破骨细胞分化调节机制的研究进展

破骨细胞起源于骨髓的单核髓性造血干细胞, 是一种具有骨吸收功能的多核巨细胞, 其在骨代谢方面起着关键性的作用, 因而机体对于破骨细胞的调控非常严格。破骨细胞动员和分化成熟过程是一个复杂而又精细的多级调控过程, 在相关调控机制中, OPG/RANKL/RANK系统起着分化调节枢纽的作用, 是调节破骨细胞分化和功能的关键信号途径。最近研究发现破骨细胞和免疫细胞在骨代谢领域相互联系紧密, 这也为骨疾病的治疗提供了新的治疗靶点。另外破骨细胞凋亡在骨代谢中的作用越来越受重视, 但其相关机制还不是很明确, 仍需要深入研究。     

Transmigration: a new property of mature multinucleated osteoclasts.

Even though it is assumed that multinucleated osteoclasts are migrating cells on the bone surface to be resorbed, we show that they can also selectively transmigrate through layers of cells usually found in the bone microenvironment. This activity is associated with c-src and MMPs and can be stimulated by bone metastatic breast cancer cells, a process blocked by bisphosphonate treatment. INTRODUCTION: Osteoclasts have an hematopoietic origin and are bone-resorbing cells. Monocytic precursors migrate to the bone surface where they fuse to form multinucleated osteoclasts able to migrate over the bone surface. We studied whether multinucleated osteoclasts were also able to transmigrate through tissues. MATERIALS AND METHODS: Murine spleen-derived and green fluorescent protein (GFP)-Raw derived osteoclasts were seeded on osteoblasts and several other cell types. The cells were fixed for 20 minutes, 4 or 12 h after osteoclast seeding, and stained with phalloidin to visualize actin using confocal microscopy. Drugs such as PP2 and GM6001, inhibitors of c-src and matrix metalloproteinases (MMPs), respectively, and risedronate were used to determine osteoclast transmigration regulating factors. RESULTS: We observed by confocal microscopy that multinucleated osteoclasts specifically transmigrate through confluent layers of various cell types present in the bone microenvironment in vitro. This is an efficient process associated with c-src and MMPs but is independent of podosomes. Moreover, conditioned medium from bone metastatic breast cancer cells stimulates osteoclast transmigration in vitro, a process inhibited by bisphosphonate treatment. CONCLUSIONS: Our data describe a new property of mature multinucleated osteoclasts to transmigrate through various cell types. The ability to control this highly regulated osteoclast transmigration process may offer new therapeutic strategies for bone diseases associated with an imbalance in bone remodeling caused by excessive osteoclast resorption.     

Osteoclast formation is related to bone matrix age

Summary Little is known about the relationship between the age of the skeleton and the development of multinucleated bone-resorbing cells, osteoclasts. It has been shown that mineralized bone implanted onto the chick chorioallantoic membrane (CAM) is effective in the recruitment and differentiation of osteoclast precursors. In studies reported here we used the CAM system to examine the influence of bone matrix age on osteoclast formation. Devitalized mineralized bone particles (75–250 μm) were prepared from rats of various ages (2, 4, 9, 12, and 16 months). The particles were implanted onto the chick chorioallantoic membrane and 8 days later implants were harvested and processed for morphometric or immunohistochemical analysis. Osteoclast number, cell area, nucleocytoplasmic ratio, and the presence of a distinctive osteoclast antigen, defined by the 121F monoclonal antibody, were determined. Bone particles of each age group resulted in the formation of osteoclast-like giant cells. Compared with multinucleated cells that formed in response to bone particles obtained from 2-month-old rats, matrix from the oldest age group (16 months) elicited significantly fewer and smaller cells which contained a smaller number of nuclei. These data suggest that with aging, bone undergoes qualitative and/or quantitative changes that affect the recruitment and differentiation of osteoclast precursor cells.     

Jaw and Long Bone Marrows Have a Different Osteoclastogenic Potential

Osteoclasts, the multinucleated bone-resorbing cells, arise through fusion of precursors from the myeloid lineage. However, not all osteoclasts are alike; osteoclasts at different bone sites appear to differ in numerous respects. We investigated whether bone marrow cells obtained from jaw and long bone differed in their osteoclastogenic potential. Bone marrow cells from murine mandible and tibiae were isolated and cultured for 4 and 6 days on plastic or 6 and 10 days on dentin. Osteoclastogenesis was assessed by counting the number of TRAP⁺ multinucleated cells. Bone marrow cell composition was analyzed by FACS. The expression of osteoclast- and osteoclastogenesis-related genes was studied by qPCR. TRAP activity and resorptive activity of osteoclasts were measured by absorbance and morphometric analyses, respectively. At day 4 more osteoclasts were formed in long bone cultures than in jaw cultures. At day 6 the difference in number was no longer observed. The jaw cultures, however, contained more large osteoclasts on plastic and on dentin. Long bone marrow contained more osteoclast precursors, in particular the myeloid blasts, and qPCR revealed that the RANKL:OPG ratio was higher in long bone cultures. TRAP expression was higher for the long bone cultures on dentin. Although jaw osteoclasts were larger than long bone osteoclasts, no differences were found between their resorptive activities. In conclusion, bone marrow cells from different skeletal locations (jaw and long bone) have different dynamics of osteoclastogenesis. We propose that this is primarily due to differences in the cellular composition of the bone site-specific marrow.     

Quantitative analyses of osteoclast changes in resorbing bone organ cultures

Summary The stimulation of bone resorption, assessed by the release of⁴⁵Ca from prelabeled bones, was associated with an increase in number of osteoclasts per bone section in parathyroid hormone (PTH)-treated bones, but not in lipopolysaccharide (LPS)-treated bones. By contrast the number of nuclei per osteoclast increased following LPS treatment, but was not affected by PTH. LPS-treated bones had more multinucleated cells, some having as many as 27 nuclei per osteoclast. More osteoclasts were adjacent to the bone collar in bones treated with LPS or PTH than in control bones. In LPS-treated bones this area also contained the largest osteoclasts, as determined by the greatest number of nuclei per osteoclast. The results suggest that LPS and PTH stimulate osteoclastic resorption by different mechanisms.     

Human mesenchymal tumour-associated macrophages differentiate into osteoclastic bone-resorbing cells

The cellular mechanisms which account for the formation of osteoclasts and bone resorption associated with enlarging benign and malignant mesenchymal tumours of bone are uncertain. Osteoclasts are marrow-derived, multinucleated, bone-resorbing cells which express a macrophage phenotype. We have determined whether tumour-associated macrophages (TAMs) isolated from benign and malignant mesenchymal tumours are capable of differentiating into osteoclasts. Macrophages were cultured on both coverslips and dentine slices for up to 21 days with UMR 106 osteoblastic cells in the presence of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) and human macrophage colony-stimulating factor (M-CSF) or, in the absence of UMR 106 cells, with M-CSF and RANK ligand. In all tumours, the formation of osteoclasts from CD14-positive macrophages was shown by the formation of tartrate-resistant-acid-phosphatase and vitronectin-receptor-positive multinucleated cells which were capable of carrying out lacunar resorption. These results indicate that the tumour osteolysis associated with the growth of mesenchymal tumours in bone is likely to be due in part to the differentiation of mononuclear phagocyte osteoclast precursors which are present in the TAM population of these lesions.     

Isolation of human osteoclasts formed in vitro: hormonal effects on the bone-resorbing activity of human osteoclasts

Osteoclasts are multinucleated cells that carry out bone resorption. Analysis of the direct effect of hormones on the bone-resorbing activity of human osteoclasts has been limited by difficulties in isolating these cells from the human skeleton. In this study, human osteoclasts formed from cultures of peripheral blood mononuclear precursors (PBMCs) on a Type-I collagen gel were isolated by collagenase treatment for investigating their resorptive activity. PBMCs were cultured in the presence of M-CSF, soluble RANKL, dexamethasone, and 1,25(OH)2D3. The isolated multinucleated cells expressed the osteoclast markers, TRAP, VNR, cathepsin K, calcitonin receptors and were capable of extensive lacunar resorption. Calcitonin inhibited the motility and resorptive activity of osteoclasts. RANKL significantly stimulated osteoclast resorption, but 1,25(OH)2D3, PTH, and OPG did not. These findings indicate that calcitonin and RANKL act directly on human osteoclasts to inhibit and stimulate osteoclast bone-resorbing activity, respectively, and that PTH, 1,25(OH)2D3, and OPG are more likely to influence osteoclast activity indirectly. This technique of human osteoclast isolation should permit the effects of cellular and hormonal/humoral factors on the bone-resorbing activity of mature human osteoclasts to be assessed independently of any effect such factors have on osteoclast formation. It should also make it possible to examine directly the resorptive activity and other characteristics of osteoclasts in specific bone disorders such as Paget's disease.     

Isolation of osteoclasts from Pagetic bone tissue: morphometry and cytochemistry on isolated cells

Giant osteoclasts and other cells were isolated from Pagetic bone tissue using 0.5 mM ethylene diamine tetraacetic acid on bone samples from 8 patients with Paget's disease. The cell suspension contained osteoclasts and osteoblasts as well as some mononuclear cells such as monocytes. The number of nuclei in isolated osteoclasts (33.85 +/- 20.92 nuclei/osteoclast) correlates fairly well (p less than 0.02) with the number of nuclei counted on histologic sections (15.88 +/- 11.80 nuclei/osteoclast) for samples from each patient. Enzyme histochemistry demonstrated acid phosphatase activity in isolated osteoclasts and in mononucleated cells, such as monocytes. Alkaline phosphatase was detected only in osteoblasts while succinate dehydrogenase was observed in osteoclasts, osteoblasts and monocytes. Esterases, such as nonspecific aliesterase and specific naphthol AS-D acetate esterase, were identified in osteoclasts and in macrophages. Inhibition of specific naphthol AS-D acetate esterase in osteoclasts by addition of sodium fluoride suggests that the enzyme could be of monocytic origin.     

(Pre-)osteoclasts induce retraction of osteoblasts before their fusion to osteoclasts.

Precursors of osteoclasts seeded on top of a confluent layer of osteoblasts/bone lining cells induced retraction of the latter cells. The (pre)osteoclasts then migrated in the formed cell-free areas and fused to form osteoclast-like cells. Retraction of the osteoblasts/bone lining cells proved to depend on activity of matrix metalloproteinases, and TGF-beta1 prevented the retraction. INTRODUCTION: It is well known that osteoblasts have a profound effect on (pre)osteoclasts in inducing the formation of bone-resorbing osteoclasts. Whether, on the other hand, (pre)osteoclasts also modulate osteoblast activity is largely unknown. Because osteoblasts/bone lining cells have to retract from the surface before resorption of bone by osteoclasts, we addressed the question of whether (pre)osteoclasts have the capacity to induce such an activity. MATERIALS AND METHODS: Rabbit calvarial osteoblasts/bone lining cells or periosteal fibroblasts were cultured until confluency, after which rabbit peripheral blood mononuclear cells (PBMCs) were seeded on top of them. The co-cultures were maintained for up to 15 days in the presence or absence of the cytokines transforming growth factor (TGF)-beta1 and TNF-alpha and selective inhibitors of matrix metalloproteinases and serine proteinases. The formation of cell-free areas and the number of TRACP+ multinucleated osteoclast-like cells were analyzed. In addition, formation of cell-free areas was analyzed in co-cultures of osteoblasts with mature osteoclasts. RESULTS: The seeding of PBMCs on a confluent layer of osteoblasts/bone lining cells resulted in the following sequence of events. (1) A low number of PBMCs strongly attached to osteoblasts. 2) At these sites of contact, the osteoblasts retracted, thus forming cell-free areas. (3) The PBMCs invaded these areas and attached to the surface of the well, after which they fused and formed multinucleated TRACP+ osteoclast-like cells. Retraction was only seen if the cells were in direct contact; conditioned media from cultured PBMCs added to osteoblasts had no effect. Mature osteoclasts seeded on osteoblasts similarly induced retraction, but this retraction occurred at a much faster rate (within 2 days) than the retraction effectuated by the osteoclast precursors (after 8 days in co-culture). Inhibition of matrix metalloproteinase activity, but not of serine proteinases, strongly reduced retraction of the osteoblasts, thus indicating that this type of cell movement depends on the activity of matrix metalloproteinases. A similar inhibitory effect was found with TGF-beta1. TNF-alpha had no effect on osteoblast retraction but enhanced the formation of multinucleated osteoclast-like cells. Addition of PBMCs to confluent layers of periosteal fibroblasts resulted in similar phenomena as observed in co-cultures with osteoblasts. However, the cell-free areas proved to be significantly smaller, and the number of multinucleated cells formed within cell-free areas was three to four times lower. CONCLUSION: Our results indicate that osteoclast precursors and mature osteoclasts have the capacity to modulate the activity of osteoblasts and that, yet unknown, membrane-bound signaling molecules are essential in inducing retraction of osteoblasts and the subsequent formation of cell-free areas.     

Osteoclastogenesis is Influenced by Modulation of Gap Junctional Communication with Antiarrhythmic Peptides

Osteoclasts are formed by the fusion of mononuclear precursor cells of the monocyte–macrophage lineage. Among several putative mechanisms, gap-junctional intercellular communication (GJC) has been proposed to have a role in osteoclast fusion and bone resorption. We examined the role of GJC in osteoclastogenesis and in vitro bone resorption with mouse bone marrow hematopoietic stem cells and RAW 264.7 cells. Blocking of gap junctions with 18-α-glycyrrhetinic acid (18GA) led to inhibition of osteoclastogenesis and in vitro bone resorption. Similarly, the GJC inhibitor GAP27 inhibited osteoclast formation. GJC modulation with the antiarrhythmic peptides (AAPs) led to increased amounts of multinuclear RAW 264.7 osteoclasts as well as increased number of nuclei per multinuclear cell. In the culture of bone marrow hematopoietic stem cells in the presence of bone marrow stromal cells AAP reduced the number of osteoclasts, and coculture of MC3T3-E1 preosteoblasts with RAW 264.7 macrophages prevented the action of AAPs to promote osteoclastogenesis. The present data indicate that AAPs modulate the fusion of the pure culture of cells of the monocyte–macrophage lineage. However, the fusion is influenced by GJC in cells of the osteoblast lineage.     

Nucleolar organizer regions (Ag-NORs) in multinuclear osteoclasts

Osteoclasts are multinuclear bone-resorbing cells. Little is known about regulation of gene expression in these cells and how it changes during the aging of an osteoclast, which is indicated by an increasing number of nuclei. Silver staining of nucleolar organizer regions, so-called Ag-NORs, allows us to assess the activity of rRNA expression. Using this technique, we show that individual nuclei of osteoclasts can be stained specifically and that the majority of individual nuclei contained Ag-NORs. The number and size of Ag-NORs varied, indicative of difference in the progression of the cell cycle through the G₁ phase. The relative activity of Ag-NORs was not synchronized on the level of individual nuclei, regardless of the number of nuclei per osteoclast. Surprisingly, the total activity of Ag-NOR appeared to be correlated to the total nuclear area of an osteoclast. We conclude that regulation of gene expression in multinuclear osteoclasts most likely occurs on the level of individual nuclei. Received: Jan. 12, 1998 / Accepted: May 25, 1998     

Polarized osteoclasts put marks of tartrate-resistant acid phosphatase on dentin slices--a simple method for identifying polarized osteoclasts

Osteoclasts form ruffled borders and sealing zones toward bone surfaces to resorb bone. Sealing zones are defined as ringed structures of F-actin dots (actin rings). Polarized osteoclasts secrete protons to bone surfaces via vacuolar proton ATPase through ruffled borders. Catabolic enzymes such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K are also secreted to bone surfaces. Here we show a simple method of identifying functional vestiges of polarized osteoclasts. Osteoclasts obtained from cocultures of mouse osteoblasts and bone marrow cells were cultured for 48 h on dentin slices. Cultures were then fixed and stained for TRAP to identify osteoclasts on the slices. Cells were removed from the slices with cotton swabs, and the slices subjected to TRAP and Mayer's hematoxylin staining. Small TRAP-positive spots (TRAP-marks) were detected in the resorption pits stained with Mayer's hematoxylin. Pitted areas were not always located in the places of osteoclasts, but osteoclasts existed on all TRAP-marks. A time course experiment showed that the number of TRAP-marks was maintained, while the number of resorption pits increased with the culture period. The position of actin rings formed in osteoclasts corresponded to that of TRAP-marks on dentin slices. Immunostaining of dentin slices showed that both cathepsin K and vacuolar proton ATPase were colocalized with the TRAP-marks. Treatment of osteoclast cultures with alendronate, a bisphosphonate, suppressed the formation of TRAP-marks and resorption pits without affecting the cell viability. Calcitonin induced the disappearance of both actin rings and TRAP-marks in osteoclast cultures. These results suggest that TRAP-marks are vestiges of proteins secreted by polarized osteoclasts.     

Osteoclast development in the coculture system of periostless metatarsal bones and hemopoietic cells studied by in situ hybridization with a probe for Y chromosomes

In the coculture system of periostless metatarsal bones of 17-day-old fetal mice and osteoclast progenitors, osteoclasts will develop. Our goal in the present report was to provide further evidence that in the coculture system of fetal metatarsal bone rudiments with hemopoietic cells, the osteoclasts developing inside the bone rudiments are exclusively derived from the cells suspended in the plasma clot and not from endogenous precursor cells of the bone explants themselves, by using the technique of in situ hybridization with a probe for the mouse Y chromosome. Osteoclast formation in unstripped male metatarsal rudiments, occurring after 3–4 days of culture, was compared with osteoclast formation in cocultures of female metatarsal rudiments and male bone marrow cells, occurring after 5–6 days of culture. Osteoclasts were recognized by their tartrate-resistant acid phosphatase activity. In paraffin sections of cultured male metatarsals, the mean percentage of microscopically identifiable osteoclast nuclei, in which the Y chromosome could be detected, was 43.1±4.2% (n=12). For cocultures of female metatarsal bones and male bone marrow cells this mean percentage was 40.9±5.7% (n=17). Statistical comparison by means of the two sample t-test indicated no significant difference in the percentages of osteoclast nuclei containing the Y chromosome for both groups. We concluded that the osteoclasts do derive from cocultured cells and not from precursor cells in the bone explant itself. Therefore, the coculture system is a reliable in vitro system for studying osteoclast formation from progenitor/precursor cells.     

New knowledge on the origin, function and fate of osteoclasts

The most recent findings on the origin, life-span and fate of the osteoclast can be summarized as follows. Osteoclasts originate from progenitor, mononuclear, lymphoid cells which reach the bone surface through the bloodstream. Osteoclasts resorb bone by secreting lysosomal enzymes and procollagenase in the osteoclast-bone interspace. The organic components of the matrix (first interfibrillary substance, then collagen fibrils) are digested in the extracellular space. Dislodged crystals and residual organic constituents are then phagocytosed and collected in cytoplasmic vacuoles where they are completely solubilized. The ruffled border and the adjacent "clear" zone constitute the resorbing apparatus, whose development is roughly proportional to osteoclast activity. Osteoclast nuclei are continuously incorporated and shed, so that individual cells are continuously renewed. This makes the life-span of the osteoclast extremely difficult to determine. The life of each individual osteoclast might theoretically continue as long as the stimulus to resorption persists and sufficient bone matrix is available. Primary abnormalities of the osteoclasts can induce pathologic skeletal changes, as in the case of osteopetrosis and Paget's disease of bone. Conversely, skeletal abnormalities may damage osteoclasts, as in the case of lead intoxication. When this happens, osteoclasts are essentially characterized by an underdeveloped ruffled border, pyknotic nuclei, detachment from the bone matrix and, finally, shrinkage and fragmentation. It is not yet known whether these changes only occur in pathologic conditions, or whether they are the alterations which lead senescent osteoclasts to death even in normal bone.     

Osteoblasts of calvaria induce higher numbers of osteoclasts than osteoblasts from long bone

Several studies have demonstrated the existence of functional differences between osteoclasts harbored in different bones. The mechanisms involved in the occurrence of such a heterogeneity are not yet understood. Since cells of the osteoblast lineage play a critical role in osteoclastogenesis, osteoclast heterogeneity may be due to osteoblasts that differ at the different bone sites. In the present study we evaluated possible differences in the capacity of calvaria and long bone osteoblasts to induce osteoclastogenesis. Osteoblasts were isolated from calvaria and long bone of mice and co-cultured with osteoclast precursors obtained from bone marrow of both types of bone, spleen and peripheral blood. Irrespective of the source of the precursors, a significantly higher number of TRACP-positive multinucleated cells were formed with calvaria osteoblasts. The expression of osteoclastogenesis related genes was analyzed by qPCR. OPG was significantly higher expressed by long bone osteoblasts. The RANKL/OPG ratio and TNF-α gene expression were significantly higher in calvaria osteoblast cultures. OPG added to the culture system inhibited osteoclastogenesis in both groups. Blocking TNF-α had no effect on osteoclastogenesis. Calvaria and long bone osteoblasts were pre-stimulated with VitD3 for 5 days. Subsequently, osteoclast precursors were added to these cultures. After a co-culture of 6 days, it was shown that VitD3 pre-stimulation of long bone osteoblasts strongly improved their capacity to induce osteoclast formation. This coincided with an increased ratio of RANKL/OPG. Taken together, the data demonstrated differences in the capacity of calvaria and long bone osteoblasts to induce osteoclastogenesis. This appeared to be due to differences in the expression of RANKL and OPG. VitD3 pre-stimulation improved the ability of long bone osteoblasts to induce osteoclast formation. Our findings demonstrate bone-site specific differences in osteoblast-mediated formation of osteoclasts. The data may suggest that the heterogeneity of osteoclasts is partially due to the way the osteoblasts induce their formation.     

CD55 Regulates Bone Mass in Mice by Modulating RANKL-Mediated Rac Signaling and Osteoclast Function

CD55 is a glycosylphosphatidylinositol (GPI)-anchored protein that regulates complement-mediated and innate and adaptive immune responses. Although CD55 is expressed in various cell types in the bone marrow, its role in bone has not been investigated. In the current study, trabecular bone volume measured by μCT in the femurs of CD55KO female mice was increased compared to wild type (WT). Paradoxically, osteoclast number was increased in CD55KO with no differences in osteoblast parameters. Osteoclasts from CD55KO mice exhibited abnormal actin-ring formation and reduced bone-resorbing activity. Moreover, macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) treatment failed to activate Rac guanosine triphosphatase (GTPase) in CD55KO bone marrow macrophage (BMM) cells. In addition, apoptotic caspases activity was enhanced in CD55KO, which led to the poor survival of mature osteoclasts. Our results imply that CD55KO mice have increased bone mass due to defective osteoclast resorbing activity resulting from reduced Rac activity in osteoclasts. We conclude that CD55 plays an important role in the survival and bone-resorption activity of osteoclasts through regulation of Rac activity. © 2019 American Society for Bone and Mineral Research.     

Advances in osteoclast biology reveal potential new drug targets and new roles for osteoclasts

Osteoclasts are multinucleated myeloid lineage cells formed in response to macrophage colony‐stimulating factor (M‐CSF) and receptor activator of NF‐κB ligand (RANKL) by fusion of bone marrow–derived precursors that circulate in the blood and are attracted to sites of bone resorption in response to factors, such as sphingosine‐1 phosphate signaling. Major advances in understanding of the molecular mechanisms regulating osteoclast functions have been made in the past 20 years, mainly from mouse and human genetic studies. These have revealed that osteoclasts express and respond to proinflammatory and anti‐inflammatory cytokines. Some of these cytokines activate NF‐κB and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling to induce osteoclast formation and activity and also regulate communication with neighboring cells through signaling proteins, including ephrins and semaphorins. Osteoclasts also positively and negatively regulate immune responses and osteoblastic bone formation. These advances have led to development of new inhibitors of bone resorption that are in clinical use or in clinical trials; and more should follow, based on these advances. This article reviews current understanding of how bone resorption is regulated both positively and negatively in normal and pathologic states. © 2013 American Society for Bone and Mineral Research.     

The increased in vitro osteoclastogenesis in patients with rheumatoid arthritis is due to increased percentage of precursors and decreased apoptosis - the In Vitro Osteoclast Differentiation in Arthritis (IODA) study

Increases in local and systemic bone resorption are hallmarks of rheumatoid arthritis (RA). Osteoclasts are implicated in these processes and their enhanced differentiation may contribute to bone destruction. We observed that in vitro osteoclastogenesis varies among healthy individuals and hypothesized that increased osteoclastogenesis could be a marker for the presence of RA. Our objective in the present study was to determine if in vitro osteoclastogenesis from peripheral blood mononuclear cells (PBMCs) was different in patients with RA compared to healthy controls and osteoarthritis (OA) patients. Expression of CD14 in PBMCs was quantified and PBMCs were incubated for 21 days in the presence of the osteoclastogenic cytokines M-CSF and RANKL. Differentiation on cortical bone slices permitted the analysis of bone resorption while apoptotic potential was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. In vitro osteoclastogenesis was higher in PBMCs from RA patients compared to controls, and a similar increase was observed in the percentage of osteoclast precursors in RA patients. Osteoclasts from RA patients showed lower apoptotic rates than osteoclasts from healthy controls. No difference was observed in bone resorption activity between RA patients and controls. Interestingly, the difference in osteoclast number and apoptosis rate allowed the implementation of an algorithm capable of distinguishing patients with RA from controls. In conclusion, our study shows that osteoclast differentiation from PBMCs is enhanced in patients with RA, and this difference can be explained by both a higher percentage of osteoclast precursors in the blood and by the reduced apoptotic potential of mature osteoclasts.