Medullary Bone Osteoclasts Research Articles

Osteoclastogenesis from bone marrow cells during estrogen-induced medullary bone formation in Japanese quails.

Osteoclasts are differentiated from hematopoietic mononuclear cells by regulation of the receptor activator of nuclear factor kappa-B (RANK)/receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) system. Medullary bone (MB) that forms in the bone marrow of female birds is remodeled under the control of circulating estrogen (E2) during the laying period. Although the osteoclasts of MB are differentiated from mononuclear cells, the mechanism of osteoclastogenesis is not known. We investigated whether MB osteoclastogenesis is regulated by the RANK/RANKL/OPG system using MB from male quails induced with E2. Bone marrow cells (BMCs) differentiate into osteoclasts that have the ability of bone resorption via stimulation of RANKL/M-CSF, but this ability is suppressed by OPG and differentiation is inhibited by calcinurin inhibitors. We found that BMCs at 3days after E2 administration had high bone osteoclastogenesis ability and colony forming unit-granulocyte/macrophage (CFU-GM)/colony forming unit-macrophage (CFU-M) formation abilities. We conclude that MB osteoclasts are differentiated from BMCs by the RANK/RANKL/OPG system, and that precursor cells of osteoclasts are increased during MB formation.

Concepts and methods for understanding bone metabolism in laying hens

Laying hens have a unique bone turnover due to the daily egg laying cycle. Laying hens have three distinctive kinds of bones related to egg formation: cortical, cancellous, and medullary bones. Cortical bone is a compact structural bone, whereas cancellous bone is the three-dimensional lattice-like honeycomb architecture at the end of long bones. Medullary bone is a highly labile woven bone lying in the marrow cavities. Medullary bone acts as Ca storage for egg shell formation. Thus, bone quality is closely related with egg production and eggshell quality. During the daily egg laying cycle, medullary bone osteoclasts alternately cease and accelerate bone resorption. Although osteoclast numbers are not changed during the daily egg laying cycle, considerable morphological changes in osteoclasts occur along with changes in calcium requirements for egg shell formation. Furthermore, the selection of proper methods is critical to obtain precise bone evaluation data, and include bone ashing, densitometric techniques, mechanical testing, or histomorphometry to evaluate bone status in laying hens. Since bone metabolism in laying hens is related to economic and animal welfare issues, better understanding of bone metabolism in laying hens would be important to enhance productivity and improve animal welfare.

培養ニワトリ骨髄骨における破骨細胞のアクチンフィラメントに対する副甲状腺ホルモンおよびカルシトニンの影響

Medullary bones dissected from hen femurs at 3 hours (when osteoclasts cease bone resorption ) and 12 hours (when osteoclasts actively resorb bone) after oviposition were cultured in BGJb media containing synthetic human parathyroid hormone (PTH) and synthetic eel calcitonin (CT), respectively, and the effects of PTH and CT on the distributional patterns of actin filaments in the medullary bone osteoclasts were observed. Three hours after oviposition, the osteoclasts displayed strong fluorescence of actin filaments in the form of amorphous bands in the apical region of the cytoplasm, corresponding to clear zones. There was no change in the distributional patterns of actin filaments in osteoclasts after culturing without PTH for 24 hours. However, after culturing with PTH for 24 hours, central parts of the actin bands in the osteoclasts became obscure, and the osteoclasts displayed actin filaments as linear forms perpendicular to the bone surface. On the other hand, 12 hours after oviposition, actin filaments appeared as lines perpendicular to the bone surface in the apical region of the cytoplasm, where ruffled borders had developed. There was no change in the distributional patterns of actin filaments in osteoclasts after culturing without CT for 24 hours. However, after culturing with PTH for 24 hours, the linear actin filaments disappeared and the osteoclasts displayed amorphous bands of actin filaments in the apical region of the cytoplasm. These results suggested that PTH and CT would regulate osteoclastic bone resorption by modifying the organization of actin filaments in osteoclasts

Estimation of intracellular calcium activity in confluent monolayers of primary cultures of quail medullary bone osteoclasts.

The present study was concerned with an investigation of the relative utilities of the fluorescent indicators fura-2 and fluo-3 for spectrofluorimetric estimation of the intracellular calcium concentration ([Ca2+]i) in confluent osteoclast monolayers that had been isolated from medullary bone of quail hens and maintained in tissue culture for 6-8 days. Additionally, we have determined the effects of raised extracellular calcium ([Ca2+]o) and chicken calcitonin (CT) on [Ca2+]i in this preparation. Relative to fura-2, fluo-3 was poorly incorporated into the osteoclasts and had a high apparent equilibrium binding constant (Kd) for Ca2+ binding (809 nM). The osteoclasts were only weakly sensitive to the calcium ionophore, ionomycin. It is concluded that fura-2 is of greater utility than furo-3 in this preparation. In contrast to its lack of effect in freshly isolated cells, elevated [Ca2+]o up to 20 mM stimulated a concentration-dependent increase in [Ca2+]i in cultured osteoclasts, but CT was without effect. These findings further support the idea that quail osteoclasts are able to acquire a Ca2+ sensor or 'receptor' and thus to respond to [Ca2+]o in a similar manner to mammalian osteoclasts when they are removed from the bone microenvironment, but retain a refractoriness to CT under these conditions.

The kinetics of actin filaments in osteoclasts on chicken medullary bone during the egg-laying cycle

Distributions of actin filaments in osteoclasts on chicken medullary bone were examined during the egg-laying cycle. When an egg was in the magnum or isthmus of the oviduct at 6 h after oviposition, the osteoclasts lacked ruffled borders and attached to medullary bone surface via clear zones. Actin filaments were localized as amorphous bands in the bone site of the cytoplasm, corresponding to clear zones in the osteoclasts. When an egg was in the shell gland of the oviduct at 15 h after oviposition, the osteoclasts had long and slender, well-developed ruffled borders. Actin filaments were localized as lines perpendicular to the bone surface at the bone site of the cytoplasm. In medullary bone osteoclasts, actin filaments changed during the egg-laying cycle. The relationship between the actin filaments and the ultrastructure of the osteoclasts are discussed in this report.

Efeect of Parathyroid Hormone on Osteoclasts in Organ-cultured Medullary Bone.

The enzyme activity and ultrastructure of osteoclasts in medullary bone cultured in the presence of parathyroid hormone (PTH) were examined using laying hens during the formative phase of the bone when an egg was in the magnum of the oviduct. Before and after the culturing in the absence of PTH, osteoclasts on the surface of medullary bone showed moderate activity for both acid phosphatase (ACP) and succinate dehydrogenase (SDH), and strong lactate dehydrogenase (LHD) activity. These cells lacked ruffled borders adjacent to the bone matrix. On the contrary, in the presence of PTH, ACP and SDH activity in osteoclasts increased after 3 hours of culture. Ruffled bordes also apperared at the peripheral cytoplasm apposed to the bone. After 12 hours of culture, the area of the ruffled borders further increased. After 24 hours of culture, almost all the osteoclasts had well developed ruffled borders. These results suggest that PTH stimulates osteoclastic resorption in the medullary bone of laying hens during the formative phase of the bone.

Effect of raised extracellular calcium on cell spread area in quail medullary bone osteoclasts

The present study reports on the effects of extracellular calcium ([Ca2+]o) elevation and ionomycin on cell spread area of medullary bone osteoclasts freshly isolated from egg-laying Japanese quail. The responses were compared with those demonstrated in osteoclasts cultured for periods of 5-8 days and also to those previously demonstrated in neonatal rat osteoclasts. Freshly isolated medullary bone osteoclasts, unlike rat osteoclasts, were refractory to 20 mM [Ca2+]o, in that they showed no change in cell spread area. They did, however, show a modest (15%) reduction in cell spread area to ionomycin (7-50 microM), applied for 15-30 min. When medullary bone osteoclasts were precultured for 5-8 days, they exhibited a well-developed response to 20 mM [Ca2+]o with a 46% reduction in cell spread area. They also showed a similar reduction in cell spread area in response to ionomycin (4 microM). It is concluded that, unlike freshly isolated neonatal rat osteoclasts, those obtained from quail medullary bone appear refractory to inhibitory factors such as [Ca2+]o. However, when the avian cells are cultured for a few days they appear to recover their ability to respond to [Ca2+]o.

Medullary Bone and Avian Calcium Regulation

ABSTRACTMedullary bone forms in egg-laying birds in response to gonadal steroids and is the most overtly oestrogen-dependent of all bone types. It acts as a labile reservoir for the supply of eggshell calcium. Previous studies indicate that feeding calcium- and vitamin-D-deficient diets to chickens results in resorption of cortical rather than medullary bone. More recent studies in calcium-stressed quail hens question this hypothesis and suggest that during the first 2 weeks of dietary calcium depletion the medullary bone is resorbed while cortical bone volume remains intact. The role of the osteoclast in bone resorption is the focus of much research that has recently included studies of medullary bone osteoclasts. The functional morphology of the avian cells, i.e. changes from quiescent to active osteoclasts with ruffled borders, reflects the rapid changes in medullary bone turnover that occur during the egg-laying cycle. Unlike mammalian osteoclasts, those from avian sources generally appear refractory to inhibitory factors such as calcitonin or raised extracellular calciumconcentration. However, medullary bone osteoclasts cultured in vitro for several days recover their ability to respond to the latter factor by increasing their levels of free cytosolic Ca2+, reducing tartrate-resistant acid phosphatase secretion and reducing their cell spread area. It is suggested that factors such as ambient calcium levels and prostaglandins may form part of a system of rapid local control for medullary bone osteoclast activity.

Inhibition of Osteoclastic Bone Resorption by Calcitonin in the Cultured Medullary Bone of Laying Hens.

The enzyme activity and ultrastructure of osteoclasts in medullary bone cultured in the presence of calcitonin were examined using laying hens during the bone resorptive phase when the egg is in the shell gland of the oviduct. Before and after culturing in the absence of calcitonin, osteoclasts on the surface of medullary bone matrix showed strong acid phosphatase (ACP) and succinate dehydrogenase (SDH) activity and moderate lactate dehydrogenase (LDH) activity. These cells had a well-developed ruffled border adjacent to the bone matrix. In the presence of calcitonin, ACP, SDH, and LDH activity of osteoclasts decreased after 12 hours of culturing. The ruffled border disappeared within 1 hour. The clear zone developed near the bone matrix after 12 hours. These results suggest that calcitonin directly inhibits osteoclastic bone resorption in the medullary bone of laying hens during the bone resorptive phase.

A histochemical study of acid phosphatases in medullary bone matrix and osteoclasts in laying Japanese quail.

Acid phosphatase activity in medullary bone matrix and osteoclasts of laying Japanese quail was examined histochemically. To avoid nonspecific staining, the reactivity of the enzyme was evaluated using both the azo dye method and the lead salt method and nonembedded thick sections and resin-embedded thin sections. The pH of the incubation medium was also varied from the acid range (pH 5.0 and 6.5) to the alkaline range (pH 8.5). Medullary bone osteoclasts contain both tartrate-resistant acid phosphatase (TRAP) activity and fluoride-resistant acid phosphatase (FRAP) activity, and no significant difference in intensity was detected between active and inactive osteoclasts. The entire matrix of medullary bone was positive for tartrate-resistant, fluoride-sensitive acid phosphatase activity. No reaction product was observed in sections incubated in substrate-free and pH 8.5 media. The results demonstrate the existence of FRAP in medullary bone osteoclasts and suggest that medullary bone matrix includes TRAP throughout the matrix.

The rapid appearance of acid phosphatase activity at the developing ruffled border of parathyroid hormone activated medullary bone osteoclasts.

The localization of acid phosphatase (ACPase) activity in and near parathyroid hormone (PTH) activated osteoclasts was investigated using electron microscopic cytochemical methods. At 3 hours after oviposition in Japanese Quail hens, medullary bone osteoclasts were highly reactive for ACPase but lacked ruffled borders. There was no evidence of extracellular ACPase activity associated with these osteoclasts. At 20 minutes after PTH administration, osteoclasts had developing ruffled borders and ACPase activity was found in the matrix and extracellular space adjacent to most of these ruffled borders. ACPase activity was seldom observed beyond the resorption zone delineated by the osteoclast clear zones. These results provide direct cytochemical evidence that the ruffled border functions in the release and/or activation of ACPase. In addition, these results show that ACPase localization is rapidly responsive to exogenous PTH.

Activation of avian medullary bone osteoclasts by oxidized synthetic parathyroid hormone (1-34).

Synthetic bovine parathyroid hormone (1-34) [bPTH(1-34)] has been treated with hydrogen peroxide and assayed for the effect of such treatment on the ability of bPTH(1-34) to activate medullary bone osteoclasts during their quiescent period in the early phase of the ovulatory cycle in Japanese quail. In addition, the same batches of oxidized and unoxidized bPTH(1-34) were assayed for their hypercalcemic activity in Japanese quail and their capacity to stimulate renal adenylate cyclase activity in the same species. Three groups, each consisting of five 5-month-old egg-laying Japanese quail (Coturnix coturnix japonica), were used. Between 4 to 5 hr after oviposition the three groups were injected intraperitoneally with acid saline (control) solution, bPTH(1-34) at 40 micrograms/bird, or oxidized bPTH(1-34) at 40 micrograms/bird, respectively. Twenty minutes after injection, the femoral bones were removed, split, fixed, and appropriately processed for examination by electron microscopy. Both oxidized and unoxidized bPTH(1-34) stimulated the development of osteoclast ruffled borders within 20 min after injection of the hormone preparations. As anticipated from previously published work from this laboratory, oxidized bPTH(1-34) retained its hypercalcemic activity and lost its capacity to stimulate renal adenylate cyclase activity in the Japanese quail. These results support, but do not prove, the contention that bPTH(1-34) exhibits its responses in the Japanese quail through the mediation of more than one type of receptor.

Morphological and ultrastructural aspects of the activation of avian medullary bone osteoclasts by parathyroid hormone.

The activation of physiologically inactive medullary bone osteoclasts by parathyroid hormone (PTH) was examined using light and electron microscopy and histomorphometric methods. Medullary bone osteoclasts are functionally inactive during the avian egg-laying cycle when an egg shell is not being calcified in the shell gland. Japanese quail hens were given 0.5 IU/g PTH and the medullary bone osteoclasts were examined up to 90 min later. Administration of PTH results in rapid changes in osteoclast morphology and ultrastructure. Within 10 min ectoplasmic regions containing condensed-appearing material are evident in areas of the cell adjacent to bone surfaces. In tannic acid-fixed specimens, these ectoplasmic regions contain bundles of filaments extending perpendicularly from the osteoclast plasma membrane into the cytoplasm. It is in these areas that ruffled border development is initiated. Even at 10 min after PTH administration, mineral crystals are seen between the developing cell surface invaginations and folds. By 15 min after PTH administration, ruffled borders have appeared next to bone surfaces. The rapid development of ruffled borders on medullary bone osteoclasts after PTH is confirmed by electron microscope histomorphometry. By 30 min after PTH administration, ruffled borders are well developed and large endocytic vacuoles are beginning to appear in the osteoclast cytoplasm. Light microscope histomorphometric measurements indicate that osteoclasts are also increasing in size and spreading along bone surfaces with time after PTH administration. This study provides a morphologic and ultrastructural description of osteoclast activation by PTH. The results indicate that osteoclasts may effect rapid changes in bone resorption and mineral metabolism due to exogenous PTH in hens.

Osteoclast cell‐surface specializations and nuclear kinetics during egg‐laying in Japanese quail

Medullary bone deposits serve as a reservoir of labile calcium for egg-shell calcification in birds. Quantitative transmission-electron-microscope methods and light-microscope autoradiographic cell-population-kinetic analyses were used to determine changes in cell-surface specializations and population dynamics of medullary bone osteoclasts during egg-laying in Japanese quail. Prior to egg-shell formation, from 0 to about 8 hours after the previous oviposition, very few osteoclast profiles had ruffled borders. The appearance of ruffled borders coincided with the beginning of egg-shell calcification, about 9-10 hours after the previous oviposition. During egg-shell calcification, about 10-21 hours after the previous oviposition, most osteoclast profiles had ruffled borders. Ruffled borders disappeared at the completion of egg-shell calcification and commencement of egg-shell pigmentation. Thus, functional activities of medullary bone osteoclasts appear to be closely synchronized with egg-shell calcification during egg-laying. From 1 to 48 hours after a single injection of 3H-thymidine (3H-TdR), very few labeled osteoclast nuclei were seen during egg-laying. Following multiple injections of 3H-TdR, the percentage of labeled nuclei reached a peak at about 170 hours after the first injection. At this peak-labeling time, relatively few of the osteoclast profiles that had labeled nuclei had two or more; although the average number of nuclei per osteoclast profile was about 3.6. These kinetic data suggest that the medullary bone osteoclast population has a prolonged rate of turnover compared to rapid changes in cell activities associated with each 24-hour egg-laying cycle; and collectively they would suggest that rapid changes in osteoclast functions occur independently of changes in cell-population dynamics.

Rapid activation of the medullary bone osteoclast cell surface by parathyroid hormone.

Quantitative transmission electron microscope methods were used to determine the response of functionally inactive avian medullary bone osteoclasts to parathyroid hormone (PTH). Egg-lying Japanese quail were used during a period of the egg cycle when medullary bone was not being resorbed for egg shell calcification and when medullary bone osteoclasts were functionally inactive. Ruffled borders adjacent to bone surfaces were rarely, if ever, found on these cells. 20 min after the administration of PTH, over 70% of the osteoclast profiles had ruffled borders adjacent to bone surfaces. These ruffled borders were bounded by filamentous-rich "clear zones" and resembled ruffled borders found on functionally active cells. There was also a marked increase in plasma calcium levels after PTH administration. This study demonstrates that PTH stimulates the de novo generation of ruffled borders on osteoclasts in vivo and suggests that osteoclasts may be involved in the acute regulation of calcium metabolism by exogenous PTH.

Osteoclast cell-surface changes during the egg-laying cycle in Japanese quail

The medullary bone serves as a source of labile calcium mobilized during calcification of the egg shell in birds. Quantitative histological methods demonstrate that the numbers of medullary bone osteoclasts and nuclei per osteoclast remain unchanged during the egg cycle in the Japanese quail (Coturnix). Therefore, cyclic changes in bone resorption cannot be explained by modulations of osteoclasts from and into other bone cells, a mechanism previously suggested for certain species of birds. Rather, dramatic changes in osteoclast cell-surface features occur during the egg cycle, which might account for cyclic variations in resorptive activity. During egg shell calcification, osteoclasts with ruffled borders are closely apposed to bone surfaces; the cytoplasm is rich in vacuoles that contain mineral crystals and seem to derive from the ruffled border. At the completion of egg shell calcification, the ruffled borders and vacuoles move away from the bone surface, although the osteoclast remains attached to the bone along the filamentous or "clear" zone. Associated with the disappearance of the ruffled borders is the appearance of extensive interdigitated cell processes along the peripheral surface of the osteoclast away from the bone. These unusual structures, which may serve as a reservoir of membrane, largely disappear when ruffled borders and associated structures reappear. Therefore, in these hens, the osteoclasts modulate their cell surface rather than their population during the egg cycle.

Medullary bone of laying hens during calcium depletion and repletion.

Bone cell activity and the composition of the femur of laying hens were studied during 7 days of calcium depletion on a 0.13% calcium diet and 7 days of calcium repletion on a 3.2% calcium diet. Histologically, only cortical bone showed clear signs of bone resorption and osteoclastic activity during the depletion period. The number of osteoclasts in medullary bone varied little from control values throughout both calcium depletion and repletion, except for a significant increase on the first day of depletion. The major histologicalchange in medullary bone was a marked increase in the number of osteoblasts on the third, fifth and, to a lesser extent, seventh, day of depletion. The number of osteoblasts in medullary bone was positively correlated with its osteoid content and negatively correlated with its degree of calcification. Alkaline phosphatase activity of medullary bone increased with the time the hens had been on the calcium-deficient diet. Returning the hens to the 3.2% calcium ration caused, within one day, a significant increase in medullary bone calcification, a decrease of osteoblast numbers to, or below, control levels, and a drastic reduction in alkaline phosphatase activity of medullary bone. The significance of these findings in relation to the control of bone cell populations and the functions of medullary bone is discussed.