Spontaneous generation of functional osteoclasts from synovial fluid mononuclear cells as a model of inflammatory osteoclastogenesis

In osteoimmunology, osteoclastogenesis is understood in the context of the immune system. Today, the in vitro model for osteoclastogenesis necessitates the addition of recombinant human receptor activator of nuclear factor kappa‐B ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF). The peripheral joints of patients with rheumatoid arthritis (RA) and spondyloarthritis (SpA) are characterized by an immune‐mediated inflammation that can lead to bone destruction. Here, we evaluate spontaneous in vitro osteoclastogenesis in cultures of synovial fluid mononuclear cells (SFMCs) activated only in vivo. SFMCs were isolated and cultured for 21 days at 0.5–1.0 × 10⁶ cells/mL in culture medium. SFMCs and healthy control peripheral blood monocytes were cultured with RANKL and M‐CSF as controls. Tartrate‐resistant acid phosphatase (TRAP) positive multinucleated cells were found in the SFMC cultures after 21 days. These cells expressed the osteoclast genes calcitonin receptor, cathepsin K, and integrin β3, formed lacunae on dentin plates and secreted matrix metalloproteinase 9 (MMP9) and TRAP. Adding RANKL and M‐CSF potentiated this secretion. In conclusion, we show that SFMCs from inflamed peripheral joints can spontaneously develop into functionally active osteoclasts ex vivo. Our study provides a simple in vitro model for studying inflammatory osteoclastogenesis.     

Association between Increased Inducible Costimulator/Inducible Costimulator Ligand Expression with Bone Destruction in Apical Periodontitis

IntroductionThe aim was to assess the association of inducible costimulator (ICOS) and ICOS ligand with bone destruction in apical periodontitis (AP).MethodsSpecimens from patients presenting with AP were obtained during apicoectomy and subjected to histopathologic analysis and molecular assessment of ICOS/ICOS ligand. In addition, the experimental AP was induced by exposing the pulp of first mandibular molars of rats. Histologic and radiographic examinations were performed to validate the periapical lesions. The immunolocalization and messenger RNA expression of ICOS/ICOS ligand were evaluated by immunofluorescence staining and quantitative real-time polymerase chain reaction. The osteoclastic activities in periapical lesions, including the lesion size and the expression of tartrate-resistant acid phosphatase and the receptor activator of nuclear factor kappa B ligand, were recorded and followed by correlation analysis with ICOS/ICOS ligand expression.ResultsIn excisional specimens from AP patients, a significantly increased expression of ICOS/ICOS ligand was found compared with the healthy control. In the experimental AP samples, the expression of ICOS/ICOS ligand, tartrate-resistant acid phosphatase, and receptor activator of nuclear factor kappa B ligand was significantly elevated in inflamed periapical tissues (AP group) when compared with the healthy control. The number of ICOS⁺/ICOS ligand⁺ cells was highly correlated with the periapical lesion size (r = 0.892, P < .01 and r = 0.930, P < .01, respectively).ConclusionsThe increased expression of ICOS/ICOS ligand in periapical lesions was associated with the inflammatory infiltration and alveolar bone destruction of AP.     

细胞因子诱导破骨细胞分化的2种实验模型的建立

目的探讨体外从单个核细胞诱导分化形成破骨细胞的2种不同方法,建立研究细胞因子对破骨细胞分化作用的2种不同实验模型。方法外周血单个核细胞在10-8mol/L LTB4和25μg/LM-CSF刺激下,经过2周的直接诱导分化;外周血单个核细胞与RAFLs共培养,加入10-8mol/L LTB4和25μg/L M-CSF,经过3周的间接诱导分化,用抗酒石酸酸性磷酸酶(TRAP)染色鉴定破骨细胞样细胞(OCL)。结果通过直接分化和间接分化均可形成破骨细胞样细胞。结论2种不同实验模型能分别用于研究细胞因子对破骨细胞的直接和间接分化作用。     

实验性骨折愈合中骨吸收活动的超微结构观察

将50只家兔造成实验性桡骨骨折,分批取骨痂标本,用光镜和电子显微镜观察表明,在骨折愈合过程中,破骨细胞是骨吸收的主要执行者,巨噬细胞能吞噬死骨,但不能吸收骨质。     

The localization of CD44 and moesin in osteoclasts after calcitonin administration in mouse tibiae

We investigated the immunohistochemical localization of CD44, hyaluronate receptor, and moesin, of the ezrinradixin-moesin (ERM) family, in osteoclasts after calcitonin adminstration using confocal laser scanning microscopy and transmission electron microscopy to clarify the role of CD44 and moesin in their cytoskeletal organization and cell polarity. We also elucidated the localization of osteopontin (OPN) to confirm its possible role in cell-matrix recognition via CD44. In untreated mice, intense immunoreactivities for CD44 and moesin were detected on the basolateral plasma membrane of osteoclasts. Rhodamine-phalloidin reactivity was seen in a bandlike pattern on the region of contact between osteoclasts and bone and was also detected moderately along their basolateral plasma membrane. At 30 min after calcitonin administration, osteoclasts did not show either clear zones or ruffled borders. The bandlike reactivity of rhodamine-phalloidin in the contact region was diminished, although labeling was seen along osteoclasts. CD44 and moesin were colocalized along their plasma membranes, including the region facing the bone surface. Electron microscopic observation revealed that the microvillus processes in the contacting region with bone surface, as well as the basolateral plasma membrane, showed immunoreactivities to CD44 and moesin. At 60 min, some osteoclasts attached to bone and showed a bandlike pattern of rhodamine-phalloidin. On the other hand, OPN was localized under CD44-positive cytoplasmic processes and the clear zone of osteoclasts. These findings suggest that calcitonin effects on the cell polarity of osteoclasts and the CD44-moesin-actin filament system in osteoclasts plays an imporant role in cell polarity and cell-matrix recognition.     

脂肪因子Chemerin对骨代谢影响的研究进展

现代社会肥胖人群逐年增多,肥胖相关代谢性骨病的患者也日益增多,严重危害人群健康。Chemerin作为一种脂肪因子,主要由脂肪细胞和肝细胞分泌,在循环中水平高于经典脂肪因子如Leptin和Adiponectin,对全身代谢和免疫发挥重要作用。近年研究发现,肥胖状态下伴随着外周脂肪组织的增多,骨髓内脂肪组织也急剧增加。骨髓内脂肪细胞分泌Chemerin,通过旁分泌或自分泌方式抑制成骨细胞分化和促进破骨细胞分化以及影响骨髓腔微环境。本文将系统回顾国内外有关Chemerin与骨代谢相关研究的文献,并探讨Chemerin对骨代谢的影响及其机制。     

不同双膦酸盐类药物抑制破骨细胞特征曲线的意义

目的比较双膦酸盐类药物唑来膦酸、伊班膦酸和帕米膦酸对体外培育破骨细胞的具体抑制作用,绘制特征性曲线并指导临床治疗。方法通过体外培育小鼠骨髓细胞,分化获得破骨细胞并将其分为唑来膦酸组、伊班膦酸组、帕米膦酸组、对照组。其中,唑来膦酸组培养液中加入唑来膦酸,药物的阶梯浓度为:10-3、10-2、10-1、1、10、102、103μmol/L。同时,伊班膦酸组与帕米膦酸组加入相应药物的阶梯浓度相同,对照组加入磷酸盐缓冲液。观察双膦酸盐类药物对破骨细胞形成、迁移、粘附、骨破坏的抑制作用特征并进行比较,绘制特征性曲线。结果随着药物浓度逐级增大,多核细胞数量随浓度增加而减少。唑来膦酸、伊班膦酸和帕米膦酸的最低有效抑制浓度分别为1、10、102μmol/L。三者50%有效浓度分别为0.66、5.58、51.9μmol/L。结论临床治疗骨质疏松症时建议参考破骨细胞抑制特征曲线,为唑来膦酸、伊班膦酸和帕米膦酸的静脉应用提供科学依据。     

Mechanistic insight of interleukin-9 induced osteoclastogenesis

Interleukin (IL)-9 is an emerging player in the pathogenesis of various chronic inflammatory diseases including bone disorders like rheumatoid arthritis (RA) and psoriatic arthritis. Recently, IL-9 was shown to enhance the osteoclast formation and their function in RA. However, the mechanisms by which IL-9 influences osteoclastogenesis are not known. Therefore, in this study we aimed to unravel the direct and indirect ways by which IL-9 can influence osteoclast formation. We used mouse bone marrow precursor cells for checking the effect of IL-9 on osteoclast differentiation and its function. Next, IL-9 induced signalling pathway were checked in the process of osteoclastogenesis. T cells play an important role in enhancing osteoclastogenesis in inflammatory conditions. We used splenic T cells to understand the impact of IL-9 on the functions of T effector (Teff) and regulatory T (Treg) cells. Furthermore, the effect of IL-9 mediated modulation of the T cell response on osteoclasts was checked using a coculture model of T cells with osteoclast precursors. We showed that IL-9 enhanced osteoclast formation and its function. We found that IL-9 activates STAT3, P38 MAPK, ERK1/2, NFκB and we hypothesize that it mediates the effect on osteoclastogenesis by accelerating mitochondrial biogenesis. Additionally, IL-9 was observed to facilitate the functions of pro-osteoclastogenic IL-17 producing T cells, but inhibits the function of anti-osteoclastogenic Treg cells. Our observations suggest that IL-9 can influence osteoclastogenesis directly by modulating the signalling cascade in the precursor cells; indirectly by enhancing IL-17 producing T cells and by reducing the functions of Treg cells.