Immunohistochemical characterization of osteoclasts and osteoclast-like cells with monoclonal antibody MB1 on paraffin-embedded tissues

In this study we provide evidence that MB1, a newly developed monoclonal antibody which reacts with B lymphocytes and a proportion of T cells and monocytes, can be successfully used for the direct immunohistochemical identification of osteoclasts on paraffin-embedded surgical specimens. The antigen(s) recognized by MB1 is present at high density in the cytoplasm of osteoclasts of fetal bone and in the multinucleated cells of human giant cell tumour of bone (osteoclastoma), but is weakly expressed or absent in the giant cells of granulomas. MB1 is thus proposed as a new immunohistochemical marker for osteoclasts on paraffin-embedded material.     

Cytoplasmic modifications at the contact zone of osteoclasts and calcified tissue in the diaphyseal growing plate of foetal guinea-pig tibia

Longitudinal sections of foetal guinea-pig tibia prepared from decalcified and undecalcified samples were examined in the electron microscope. Osteoclasts in contact with calcified tissue in the growing plate of the diaphysis showed a modified oval-shaped cytoplasmic zone, situated at the brim of the cup-like depression containing the ruffled border, with dense material arranged either in parallel bands or in a loose network according to the section observed. This modified zone, interpreted as roughly ring-shaped around the edge of the resorption region, probably participates in the development of the ruffled border and thus contributes to the extension of the surface of active resorption.     

Matricellular proteins: Extracellular modulators of bone development, remodeling, and regeneration

Matricellular proteins are components of the extracellular matrix which are highly expressed in the developing and mature skeleton. Members of this protein class serve as biological mediators of cell function by interacting directly with cells or by modulating the activity of growth factors, proteases, and other extracellular matrix proteins. Although skeletons of matricellular protein-null mice are grossly normal, they each display unique deficiencies that are often magnified under pathological conditions. In addition, bone cells from wild-type and matricellular protein-null mice behave differently in various in vitro models of bone matrix synthesis and turnover. In this review, osteopontin, bone sialoprotein, tenascin C, SPARC, and thrombospondins 1 and 2 will each be discussed in the context of bone cell biology. Because the biological effects of matricellular proteins are largely context dependent, in vivo and in vitro results must be considered together in order to fully appreciate the specific contributions that matricellular proteins make to bone physiology and pathophysiology. In particular, it is clear that although matricellular proteins are not required for bone development and function, the proteins act to modulate post-natal bone structure in response to aging, ovariectomy, mechanical loading, and bone regeneration.     

Bone acid phosphatase: Tartrate-resistant acid phosphatase as a marker of osteoclast function

Summary Organ cultures of newborn mouse calvaria were used to test the hypothesis that tartrate-resistant acid phosphatase might serve as a biochemical marker for osteoclast function. When bone resorption was stimulatedin vitro with either parathyroid hormone or 1,25(OH)₂D₃, there was a significant increase in both tartrate-resistant and tartrate-sensitive acid phosphatase activity in the medium relative to cultured controls. Tartrate-resistant activity was localized histochemically primarily over the osteoclast and appeared as three distinct activity bands when electrophoresed on polyacrylamide gels. The tartrate-sensitive activity was found primarily associated with bone cells other than the osteoclast using histochemical techniques, and was resolved into five bands on polyacrylamide gels. The results obtained from biochemical assays, histochemical observations, and polyacrylamide gel electrophoresis suggest that bone resorptionin vitro results in the release of tartrate-resistant acid phosphatase from osteoclasts and tartrate-sensitive acid phosphatase from other bone cells as well as osteoclasts. Tartrate-resistant acid phosphatases of bone may be suitable biochemical probes for osteoclast function, but it will be necessary to achieve further purification in order to develop analytical methods with sufficient sensitivity and specificity (e.g., immunochemical) to ensure precise localization and quantitation.     

Inhibition of osteoclast differentiation by polycyclic aryl hydrocarbons is dependent on cell density and RANKL concentration

We investigated the effect of representative polycyclic aryl hydrocarbons (PAHs), benzo[a]pyrene (BaP), and 7,12-dimethylbenz[a]anthracene (DMBA) on osteoclast differentiation and function by using dispersed cancellous bone derived rabbit osteoclasts and the RAW264.7 cells. These cells differentiate into osteoclasts when exposed to receptor activator of NF-kappaB ligand (RANKL). The rabbit osteoclasts were exposed to 10(-6) to 10(-9)M BaP or DMBA and the tartrate-resistant acid phosphatase (TRAP)-positive cells were counted. The effect of PAHs on osteoclast differentiation in dispersed rabbit osteoclast-containing stromal cell populations was cell density dependent, suggesting that the cell density of stromal cells, osteoclast precursors, and/or mature osteoclasts are factors regulating the effect of PAHs. To investigate the direct effect of BaP on osteoclast differentiation, RAW264.7 cells were exposed to 10(-5) to 10(-6) M BaP. Treatment of RAW264.7 cells cultured with 25 ng/ml soluble RANKL and 10(-5)M BaP for 5 days decreased osteoclast differentiation, TRAP activity levels, and resorption of bone-like substrata. The inhibition was prevented by 10(-6) to 10(-7) M resveratrol, an aryl hydrocarbon receptor (AhR) antagonist, and by higher concentrations of RANKL. To investigate the ability of RANKL to reverse BaP-mediated inhibition, gene expression was determined by RT-PCR. Cytochrome P450 1B1 (CYP1B1) mRNA, one of the genes activated by BaP, was present only in the groups exposed to BaP; the levels of CYP1B1 mRNA decreased in the presence of increasing concentrations of RANKL. These results suggest that the inhibitory effects of PAHs on osteoclastogenesis are direct and likely involve interaction of the RANKL and PAH signaling pathways.     

微小RNAs在骨与软骨组织中的调节作用

目的对微小RNAs(microRNAs,miRNAs)在骨与软骨组织中的调控作用和作用机制作一综述。方法广泛查阅近年来有关miRNAs在骨与软骨组织的调控作用及作用机制的文献,进行总结与分析。结果 miRNAs是近年来骨关节疾病研究的热点,越来越多研究显示其在骨与软骨组织形成和代谢过程中,对于细胞分化、细胞基质分泌等方面发挥重要的调控作用,但确切机制尚不清楚。结论 通过对miRNAs在骨与软骨组织中的调控作用及作用机制的研究,可能为了解退行性骨关节疾病开辟新的领域。     

类风湿关节炎滑膜成纤维细胞通过高表达RANKL促进破骨细胞分化及活化

目的探讨类风湿关节炎滑膜成纤维细胞（RA-FLS）对破骨细胞（Oc）分化和活化的作用及机制。方法活动期RA滑膜体外分离培养FLS,以骨关节炎（OA）-FLS为对照,分别与健康人外周血单核细胞（MNC）共培养后,抗酒石酸酸性磷酸酶（TRAP）染色鉴定Oc并计数、甲苯胺蓝染色观察骨吸收陷窝情况。细胞免疫荧光染色检测FLS RANKL表达,Real-time PCR及W estern blot检测RANKL和OPG mRNA、蛋白表达。结果 RA-FLS与MNC共培养7 d时TRAP＋且细胞核≥3个的Oc很少,14 d时见较多的Oc,21 d甲苯胺蓝染色示清晰的骨吸收陷窝,而OA-FLS与MNC共培养后未见Oc,也未见骨吸收陷窝。细胞免疫荧光染色示RA-FLS较OA-FLS高表达RANKL（P〈0.05）。RA-FLS RANKL mRNA和蛋白表达较OA-FLS明显增高,而OPG mRNA和蛋白表达则明显降低,RANKL/OPG mRNA和蛋白比率较OA-FLS明显增高（均P〈0.05）。结论 RA-FLS可能通过高表达RANKL,促进外周血MNC向Oc分化,并促进Oc的骨吸收功能。     

牙周膜成纤维细胞对外周血单个核细胞分化的影响

目的：探讨牙周膜成纤维细胞在破骨细胞形成过程中作用;观察破骨样细胞的生长过程。方法：本实验以含有1α,25（OH）2D3和地塞米松的培养基将牙周膜成纤维细胞、单个核细胞分别进行单独或直接共培养,每3d对TRAP阳性多核破骨细胞的数量及牙本质磨片的吸收陷窝数目和面积分别进行记录、计算。结果：不同时间段间的TRAP阳性单个核细胞与TRAP阳性多核细胞数目相比较,差异具有统计学意义（P〈0.05）;同时,不同组间的吸收陷窝数目和面积比较,差异具有显著性（P〈0.001）。牙周膜成纤维细胞明显增加了共培养组TRAP阳性多核细胞数量、吸收陷窝数目和面积。然而牙周膜成纤维细胞组与单个核细胞组之间的吸收陷窝数目与吸收陷窝面积差异无统计学意义。结论：末梢血单个核细胞需在牙周膜成纤维细胞存在的条件下,才能形成多核的破骨样细胞。同时,在共培养中,可以发现破骨样细胞在体外存活的时间短暂。     

Osteal macrophages promote in vivo intramembranous bone healing in a mouse tibial injury model

Bone‐lining tissues contain a population of resident macrophages termed osteomacs that interact with osteoblasts in vivo and control mineralization in vitro. The role of osteomacs in bone repair was investigated using a mouse tibial bone injury model that heals primarily through intramembranous ossification and progresses through all major phases of stabilized fracture repair. Immunohistochemical studies revealed that at least two macrophage populations, F4/80⁺Mac‐2^?/lowTRACP^? osteomacs and F4/80⁺Mac‐2^hiTRACP^? inflammatory macrophages, were present within the bone injury site and persisted throughout the healing time course. In vivo depletion of osteomacs/macrophages (either using the Mafia transgenic mouse model or clodronate liposome delivery) or osteoclasts (recombinant osteoprotegerin treatment) established that osteomacs were required for deposition of collagen type 1⁺ (CT1⁺) matrix and bone mineralization in the tibial injury model, as assessed by quantitative immunohistology and micro–computed tomography. Conversely, administration of the macrophage growth factor colony‐stimulating factor 1 (CSF‐1) increased the number of osteomacs/macrophages at the injury site significantly with a concurrent increase in new CT1⁺ matrix deposition and enhanced mineralization. This study establishes osteomacs as participants in intramembranous bone healing and as targets for primary anabolic bone therapies. © 2011 American Society for Bone and Mineral Research.     

Parathyroid hormone regulates the distribution and osteoclastogenic potential of hematopoietic progenitors in the bone marrow

Parathyroid hormone (PTH) increases both the number of osteoclast in bone and the number of early hematopoietic stem cells (HSCs) in bone marrow. We previously characterized the phenotype of multiple populations of bone marrow cells with in vitro osteoclastogenic potential in mice. Here we examined whether intermittent administration of PTH influences these osteoclast progenitor (OCP) populations. C57BL/6 mice were treated with daily injections of bPTH(1–34) (80 µg/kg/day) for 7 or 14 days. We found that PTH caused a significant increase in the percentage of TN/CD115⁺CD117^high and TN/CD115⁺CD117^int cells (p < .05) in bone marrow on day 7. In contrast, PTH decreased the absolute number of TN/CD115⁺CD117^low cells by 39% on day 7 (p < .05). On day 14, there was no effect of PTH on osteoclast progenitor distribution in vivo. However, PTH treatment for 7 and 14 days did increase receptor activator of NF‐κB ligand (RANKL)– and macrophage colony‐stimulating factor (M‐CSF)–stimulated in vitro osteoclastogenesis and bone resorption in TN/CD115⁺ cells. In the periphery, 14 days of treatment increased the percentage and absolute numbers of HSCs (Lin^?CD117⁺Sca‐1⁺) in the spleen (p < .05). These data correlated with an increase in the percent and absolute numbers of HSCs in bone marrow on day 14 (p < .05). Interestingly, the effects on hematopoietic progenitors do not depend on osteoclast resorption activity. These results suggest that in vivo PTH treatment increased in vitro osteoclastogenesis and resorption without altering the number of osteoclast precursors. This implies that in vivo PTH induces sustained changes, possibly through an epigenetic mechanism, in the in vitro responsiveness of the cells to M‐CSF and RANKL. © 2011 American Society for Bone and Mineral Research.