载荷作用下骨细胞分泌因子对成骨细胞和破骨细胞的调节

骨细胞是骨组织主要的力学感受及转导细胞,它们通过众多突触结构相互连接,形成庞大的骨稳态细胞调控网络,联系着成骨细胞、破骨细胞等骨基质表面细胞。骨细胞通过旁分泌途径影响成骨细胞骨形成和破骨细胞骨吸收来调节骨代谢,维持骨更新。针对骨细胞在受到力学刺激后分泌或释放的一些信号分子或蛋白因子对成骨细胞和破骨细胞生长分化的影响,本文综述近年来关于受力学刺激的骨细胞如何与成骨/破骨细胞进行通讯,为骨细胞生物力学研究提供新思路。     

PTH对破骨细胞骨吸收功能的影响及成骨细胞介导作用

采用分离、培养兔破骨细胞和成骨细胞的方法，体外研究甲状旁腺激素（ＰＴＨ）对破骨细胞骨吸收功能的影响，以及成骨细胞和破骨细胞之间的相互作用。结果表明，ＰＴＨ对破骨细胞的骨吸收功能无直接影响，但在成骨细胞参与下，ＰＴＨ对破骨细胞性骨吸收有明显的促进作用。说明成骨细胞在ＰＴＨ调节破骨细胞功能活动中有着重要的介导作用。     

GM-CSF和其诱导的破骨细胞对成骨细胞的影响

目的：探讨GM-CSF和其诱导的破骨细胞对成骨细胞生长的影响。方法：采用分组对照、细胞计数等方法进行研究。结果：GM-CSF和其诱导的破骨细胞可以促进成骨细胞生长。结论：GM-CSF和其诱导的破骨细胞促进成骨细胞增殖、分化，可能是通过破骨细胞内GM-CSF介导的信号通路而发挥作用。     

成骨细胞参与白细胞介素—1调节破骨细胞功能的实验研究

建立了成骨细胞和破骨细胞共同培养体系，探讨了白细胞介素－１（ＩＬ１）促进骨吸收的作用机理。研究发现，破骨细胞＋ＩＬ１组的骨吸收陷窝数目和面积，与单纯破骨细胞组比较，差异无显著性意义。破骨细胞＋成骨细胞＋ＩＬ１组，骨吸收陷窝数目和面积均显著增加，与破骨细胞＋ＩＬ１组及单纯破骨细胞对照组比较，差异均有显著性意义。提示ＩＬ１对破骨细胞缺乏直接作用，而是在成骨细胞介导下，发挥调节破骨细胞的骨吸收作用。     

力学载荷对破骨细胞凋亡的影响及其调节机制

破骨细胞是参与骨代谢的基本功能细胞之一.破骨细胞在骨重建过程中主要承担旧骨组织的破坏和吸收,因此,破骨细胞凋亡的微小变化都可能会改变骨重建的进程.调节破骨细胞凋亡的因素有很多,如雌激素、二磷酸盐等生物化学因素,但力学载荷对于破骨细胞生物学活性影响的研究相对较少.综述了力学载荷对破骨细胞生物学活性的影响以及细胞凋亡与破骨细胞凋亡的调节.     

从细胞水平研究龟鹿二仙含药血清在治疗骨质疏松中的作用

从细胞水平研究龟鹿二仙含药血清在治疗骨质疏松中的作用.(1)用酶消化法获得新生大鼠的成骨细胞.碱性磷酸酶(ALP)染色进行鉴定,通过对成骨细胞增殖、胰岛素样生长因子(IGF-1)分泌的检测来考察成骨细胞功能.(2)用1,25(OH)2D3诱导骨髓单核细胞获得破骨细胞,抗酒石酸酸性磷酸酶(TRAP)染色鉴定破骨细胞.电镜扫描观察破骨细胞形成的骨凹陷情况;同时检测TRAP( )细胞数和破骨细胞TRAP活性.(3)用血清药理学方法制备龟鹿二仙含药血清,分别加入不同剂量组10%含药血清进行干预,观察该中药血清对上述成骨细胞和破骨细胞功能指标的影响.龟鹿二仙高剂量组血清可促进成骨细胞增殖,增加IGF-1的分泌量.而中剂量组血清可明显抑制骨髓细胞向破骨细胞的转化,抑制骨凹陷形成,降低细胞内TRAP的活性.本研究表明龟鹿二仙具有显著的抗骨质疏松作用.     

建立小鼠成骨与破骨细胞相互作用的共育新体系

背景：众所周知,骨重建是骨组织中重要的生物学反应过程,其中成骨细胞与破骨细胞发挥了关键作用。但目前,关于骨重建中成骨与破骨细胞间信号传递的深层机制还不清楚。 目的：利用transwell技术,在体外建立一种成骨与破骨细胞的新型共育体系,为深入研究骨重建中成骨与破骨细胞的相互作用提供成熟的实验模型。 方法：采用MC3T3-E1成骨样细胞株与RAW264.7破骨前体细胞株,进行体外成骨与破骨细胞的诱导分化,并利用Transwell共培养板(0.4 µm聚酯膜)建立成骨与破骨细胞的共育体系。共培养6 d后,通过测定细胞活性和碱性磷酸酶(ALP)活力分析成骨细胞的增殖和分化活性,利用抗酒石酸酸性磷酸酶(TRAP)染色、甲苯胺蓝(TB)染色、TRAP活性测定及扫描电镜技术观察破骨细胞的分化及骨吸收功能。 结果与结论：共培养体系中成骨样细胞的无限增殖能力减弱,而分化活性明显增强,同时破骨前体细胞被诱导分化为成熟的破骨细胞,并具有一定的骨吸收功能。因此,该共培养体系可用于骨重建中成骨与破骨细胞间信号通路的深层研究。     

应用破骨细胞-颅骨联合培养体系研究先天性成骨不全的骨再建过程

目的　采用先天性成骨不全(OI)小鼠,oim/oim为动物模型,应用破骨细胞-颅骨联合培养体系研究OB和OC两种细胞在OI骨再建过程中的功能改变和相互作用。 方法　实验采用小鼠颅骨（CAL）组织培养,实验设两组：WTCAL-WTOC组：联合培养对照组颅（WTCAL） 与对照破骨细胞（WTOC）;OICAL-OIOC组：联合培养OI颅骨（OICAL）与OI破骨细胞（OIOC）。以免疫组化染色方法　-TRAP识别破骨细胞,ALP免疫组化染色方法识别成骨细胞。破骨细胞骨吸收活性为骨吸收陷窝占颅骨表面百分比。单位OC吸收面积为总骨吸收陷窝除以破骨细胞数。 结果　于7d,OICAL-OIOC组破骨细胞数低于WTCAL-WTOC组;OICAL-OIOC组的OC/OB比例低WTCAL-WTOC组;OICAL-OIOC组单位破骨细胞吸收能力高于WTCAL-WTOC组。 结论　OI的小鼠模型骨再建中骨量丢失一方面由于其成骨细胞功能异常,另一方面也可能因为其破骨细胞的代偿性功能活跃。     

补肾接骨中药对骨折修复的成骨作用及机制

背景：骨折愈合是成骨细胞与破骨细胞耦联相互作用相互影响促进骨质生长的过程,其中成骨细胞介导骨的形成,破骨细胞介导骨吸收,使骨重建处于一种动态平衡中,于动态平衡环境中促进骨生长,而目前研究多数倾向于单独的成骨或者破骨机制,但会忽略这两种细胞共同存在的环境下相互作用机制。 目的：观察补肾接骨中药对成骨与破骨细胞耦联中骨护骨素-核因子κB受体激活剂的配基-核因κB受体活化因子的影响及其在骨折治疗中的作用机制。 方法：分离小鼠成骨、破骨细胞并体外细胞培养,建立小鼠“成骨-破骨细胞共育系”作为研究平台,补肾接骨中药1.25,2.5,6.25 g/(kg•d)灌胃,空白对照组给予同等体积的生理盐水灌胃,每日1次,连续7 d。 结果与结论：共育培养24 h后,成骨细胞内碱性磷酸酶水平明显高于单纯培养的成骨细胞(P < 0.05)。实时PCR检测显示,共育体系细胞中碱性磷酸酶、Runx2及骨护骨素表达增加(P < 0.05),呈剂量依赖性(P < 0.05)。Western-blot检测显示,高浓度[6.25 g/(kg•d)]中药能明显促进骨护骨素、核因子κB受体激活剂的配基的表达,抑制核因子κB受体活化因子蛋白表达(P < 0.05)。结果证实,补肾接骨中药可动态调节骨护骨素-核因子κB受体激活剂的配基-核因子κB受体活化因子信号通路,对促进骨组织重建与恢复有着积极的作用。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

抗酒石酸酸性磷酸酶染色方法的改良

<正>破骨细胞是一种执行骨吸收功能的细胞,其清晰辨识对骨组织生理病理学研究及代谢性骨病的研究具有十分重要的意义。当机体内破骨细胞功能活跃时,胞质内产生大量的抗酒石酸酸性磷酸酶(tartrate-resistnt acid phosphatase,TRAP)。因此,鉴定破骨细胞的TRAP染色技术在病理诊断中不可或缺。TRAP作为破骨细胞的特异性标志酶,是鉴别破骨细胞的重要标志~([1]),应用TRAP染色法可显示破骨细胞     

Bone stromal cells in pagetic bone and Paget's sarcoma express RANKL and support human osteoclast formation

Paget's disease is a focal disorder of bone remodelling, in which there is an increase in osteoclast formation. A rare complication of Paget's disease is the development of a sarcoma, most commonly an osteosarcoma. Osteoclast formation occurs in the presence of macrophage-colony stimulating factor and receptor activator for nuclear factor-kappaB ligand (RANKL), and it has been shown that bone stromal cells in Paget's disease can influence osteoclast formation by modulating the expression of RANKL and its decoy receptor, osteoprotegerin (OPG). In this study we show that pagetic bone stromal cells express RANKL and that these cells promote osteoclast formation by a RANKL-dependent mechanism. Osteoclast formation in co-cultures of monocytes and either pagetic bone stromal cells or Paget's sarcoma stromal cells was not only induced by a contact-dependent mechanism but also occurred via the release of a soluble factor. In contrast to bone stromal cells isolated from normal controls, stromal cells isolated from morphologically normal bone in one patient with Paget's disease also stimulated osteoclast formation in this way; this osteoclastogenesis was inhibited by OPG. Our results indicate that Paget's bone stromal cells support osteoclast formation by a RANKL-dependent process which involves not only cell-cell contact but also secretion of soluble RANKL.     

Ewing sarcoma cells express RANKL and support osteoclastogenesis

Ewing sarcoma (ES) is a primary malignant round cell tumour of bone characterized by rapid and extensive osteolysis. Cellular mechanisms underlying the rapid bone resorption in ES have not been characterized. Osteoclasts are marrow-derived multinucleated cells that effect tumour osteolysis. The role of ES tumour cells in influencing osteoclast formation and/or directly contributing to the osteolysis in ES has not been determined. Using a tissue culture bioassay, we found that lacunar resorption is not carried out by (CD99(+) ) ES tumour cells, but by (CD68(+) ) macrophage/osteoclast-like cells; this resorption occurred in the absence of the osteoclastogenic factor, receptor activator of nuclear factor κB ligand (RANKL). ES cell lines cultured directly on dentine slices did not resorb the mineral or organic components of the bone matrix. Immunohistochemistry of ES tissue microarrays, western blotting, and RT-PCR studies showed that ES cells strongly expressed both RANKL and macrophage-colony stimulating factor (M-CSF), two major osteoclastogenic factors. When co-cultured with human monocytes, ES cells induced the formation of TRAP(+) osteoclastic cells. Conditioned medium from cultured ES cells did not result in osteoclast formation, indicating that cell-cell contact is required for ES-induced osteoclastogenesis. Our findings indicate that ES cells do not resorb bone directly but that they may support osteoclast formation by a RANKL-dependent mechanism.     

Hepatocellular carcinoma presenting with multiple bone and soft tissue metastases and atypical cytomorphological features—A rare case report

Hepatocellular carcinoma (HCC) with atypical cytomorphological features and presenting with bone and soft tissue metastasis is very rare. We report a 65‐year‐old male patient of HCC who presented with bone and soft tissue metastases and was clinically and radiologically suspected to have a soft tissue sarcoma. The patient presented with severe cervical pain with palpable masses in right scapular, nape of neck, and occiput area of scalp. Radiologically, these were large, bulky soft tissue masses expansile, destructive, and lytic in nature. Cytomorphologic studies revealed HCC with uncommon features of multinucleated osteoclast‐like giant cell and very prominent intracytoplasmic hyaline bodies (IHBs). Cytology, immunohistochemistry on cell block preparation, rising serum α‐fetoprotein (AFP) levels (1121.93–5000 ng/ml), and PIVKA II levels confirmed the diagnosis. The patient has been on follow‐up on sorafinib for 2 months and is doing well. This case emphasizes the need for systematic approach in cases of HCC with atypical clinical presentation and unusual cytomorphology. Diagn. Cytopathol. 2013. © 2011 Wiley Periodicals, Inc.     

Multiple increased osteoclast functions in individuals with neurofibromatosis type 1

Skeletal abnormalities including scoliosis, tibial dysplasia, sphenoid wing dysplasia, and decreased bone mineral density (BMD) are associated with neurofibromatosis type 1 (NF1). We report the cellular phenotype of NF1 human‐derived osteoclasts and compare the in vitro findings with the clinical phenotype. Functional characteristics (e.g., osteoclast formation, migration, adhesion, resorptive capacity) and cellular mechanistic alterations (e.g., F‐actin polymerization, MAPK phosphorylation, RhoGTPase activity) from osteoclasts cultured from peripheral blood of individuals with NF1 (N = 75) were assessed. Osteoclast formation was compared to phenotypic, radiologic, and biochemical data. NF1 osteoprogenitor cells demonstrated increased osteoclast forming capacity. Human NF1‐derived osteoclasts demonstrated increased migration, adhesion, and in vitro bone resorption. These activities coincided with increased actin belt formation and hyperactivity in MAPK and RhoGTPase pathways. Although osteoclast formation was increased, no direct correlation of osteoclast formation with BMD, markers of bone resorption, or the clinical skeletal phenotype was observed suggesting that osteoclast formation in vitro cannot directly predict NF1 skeletal phenotypes. While NF1 haploinsufficiency produces a generalized osteoclast gain‐in‐function and may contribute to increased bone resorption, reduced BMD, and focal skeletal defects associated with NF1, additional and perhaps local modifiers are likely required for the development of skeletal abnormalities in NF1. © 2011 Wiley‐Liss, Inc.     

Osteoblast: Osteoclast co-cultures on silk fibroin, chitosan and PLLA films

This study investigates the growth of a co-culture of osteoblasts and osteoclasts on four different types of degradable biomaterials with bone tissue engineering potential. Single or co-cultures of osteoblasts and osteoclasts (used at a ratio of 1:100 osteoblast:osteoclasts) were cultured on vapour stabilised silk fibroin, methanol stabilised silk fibroin, chitosan and poly (l lactic acid) (PLLA) films for 10 days. Osteoclast differentiation was determined by tartrate resistant acid phosphatase (TRAP) staining, total cell number by a picogreen DNA assay, cell morphology by scanning electron microscopy (SEM) and the material topography by atomic force microscopy (AFM). Samples were also monitored for degradation by differential scanning calorimetry (DSC) and fourier transform infrared (FTIR). Results demonstrated that vapour stabilised silk fibroin, methanol stabilised silk fibroin and chitosan all support the growth of osteoblasts and osteoclasts in both single and co-cultures. PLLA showed poor osteoclast differentiation in both single and co-cultures but supported osteoblast attachment and proliferation. Both silk fibroin materials showed sign of early degradation in the ten-day period, but very little change was seen in chitosan and PLLA samples. This study indicates that this novel co-culture approach for bone tissue engineering may be possible if scaffolds are created from silk fibroin or chitosan.     

Paeoniflorin ameliorates collagen‐induced arthritis via suppressing nuclear factor‐κB signalling pathway in osteoclast differentiation

Paeoniflorin (PF), extracted from the root of Paeonia lactiflora Pall, exhibits anti‐inflammatory properties in several autoimmune diseases. Osteoclast, the only somatic cell with bone resorbing capacity, was the direct cause of bone destruction in rheumatoid arthritis (RA) and its mouse model, collagen‐induced arthritis (CIA). The objective of this study was to estimate the effect of PF on CIA mice, and explore the mechanism of PF in bone destruction. We demonstrated that PF treatment significantly ameliorated CIA through inflammatory response inhibition and bone destruction suppression. Furthermore, PF treatment markedly decreased osteoclast number through the altered RANKL/RANK/OPG ratio and inflammatory cytokines profile. Consistently, we found that osteoclast differentiation was significantly inhibited by PF through down‐regulation of nuclear factor‐κB activation in vitro. Moreover, we found that PF suppressed nuclear factor‐κB activation by decreasing its translocation to the nucleus in osteoclast precursor cells. Taken together, our new findings provide insights into a novel function of PF in osteoclastogenesis and demonstrate that PF would be a new therapeutic modality as a natural agent for RA treatment and other autoimmune conditions with bone erosion.     

Regulation of the differentiation and function of osteoclasts

The osteoclast is the cell that resorbs bone. It has been known for many years that its formation and function are regulated by cells of the osteoblastic lineage. Recently the molecular basis for this regulation was identified; osteoblastic cells induce osteoclastic differentiation and resorptive activity through expression of tumour necrosis factor (TNF) activation-induced cytokine (TRANCE) (also known as RANKL, ODF, OPGL, and TNFSF11), a novel membrane-inserted member of the TNF superfamily. Osteoclastic regulation is assisted through secretion of an inhibitor, osteoprotegerin (OPG) (OCIF, TNFRSF11B), a soluble (decoy) receptor for TRANCE. Osteoclast formation and survival also depend on and are substantially enhanced by transforming growth factor-beta (TGF-beta), which is abundant in bone matrix. Surprisingly, not only TRANCE but also TNF-alpha can induce osteoclast formation in vitro from bone marrow-derived mononuclear phagocytes, especially in the presence of TGF-beta. Whether or not TNF-alpha does the same in vivo, its ability to generate osteoclasts in vitro has significant implications regarding the nature of osteoclasts and their relationship to other mononuclear phagocytes, and a possible wider role for TRANCE in macrophage pathobiology. A hypothesis is presented in which the osteoclast is a mononuclear phagocyte directed towards a debriding function by TGF-beta, activated for this function by TRANCE, and induced to become specifically osteoclastic by the characteristics of the substrate or signals from bone cells that betoken such characteristics.     

破骨细胞功能调控与骨质疏松症

Osteoclast(OC)are bone resorbing cells whose development and activity are under the influence of osteoblast(OB).The potential association between those two cell lineages is an area of significant interest.It was not until 1998 that osteoclast differentiation factor(ODF)and osteoprotegerin(OPG)was clonedl.The two novel TNF superfamily members have been recently demonstrate to be the key extracellular regulators of osteoclasteogenesis and bone resorption both in vivo and in vitro,and they play important roles in osteoporosis development,diganosis and treatment.     

OSTEOCLASTS ARE CAPABLE OF PARTICLE PHAGOCYTOSIS AND BONE RESORPTION

Osteoclasts are multinucleated cells specialized for the function of lacunar bone resorption. Although they are known to be capable of phagocytosis of inert particles, it is not known whether this abolishes their ability to respond to hormones or to form resorption lacunae. Human and rat osteoclasts were isolated from giant cell tumours of bone and rat long bones, respectively, and cultured on coverslips and cortical bone slices, both in the presence and in the absence of particles of latex (1 μm diameter) and polymethylmethacrylate (PMMA) (<50 μm). By light microscopy, it was evident that osteoclasts which had phagocytosed both latex and PMMA particles remained responsive to calcitonin. Osteoclast phagocytosis of particles was also evident on scanning electron microscopy, where it could also be seen that these cells were associated with the formation of resorption lacunae. These findings underline the fact that the osteoclast is a true member of the mononuclear phagocyte system and that phagocytosis does not abrogate either its hormonal response to calcitonin or its highly specialized function of bone resorption. That osteoclasts which have phagocytosed biomaterial particles such as PMMA are still able to carry out lacunar bone resorption is of interest in clinical conditions such as aseptic loosening, where a heavy foreign body particle load is often associated with extensive bone resorption. © 1997 John Wiley & Sons, Ltd.