RESUMEN
The development of the synovial joint cavity between the cartilage anlagen of the long bones is thought to be mediated by differential matrix synthesis at the developing articular surfaces. In addition, many studies have shown that removal of movement-induced mechanical stimuli from developing diarthrodial joints prevents cavity formation or produces a secondary fusion of previously cavitated joints. Herein, we describe an inductive influence of mechanical strain on hyaluronan metabolism and the expression of hyaluronan-binding proteins in cultured cells isolated from the articular surface of the distal tibial condyles of 18-day chick embryos. The effect of 10 min of mechanical strain on hyaluronan release into culture media, intracellular uridine diphospho-glucose dehydrogenase activity (an enzyme required for hyaluronan saccharide precursor production), cell surface hyaluronan-binding protein expression and HA synthase mRNA expression were analysed up to 24 h later. Six hours after the application of strain, there was a significant increase in the accumulation of hyaluronan released into tissue culture media by strained fibrocartilage cells compared with controls, an effect still detectable after 24 h. Strained cells also showed increased activity for uridine diphospho-glucose dehydrogenase and expressed higher levels of the hyaluronan-binding protein CD44 at 24 h. In addition, at 24 h mRNA for HA synthase 2 was expressed in all samples whereas mRNA for HA synthase 3 was only expressed in strained cells. These results further highlight the role for movement-induced stimuli in differential extracellular matrix metabolism during joint development and also show that strain may facilitate differential HA synthase gene expression.
Asunto(s)
Cartílago Articular/citología , Cartílago Articular/metabolismo , Glicosiltransferasas , Ácido Hialurónico/metabolismo , Proteínas de la Membrana , Transferasas , Proteínas de Xenopus , Animales , Secuencia de Bases , Cartílago Articular/embriología , Células Cultivadas , Embrión de Pollo , Cartilla de ADN/genética , Matriz Extracelular/metabolismo , Expresión Génica , Glucuronosiltransferasa/genética , Receptores de Hialuranos/metabolismo , Hialuronano Sintasas , Inmunohistoquímica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estrés Mecánico , Uridina Difosfato Glucosa Deshidrogenasa/metabolismoRESUMEN
Previous studies have indicated that physiological levels of dynamic mechanical strain produce rapid increases in nitric oxide (NO) release from rat ulna explants and primary cultures of osteoblast-like cells and embryonic chick osteocytes derived from long bones. To establish the mechanism by which loading-induced NO production may be regulated, we have examined: nitric oxide synthase (NOS) isoform mRNA and protein expression, the effect of mechanical loading in vivo on NOS mRNA expression, and the effect of mechanical strain on NO production by bone cells in culture. Using Northern blot analyses, in situ hybridization, and immunocytochemistry we have established that the predominant NOS isoform expressed in rat long bone periosteal osteoblasts and in a distinct population of cortical bone osteocytes is the endothelial form of NOS (eNOS), with little or no expression of the inducible NOS or neuronal NOS isoforms. In contrast, in non-load-bearing calvariae there are no detectable levels of eNOS in osteocytes and little in osteoblasts. Consistent with these observations, ulnar explants release NO rapidly in response to loading in vitro, presumably through the activation of eNOS, whereas calvarial explants do not. The relative contribution of different bone cells to these rapid increases in strain-induced NO release was established by assessment of medium nitrite (stable NO metabolite) concentration, which showed that purified populations of osteocytes produce significantly greater quantities of NO per cell in response to mechanical strain than osteoblast-like cells derived from the same bones. Using Northern blot hybridization, we have also shown that neither a single nor five consecutive daily periods of in vivo mechanical loading produced any significant effect on different NOS isoform mRNA expression in rat ulnae. In conclusion, our results indicate that eNOS is the prevailing isoform expressed by cells of the osteoblast/osteocyte lineage and that strain produces increases in the activity of eNOS without apparently altering the levels of eNOS mRNA.
Asunto(s)
Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/biosíntesis , Osteoblastos/metabolismo , Animales , Animales Recién Nacidos , Northern Blotting , Células Cultivadas , Embrión de Pollo , Técnicas de Cultivo , Inmunohistoquímica , Hibridación in Situ , Isoenzimas/biosíntesis , Isoenzimas/metabolismo , Masculino , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo III , Osteocitos/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Cráneo/metabolismo , Estrés Mecánico , Tibia/metabolismo , Cúbito/metabolismoRESUMEN
Explants of ulnae from 5-week-old male and female rats were cleaned of marrow and soft tissue and, in the presence and absence of 10(-8) M 17 beta-estradiol (E2) or 5 alpha-dihydrotestosterone (DHT), mechanically loaded or treated with exogenous prostanoids previously shown to be produced during loading. Over an 18-h period, mechanical loading (peak strain 1300 mu epsilon, 1 Hz, 8 minutes, maximum strain rate 25,000 mu epsilon/s), prostaglandin E2 (PGE2) and prostacyclin (PGI2) (10(-6) M), each separately produced quantitatively similar increases in cell proliferation and matrix production in bones from males and females, as indicated by incorporation of [3H]thymidine into DNA and [3H]proline into collagen. E2 and DHT both increased [3H]thymidine and [3H]proline incorporations, E2 producing greater increases in females than in males. Indomethacin abrogated the effects of loading, but had no effects on those of sex hormones. Loading, or prostanoids, together with sex hormones, produced responses generally equal to or greater than the addition of the individual influences acting independently. In females there was a synergistic response in [3H]thymidine incorporation between loading and E2, which was quantitatively similar to the interaction between E2 and PGE2 or PGI2. The interaction between loading and E2 for [3H]proline incorporation was not mimicked by these prostanoids. In males the synergism in [3H]proline incorporation seen between loading and DHT was mimicked by that between PGI2 and DHT. We conclude that loading stimulates increased bone cell proliferation and matrix production in situ through a prostanoid-dependent mechanism. This response is equal in size in males and females. Estrogen and testosterone increase proliferation and matrix production through a mechanism independent of prostanoid production. The interactions between loading and hormones are reproduced in some but not all cases by E2 and prostaglandins. E2 with loading and prostaglandins has greater effects in female bones, while DHT with loading and prostaglandins has greater effects in males.
Asunto(s)
Hormonas Esteroides Gonadales/fisiología , Prostaglandinas/fisiología , Cúbito/fisiología , Animales , Antiinflamatorios no Esteroideos/farmacología , División Celular/efectos de los fármacos , Dihidrotestosterona/farmacología , Dinoprostona/farmacología , Interacciones Farmacológicas , Epoprostenol/farmacología , Estradiol/farmacología , Femenino , Técnicas In Vitro , Indometacina/farmacología , Masculino , Prolina/metabolismo , Ratas , Ratas Sprague-Dawley , Estrés Mecánico , Timidina/metabolismo , Cúbito/efectos de los fármacos , Cúbito/metabolismoRESUMEN
Mechanical loading of bone explants stimulates prostaglandin E2 (PGE2) and prostacyclin (PGI2) release and increases glucose 6-phosphate dehydrogenase (G6PD) activity. This response is blocked by indomethacin and imitated by exogenous PGs. In the experiments reported here, primary cultures of rat long bone-derived osteoblast-like cells were exposed to a dynamic strain and exogenous PGs in the culture dish. Strain (3400 mu epsilon, 600 cycles, 1 Hz) caused an immediate release of PGI2 into the culture medium but had no effect on PGE2. Strain also caused an increase in G6PD activity per cell and an increase in the smallest transcript of insulin-like growth factor II (IGF-II) (IGF-II T3) but had no effect on the expression of transforming growth factor-beta1 (TGF-beta1). Indomethacin inhibited strain-induced release of PGI2 and suppressed strain-induced stimulation of IGF-II T3 transcript. PGI2 (1 microM) increased G6PD activity and mRNA levels of all three transcripts of IGF-II but had no effect on the mRNA levels of IGF-I or TGF-beta1. PGE2 (1 microM) stimulated G6PD activity and caused a marked increase in IGF-I and the largest transcript of IGF-II (IGF-II T1) but had no effect on the IGF-II transcripts T2 and T3 or on TGF-beta1 mRNA levels. These findings show similarities in response between osteoblast-like cells strained in monolayer culture and bone cells in loaded bone explants in situ. They provide support for a role for IGF-II and PGI2 in the early strain-related response of osteoblasts in loading-related bone modeling/remodeling.
Asunto(s)
Huesos/metabolismo , Dinoprostona/metabolismo , Epoprostenol/metabolismo , Glucosafosfato Deshidrogenasa/metabolismo , Animales , Huesos/citología , Células Cultivadas , Masculino , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Soporte de PesoRESUMEN
The separate and combined effects of loading and 17 beta-estradiol (E2) or 5 alpha-dihydrotestosterone (DHT) on [3H]thymidine and [3H]proline incorporation were investigated in cultured ulna shafts from male and female rats. Ulnae were cultured and loaded to produce physiological strains in the presence or absence of 10(-8) M E2 or DHT. Loading engendered similar increases in incorporation of [3H]thymidine and [3H]proline in male and female bones. E2 engendered greater increases in incorporation in females than in males, and DHT greater increases in males than in females. In males E2 with loading produced increases in both [3H]thymidine and [3H]proline incorporation, which approximated to the arithmetic addition of the increases due to E2 and loading separately. In females E2 with loading produced increases greater than those in males, and substantially greater than the addition of the effects of E2 and loading separately. Loading with DHT in males also showed additional [3H]thymidine and [3H]proline incorporation. In females there was additional incorporation of [3H]proline, but not [3H]thymidine. The location of incorporation of [3H]thymidine and [3H] proline was consistent with their level of incorporation reflecting periosteal osteogenesis, in which case the early osteogenic effects of sex hormones are gender-specific when acting alone and in combination with loading. In males the effects of estrogen and testosterone add to, but do not enhance, the osteogenic responses to loading. In females testosterone with loading produces an additional effect on [3H]proline incorporation but no greater effect than loading alone on that of [3H]thymidine. In contrast, estrogen and loading together produce a greater effect than the sum of the two influences separately. Because premenopausal bone mass will have been achieved under the influence of loading and estrogen acting together, these findings suggest that the bone loss which follows estrogen withdrawal may result, at least in part, from reduction in the effectiveness of the loading-related stimulus on bone cell activity. This stimulus is normally responsible for maintaining bone mass and architecture.
Asunto(s)
Dihidrotestosterona/farmacología , Estradiol/farmacología , Cúbito/efectos de los fármacos , Soporte de Peso , Animales , Autorradiografía , Interacciones Farmacológicas , Femenino , Marcaje Isotópico , Masculino , Técnicas de Cultivo de Órganos , Prolina/metabolismo , Ratas , Ratas Sprague-Dawley , Timidina/metabolismo , Tritio/metabolismo , Cúbito/metabolismo , Cúbito/fisiologíaRESUMEN
The structural competence of the skeleton is maintained by an adaptive mechanism in which resident bone cells respond to load-induced strains. To investigate the possible role of the messenger molecule nitric oxide (NO) in this response, we studied NO production in well-characterized organ culture systems, rat long bone-derived osteoblast-like (LOBs) cells, and embryonic chick osteocytes (LOCYs) in monolayer culture. In superfused cancellous bone cores, loading (for 15 min) produces increases in NO2- (stable NO metabolite) release during the loading period, which paralleled those in PGI2 and PGE2. Loading of rat vertebrae and ulnae produces increases in NO2- release, and in ulnae NO synthase inhibitors diminish these responses. Transient rapid increases in NO release are stimulated by strain in both LOBs and LOCYs. Polymerase chain reaction amplification of extracted mRNA shows that rat ulnae, LOBs, and LOCYs express both the inducible and neuronal (constitutive) isoforms of NO synthase. Adaptability to mechanical strain relies on assessment of the strain environment followed by modification of bone architecture. Immediate increases in NO production induced by loading suggest the involvement of NO in strain measurement and cellular communication to establish strain distribution, as well as potentially in adaptive changes in bone cell behavior.
Asunto(s)
Adaptación Fisiológica , Remodelación Ósea/fisiología , Isoenzimas/biosíntesis , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico/fisiología , Osteoblastos/metabolismo , Estrés Mecánico , Animales , Secuencia de Bases , Células Cultivadas , Embrión de Pollo , Perros , Inducción Enzimática , Femenino , Fémur/citología , Isoenzimas/genética , Masculino , Datos de Secuencia Molecular , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/genética , Ratas , Ratas Sprague-Dawley , Columna Vertebral/citología , Cúbito/citologíaRESUMEN
Responses to mechanical strain in calvaria and limb bone organ cultures were compared by measuring cellular glucose 6-phosphate dehydrogenase (G6PD) activity in situ and prostaglandin release. Normal functional strains were recorded in the ulnae (1000 mu epsilon) and calvarium (30 mu epsilon) in vivo in 110 g rats. Organ cultures of ulnae and calvaria from similar animals were loaded to produce dynamic strains (600 cycles, 1 Hz) of 1000 mu epsilon in the ulna, and 100 or 1000 mu epsilon in calvaria. In ulnae, both PGE2 and PGI2 were released and resident osteocytes and osteoblasts showed increased G6PD activity. Neither response was seen in calvaria. However, exogenous PGI2 (10(-5)-10(-9) M) stimulated G6PD activity in osteocytes and osteoblasts in organ cultures of both calvaria and ulnae. In ulnar cells the response was linear, in calvarial cells it was biphasic with maximum activity at 10(-7) M. Osteoblasts derived from ulnae and cultured on plastic plates subjected to dynamic strain (600 cycles, 1 Hz, 4000 mu epsilon) showed increased G6PD activity. There was no such response in similarly treated calvarial-derived cells. Calvarial bone cells differ from those of the ulna in that they do not respond to physiological strains in their locality with increased prostanoid release or G6PD activity either in situ or when seeded onto dynamically strained plastic plates. Cells from both sites in organ culture show increased G6PD activity in response to exogenous PGI2, but their dose:responses differ in shape. These differences may reflect the extent to which functional loading influences bone architecture in these two sites.
Asunto(s)
Epoprostenol/farmacología , Glucosafosfato Deshidrogenasa/metabolismo , Cráneo/enzimología , Cúbito/enzimología , Animales , Células Cultivadas , Estimulación Eléctrica , Glucosafosfato Deshidrogenasa/efectos de los fármacos , Miembro Posterior , Técnicas de Cultivo de Órganos , Osteoblastos/efectos de los fármacos , Osteoblastos/enzimología , Osteocitos/efectos de los fármacos , Osteocitos/enzimología , Hueso Parietal/citología , Hueso Parietal/efectos de los fármacos , Hueso Parietal/fisiología , Ratas , Cráneo/fisiología , Estrés Mecánico , Resistencia a la Tracción/fisiología , Cúbito/fisiologíaRESUMEN
Adaptive changes in bone modeling in response to noninvasive, cyclic axial loading of the rat ulna were compared with those using 4-point bending of the tibia. Twenty cycles daily of 4-point bending for 10 days were applied to rat tibiae through loading points 23 and 11 mm apart. Control bones received nonbending loads through loading points 11 mm apart. As woven bone was produced in both situations, any strain-related response was confounded by the response to direct periosteal pressure. Four-point bending is not, therefore, an ideal mode of loading for the investigation of strain-related adaptive modeling. The ulna's adaptive response to daily axial loading over 9 days was investigated in 30 rats. Groups 1-3 were loaded for 1200 cycles: Group 1 at 10 Hz and 20 N, Group 2 at 10 Hz and 15 N, and Group 3 at 20 Hz and 15 N. Groups 4 and 5 received 12,000 cycles of 20 N and 15 N at 10 Hz. Groups 1 and 4 showed a similar amount of new bone formation. Group 5 showed the same pattern of response but in reduced amount. The responses in Groups 2 and 3 were either small or absent. Strains were measured with single-element, miniature strain gauges bonded around the circumference of dissected bones. The 20 N loading induced peak strains of 3500-4500 mustrain. The width of the periosteal new bone response was proportional to the longitudinal strain at each point around the bone's circumference. It appears that when a bone is loaded in a normal strain distribution, an osteogenic response occurs when peak physiological strains are exceeded. In this situation the amount of new bone formed at each location is proportional to the local surface strain. Cycle numbers between 1200 and 12,000, and cycle frequencies between 10 and 20 Hz have no effect on the bone's adaptive response.
Asunto(s)
Osteogénesis , Periostio/fisiología , Tibia/fisiología , Cúbito/fisiología , Animales , Fenómenos Biomecánicos , Calibración , Femenino , Presión , Ratas , Ratas Sprague-Dawley , Soporte de PesoRESUMEN
A new light microscopy classification of spermatogenesis in the bull and the boar was formulated to be used in conjunction with electron microscopy. It uses information available from all cell types and allows for very small portions of testicular epithelium at any orientation to be evaluated. The classification devised, being simple in outline, allows for the natural overlap of cell types. Furthermore, it emphasises the direct comparability of the morphological events occurring during spermatogenesis in the bull and the boar.