP (P < 0.001). In contrast, ATP levels were not affected by oligomycin. Peroxide treatment blocked rotenone effects on ATP, while treatment with MitoQ significantly suppressed ATP levels. Rotenone and 2-FG caused a significant decline in pyruvate, but not in lactate production. NADH:NAD+ ratios decreased significantly in both rotenone and 2-FG-treated explants (P < 0.05). Rotenone also significantly reduced superoxide production. CONCLUSIONS: These findings showing a link between glycolysis and ET are consistent with previous reports on the critical need for oxidants to support normal chondrocyte metabolism. They suggest a novel role for mitochondria in cartilage homeostasis that is independent of oxidative phosphorylation.
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Cartílago Articular/metabolismo , Mitocondrias/metabolismo , Adenosina Trifosfato/biosíntesis , Animales , Bovinos , Condrocitos/metabolismo , Transporte de Electrón/efectos de los fármacos , Transporte de Electrón/fisiología , Fluorodesoxiglucosa F18/farmacología , Glucólisis/efectos de los fármacos , Glucólisis/fisiología , Mitocondrias/efectos de los fármacos , Rotenona/farmacología , Superóxidos/metabolismo , Técnicas de Cultivo de Tejidos , Desacopladores/farmacologíaRESUMEN
A small-scale biomechanical disc culture system was designed to stimulate intervertebral disc (IVD) 'motion segment' in culture environment with load-controlled compression and combined load (compression+shear). After 7 days of diurnal mechanical loading, cell viability of discs stimulated with static compression load (0.25 MPa) and static combined load (compression (0.25 MPa)+shear (1.5N)) were similar (>90 per cent) to unloaded controls. Mechanically stimulated discs showed decrease in static/dynamic moduli, early stress relaxation, and loss of disc height after 7 days of diurnal loading. Histological data of discs indicated load-induced transformations that were not apparent in controls. The feasibility of studying the mechanobiology of intact IVD as a motion segment was demonstrated. Media conditioning (improve tissue stability in long-term culture) and application of biochemical gene expression assays (differential tissue response to types of mechanical stimulation) are proposed as future improvements. The study suggests that the limitations in studying mechanobiology of IVD pathology in vitro can be overcome and it is possible to understand the physiologically relevant mechanism of IVD pathology.
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Fenómenos Biomecánicos/fisiología , Disco Intervertebral , Técnicas de Cultivo de Órganos/métodos , Animales , Supervivencia Celular/fisiología , Fuerza Compresiva/fisiología , Estudios de Factibilidad , Disco Intervertebral/citología , Disco Intervertebral/fisiología , Técnicas de Cultivo de Órganos/instrumentación , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: To determine the activation of Mitogen activated protein (MAP) kinases in and around cartilage subjected to mechanical damage and to determine the effects of their inhibitors on impaction-induced chondrocyte death and cartilage degeneration. DESIGN: The phosphorylation of MAP kinases was examined with confocal microscopy and immunoblotting. The effects of MAP kinase inhibitors on impaction-induced chondrocyte death and proteoglycan (PG) loss were determined with fluorescent microscopy and 1, 9-Dimethyl-Methylene Blue (DMMB) assay. The expression of catabolic genes at mRNA levels was examined with quantitative real-time PCR. RESULTS: Early p38 activation was detected at 20 min and 1h post-impaction. At 24h, enhanced phosphorylation of p38 and extracellular signal-regulated protein kinase (ERK)1/2 was visualized in chondrocytes from in and around impact sites. The phosphorylation of p38 was increased by 3.0-fold in impact sites and 3.3-fold in adjacent cartilage. The phosphorylation of ERK-1 was increased by 5.8-fold in impact zone and 5.4-fold in adjacent cartilage; the phosphorylation of ERK-2 increased by 4.0-fold in impacted zone and 3.6-fold in adjacent cartilage. Furthermore, the blocking of p38 pathway did not inhibit impaction-induced ERK activation. The inhibition of p38 or ERK pathway significantly reduced injury-related chondrocyte death and PG losses. Quantitative Real-time PCR analysis revealed that blunt impaction significantly up-regulated matrix metalloproteinase (MMP)-13, Tumor necrosis factor (TNF)-α, and ADAMTS-5 expression. CONCLUSION: These findings implicate p38 and ERK mitogen activated protein kinases (MAPKs) in the post-injury spread of cartilage degeneration and suggest that the risk of post-traumatic osteoarthritis (PTOA) following joint trauma could be decreased by blocking their activities, which might be involved in up-regulating expressions of MMP-13, ADAMTS-5, and TNF-α.
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Cartílago Articular/lesiones , Cartílago Articular/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Cartílago Articular/enzimología , Bovinos , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Supervivencia Celular/fisiología , Condrocitos/enzimología , Condrocitos/fisiología , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Immunoblotting , Reacción en Cadena de la Polimerasa , Proteoglicanos/análisis , Estrés Mecánico , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The role of the primate retrosplenial cortex (RSC) in memory processing and spatial navigation has been well established. Recently, processing emotionally salient information has been attributed to the RSC as well. Little anatomical data, however, exist linking the RSC with known emotional processing centers within the brain. The amygdala has been implicated as a substrate for modulating memory for emotionally salient events; yet no study to date has demonstrated that this area has a direct connection in the primate brain. With modern retrograde tracer injections into the RSC and adjacent cortical areas of the monkey (Macaca fascicularis), we demonstrate that there are efferent projections from the basal nucleus of the amygdala to the RSC and area 31. These projections offer anatomical data supporting the hypothesis that the RSC might receive emotionally salient input directly from the amygdala and suggest a role for the RSC as a node within a neural system potentially capable of integrating emotional information for use in memory or other cognitive processes.
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Amígdala del Cerebelo/anatomía & histología , Corteza Cerebral/anatomía & histología , Amígdala del Cerebelo/fisiología , Animales , Peso Corporal , Mapeo Encefálico , Corteza Cerebral/fisiología , Femenino , Macaca fascicularis , Macaca mulatta , Masculino , Memoria/fisiología , Tamaño de los Órganos , Percepción Espacial/fisiologíaRESUMEN
Articular cartilage in congruous joints benefits from the moderate stresses and strains associated with normal cyclic loading. However, loading of joints with surface incongruities can lead to local stress and strain elevation at "step-off' sites where cartilage is not fully buttressed b ysurrounding matrix. Excessive stresses and strains predicted to occur at such sites may induce apoptosis, a process thought to promote cartilage degeneration and osteoarthritis (OA) through chondrocyte attrition. We hypothesized that the induction of apoptosis is mediated by oxidants, and that antioxidants can reduce elevated stress-induced chondrocyte attrition. To test this we exposed cylindrical cartilage explants from human articular cartilage to radially unconfined cyclic axial compression (3600 cycles, 1 Hz, 50% duty cycle) using two different physiologic loads (2MPa and 5 MPa). We found that 30% of chondrocytes in the superficial zone died within 24 hours of exposure to loading with 5 MPa axial compression, whereas mortality was limited to less than 15% with 2 MPa axial compression. Similarly, lactate accumulation in the medium was suppressed by compression with 5 MPa, but not 2 MPa. Approximately 80% of cell death induced by 5 MPa compression was blocked by pre-incubation of the explants in a variety of anti-oxidants including vitamin E, n-acetyl cysteine (NAC), and a superoxide dismutase mimetic (SOD). SOD and NAC also prevented the suppression of lactate secretion after 5 MPa compression. These observations support the hypothesis that the harmful effects of abnormal cyclic loading are mediated by oxidants and suggest that treatments to prevent OA may include methods of minimizing oxidative damage to chondrocytes.
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Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Cartílago Articular/citología , Cartílago Articular/patología , Condrocitos/patología , Humanos , Etiquetado Corte-Fin in Situ , Ácido Láctico/análisis , Osteoartritis/fisiopatología , Osteoartritis/prevención & control , Estrés Mecánico , Técnicas de Cultivo de TejidosRESUMEN
Post-traumatic osteoarthritis is the form of osteoarthritis (OA) that develops following joint injury. Although its end-stage is indistinguishable from idiopathic OA, many patients with post-traumatic OA are younger than those with idiopathic OA, and they have a well-defined precipitating insult. Clinical and experimental studies suggest that excessive acute impact energy or chronic mechanical overload cause the degeneration of the articular surface responsible for post-traumatic OA. Yet, the mechanisms by which excessive mechanical force causes OA remain unknown. For these reasons it has not been possible to develop effective methods of preventing or decreasing the risk of post-traumatic OA. We hypothesized that mechanical loading that exceeds the tolerance of the articular surface causes chondrocyte damage due to oxidative stress. Our in vitro tests of human articular cartilage samples showed that shear stress causes chondrocyte death and that anti-oxidants decrease the shear stress induced cell death. These observations suggest that specific patterns of loading are particularly damaging to articular surfaces and that improved treatments of joint injuries may include mechanical methods of minimizing shear stresses and biologic methods of minimizing oxidative damage.
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Cartílago Articular/patología , Condrocitos/patología , Articulaciones/lesiones , Mecanotransducción Celular/fisiología , Osteoartritis/etiología , Antioxidantes/farmacología , Apoptosis/fisiología , Reactores Biológicos , Cartílago Articular/efectos de los fármacos , Cartílago Articular/fisiopatología , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Condrocitos/efectos de los fármacos , Condrocitos/fisiología , Humanos , Osteoartritis/patología , Osteoartritis/fisiopatología , Estrés Oxidativo/fisiología , Estrés Mecánico , Técnicas de Cultivo de Tejidos , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
A family consisting of a mother, a father with probable pseudopseudohypoparathyroidism (PPHP), two normal daughters, and four daughters with pseudohypoparathyroidism (PHP) have been observed for more than 15 years at North Carolina Memorial Hospital (NCMH). The studies performed on family members included (1) roentgenographic examinations of the chest, skull, hands, and soft tissues; (2) serum calcium, phosphorus, and immunoreactive parathyroid hormone measurements; (3) urinary cyclic adenosine 3'5'-monophosphate determinations following parathyroid injection; and (4) HLA and blood-type determinations. We review the genetic aspects of PHP. The findings in this family suggest an autosomal dominant mode of transmission in PHP.
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Seudohipoparatiroidismo/genética , Adolescente , Adulto , Antígenos de Grupos Sanguíneos , Calcio/sangre , AMP Cíclico/orina , Femenino , Mano/diagnóstico por imagen , Humanos , Masculino , Hormona Paratiroidea/sangre , Linaje , Fósforo/sangre , Seudohipoparatiroidismo/diagnóstico , Seudoseudohipoparatiroidismo/diagnóstico , Seudoseudohipoparatiroidismo/genética , Radiografía Torácica , Cráneo/diagnóstico por imagenRESUMEN
Data on 121 primary above-knee amputations and 12 stump revisions performed on a population of nonambulatory elderly psychiatric inpatients showed that the overall morbidity and mortality rates compared favorably with those in previously published reports on other groups of patients who received the same type of amputations, despite the precarious health of the psychiatric patients. By use of spinal anesthesia; meticulous attention to the prevention, early detection, and prompt treatment of wound complications; and identification of patients at high risk, this often desperately needed operation can be performed with good results.
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Amputación Quirúrgica , Hospitalización , Trastornos Mentales/complicaciones , Adulto , Anciano , Amputación Quirúrgica/métodos , Amputación Quirúrgica/mortalidad , Anestesia Raquidea , Arteriopatías Oclusivas/complicaciones , Arteriopatías Oclusivas/cirugía , Angiopatías Diabéticas/complicaciones , Angiopatías Diabéticas/cirugía , Femenino , Humanos , Pierna/irrigación sanguínea , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/prevención & controlRESUMEN
A common question posed to rheumatologists by both patients and physicians is 'What is the role of exercise in arthritis?' Practitioners and patients want to know if regular exercise is a risk factor for the later development of osteoarthritis. This article briefly reviews the epidemiology of osteoarthritis and exercise and risk factors associated with the development of osteoarthritis in athletes. It also reviews how musculoskeletal tissues respond to loading in normal and injured states. Finally, the topic of exercise for arthritic patients is reviewed, and recommendations are given.
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Osteoartritis/etiología , Esfuerzo Físico , Adaptación Fisiológica , Animales , Cartílago Articular/lesiones , Cartílago Articular/fisiología , Terapia por Ejercicio , Humanos , Movimiento (Física) , Fenómenos Fisiológicos Musculoesqueléticos , Osteoartritis/patología , Osteoartritis/terapia , Valores de Referencia , Estrés Mecánico , Heridas y Lesiones/fisiopatologíaRESUMEN
Biochemical and biophysical studies have shown that the composition and sedimentation velocity of cartilage proteoglycans change with age, but these investigations cannot demonstrate the alterations in molecular structure responsible for these changes. Development of quantitative electron microscopic methods has made it possible to define the age-related structural changes in aggregating proteoglycans and to correlate the alterations in their structure with changes in tissue composition and morphology. Electron microscopic measurement of human and animal hyaline cartilage proteoglycans has shown that with increasing age the length of the chondroitin sulfate-rich region of aggregating proteoglycan monomers (aggrecan molecules) decreases, the variability in aggrecan length increases, the density of aggrecan keratan sulfate chains increases, the number of monomers per aggregate decreases, and the proportion of monomers that aggregate declines. Proteoglycans from the nucleus pulposus of the intervertebral disc show similar but more dramatic age-related alterations. At birth, nucleus pulposus aggrecan molecules are smaller and more variable in length than those found in articular cartilage. Within the first year of human life, the populations of aggregates and large aggrecan molecules analogous to those found in articular cartilage decline until few if any of these molecules remain in the central disc tissues of skeletally mature individuals. The mechanisms of the age-related changes in cartilage proteoglycans have not been fully explained, but measurement of proteoglycans synthesized by chondrocytes of different ages suggests that alterations in synthesis produce at least some of the age-related changes in aggrecan molecules. Degradation of aggrecan chondroitin sulfate-rich regions in the matrix probably also contributes to the structural changes seen by electron microscopy. Age-related changes in proteoglycan aggregation may be due to alterations in link protein function or inhibition of aggregation of newly synthesized aggrecan molecules by accumulation of degraded aggrecan molecules.
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Envejecimiento/fisiología , Cartílago Articular/metabolismo , Disco Intervertebral/metabolismo , Tabique Nasal/metabolismo , Proteoglicanos/metabolismo , Adolescente , Adulto , Anciano , Animales , Cartílago Articular/ultraestructura , Bovinos , Feto , Humanos , Recién Nacido , Disco Intervertebral/ultraestructura , Microscopía Electrónica , Tabique Nasal/ultraestructura , Papio , Proteoglicanos/química , Proteoglicanos/ultraestructuraRESUMEN
A leak from the stomach is the most serious complication that occurs after a gastric bariatric operation. The experience with 19 leaks that occurred after 791 gastric bariatric operations performed at North Carolina Memorial Hospital from 1975 to 1986 is described. The incidence of leaks was higher (8.3%) after a second than after a first gastric bariatric operation (2.0%). Ten leaks were life threatening and nine were not. There were no deaths. The diagnosis was made on the basis of Gastrografin swallow in seven patients, clinical findings in six, oral dye studies in three, barium study, a sinogram, and operation in one patient each. Thirteen of 16 patients were operated on within 4 hours of the diagnosis of a leak. One patient with a life-threatening leak and two with non-life-threatening leaks were managed without operations. Three patients were discharged after uncomplicated courses and readmitted, and the diagnosis of a leak was established. To minimize morbidity and mortality related to a leak, it is imperative to (1) recognize that a leak can occur after any gastric bariatric operation, (2) perform a Gastrografin swallow when a leak is suspected, and (3) operate on the patient if the Gastrografin swallow is positive or if the clinical findings suggest a leak.
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Fístula Intestinal/diagnóstico , Obesidad/terapia , Complicaciones Posoperatorias/diagnóstico , Estómago/cirugía , Adulto , Colorantes , Diatrizoato de Meglumina , Femenino , Humanos , Fístula Intestinal/cirugía , Derivación Yeyunoileal , Yeyuno/cirugía , Masculino , Persona de Mediana Edad , Peritonitis/etiología , ReoperaciónRESUMEN
Gastric bariatric operation has been advocated to ameliorate adult-onset diabetes mellitus in the morbidly obese patient. However, there are few data to support this. In this clinical study we report the cases of 23 insulin-treated, morbidly obese, adult-onset diabetes (type II) patients who underwent gastric bariatric operations for weight reduction. Preoperative insulin requirements ranged from 10 to 230 U/day (mean 74 U/day). After operation, 14 patients discontinued insulin, and seven patients decreased their insulin requirement by 72% (100 to 28 U/day). Two patients were lost to follow-up. Six patients were admitted to the Clinical Research Unit before and after operation to evaluate the effects of surgically induced weight loss on fasting blood glucose levels, hemoglobin A1C insulin resistance, and glucose tolerance. Weight loss at the time of postoperative studies ranged between 22.5 and 49.1 kg (mean 30.6 kg). Fasting blood glucose levels decreased 48% (mean 321 mg/dl before operation to 166 mg/dl after operation). Hemoglobin A1C levels improved from a mean of 11.8% to 7.9%. Insulin resistance assessed in four patients by measurement of glucose clearance during insulin and glucose infusion improved from a mean of 77 ml/min before operation to 228 ml/min after operation. Glucose tolerance was quantitated by determination of glucose disappearance during intravenous glucose tolerance test. The mean glucose disappearance improved from 0.176% to 0.385%/min following operation. This study provides clinical and laboratory evidence indicating significant improvement of insulin-treated diabetes mellitus in morbidly obese patients who have lost weight following gastric bariatric operation.
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Diabetes Mellitus Tipo 2/terapia , Diabetes Mellitus/terapia , Insulina/uso terapéutico , Obesidad , Estómago/cirugía , Adulto , Glucemia/análisis , Peso Corporal , Femenino , Prueba de Tolerancia a la Glucosa , Hemoglobina Glucada/análisis , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Aging and the degeneration of articular cartilage in osteoarthritis are distinct processes, but a strong association exists between age and the incidence and prevalence of osteoarthritis. We hypothesized that this association is due to in vivo replicative senescence, which causes age-related declines in the ability of chondrocytes to maintain articular cartilage. For this hypothesis to be tested, senescence-associated markers were measured in human articular chondrocytes from donors ranging in age from 1 to 87 years. These measures included in situ staining for senescence-associated beta-galactosidase activity, (3)H-thymidine incorporation assays for mitotic activity, and Southern blots for telomere length determinations. We found that senescence-associated beta-galactosidase activity increased with age, whereas both mitotic activity and mean telomere length declined. These findings indicate that chondrocyte replicative senescence occurs in vivo and support the hypothesis that the association between osteoarthritis and aging is due in part to replicative senescence. The data also imply that transplantation procedures performed to restore damaged articular surfaces could be limited by the inability of older chondrocytes to form new cartilage after transplantation.
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Envejecimiento/fisiología , Cartílago Articular/patología , Cartílago Articular/fisiopatología , Condrocitos/patología , Condrocitos/fisiología , Telómero/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Células Cultivadas , Niño , Preescolar , Humanos , Lactante , Persona de Mediana Edad , Mitosis , Timidina/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
To investigate possible structural changes in reassembled proteoglycan aggregates during cartilage mineralization, we examined the molecular architecture and dimensions of growth plate proteoglycan aggregates by electron microscopy. The ends of fetal bovine femurs and tibias were separated into three regions: the epiphysis; the cartilage growth plate, consisting of the proliferative zone and the unmineralized portion of the hypertrophic zone; and the calcified portion of the hypertrophic zone along with part of the metaphysis. Aggregates from all three regions had the same molecular architecture. They consisted of central hyaluronic filaments with multiple attached monomers. Monomers consisted of two segments: a peripheral thick segment, which represents primarily the chondroitin sulfate-rich region, and a thin segment attached directly to the hyaluronic acid filament. The length of aggregated monomers did not differ between the growth plate cartilage and the metaphysis, nor did the lengths of the thin and thick segments, indicating that the chondroitin sulfate-rich region of aggregated monomers is not degraded during cartilage mineralization. Between the growth plate cartilage and the metaphysis, aggregates became shorter and had fewer monomers and wider spacing between monomers. These structural alterations in proteoglycan aggregates may be one of the events that prepares the matrix for mineralization.
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Placa de Crecimiento/ultraestructura , Animales , Calcificación Fisiológica , Bovinos , Epífisis/análisis , Epífisis/ultraestructura , Feto , Placa de Crecimiento/análisis , Ácido Hialurónico/análisis , Microscopía Electrónica , Proteoglicanos/análisis , Proteoglicanos/aislamiento & purificaciónRESUMEN
Fibronectin, a ubiquitous glycoprotein of the extracellular matrix, serves as a substrate for cell attachment. Binding to fibronectin through cell-surface receptors promotes a flattened cell shape, stimulates the phosphorylation of intracellular protein, and changes the pattern of gene expression. Although fibronectin is abundant in normal articular cartilage, its effects on chondrocytes are not well understood. Proteolytic fragments of fibronectin stimulate the catabolism of matrix in articular cartilage and may promote the degeneration of cartilage in osteoarthritis; however, intact fibronectin may regulate other aspects of matrix metabolism, including matrix synthesis. To determine whether intact fibronectin affects the synthetic activity of chondrocytes, as well as to determine the responses of chondrocytes to the anabolic growth factor insulin-like growth factor-I, we compared the incorporation of [35S] by articular chondrocytes of the rat cultured in the presence and absence of commercially prepared cellular fibronectin and 0, 10, or 100 ng/ml recombinant human insulin-like growth factor-I. Monolayer and alginate suspension cultures were compared to determine whether responses differed under conditions in which fibronectin promoted a flattened cell shape (monolayer culture) and under those in which cells maintained a spherical shape (alginate culture). In alginate cultures, fibronectin alone stimulated the incorporation of [35S]. Fibronectin with 10 ng/ml insulin-like growth factor-I had additive effects in alginate culture, producing the maximum incorporation of [35S]. In monolayer cultures, fibronectin did not stimulate incorporation and blocked stimulation by 100 ng/ml insulin-like growth factor-I. The cells from the monolayer culture were much less active synthetically (at all doses of the growth factor) than those cultured in alginate. Thus, fibronectin enhanced proteoglycan synthesis and the response to insulin-like growth factor-I in alginate but inhibited the response to the growth factor in monolayers. These observations suggest intact fibronectin may contribute to the maintenance or repair of the matrix of articular cartilage by stimulating proteoglycan synthesis.
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Cartílago Articular/metabolismo , Condrocitos/metabolismo , Fibronectinas/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteoglicanos/biosíntesis , Alginatos/farmacología , Animales , Células Cultivadas , Ácido Glucurónico , Ácidos Hexurónicos , Humanos , Ratas , Ratas Sprague-DawleyRESUMEN
We studied the proximal tibial growth plates of 15-day-old mice to determine if matrix vesicle concentration varies among growth plate zones or between the pericellular and territorial matrix compartment and the interterritorial matrix compartment. Growth plates were examined by electron microscopy and divided into five zones: reserve zone (RZ), upper proliferative zone (UPZ), lower proliferative zone (LPZ), upper hypertrophic zone (UHZ), and lower hypertrophic zone (LHZ) which included the calcifying zone. We measured the diameter and volume fraction of matrix vesicles and calculated their numerical density and volume per cell and number per cell in the pericellular and territorial matrix and in the interterritorial matrix of each zone. In the pericellular and territorial matrix compartment, the matrix vesicle concentration progressively decreased from the RZ to the LHZ. Changes in matrix vesicle concentration in the interterritorial matrix followed a different pattern. Between the RZ and the UPZ, matrix vesicle numerical density declined slightly and then increased to peak values in the LPZ and UHZ, followed by a decline between the UHZ and the LHZ. These changes in matrix vesicle concentration paralleled previously reported changes in intramitochondrial calcium content, suggesting that matrix vesicle production in growth plate may be related to intracellular calcium concentration. The existence of the maximum concentration of matrix vesicles in the LPZ and UHZ longitudinal septa which do not mineralize followed by a decline in matrix vesicle concentration in the LHZ longitudinal septa which mineralize suggests that a high concentration of matrix vesicles may be needed to prepare the matrix for mineralization or to initiate mineralization and that matrix vesicles are depleted during mineralization.
Asunto(s)
Matriz Ósea/citología , Placa de Crecimiento/citología , Tibia/citología , Animales , Matriz Ósea/ultraestructura , División Celular , Técnicas de Cultivo , Placa de Crecimiento/ultraestructura , Ratones , Microscopía ElectrónicaRESUMEN
To define the contributions of changes in cell, matrix compartment, and fibrillar collagen volumes to longitudinal bone growth, we measured the differences in cell, pericellular/territorial matrix and interterritorial matrix volumes, and fibrillar collagen concentrations between the upper proliferative and lower hypertrophic zones of the proximal tibial physes of six miniature pigs. The mean numerical density of cells decreased from 110,000 cells/mm3 in the upper proliferative zone to 59,900 cells/mm3 in the lower hypertrophic zone. The mean cell volume increased nearly 5-fold (from 1,174 to 5,530 microm3), and the total matrix volume per cell increased 46% (from 8,040 to 11,760 microm3/cell) between the upper proliferative and lower hypertrophic zones. Both the pericellular/territorial matrix volume per cell and the interterritorial matrix volume per cell increased between the upper proliferative and lower hypertrophic zones; the pericellular/territorial matrix volume per cell increased 61% (from 4,580 to 7,390 microm3/cell), whereas the interterritorial matrix volume per cell increased 26% (from 3,460 to 4,370 microm3/cell). The total increase in mean cell volume of 4,356 microm3 exceeded the total increase in mean matrix volume per cell of 3,720 microm3; the total mean pericellular/territorial matrix volume per cell increased more than the total mean interterritorial matrix volume per cell (2,810 compared with 910 microm3/cell). Fibrillar collagen concentration was greater in the interterritorial matrix than in the pericellular/territorial matrix in both zones and increased in both matrix compartments between the upper proliferative and lower hypertrophic zones. The amount of fibrillar collagen per cell also increased in both matrix compartments between the upper proliferative and lower hypertrophic zones (from 1,720 to 3,100 microm3/cell in the pericellular/territorial matrix and from 1,490 to 2,230 microm3/cell in the interterritorial matrix; thus, the total amount of fibrillar collagen per cell increased from 3,210 to 5,530 microm3/cell). Growth rate was inversely related to the cell numerical density in the upper proliferative and lower hypertrophic zones and was directly related to interterritorial matrix volume per cell in the upper proliferative zone and to pericellular/territorial matrix volume per cell in the lower hypertrophic zone. These results show that cell enlargement contributes more to longitudinal bone growth than does increased matrix volume, that increased pericellular/territorial matrix volume makes a greater contribution to growth than does increased interterritorial matrix volume, and that the total amount of fibrillar collagen per cell increases between the upper proliferative and lower hypertrophic zones. The differences between the two matrix compartments in increase in volume, fibrillar collagen concentration, and amount of fibrillar collagen per cell strongly suggest that they differ not only in matrix organization but in rate of matrix accumulation and assembly and that these differences give the two compartments different roles in skeletal growth.
Asunto(s)
Colágeno/metabolismo , Matriz Extracelular/metabolismo , Placa de Crecimiento/crecimiento & desarrollo , Animales , Recuento de Células , Tamaño de la Célula , Condrocitos/citología , Condrocitos/metabolismo , Colágeno/ultraestructura , Matriz Extracelular/ultraestructura , Fluoresceínas/farmacología , Colorantes Fluorescentes/farmacología , Placa de Crecimiento/citología , Placa de Crecimiento/metabolismo , Porcinos , TibiaRESUMEN
Biochemical and biophysical studies have demonstrated that proteoglycan monomers from immature and adult articular cartilage differ in composition and size. To investigate the structural basis of age-related differences in articular cartilage proteoglycan monomers and aggregates, we isolated and purified high buoyant density proteoglycans from the articular cartilages of 2- to 3-month-old calves and 18-month-old steers. The molecular architecture and dimensions of the proteoglycans were examined using the electron microscope monolayer method. Aggregated and nonaggregated monomers from calf cartilage were longer and less variable in length than the corresponding monomers from steer articular cartilage. Calf monomer lengths had unimodal frequency distributions whereas nonaggregated steer monomer lengths had a bimodal distribution. These observations were confirmed by acrylamide-agarose electrophoresis, which demonstrated that the samples contained only one species of proteoglycan monomer in calf but two species in steer. In addition, calf aggregated monomers had longer thin segments indicating that calf and steer monomers differed in structure as well as in size. Steer proteoglycan aggregates were shorter and had fewer monomers than those from calf. These observations demonstrate the existence of significant age-related structural differences in articular cartilage proteoglycans and form the basis for future study of the mechanisms responsible for these differences.