RESUMEN
UNLABELLED: OVX increased the percentage of AP-positive CFU-F in healing rat mandible. The increase of the number of osteoprogenitors was not significant in rat mandible-derived cultures but was in femur-derived ones. This suggests that the effect of OVX on osteoprogenitors is either smaller or develops later in mandible relative to femur. INTRODUCTION: Osteoprogenitors play an essential role in the regeneration process that leads to the successful integration of dental implants. However, it is unclear how systemic osteoporosis affects osteoprogenitors in oral bone. The present study was designed to determine the short-term effects of ovariectomy (OVX) on osteoprogenitors from the healing extraction socket in rat mandible. METHODS: Six-month-old rats were ovariectomized (n=8) and control rats were left intact (n=8). Two weeks post-OVX, the right mandibular incisor was extracted. Four weeks post-extraction, the basal mandibular bone between the 1st and 3rd molar in the healing extraction socket was used to determine the number of fibroblastic progenitors (CFU-F), alkaline phosphatase-positive fibroblastic progenitors (AP-positive CFU-F), Dex-dependent osteoprogenitors (CFU-O Dex) and Prog-dependent osteoprogenitors (CFU-O Prog) using colony assays (n=5). Osteocalcin mRNA expression was evaluated using in situ hybridization (n=3). Data were analyzed using two-way ANOVA or Student's t-test. RESULTS: OVX increased the percentage of AP-positive CFU-F in both mandible and femur. The number of CFU-O was increased only in femur. Osteocalcin mRNA expression in regenerating mandible was not statistically different between control and OVX animals. CONCLUSION: Our results suggest that the effect of OVX on osteoprogenitors is either smaller or develops later in mandible relative to femur.
Asunto(s)
Regeneración Ósea , Osteoporosis/fisiopatología , Ovariectomía , Células Madre/fisiología , Extracción Dental , Cicatrización de Heridas , Animales , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Modelos Animales de Enfermedad , Femenino , Fémur/fisiopatología , Regulación de la Expresión Génica , Incisivo , Mandíbula/metabolismo , Mandíbula/fisiopatología , Osteocalcina/biosíntesis , Osteocalcina/genética , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Alveolo Dental/metabolismo , Alveolo Dental/fisiopatologíaRESUMEN
Ovariectomy (OVX) in rats results in increased bone turnover and decreased bone volume and bone mineral density when measured in the metaphyses of long bones. We have investigated the effects of OVX on changes in the number of progenitors in cell populations derived from the metaphyseal bone of femurs of ovariectomized rats at 12 months of age, by using colony assays, bone nodule assays, and limiting dilution analysis at 1.5 and 9 months post-OVX. We have also measured histomorphometric parameters of bone formation and resorption in the corresponding tibia at the same time-points. A significant increase, as shown by bone nodule assays and limiting dilution analysis, occurs in the number of progesterone- and dexamethasone-responsive osteoprogenitors in cell populations isolated from ovariectomized rats at the 9-month post-OVX time-point. Progesterone-responsive osteoprogenitors are also increased at 1.5 months post-OVX. The number of fibroblast colony-forming units does not change. Histomorphometry has shown that OVX causes an increase in osteoblast surfaces, mineralizing surfaces, and bone formation rate at both 1.5 and 9 months post-OVX. The mineral apposition rate is increased at 1.5 months post-OVX. OVX also increases parameters of bone resorption at both time-points, the net result being a decrease in bone mineral density and cancellous bone volume at 9 months post-OVX. Thus, OVX in rats at 12 months of age is associated with an increase in the number of both progesterone- and dexamethasone-responsive osteoprogenitors 9 months post-OVX; this corresponds with increases in the histomorphometric parameters of bone formation.
Asunto(s)
Remodelación Ósea , Fémur/citología , Osteoblastos/citología , Ovariectomía , Células Madre/citología , Tibia/citología , Animales , Densidad Ósea , Células Cultivadas , Femenino , Fémur/metabolismo , Ratas , Ratas Sprague-Dawley , Tibia/metabolismo , Factores de TiempoRESUMEN
We investigated the effects of insulin (1-1,000 nM), insulin-like growth factor (IGF)-I, and IGF-II (3-100 nM each) alone or together with 10 nM dexamethasone (DEX) or 10 nM 1,25-dihydroxyvitamin D(3) (1,25[OH](2)D(3)) on proliferation and differentiation of adipocyte and osteoblast progenitors in bone cell populations derived from fetal rat calvaria. The effects on differentiation were evaluated by counting the number of bone or osteoid nodules and adipocyte colonies and the effects on proliferation, by measuring their size by image analysis. The types of cells studied were 1,25(OH)(2)D(3)- and DEX-responsive adipocyte progenitors and DEX-dependent and independent osteoprogenitors. Both IGF-I and IGF-II stimulated osteoprogenitor differentiation both alone and in the presence of DEX, while insulin stimulated osteoprogenitor differentiation only in the absence of DEX. Neither IGF-I/-II nor insulin affected proliferation of osteoprogenitors. Insulin had little effect on adipocyte differentiation by itself but strongly stimulated differentiation in the presence of either 1,25(OH)(2)D(3) or DEX, while IGF-II stimulated adipocyte differentiation in both the absence and presence of 1,25(OH)(2)D(3) or DEX. IGF-I by itself or in the presence of DEX strongly stimulated adipocyte cell differentiation but had little effect in the presence of 1,25(OH)(2)D(3). Our results demonstrate that insulin, IGF-II, and IGF-I have specific and different effects on the differentiation and proliferation of different groups of progenitor cells.
Asunto(s)
Adipocitos/efectos de los fármacos , Huesos/embriología , Hipoglucemiantes/farmacología , Factor II del Crecimiento Similar a la Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Insulina/farmacología , Osteoblastos/efectos de los fármacos , Adipocitos/citología , Animales , Huesos/citología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Osteoblastos/citología , Embarazo , Ratas , Ratas WistarRESUMEN
We investigated the effect of representative polycyclic aryl hydrocarbons (PAHs), benzo[a]pyrene (BaP), and 7,12-dimethylbenz[a]anthracene (DMBA) on osteoclast differentiation and function by using dispersed cancellous bone derived rabbit osteoclasts and the RAW264.7 cells. These cells differentiate into osteoclasts when exposed to receptor activator of NF-kappaB ligand (RANKL). The rabbit osteoclasts were exposed to 10(-6) to 10(-9)M BaP or DMBA and the tartrate-resistant acid phosphatase (TRAP)-positive cells were counted. The effect of PAHs on osteoclast differentiation in dispersed rabbit osteoclast-containing stromal cell populations was cell density dependent, suggesting that the cell density of stromal cells, osteoclast precursors, and/or mature osteoclasts are factors regulating the effect of PAHs. To investigate the direct effect of BaP on osteoclast differentiation, RAW264.7 cells were exposed to 10(-5) to 10(-6) M BaP. Treatment of RAW264.7 cells cultured with 25 ng/ml soluble RANKL and 10(-5)M BaP for 5 days decreased osteoclast differentiation, TRAP activity levels, and resorption of bone-like substrata. The inhibition was prevented by 10(-6) to 10(-7) M resveratrol, an aryl hydrocarbon receptor (AhR) antagonist, and by higher concentrations of RANKL. To investigate the ability of RANKL to reverse BaP-mediated inhibition, gene expression was determined by RT-PCR. Cytochrome P450 1B1 (CYP1B1) mRNA, one of the genes activated by BaP, was present only in the groups exposed to BaP; the levels of CYP1B1 mRNA decreased in the presence of increasing concentrations of RANKL. These results suggest that the inhibitory effects of PAHs on osteoclastogenesis are direct and likely involve interaction of the RANKL and PAH signaling pathways.
Asunto(s)
Proteínas Portadoras/metabolismo , Diferenciación Celular/efectos de los fármacos , Inhibidores de Crecimiento/farmacología , Glicoproteínas de Membrana/metabolismo , Osteoclastos/efectos de los fármacos , Compuestos Policíclicos/farmacología , Animales , Proteínas Portadoras/fisiología , Recuento de Células/métodos , Diferenciación Celular/fisiología , Línea Celular , Relación Dosis-Respuesta a Droga , Glicoproteínas de Membrana/fisiología , Ratones , Osteoclastos/citología , Osteoclastos/fisiología , Ligando RANK , Conejos , Receptor Activador del Factor Nuclear kappa-BRESUMEN
This study was initiated to determine whether partially dissected bones of rats could be refrigerated for 24 h in saline without losing viability of progenitor cells, specifically osteoprogenitors. This is directly applicable to studies involving bone tissue requiring overnight shipment, for example, studies involving space flown animals, grafting experiments, or transplantation. We evaluated cell populations isolated from the proximal femur of 6-week-old male Fisher 344 rats. Explants from the left femur were prepared and placed into culture immediately following dissection, while the right femur was cleaned, fragmented, and stored in saline at 4 degrees C for 24 h, after which explant cultures were initiated. After 11 days of explant culture, cells were collected from outgrowths, counted, and plated to initiate experiments. Plated cells were grown for either 15 or 21 days. To determine if storage affected the total number of colony forming progenitors, alkaline phosphatase positive colonies, or the number of osteoprogenitors, were counted. There was no significant difference in any of the types of colony forming units examined between cell populations derived from freshly prepared samples or those stored for 24 h, indicating that storage at 4 degrees C of bone tissue for 24 h in saline does not affect the osteogenic potential or the number of osteoprogenitors of the cell populations isolated.
Asunto(s)
Supervivencia Celular , Criopreservación , Fémur/citología , Osteoblastos/citología , Células Madre/citología , Fosfatasa Alcalina/biosíntesis , Animales , Ensayo de Unidades Formadoras de Colonias , Osteoblastos/enzimología , Ratas , Ratas Endogámicas F344 , Células Madre/enzimologíaRESUMEN
Experiments with rats flown in space or hind limb unloaded (HU) indicate that bone loss in both conditions is associated with a decrease in bone volume and osteoblast surface in cancellous and cortical bone. We hypothesize that the decrease in osteoblastic bone formation and osteoblast surface is related to a decrease in the number of osteoprogenitors and/or decreased proliferation of their progeny. We tested this hypothesis by evaluating the effect of 14 days of HU on the number of osteoprogenitors (osteoblast colony forming units; CFU-O), fibroblastic colony forming units (CFU-F), and alkaline phosphatase-positive CFU (CFU-AP) in cell populations derived from the proximal femur (unloaded) and the proximal humerus (normally loaded) in 6-week-old and 6-month-old rats. To confirm the effect of unloading on bone volume and structure, static histomorphometric parameters were measured in the proximal tibial metaphysis. Effects of HU on proliferation of osteoprogenitors were evaluated by measuring the size of CFU-O. HU did not affect the total number of progenitors (CFU-F) in young or adult rats in any of the cell populations. In femoral populations of young rats, HU decreased CFU-O by 71.0% and mean colony size was reduced by 20%. HU decreased CFU-AP by 31.3%. As expected, no changes in CFU-O or CFU-AP were seen in cell populations from the humerus. In femoral cell populations of adult rats, HU decreased CFU-O and CFU-AP by 16.6% and 36.6%, respectively. Again, no effects were seen in cell populations from the humerus. In 6-week-old rats, there was a greater decrease in bone volume, osteoblast number, and osteoblast surface in the proximal tibial metaphysis than that observed in adult rats. Both trabecular thickness and trabecular number were decreased in young rats but remained unaffected in adults. Neither osteoclast number nor surface was affected by unloading. Our results show that the HU-induced decrease in the number of osteoprogenitors observed in vitro parallels the effects of HU on bone volume and osteoblast number in young and old rats in vivo, suggesting that the two may be interdependent. HU also reduced CFU-O colony size in femoral populations indicating a diminished proliferative capacity of osteoblastic colonies.
Asunto(s)
Diferenciación Celular/fisiología , Osteoblastos/citología , Células Madre/citología , Ingravidez , Animales , Suspensión Trasera , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
It has been suggested that functional heterogeneity exists between osteoclasts from different bone sites. This could be exploited to design therapeutics that would selectively inhibit bone resorption only at compromised sites. To further investigate the existence of functional differences between osteoclasts from different bone sites we assessed whether osteoclasts isolated from intramembranous bone differ from osteoclasts isolated from endochondral bone in the extent that they utilize cysteine proteinases and matrix metalloproteinases to degrade the organic matrix of bone. The differential involvement of the two classes of proteases was assessed by analyzing dose-dependent effects of the matrix metalloproteinase inhibitor, CT-1746, and of the cathepsin inhibitor, E64, on bone resorption. Osteoclasts isolated from the scapula (intramembranous) and long bones (endochondral) of newborn New Zealand white rabbits were seeded on cortical bovine bone slices in the presence or absence of inhibitors. Resorptive activity was evaluated by measuring the number and area of resorption pits and by measuring the release of collagen degradation products in the culture medium. In the absence of inhibitors, scapular osteoclasts and long bone osteoclasts had similar activity based on these criteria. The resorptive activity of scapular osteoclasts was inhibited to a greater extent by the MMP inhibitor CT-1746 than by the cysteine proteinase inhibitor E64. Conversely, resorption by osteoclasts derived from long bones was inhibited to a greater degree by the cysteine proteinase inhibitor. These results strongly suggest that there are functional differences between dispersed osteoclasts derived from the scapula and long bones, with scapular osteoclasts utilizing matrix metalloproteinases to a greater extent than cysteine proteinases and long bone osteoclasts using cysteine proteinases to a greater extent than matrix metalloproteinases.
Asunto(s)
Resorción Ósea/enzimología , Huesos/patología , Cisteína Endopeptidasas/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Osteoclastos/enzimología , Escápula/patología , Amidas/farmacología , Animales , Animales Recién Nacidos , Huesos/anatomía & histología , Huesos/enzimología , Recuento de Células , Células Cultivadas , Inhibidores de la Metaloproteinasa de la Matriz , Osteoclastos/efectos de los fármacos , Osteoclastos/patología , Inhibidores de Proteasas/farmacología , Conejos , Escápula/enzimologíaRESUMEN
Endothelin-1 (ET-1), a peptide produced by vascular endothelial cells, has been suggested to be one of the signaling factors between vascular and osteoblastic cells during bone growth and remodeling. The osteoinductive effects of ET-1 were tested on fetal rat calvaria which have the ability to form bone nodules in culture. ET-1 (10(-10) to 10(-6) M) dose-dependently increased cell proliferation. The effect of ET-1 (10(-8) M) on proliferation was greater than that of dexamethasone (Dex; 10(-8) M). ET-1 also increased the number of bone nodules by 146% over untreated cells, which coincided with a 3.1-fold increase in alkaline phosphatase activity. Limiting dilution assays showed that ET-1 treatment increased the number of osteoprogenitors (CFU-AP and CFU-OB) beyond what would be expected by a proliferative effect alone, indicating that ET-1 also stimulated osteoblast differentiation. Osteocalcin mRNA expression was upregulated as shown by Northern blot analysis. Using cDNA microarray analysis, ET-1 treatment resulted in an expression profile that included an upregulation of 163 genes and expressed sequence tags. Simultaneous addition of ET-1 and Dex to the medium further increased the number of bone nodules and alkaline phosphatase activity over either treatment alone. Our results show that ET-1 promotes both osteoblastic proliferation and differentiation and that the effects of ET-1 and Dex on differentiation are cooperative.
Asunto(s)
Endotelina-1/farmacología , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Células Madre/citología , Células Madre/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Dexametasona/farmacología , Feto/citología , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteoblastos/enzimología , Osteocalcina/genética , Osteogénesis/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Cráneo/citología , Cráneo/efectos de los fármacos , Cráneo/enzimología , Células Madre/enzimologíaRESUMEN
A significant contribution to the bone loss associated with aging is likely to be a decline in bone formation. We have characterized and compared the number, capacity for proliferation and differentiation and the self-renewal ability of osteoprogenitors of aged (17-26-month-old) and young (1.5-month-old) female Wistar rats using limiting dilution analyses and continuous subculture experiments. Cells were obtained from outgrowths of explants of lumbar vertebrae (L1-L6) and grown in alpha-minimal essential medium (alpha-MEM), 10% FBS and 50 microg/ml ascorbic acid with or without dexamethasone (Dex; 0.3-300 nM) or progesterone (Prog; 0.01-10 microM). Growth curves for cell populations of both age groups were similar with population doubling times of 27.1 and 26.7 h for the aged and young animals, respectively. Osteoprogenitors from both age groups formed bone nodules when cultured in the presence of either Dex or Prog. Limiting dilution analysis in the presence of 10 nM Dex showed no difference between the aged and young rats in the number of colony forming units-fibroblast (CFU-F), alkaline phosphatase-positive colony forming units-fibroblast (AP+ CFU-F) or colony forming units-osteoblast (CFU-O). No differences were also found for any progenitor within the aged group. Limiting dilution analysis in the presence of 3 microM Prog showed no differences in the numbers of CFU-F, AP+ CFU-F or CFU-O between the aged and young groups or within the aged group. Continuous subculture of cells in the presence of 10 nM Dex revealed that the number of nodules per 10(4) plated cells increased in second subculture over first subculture cells in the young group but decreased in the aged group. Also, in third to fifth subculture cells, the number of nodules was lower in the aged group than in the young group. A similar pattern was observed in the presence of 3 microM Prog. Results indicate that the cell population doubling times, growth characteristics, and the number of CFU-F and osteoprogenitors in vertebral bone cell populations from aged rats and young rats are similar. This suggests that the bone loss associated with aging is not caused by a decrease in osteoprogenitor cell number. However, cell populations from the aged rats showed a reduced capacity for self-renewal in vitro, which would ultimately translate into a reduced number of osteoblasts and might be partly responsible for a decrease in bone formation in aged animals.
Asunto(s)
Envejecimiento/fisiología , Vértebras Lumbares/citología , Osteoblastos/citología , Células Madre/citología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Dexametasona/farmacología , Femenino , Glucocorticoides/farmacología , Técnicas de Dilución del Indicador , Progesterona/farmacología , Ratas , Ratas WistarRESUMEN
The purpose of this investigation was to establish whether or not dexamethasone (Dex)-dependent osteoprogenitors with sufficient proliferative capacity to form a colony of bone-forming osteoblasts could be identified in cell populations isolated from adult human bone. This question is relevant because of the ongoing controversy regarding the effects of dexamethasone on bone formation in humans, the clearly different effects of dexamethasone on osteoprogenitor differentiation in mouse vs. rat bone cell populations, and the related question of whether observations in either rat or mouse systems are applicable to human systems. To answer the question, we isolated cell populations from distal femoral cancellous bone of 8 female patients with osteoarthritis and quantitated the number of Dex-dependent osteoprogenitors in these populations by counting the number of osteoblastic colonies forming bone (bone nodules) or unmineralized bone matrix (osteoid nodules). Dex increased alkaline phosphatase (AP) content in all populations, induced bone nodule formation in 2 of the 8 populations, and induced formation of AP-positive clusters of cells with osteoblastic morphology in one. Treatment with 1,25-dihydroxyvitamin D3 increased osteocalcin (OC) production in the nodule forming populations, but not in the non-nodule-forming populations. Our results thus establish that Dex-dependent osteoprogenitors with sufficient proliferative capacity to form bone or osteoid nodules are present in cell populations derived from adult human bone. They also show that frozen primary human bone cell populations that have been characterized previously in terms of the number of Dex-dependent osteoprogenitors present can be used to further study the characteristics of such progenitors.
Asunto(s)
Huesos/citología , Dexametasona/farmacología , Osteoblastos/citología , Células Madre/fisiología , Adulto , Anciano , Fosfatasa Alcalina/metabolismo , Diferenciación Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Persona de Mediana Edad , Osteoblastos/efectos de los fármacos , Osteocalcina/biosíntesis , Osteogénesis , Células Madre/citología , Células Madre/efectos de los fármacosRESUMEN
A number of studies suggest that progestagens may have beneficial effects on bone metabolism. C(21) Progestin medroxyprogesterone acetate (MPA) is one of the most commonly prescribed progestins for hormone replacement therapy and in gynecologic practice. However, it appears that MPA with significant glucocorticoid (GC) activity may decrease bone density. In this review, we argue that bone loss associated with MPA administration is caused by decreased osteoblast differentiation as a result of MPA occupying the GC receptor, since increasing GC receptor occupancy beyond that reached at normal (= optimal) GC concentrations attenuates osteoblast differentiation. We propose that progestins with no GC activity may be a better choice for progestagen therapy to achieve more beneficial effects on bone metabolism.
Asunto(s)
Densidad Ósea/efectos de los fármacos , Acetato de Medroxiprogesterona/efectos adversos , Congéneres de la Progesterona/efectos adversos , Receptores de Glucocorticoides/agonistas , Adolescente , Animales , Femenino , Glucocorticoides/efectos adversos , Terapia de Reemplazo de Hormonas/efectos adversos , Humanos , Acetato de Medroxiprogesterona/sangre , Acetato de Medroxiprogesterona/farmacocinética , Osteoporosis/inducido químicamente , Posmenopausia/fisiología , Congéneres de la Progesterona/sangre , Congéneres de la Progesterona/farmacocinética , RatasRESUMEN
Studies in vitro and in vivo have shown that glucocorticoids and sex steroids play an important role in bone physiology and pathophysiology. In this study we investigated glucocorticoid and sex steroid conversion in osteoblasts derived from lumbar vertebrae of adult male and female rats. Progesterone was converted to inactive 20alpha-OH-progesterone and the conversion at day 5 was 16-fold greater than that at day 13 in both sexes (male/ female, 2.7/1.7 and 0.16/0.10 nM/10(5)cells/24 h, respectively). The conversion of inactive androstenedione to active androgen testosterone in males and females was 1.2- and 2.4-fold greater at day 5 than at day 13, respectively (male/female, 0.40/0.70 and 0.34/0.30 nM/ 10 cells/24 h, respectively). These results suggest that osteoblasts possess 20alpha-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD and that their activities are dependent on the stage of cell differentiation. At day 5, dehydroepiandrosterone was converted to androstenedione (male/female, 0.25/0.098 nM/10(5)cells/24 h), to 7alpha-OH-dehydroepiandrosterone (male/female, 0.49/0.39 nM/10(5)cells/24 h) and to 5-androstene-3beta,17beta-diol (male/female, 0.18/0.37 nM/10(5)cells/24 h), indicating the presence of 3beta-HSD, 7alpha-hydroxylase and 17beta-HSD, respectively. Both 3beta-HSD and 7alpha-hydroxylase activities declined with cell differentiation. Hormonally inactive cortisone was converted to active cortisol (male/female, 0.34/0.29 microM/10(6)cells/6 h) while conversion of cortisol to cortisone was not detectable, suggesting the presence of oxoreductase activity of 11beta-HSD-1. These results show, for the first time, the presence of 7alpha-hydroxylase and 20alpha-HSD in osteoblasts, and provide further evidence that osteoblasts metabolize a variety of steroid hormones and can thus regulate tissue responsiveness to them.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/metabolismo , Hidroxiesteroide Deshidrogenasas/metabolismo , Osteoblastos/enzimología , Columna Vertebral/enzimología , Esteroide Hidroxilasas/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasas , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , 20-Hidroxiesteroide Deshidrogenasas/metabolismo , 20-alfa-Hidroxiesteroide Deshidrogenasa , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Femenino , Masculino , Osteoblastos/citología , Ratas , Ratas Wistar , Columna Vertebral/citologíaRESUMEN
Normal loading strains of 200-2000 (mu)epsilon to bone result in bending forces, generating mechanical stretch and pressure gradients in canaliculi that drive extracellular fluid flow, resulting in stress on the membranes of osteocytes, lining cells, and osteoblasts. Under excess loading, as well as during unloading (e.g., microgravity, bed rest), the fluid shift and resultant change in interstitial fluid flow may play a larger role in bone remodeling than mechanical stretch. The in vitro model systems used to investigate mechanical loading of bone generate either fluid shear, hydrostatic compression, biaxial stretch, uniaxial stretch, or a combination of two or more of these forces. The results of in vitro experiments suggest that fluid shear is a major factor affecting bone cell metabolism. Both the flow-loop apparatus (which produces pulsatile flow and uses fluid shear as its principal stimulus) and the uniaxial silicone plate stretching apparatus (which generates cyclic stretch) create a reproducible and consistent stimulus. Endpoints measured in flow experiments, however, are short term and usually short lived, and it is unknown whether these changes impact the function of differentiated osteoblasts. Endpoints measured in uniaxial stretch experiments are generally long-term-sustained effects of mechanical perturbation and more easily relatable to changes in osteoblastic activity. Biaxial stretch devices create both bending and compressive forces, resulting in different types of force on the cells, with the relative amount of each depending on the position of the cell in the device. Therefore, systems that incorporate pulsatile fluid flow or uniaxial stretch as the principal stimulus should be further developed and implemented in the study of the relationship between mechanical loading and bone response.
Asunto(s)
Huesos/citología , Huesos/fisiología , Osteoclastos/fisiología , Soporte de Peso/fisiologíaRESUMEN
In fetal rat calvaria (RC) cell populations, adipocyte differentiation is stimulated by both dexamethasone (Dex) and 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], whereas osteoblast differentiation is stimulated by Dex but inhibited by 1,25(OH)2D3. We examined whether the osteoblastic and adipocytic colonies were derived from a common progenitor, from committed and restricted adipocyte and osteoblast progenitors, or from both and whether the adipocyte progenitors stimulated by 1,25(OH)2D3 constitute a population of progenitors that is different from that stimulated by Dex. RC cells were isolated by sequential enzyme digestion yielding five populations designated I-V. In population I the effect of Dex on adipocyte formation was greater than that of 1,25(OH)2D3, whereas the effect of 1,25(OH)2D3 was greater than that of Dex in populations III-V. We next applied replica plating techniques to further investigate the response characteristics of individual osteoprogenitors and adipocyte progenitors by looking at the fate of duplicate colonies derived from the same progenitor under different culture conditions. RC cells were plated at 1,000-1,500 cells/100 mm culture dish and a 17-microm mesh polyester membrane overlaid onto master dishes on day 4 or day 5 and removed on day 11 or day 12. Then, replicas and master dishes were cultured separately in medium containing either Dex, 1,25(OH)2D3, or Dex plus 1,25(OH)2D3 for a further 17-21 days and then fixed and stained with both Sudan IV and the von Kossa technique. Nine hundred twenty-seven matched colonies present on both master dishes and replica membranes were screened and colonies were classified as either adipocytic, osteoblastic (bone or osteoid), or fibroblastic. Results show convincingly that most of the osteoprogenitors present in fetal RC cells are committed and restricted to the osteoblastic cell lineage (95.29%); that the 1,25(OH)2D3-responsive adipocyte progenitors are different from the Dex-responsive adipocyte progenitors, but both are restricted to form adipocytes and finally; and that a common osteoblastladipocyte progenitor is present in a low frequency (4.71% of osteoprogenitors).
Asunto(s)
Adipocitos/citología , Osteoblastos/citología , Cráneo/citología , Células Madre/citología , Adipocitos/efectos de los fármacos , Animales , Calcitriol/farmacología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Dexametasona/farmacología , Feto/citología , Osteoblastos/efectos de los fármacos , Ratas , Células Madre/clasificación , Células Madre/efectos de los fármacosRESUMEN
Diseases exhibiting excessive bone loss are often characterized by an increase in the size and number of osteoclasts in affected areas, suggesting that osteoclast size is associated with increased resorptive activity or efficiency. Because osteoclastic bone resorption depends on proton extrusion via a bafilomycin A1-sensitive vacuolar type H+ ATPase (V-ATPase), we investigated the relationship between osteoclast size and state of activity on the one hand, and proton-extruding mechanisms (bafilomycin A1-sensitive V-ATPase and amiloride-sensitive Na+/H+ exchange) on the other. In determining resorptive activities of individual osteoclasts, osteoclast-containing cell suspensions obtained from newborn rabbit long bones were cultured on apatite-collagen complex (ACC)-coated coverslips. Large osteoclasts resorbed 2.5 times more per cell than small osteoclasts, but the amount resorbed per nucleus was the same for the two categories. However, a much larger percentage of large osteoclasts was resorbing compared with small osteoclasts. To study pH regulatory mechanisms in individual large and small osteoclasts, the cells were loaded with the pH-sensitive indicator BCECF and analyzed by single-cell fluorescence. Small and large resorbing osteoclasts had significantly higher basal pH(i) than their nonresorbing counterparts. Also, small nonresorbing osteoclasts were insensitive to bafilomycin A1 addition or Na+ removal from the medium, large nonresorbing osteoclasts responded slightly, and all resorbing osteoclasts (small and large) responded strongly. Differences were also seen in the recovery from an acid load: both small and large nonresorbing osteoclasts were more sensitive to amiloride inhibition, while large resorbing cells were more sensitive to bafilomycin A1 inhibition. Small resorbing cells were inhibited equally by bafilomycin A1 and amiloride. These results clearly show that a greater proportion of large osteoclasts are active in resorption and that pH(i) regulation is associated with enhanced proton pump activity in actively resorbing osteoclasts. Thus, large and small osteoclasts differ in the proportion of cells that are resorbing, while pH regulatory mechanisms differ mainly between resorbing and nonresorbing cells.
Asunto(s)
Colágeno/farmacología , Durapatita/farmacología , Concentración de Iones de Hidrógeno/efectos de los fármacos , Macrólidos , Osteoclastos/citología , Osteoclastos/enzimología , ATPasas de Translocación de Protón Vacuolares , Fosfatasa Ácida/análisis , Ácidos/farmacología , Amilorida/farmacología , Animales , Antibacterianos/farmacología , Técnicas de Cultivo de Célula/métodos , Núcleo Celular/fisiología , Tamaño de la Célula/efectos de los fármacos , Tamaño de la Célula/fisiología , Células Cultivadas , Diuréticos/farmacología , Inhibidores Enzimáticos/farmacología , Espacio Extracelular/metabolismo , Isoenzimas/análisis , Membranas Artificiales , ATPasas de Translocación de Protón/metabolismo , Conejos , Sodio/farmacocinética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Fosfatasa Ácida TartratorresistenteRESUMEN
In the present study, we test the hypothesis that the stimulation of osteoprogenitor differentiation by the glucocorticoid dexamethasone (Dex) is mediated, at least in part, through components of the insulin-like growth factor (IGF) system. Because it has been suggested that osteoprogenitors and adipocyte progenitors originate from the same precursor cells, and that their differentiation in many systems is reciprocally regulated, the effects of Dex and IGF on adipocyte formation were also evaluated in the same cultures. In view of the presence of IGFs and their binding proteins in serum, we also evaluated to what degree the effects of IGF-1 and IGF-2 on differentiation of osteoblasts and adipocytes were affected by the serum concentration of the culture media. Bone cell populations were isolated from vertebrae of 3-month-old female Wistar rats using an explant culture technique. Osteoprogenitor differentiation was evaluated by a colony assay: Bone-forming osteoblastic colonies (bone nodules) derived from single osteoprogenitors were identified by alkaline phosphatase (ALP) staining and/or staining for mineralized matrix according to the von Kossa technique. Unmineralized nodules and osteoblastic colonies were subsequently identified by their distinctive color and morphology after methylene blue counterstaining. Differentiated adipocytes were identified by Sudan IV staining. IGF-1 and IGF-2 stimulated both osteoprogenitor and adipocyte progenitor differentiation in a dose-dependent pattern. The stimulation of osteoprogenitor differentiation by IGF was not dependent on Dex, but differentiation of adipocytes was. The stimulatory effects of IGF-1 and IGF-2 on osteoprogenitor differentiation were greater in media containing 2.5% fetal bovine serum (FBS) than in media containing 5% or 10% FBS, whereas stimulation of adipocyte formation was greater in media containing 10% FBS. Neutralizing antibody against the type 1 IGF receptor (IGF-1R) partially blocked IGF- and Dex-induced osteoprogenitor differentiation, but did not affect IGF-induced adipocyte formation. This suggests that IGF-stimulated osteoprogenitor differentiation is mediated through IGF-1R, that the stimulation of adipocyte formation by IGF is not, that the stimulatory effects of Dex on osteoprogenitor differentiation are partially mediated through IGF-1R, and that the effects on adipocyte differentiation appear to be mediated through signaling pathways other than the IGF-1R.
Asunto(s)
Adipocitos/citología , Factor II del Crecimiento Similar a la Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Vértebras Lumbares/citología , Células Madre/citología , Adipocitos/efectos de los fármacos , Factores de Edad , Animales , Anticuerpos/farmacología , Proteínas Sanguíneas/farmacología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Dexametasona/farmacología , Femenino , Glucocorticoides/farmacología , Ratas , Ratas Wistar , Receptor IGF Tipo 1/inmunología , Células Madre/efectos de los fármacosRESUMEN
Osteoclasts are multinucleated cells that resorb bone by extrusion of protons and proteolytic enzymes. They display marked heterogeneity in cell size, shape, and resorptive activity. Because high resorptive activity in vivo is associated with an increase in the average size of osteoclasts in areas of greater resorption and because of the importance of proton extrusion in resorption, we investigated whether the activity of the bafilomycin A(1)-sensitive vacuolar-type H(+)-ATPase (V-ATPase) and amiloride-sensitive Na(+)/H(+) exchanger differed between large and small osteoclasts. Osteoclasts were obtained from newborn rabbit bones, cultured on glass coverslips, and loaded with the pH-sensitive indicator 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Intracellular pH (pH(i)) was recorded in single osteoclasts by monitoring fluorescence. Large (>/=10 nuclei) and small (
Asunto(s)
Hidrógeno/metabolismo , Membranas Intracelulares/metabolismo , Macrólidos , Osteoclastos/citología , Osteoclastos/metabolismo , ATPasas de Translocación de Protón Vacuolares , Ácidos/farmacología , Amilorida/farmacología , Cloruro de Amonio/farmacología , Animales , Antibacterianos/farmacología , Calibración , Tamaño de la Célula , Células Cultivadas , Canales de Cloruro/metabolismo , Inhibidores Enzimáticos/farmacología , Espacio Extracelular/metabolismo , Concentración de Iones de Hidrógeno , Osteoclastos/efectos de los fármacos , ATPasas de Translocación de Protón/metabolismo , Conejos , Sodio/metabolismoRESUMEN
We have used in situ hybridization to evaluate the effects of 1,25 dihydroxyvitamin D3 (1,25 (OH)2 D3) on the expression of mRNA for bone-matrix proteins and to determine whether mature osteoblasts respond differently to 1,25 (OH)2 D3 than younger, newly differentiated osteoblasts. Rat calvaria cells were cultured for 7, 12, 15, and 19 days to obtain a range of nodules from very young to very mature. At each time point, some cultures were treated with 10 nM 1,25 (OH)2 D3 for 24 h prior to fixation. In control cultures, type-I collagen mRNA was detectable in osteoblastic cells in very young nodules and increased with increasing maturity of the nodules and the osteoblasts lining them. The bone sialoprotein mRNA signal was weak in young osteoblasts, increased in older osteoblasts, and decreased in mature osteoblasts. Weak osteocalcin and osteopontin signals were seen only in osteoblasts of intermediate and mature nodules. 1,25 (OH)2 D3 treatment markedly upregulated osteocalcin and osteopontin mRNAs and downregulated mRNA levels of bone sialoprotein and, to a lesser extent, type-I collagen in both young and mature osteoblasts. However, a marked diversity of signal levels for bone sialoprotein, osteocalcin, and osteopontin existed between neighboring mature osteoblasts, particularly after 1,25 (OH)2 D3 treatment, which may therefore selectively affect mature osteoblasts, depending on their differentiation status or functional stage of activity.
Asunto(s)
Calcitriol/fisiología , Colágeno/genética , Osteoblastos/metabolismo , Osteocalcina/genética , ARN Mensajero/biosíntesis , Sialoglicoproteínas/genética , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Sialoproteína de Unión a Integrina , Osteoblastos/citología , Osteopontina , Ratas , Ratas WistarRESUMEN
We have examined the effects of aluminum (Al) on osteoprogenitor proliferation and differentiation, cell survival, and bone formation in long-term rat calvaria (RC) cell cultures. RC cells were grown in alpha minimal essential medium containing 10% fetal bovine serum, 50 microg/ml ascorbic acid, and 10 mM beta-glycerophosphate with or without Al added to final concentrations of 1 microM-1 mM. Al caused a dose-dependent increase in the number of bone nodules present at early times (day 11) but had no significant effect on nodule numbers at later times (day 17). Time course experiments showed that Al increased nodule number beginning from day 7. Alkaline phosphatase activity, assessed at four stages during the differentiation sequence of RC cell cultures (from 4 to 13 days) was stimulated by Al at all times. However, Al decreased colony formation, inhibited cell growth in late log phase, and decreased saturation density of the treated cultures. Al concentrations of 30 microM and above resulted in degeneration of the cell layer and an increasing fibrillar appearance of the matrix present in between or adjacent to nodules when cultures were maintained for more than 15 days. The presence of Al significantly decreased the viability of cells obtained from 13-17 days cultures, as determined by plating efficiency and trypan blue exclusion. We frequently observed cellular toxicity (in 8 of 10 experiments) in cultures containing 300 microM Al, and by days 17-19, cells, nodules, and matrix were disintegrating in these cultures. We conclude that Al accelerates the rate of osteoprogenitor cell differentiation and the formation of bone nodules while concomitantly inhibiting nodule mineralization. However, concentrations that accelerate differentiation appear to be cytotoxic in long-term cultures.
Asunto(s)
Aluminio/toxicidad , Osteoblastos/citología , Cráneo/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Animales , Calcificación Fisiológica/efectos de los fármacos , Bovinos , Recuento de Células/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Osteoblastos/enzimología , Embarazo , Ratas , Ratas Wistar , Cráneo/citología , Cráneo/enzimologíaRESUMEN
Previous experiments have demonstrated that bone cell populations derived from explants of lumbar vertebral bone of adult female rats contain osteoprogenitors that require dexamethasone (Dex) or progesterone (Prog) to proliferate and differentiate into fully differentiated bone-forming osteoblasts. We now show that the Prog-dependent population cannot be detected in male rats after sexual maturation, but is present in prepubertal rats of both sexes and can be induced in adult male-derived populations by culturing the explants in medium containing 17beta-estradiol (10(-9)-10(-8) M). This suggested that the Prog- and Dex-dependent osteoprogenitors in adult female-derived populations were probably distinct populations and that the survival of the Prog-dependent osteoprogenitors and/or their ability to proliferate are dependent on the presence of estrogen. We then proceeded to prove this by using replica plating. When one of the paired colonies duplicated was cultured in medium containing Dex (10(-8) M) and the other in medium containing Prog (10(-5) M), 5.0% of duplicates formed bone in Prog only, 11.1% formed bone in Dex only, and 3.4% formed bone in both Prog and Dex. In all cases the size of the bone-forming colonies in Dex-treated cultures was larger than that in Prog-treated cultures, indicating that the effects of Dex on osteoprogenitor proliferation are greater than those of Prog. The results demonstrate the existence of three classes ofosteoprogenitors in adult female rat-derived bone cell populations: a class responding to Dex only, a class responding to Prog only, and a class responding to both Dex and Prog. The results also indicate that the effects of Prog are not mediated by Prog binding to the glucocorticoid receptor and imply that Prog plays an important role in maintaining bone mass through regulating the class of osteoprogenitors responsive to Prog.