Osteoclast Fusion and Fission

Osteoclasts are specialized multinucleated cells with the unique capacity to resorb bone. Despite insight into the various steps of the interaction of osteoclast precursors leading to osteoclast formation, surprisingly little is known about what happens with the multinucleated cell itself after it has been formed. Is fusion limited to the short period of its formation, or do osteoclasts have the capacity to change their size and number of nuclei at a later stage? To visualize these processes we analyzed osteoclasts generated in vitro with M-CSF and RANKL from mouse bone marrow and native osteoclasts isolated from rabbit bones by live cell microscopy. We show that osteoclasts fuse not only with mononuclear cells but also with other multinucleated cells. The most intriguing finding was fission of the osteoclasts. Osteoclasts were shown to have the capacity to generate functional multinucleated compartments as well as compartments that contained apoptotic nuclei. These compartments were separated from each other, each giving rise to a novel functional osteoclast or to a compartment that contained apoptotic nuclei. Our findings suggest that osteoclasts have the capacity to regulate their own population in number and function, probably to adapt quickly to changing situations.     

破骨细胞与强直性脊柱炎

破骨细胞是专职性骨吸收细胞,在骨重塑中发挥着重要的作用,其来源于骨髓单核巨噬细胞、外周血单核细胞、肺泡巨噬细胞及牙槽骨巨噬细胞,在成骨/基质细胞或OPG/RANKL/RANK、TNF-α、IL-1、M-CSF、VEGF等促分化因子的作用下分化成熟,进而发挥骨质破坏作用。骨、软骨的破坏及局部骨质疏松是强直性脊柱炎的主要病理改变之一,其中破骨细胞发挥着重要的作用,针对破骨细胞靶向治疗是阻止强直性脊柱炎骨与软骨破坏的研究方向之一。     

负载丹酚酸B的破骨前体细胞膜纳米颗粒对成骨细胞和破骨细胞分化的影响

目的 通过制备破骨前体细胞膜纳米颗粒（nanoparticles,NPs）负载丹酚酸B（salvianolic acid,SalB）,构建载药纳米颗粒SalB-NPs,观察其对破骨细胞及成骨细胞分化的影响。方法 采用超声裂解、挤膜的方法制备NPs,并将NPs与SalB共孵育后,使用200 nm聚碳酸酯膜挤出,获得SalB-NPs。在诱导小鼠原代破骨细胞分化和成骨前体细胞（MC3T3-E1）成骨分化的过程中,按照处理方式不同,分为对照组、SalB组、SalB-NPs组。采用透射电镜、纳米粒度及ZETA电位仪和Western Blot对材料进行表征,采用噻唑蓝检测试剂盒检测材料对RAW 264.7和MC3T3-E1细胞活力的影响,采用高效液相色谱法检测SalB的释放率和装载率。采用抗酒石酸酸性磷酸酶（TRAP）染色评价破骨细胞分化能力,通过碱性磷酸酶（ALP）染色和茜素红染色评估成骨分化能力,采用Real time-PCR检测破骨细胞分化及成骨分化相关基因表达水平。结果 制备的纳米颗粒直径在200 nm左右,同时表达RANK蛋白。TRAP染色显示SalB-NPs显著抑制破骨细胞的形成,下调破骨分化相关基因水平,与对照组和SalB组相比,差异有统计学意义（P＜0.05）。使用成骨诱导培养基诱导MC3T3-E1细胞成骨分化,14 d ALP染色和21 d茜素红染色均显示SalB-NPs组ALP活性和钙盐沉积量较SalB组明显增加,差异有统计学意义（P＜0.05）。结论 SalB-NPs体外发挥促进成骨分化、抑制破骨细胞形成双重功效,作为骨质疏松的治疗药物开发,有很好的应用前景,未来仍需在骨质疏松模型动物进一步验证其治疗效果。     

Microcracks and Osteoclast Resorption Activity In Vitro

During bone remodeling osteoclasts resorb bone, thus removing material, e.g., damaged by microcracks, which arises as a result of physiological loading and could reduce bone strength. Such a process needs targeted bone resorption exactly at damaged sites. Osteocytic signaling plays a key role in this process, but it is not excluded that osteoclasts per se may possess toposensitivity to recognize and resorb damaged bone since it has been shown that resorption spaces are associated with microcracks. To address this question, we used an in vitro setup of a pure osteoclast culture and mineralized substrates with artificially introduced microcracks and microscratches. Histomorphometric analyses and statistical evaluation clearly showed that these defects had no effect on osteoclast resorption behavior. Osteoclasts did not resorb along microcracks, even when resorption started right beside these damages. Furthermore, quantification of resorption on three different mineralized substrates, cortical bone, bleached bone (bone after partial removal of the organic matrix), and dentin, revealed lowest resorption on bone, significantly higher resorption on bleached bone, and highest resorption on dentin. The difference between native and bleached bone may be interpreted as an inhibitory impact of the organic matrix. However, the collagen-based matrix could not be the responsible part as resorption was highest on dentin, which contains collagen. It seems that osteocytic proteins, stored in bone but not present in dentin, affect osteoclastic action. This demonstrates that osteoclasts per se do not possess a toposensitivity to remove microcracks but may be influenced by components of the organic bone matrix.     

Osteoclast enlargement in endstage renal disease

In normal subjects and patients with endstage renal disease, osteoclast cell volumes were constructed using serial 2-micron thick plastic embedded sections from iliac crest bone biopsy specimens. Four cells randomly selected from each of the subjects were analyzed to give both the cell volume from the cumulative areas and thickness of each cell slice and also the cell axes taking the vector along the bone face as width or Y, thickness from the bone surface as breadth or X and vertical dimension length or Z. The mean cell volume was 6,230 microns3 in the control subjects and was significantly larger being 11,730 microns3 and 13,680 microns3 in the two patient groups. The cells showed polarity with the largest axes, Y and Z, being those in apposition to the bone surface. Howship's lacunae were enlarged in the patients and the cross-sectional area of an individual lacuna corresponded to the area of the contiguous osteoclast, r = 0.62, P less than 0.001. All patients had secondary hyperparathyroidism and osteoclast numbers were increased. There was no correlation between osteoclast size and duration of renal failure, previous vitamin D intake, or aluminum exposure. It is suggested that in ESRD, osteoclasts undergo both an increase in number and size and that these cells, being larger, remove more bone than the smaller cells in normal subjects.     

Osteoclast precursors circulate in avian blood

Summary The osteoclast is known to be derived from a marrow-residing precursor that is a member of the mononuclear phagocyte family, but the means by which this cell moves from marrow to bone is unknown. We herein demonstrate that mononuclear progenitors capable of differentiating, in vitro, into cells exhibiting the osteoclast phenotype circulate in chickens. The mononuclear fraction was isolated on a density gradient from blood drawn from calcium-deprived laying hens and the plastic-adherent population was obtained. These cells are members of the mononuclear phagocyte family, as demonstrated by nonspecific esterase and tartrate-resistant acid phosphatase (TRAP) activities, expression of the macrophage-specific mannose receptor, and their ability to phagocytose latex particles. When cultured in the presence of devitalized bone, these cells undergo progressive multinucleation and ultimately become essentially indistinguishable from isolated osteoclasts and those generated from bone marrow precursors. Specifically, the blood-derived polykaryons are TRAP-positive, exhibit characteristic ruffled membranes, and express the osteoclast antigens 121F and 23C6. When placed on bone slices, these cells from typical resorptive pits. Moreover, when cultured with ³H-proline-labeled bone, the blood monocytegenerated osteoclasts mobilize matrix as effectively as those derived from marrow. Thus, osteoclast precursors circulate in the blood of laying hens and can be induced to differentiate in vitro.     

Osteoclast inhibition impairs chondrosarcoma growth and bone destruction

Because Chondrosarcoma is resistant to available chemotherapy and radiation regimens, wide resection is the mainstay in treatment, which frequently results in high morbidity and which may not prevent local recurrence. There is a clear need for improved adjuvant treatment of this malignancy. We have observed the presence of osteoclasts in the microenvironment of chondrosarcoma in human pathological specimens. We utilized the Swarm rat chondrosarcoma (SRC) model to test the hypothesis that osteoclasts affect chondrosarcoma pathogenesis. We implanted SRC tumors in tibia of Sprague‐Dawley rats and analyzed bone histologically and radiographically for bone destruction and tumor growth. At three weeks, tumors invaded local bone causing cortical disruption and trabecular resorption. Bone destruction was accompanied by increased osteoclast number and resorbed bone surface. Treatment of rats with the zoledronic acid prevented cortical destruction, inhibited trabecular resorption, and resulted in decreased tumor volume in bone. To confirm that inhibition of osteoclasts per se, and not off‐target effects of drug, was responsible for the prevention of tumor growth and bone destruction, we implanted SRC into osteopetrotic rat tibia. SRC‐induced bone destruction and tumor growth were impaired in osteopetrotic bone compared with control bone. The results from our animal model demonstrate that osteoclasts contribute to chondrosarcoma‐mediated bone destruction and tumor growth and may represent a therapeutic target in particular chondrosarcoma patients. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1562–1571, 2014.     

Mechanically Loaded Myotubes Affect Osteoclast Formation

In response to mechanical loading skeletal muscle produces numerous growth factors and cytokines that enter the circulation. We hypothesized that myotubes produce soluble factors that affect osteoclast formation and aimed to identify which osteoclastogenesis-modulating factors are differentially produced by mechanically stimulated myotubes. C2C12 myotubes were subjected to mechanical loading by cyclic strain for 1 h, and postincubated with or without cyclic strain for 24 h. The effect of cyclic strain on gene expression in myotubes was determined by PCR. Conditioned medium (CM) was collected from cultures of unloaded and loaded myotubes and from MLO-Y4 osteocytes. CM was added to mouse bone marrow cells containing osteoclast precursors, and after 6 days osteoclasts were counted. Compared to unconditioned medium, CM from unloaded osteocytes increased osteoclast formation, while CM from unloaded myotubes decreased osteoclast formation. Cyclic strain strongly enhanced IL-6 expression in myotubes. CM from cyclically strained myotubes increased osteoclast formation compared to CM from unloaded myotubes, but this effect did not occur in the presence of an IL-6 antibody. In conclusion, mechanically loaded myotubes secrete soluble factors, among others IL-6, which affect osteoclast formation. These results suggest that muscle could potentially affect bone homeostasis in vivo via production of growth factors and/or cytokines.     

Osteoclast formation and bone resorption are inhibited by megakaryocytes

It has been previously reported that addition of megakaryocytes (MKs) to osteoblasts in vitro results in increased osteoblastic collagen and osteoprotegerin (OPG) production, suggesting a role for MKs in bone formation. To further investigate this role, we have studied the effects of MKs on osteoclast formation and activity. Human osteoclasts were generated from CD14 monocytes isolated from peripheral blood and cultured in the presence of M-CSF and sRANKL on dentine and calcium phosphate substrates. MKs were generated from CD34+ cells isolated from either human peripheral blood or cord blood and cultured in liquid medium for 6 days, after which time maturing MKs (CD61-positive cells) were isolated and added to monocyte cultures. After 6 and 9 days of culture, the number of osteoclasts identified morphologically and by TRAP staining was counted. Cells were removed and the area of resorption was identified by von Kossa staining and quantitatively assessed by image analysis. The addition of MKs to osteoclast cultures at day 0 inhibited the number of osteoclasts formed 1.9-fold (p>0.003), whereas addition at 3 days had no effect on osteoclast number. The presence of MKs inhibited resorption 8.7-fold when co-cultured with osteoclasts from day 0 (p>0.004), but only by 3.1-fold when co-cultured from day 3 (p>0.01). In dose-response experiments, it was found that 1-10% of MKs added to monocyte cultures elicited the greatest inhibition of resorption. Similar osteoclast cultures were treated with CD61-negative cells (non-MKs) to confirm that the inhibition of osteoclast formation and activity was specifically due to MKs. Experiments with a cell-impermeable membrane indicated that both cell to cell contact and release of soluble factor(s) were involved in mediating these effects. These results show that MKs inhibit osteoclast formation and activity. The most pronounced effects were seen when MKs and osteoclasts were co-cultured from day 0, suggesting that MKs act primarily on osteoclast precursors.     

Secreted Tartrate-Resistant Acid Phosphatase 5b is a Marker of Osteoclast Number in Human Osteoclast Cultures and the Rat Ovariectomy Model

Purpose: To study the effects of estrogen withdrawal on osteoclast number and osteoclast activity in the rat ovariectomy (OVX) model. Methods: We first cultured human CD34+ osteoclast precursor cells on bovine bone slices, allowing them to differentiate into mature resorbing osteoclasts. Secreted tartrate-resistant acid phosphatase 5b (TRACP 5b) and C-terminal cross-linked telopeptides of type I collagen (CTX) were determined from the culture medium. TRACP 5b correlated strongly with osteoclast number and CTX with osteoclast activity, facilitating their subsequent use in the rat OVX model. An 8 week OVX study was then performed including sham-operated rats receiving vehicle, OVX rats receiving vehicle, and OVX rats receiving 10 μg/kg/day 17β-estradiol (E2). Trabecular bone parameters were determined from the tibial metaphysis using peripheral quantitative computed tomography and histomorphometry. Osteoclast number was normalized with bone perimeter (N.Oc/B.Pm) and tissue area (N.Oc/T.Ar, indicating absolute number of osteoclasts). TRACP 5b and CTX were determined from fasting serum samples. Results: Trabecular bone parameters indicated substantial bone loss after OVX that was prevented by E2. N.Oc/B.Pm increased after OVX, while N.Oc/T.Ar and TRACP 5b decreased, and TRACP 5b correlated strongly with N.Oc/T.Ar. However, CTX values increased after OVX, and the “resorption index” CTX/TRACP 5b showed more substantial changes than either CTX or TRACP 5b alone. Conclusion: These results show that TRACP 5b is a reliable marker of osteoclast number, and the index CTX/TRACP 5b is a useful parameter in rat OVX model. The high elevation of CTX/TRACP 5b values by OVX demonstrates that estrogen withdrawal generates high activity of osteoclasts in the rat OVX model. Disclosure statement: Jukka Rissanen, Mari Suominen, and Zhiqi Peng have nothing to disclose; Jussi Halleen receives royalties from and works as a consultant of SBA Sciences, a company owned by IDS Ltd.     

Osteoclast ultrastructure in Paget’s disease

The ultrastructural study of osteoclasts in biopsies from 12 patients with Paget’s disease reveals several cytological anomalies. In particular, nuclear inclusions, consisting essentially of striated filaments about 150 Å in diameter, often organized in bundles and sometimes in paracrystalline arrays, appear to be specific to the disease. Morphologically, the inclusions are remarkably similar to those observed in various cases of viral attack, and the hypothesis of the action of a possible external agent in Paget’s disease cannot be ruled out.     

Osteoclast targeted therapy for prostate cancer: bisphosphonates and beyond

Bone metastases are a major cause of morbidity for men with prostate cancer. Although typical bone metastases from prostate cancer appear osteoblastic by radiographic imaging, excess number and activity of both osteoblasts and osteoclasts characterize most "osteoblastic" bone metastases. Additionally, pathological osteoclast activation is associated with increased risk of skeletal complications, disease progression, and death. Zoledronic acid, a potent intravenous bisphosphonate, reduces markers of osteoclast activity and significantly decreases the risk of skeletal complications in men with androgen-independent prostate cancer and bone metastases. Additional studies are needed to determine the optimal timing, schedule, and duration of bisphosphonate treatment in men with bone metastases as well as the potential role of bisphosphonates in other settings, including the prevention of bone metastases. Denosumab is a human monoclonal antibody that binds and neutralizes human receptor activator of NF-kappaB ligand (RANKL), a critical mediator of osteoclast activation, differentiation, and survival. Three ongoing pivotal studies involving more than 4,500 subjects will evaluate the role of denosumab for prevention of treatment-related fractures, bone metastases, and disease-related skeletal complications in men with prostate cancer.