Resorption of vital or devitalized bone by isolated osteoclasts in vitro

Summary The maintainance of resorptive capability towards vital or devitalized bone in osteoclasts isolated from the medullary bone of laying hens and cultured for five days in vitro has been investigated morphologically with the aid of light and transmission electron microscopy. Devitalized bone particles ranging in size from 50 to 100 m, added to cultures of osteoclasts, were rapidly surrounded by the osteoclasts which, in transmission electron microscopy, showed ruffled borders and clear zones at the surfaces of contact with bone — features typical of resorptive activity. Alternatively osteoclasts were added onto the endosteal surfaces of vital or devitalized diaphyses of quail femurs after removal of the endosteal and periosteal cell layers. The results indicated that, when the vital or devitalized bone surfaces were devoid of cells, the osteoclasts adhered and resorbed bone (as confirmed by transmission electron microscopy). When vital bone of quail was cultured for 24 h before the addition of osteoclasts a new cell layer was formed; it enveloped all bone surfaces and precluded the access of osteoclasts to bone. The role of these lining cells, ultrastructurally indistinguishable from resting osteoblasts, is discussed.     

Immune signals in the context of secondary osteoporosis

Bone homeostasis is maintained by a balance between bone resorption by osteoclasts and bone formation by osteoblasts, and alterations in bone metabolism can lead to diseases such as osteoporosis. Inter-cellular and intra-cellular signaling, originating from the immune system, the largest source of cell-derived regulatory signals, are involved in these processes. Immune-competent cells such as macrophages and lymphocytes deliver cell-cell signaling through soluble factors such as cytokines and through direct contact with the cells. Such immunological signals to the bone are transmitted primarily through osteoblasts or direct stimulation of osteoclasts to induce osteoclast maturation or bone resorption, which may in turn lead to the disequilibrium of bone metabolism. Inflammatory diseases such as rheumatoid arthritis are good examples of such a process, in which immunological signals play a central role in the pathogenesis of the accompanying secondary osteoporosis. We will achieve a better understanding of the pathogenesis of bone metabolism in osteoporosis through immune signaling, and thereby develop improved therapeutic strategies for these conditions.     

Osteoclasts secrete non-bone derived signals that induce bone formation

Bone turnover is a highly regulated process, where bone resorption in the normal healthy individual always is followed by bone formation in a manner referred to as coupling. Patients with osteopetrosis caused by defective acidification of the resorption lacuna have severely decreased resorption, in face of normal or even increased bone formation. This suggests that osteoclasts, not their resorptive activity, are important for sustaining bone formation. To investigate whether osteoclasts mediate control of bone formation by production of bone anabolic signals, we collected conditioned media (CM) from human osteoclasts cultured on either bone or plastic, and tested their effects on bone nodule formation by osteoblasts. Both types of CM were shown to dose-dependently induce bone nodule formation, whereas non-conditioned osteoclast culture medium had no effects. These data show that osteoclasts secrete non-bone derived factors, which induce preosteoblasts to form bone-like nodules, potentially explaining the imbalanced coupling seen in osteopetrotic patients.     

Bone remodeling: Multiple cellular interactions required for coupling of bone formation and resorption

The dynamic nature of the skeleton is achieved by a process called "remodeling" which involves the co-ordinated actions of osteoclasts, osteoblasts, osteocytes within the bone matrix and osteoblast-derived lining cells that cover the surface of bone. Remodeling commences with signals that initiate osteoclast formation followed by osteoclast-mediated bone resorption, a reversal period, and then a long period of bone matrix formation mediated by osteoblasts, followed by mineralisation of the matrix. This review will discuss each of these steps with particular emphasis on the communication pathways between each cell type involved and the roles of ephrins, sclerostin, RANKL and PTHrP.     

Role of vascular endothelial cells in bone biology

Bone development and remodeling depend on complex interactions between bone-forming osteoblasts, bone-degrading osteoclasts, and other cells present within the bone microenvironment. Balanced control of bone formative and degradative processes is normally carefully maintained in the adult skeleton but becomes uncoupled in the course of aging or in various pathological disease states. Systemic regulators of bone metabolism and local mediators, including matrix molecules, cytokines, prostaglandins, leukotrienes, and other autocrine or paracrine factors, regulate the recruitment, differentiation, and function of cells participating in bone formation and turnover. Although some of these interactions are now understood, many yet remain to be elucidated. Recent studies have begun exploring in detail how vascular endothelial cells and their products function in bone physiology. The findings are revealing that bone vascular endothelial cells may be members of a complex communication network in bone which operates between endothelial cells, osteoblasts, osteoclasts, macrophages, stromal cells, and perhaps other cell types found in bone as well. Therefore, multiple systemic and locally produced signals may be received, transduced, and integrated by individual cells and then propagated by the release from these cells of further signals targeted to other members of the bone cell network. In this manner, bone cell activities may be continuously coordinated to afford concerted actions and rapid responses to physiological changes. The bone microvasculature may play a pivotal role in these processes, both in linking circulatory and local signals with cells of the bone microenvironment and in actively contributing itself to the regulation of bone cell physiology. Thus, skeletal homeostasis and the coupling observed between bone resorption and bone formation during normal bone remodeling may be manifestations of this dynamic interactive communication network, operating via diverse signals not only between osteoblasts and osteoclasts but between many cell types residing within bone. © 1994 Wiley-Liss, Inc.     

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.     

Short-range intercellular calcium signaling in bone

The regulation of bone turnover is a complex and finely tuned process. Many factors regulate bone remodeling, including hormones, growth factors, cytokines etc. However, little is known about the signals coupling bone formation to bone resorption, and how mechanical forces are translated into biological effects in bone. Intercellular calcium waves are increases in intracellular calcium concentration in single cells, subsequently propagating to adjacent cells, and can be a possible mechanism for the coupling of bone formation to bone resorption. The aim of the present studies was to investigate whether bone cells are capable of communicating via intercellular calcium signals, and determine by which mechanisms the cells propagate the signals. First, we found that osteoblastic cells can propagate intercellular calcium transients upon mechanical stimulation, and that there are two principally different mechanisms for this propagation. One mechanism involves the secretion of a nucleotide, possibly ATP, acting in an autocrine action to purinergic P2Y2 receptors on the neighboring cells, leading to intracellular IP3 generation and subsequent release of calcium from intracellular stores. The other mechanism involves the passage of a small messenger through gap junctions to the cytoplasm of the neighboring cells, inducing depolarization of the plasma membrane with subsequent opening of membrane bound voltage-operated calcium channels. Next, we found that osteoblasts can propagate these signals to osteoclasts as well. We demonstrated that paracrine action of ATP was responsible for the wave propagation, but now the purinergic P2X7 receptor was involved. Thus, the studies demonstrate that calcium signals can be propagated not only among osteoblasts, but also between osteoblasts and osteoclasts in response to mechanical stimulation. Thus, intercellular calcium signaling can be a mechanism by which mechanical stimuli on bone are translated into biological signals in bone cells, and propagated through the network of cells in bone. Further, the observations offer new pharmacological targets for the modulation of bone turnover, and perhaps even for the treatment of bone metabolic disorders.     

Membrane trafficking in osteoblasts and osteoclasts: new avenues for understanding and treating skeletal diseases

The endocytic and exocytic/secretory pathways are two major intracellular membrane trafficking routes that regulate numerous cellular functions in a variety of cell types. Osteoblasts and osteoclasts, two major bone cells responsible for bone remodeling and homeostasis, are no exceptions. During the past few years, emerging evidence has pinpointed a critical role for endocytic and secretory pathways in osteoblast and osteoclast differentiation and function. The endosomal membrane provides a platform to integrate bone tropic signals of hormones and growth factors in osteoblasts. In osteoclasts, endocytosis, followed by transcytosis, of degraded bone matrix promotes bone resorption. Secretory pathways, especially lysosome secretion, not only participate in bone matrix deposition by osteoblasts and degradation of mineralized bone matrix by osteoclasts; they may also be involved in the coupling of bone resorption and bone formation during bone remodeling. More importantly, mutations in genes encoding regulatory factors within the endocytic and secretory pathways have been identified as causes for bone diseases. Identification of the molecular mechanisms of these genes in bone cells may provide new therapeutic targets for skeletal disorders.     

Calcium and bone disease

Calcium transport and calcium signaling are of basic importance in bone cells. Bone is the major store of calcium and a key regulatory organ for calcium homeostasis. Bone, in major part, responds to calcium-dependent signals from the parathyroids and via vitamin D metabolites, although bone retains direct response to extracellular calcium if parathyroid regulation is lost. Improved understanding of calcium transporters and calcium-regulated cellular processes has resulted from analysis of genetic defects, including several defects with low or high bone mass. Osteoblasts deposit calcium by mechanisms including phosphate and calcium transport with alkalinization to absorb acid created by mineral deposition; cartilage calcium mineralization occurs by passive diffusion and phosphate production. Calcium mobilization by osteoclasts is mediated by acid secretion. Both bone forming and bone resorbing cells use calcium signals as regulators of differentiation and activity. This has been studied in more detail in osteoclasts, where both osteoclast differentiation and motility are regulated by calcium.     

The a3 isoform of the 100-kDa V-ATPase subunit is highly but differentially expressed in large (>or=10 nuclei) and small (<or= nuclei) osteoclasts

Osteoclasts dissolve bone through acidification of an extracellular compartment by means of a multimeric vacuolar type H+-ATPase (V-ATPase). In mammals, there are four isoforms of the 100-kDa V-ATPase "a" subunit. Mutations in the a3 isoform result in deficient bone resorption and osteopetrosis, suggesting that a3 has a unique function in osteoclasts. It is thus surprising that several studies show a basal level of a3 expression in most tissues. To address this issue, we have compared a3 expression in bone with expression in other tissues. RNA blots revealed that the a3 isoform was expressed highest in bone and confirmed its expression (in decreasing order) in liver, kidney, brain, lung, spleen, and muscle. In situ hybridization on bone tissue sections revealed that the a3 isoform was highly expressed in multinucleated osteoclasts but not in mononuclear stromal cells, whereas the a1 isoform was expressed in both cell types at about the same level. We also found that a3 expression was greater in osteoclasts with 10 or more nuclei as compared with osteoclasts with five or fewer nuclei. We hypothesize that these differences in a3 expression may be associated with previously demonstrated differences between large and small osteoclasts with reference to their resorptive activity.     

Comparison of bone and parathyroid hormone as stimulators of osteoclast development and activity in calvarial cell cultures from normal and osteopetrotic (mi/mi) mice

Osteoclast development was studied in cell cultures prepared from calvaria of neonatal osteopetrotic (mi/mi) mice or their normal littermates, using tartrate-resistant acid phosphatase (TRAPase), as an osteoclast marker. In cultures from normal mice, treatment with 10 nM PTH for 4-5 days stimulated the formation of osteoclasts. However in cultures from mi/mi mice, this response was only 7% +/- 5% that of normal mice and they were significantly smaller than osteoclasts of normal mice. Mineralized bone particles elicited osteoclast development in cultures from both normal and mi/mi mice, and osteoclast size was identical for both genotypes. Seventy-eight to 96% of the TRAPase-positive cells bound 125I-CT, as demonstrated by autoradiography. 125I-CT binding characteristics were identical in cultures from both genotypes treated with bone particles, exhibiting a Kd of 3.3-3.6 x 10(-10) M. Addition of PTH stimulated 45Ca release from the added bone particles only in the case of cultures prepared from normal mice, and CT inhibited this response. Cells from normal mice were capable of excavating bone from the surface of smooth cortical bone wafers, but such excavations were rarely seen in the case of calvarial cells from mi/mi mice. Thus, PTH-driven differentiation of osteoclasts is arrested in calvarial cell cultures from mi/mi mice, but mi/mi preosteoclasts retain the ability to express certain osteoclast markers in response to bone derived signals. We hypothesize that the lack of activity of mi/mi osteoclasts is due to the failure of mi/mi preosteoclasts to respond appropriately to resorptive agents, or to cytokines elicited by these agents.     

Expression of connexin 43 mRNA in microisolated murine osteoclasts and regulation of bone resorption in vitro by gap junction inhibitors

Several studies have demonstrated that connexin 43 (Cx43) mediates signals important for osteoblast function and osteogenesis. The role of gap junctional communication in bone resorption is less clear. We have investigated the expression of Cx43 mRNA in osteoclasts and bone resorption cultures and furthermore, the functional importance of gap junctional communication in bone resorption. RT-PCR analysis demonstrated Cx43 mRNA expression in mouse bone marrow cultures and in osteoclasts microisolated from the marrow cultures. Cx43 mRNA was also expressed in bone resorption cultures with osteoclasts and osteoblasts/stromal cells incubated for 48h on devitalized bone slices. An up-regulation of Cx43 mRNA was detected in parathyroid (PTH)-stimulated (0.1 nM) bone resorption. Two inhibitors of gap junction communication, 18alpha-glycyrrhetinic acid (30 microM) and oleamide (100 microM), significantly inhibited PTH- and 1,25-(OH)(2)D(3)-stimulated osteoclastic pit formation. In conclusion, our data indicate a functional role for gap junction communication in bone resorption.     

Lactoferrin and bone; structure-activity relationships.

The maintenance of the mechanical integrity of the skeleton depends on bone remodeling, the well-coordinated balance between bone formation by osteoblasts and bone resorption by osteoclasts. The coupled action of osteoblasts and osteoclasts is regulated by the action of many local and circulating hormones and factors as well as central regulation by a neurological mechanism. We have previously shown that lactoferrin can promote bone growth. At physiological concentrations, lactoferrin potently stimulates the proliferation and differentiation of primary osteoblasts and acts as a survival factor. Lactoferrin also affects osteoclasts, potently inhibiting their formation. In vivo, local injection of lactoferrin results in substantial increases in bone formation and bone area. In a critical bone-defect model in vivo, lactoferrin was also seen to promote bone growth. The mitogenic effect of lactoferrin in osteoblast-like cells is mediated mainly through low-density lipoprotein-receptor protein-1 (LRP1), a member of the low-density lipoprotein-receptor-related proteins that are primarily known as endocytic receptors; however, LRP1 is not necessary for the anti-apoptotic actions of lactoferrin. Lactoferrin also induces the activation of p42/44 mitogen-activated protein kinase (MAPK) signalling and the PI3-kinase-dependent phosphorylation of Akt in osteoblasts. In this study, we examined other properties of lactoferrin and the way they affect osteogenic activity. The degree of glycosylation, iron-binding, and the structure-activity relationships indicate that lactoferrin maintains osteogenic activity in deglycosylated, holo, and apo forms, and in with various small fragments of the molecule. These data suggest that lactoferrin signals through more than 1 membrane-bound receptor to produce its anabolic skeletal effects, and that it signals through diverse pathways. We conclude that lactoferrin might have a physiological role in bone growth and healing and a potential therapeutic role as an anabolic factor in osteoporosis.     

Morphological and histochemical comparison of the cells elicited by ectopic bone implants and tibial osteoclasts.

Pellets of mineralized and demineralized bone and a composite mixture of mineralized and demineralized, devitalized bone particles were implanted subcutaneously on the dorsal body wall of young adult rats. Two weeks post-implantation, the pellets were removed and processed for histochemical and morphological analyses. Rat proximal tibia was also processed for evaluation. The levels of tartrate-resistant acid phosphatase (TRAP) activity in the multinucleated giant cells (MNGCs) from each of the three implants and from osteoclasts were assessed using an image analyzer. The osteoclasts from the proximal tibia and the majority of MNGCs from the demineralized implants demonstrated high levels of TRAP activity. MNGCs from the mineralized implants showed either a low level or absence of TRAP activity. Most MNGCs from the composite implants exhibited a low level of TRAP activity; however, there was a population of cells that demonstrated a high level of reaction product, similar to that seen in the tibia and demineralized implant. Morphologically, osteoclasts from the proximal tibia and from the osteogenic demineralized implant exhibited ruffled borders. A small population of MNGCs from the composite implant also revealed osteoclastic features. In summary, MNGCs from the mineralized implant did not exhibit a level of TRAP reaction product or morphology similar to osteoclasts, while the majority of cells from the demineralized implant and a subpopulation of the MNGCs elicited by the composite implant did demonstrate TRAP expression and morphology similar to osteoclasts. The expression of osteoclastic characteristics in cells at an ectopic site may be dependent on accessory signals from the skeletal microenvironment; such signals appear to be absent from or incomplete in the mineralized implants but appear to be present when demineralized bone particles are implanted.     

Osteoclasts control osteoblast chemotaxis via PDGF-BB/PDGF receptor beta signaling

Background

Bone remodeling relies on the tightly regulated interplay between bone forming osteoblasts and bone digesting osteoclasts. Several studies have now described the molecular mechanisms by which osteoblasts control osteoclastogenesis and bone degradation. It is currently unclear whether osteoclasts can influence bone rebuilding.

Methodology/Principal Findings

Using in vitro cell systems, we show here that mature osteoclasts, but not their precursors, secrete chemotactic factors recognized by both mature osteoblasts and their precursors. Several growth factors whose expression is upregulated during osteoclastogenesis were identified by DNA microarrays as candidates mediating osteoblast chemotaxis. Our subsequent functional analyses demonstrate that mature osteoclasts, whose platelet-derived growth factor bb (PDGF-bb) expression is reduced by siRNAs, exhibit a reduced capability of attracting osteoblasts. Conversely, osteoblasts whose platelet-derived growth factor receptor β (PDGFR-β) expression is reduced by siRNAs exhibit a lower capability of responding to chemotactic factors secreted by osteoclasts.

Conclusions/Significance

We conclude that, in vitro mature osteoclasts control osteoblast chemotaxis via PDGF-bb/PDGFR-β signaling. This may provide one key mechanism by which osteoclasts control bone formation in vivo.     

Regulation of apoptosis in osteoclasts and osteoblastic cells

In postnatal life, the skeleton undergoes continuous remodeling in which osteoclasts resorb aged or damaged bone, leaving space for osteoblasts to make new bone. The balance of proliferation, differentiation, and apoptosis of bone cells determines the size of osteoclast or osteoblast populations at any given time. Bone cells constantly receive signals from adjacent cells, hormones, and bone matrix that regulate their proliferation, activity, and survival. Thus, the amount of bone and its microarchitecture before and after the menopause or following therapeutic intervention with drugs, such as sex hormones, glucocorticoids, parathyroid hormone, and bisphosphonates, is determined in part by effects of these on survival of osteoclasts, osteoblasts, and osteocytes. Understanding the mechanisms and regulation of bone cell apoptosis will enhance our knowledge of bone cell function and help us to develop better therapeutics for the management of osteoporosis and other bone diseases.     

Cells of the synovium in rheumatoid arthritis. Osteoclasts

Osteoclasts are multinucleated cells of hematopoietic origin and are the primary bone resorbing cells. Numerous osteoclasts are found within the synovial tissue at sites adjacent to bone, creating resorption pits and local bone destruction. They are equipped with specific enzymes and a proton pump that enable them to degrade bone matrix and solubilize calcium, respectively. The synovial tissue of inflamed joints has a particularly high potential to accumulate osteoclasts because it harbors monocytes/macrophages, which function as osteoclast precursors, as well as cells that provide the specific molecular signals that drive osteoclast formation. Osteoclasts thus represent a link between joint inflammation and structural damage since they resorb mineralized tissue adjacent to the joint and destroy the joint architecture.     

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.