低氧环境中PADI4促进破骨细胞分化和骨吸收功能的研究

为探讨低氧环境中肽酰基精氨酸脱亚胺酶4(peptidyl arginine deiminase 4,PADI4)对破骨细胞分化和功能的影响,采用巨噬细胞集落刺激因子(macrophage colony-stimulating factor, M-CSF)和核因子κB受体活化因子配体(receptor activator of nuclear factor-κB ligand, RANKL)诱导单核巨噬细胞白血病细胞RAW264.7向破骨细胞分化,采用PADI4抑制剂Cl-amidine和PADI4-siRNA-MSN抑制PADI4表达,采用抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase, TRAP)染色试验、TRAP活性试验和骨吸收试验鉴定破骨细胞的分化和功能,用qRT-PCR和Western blotting分别检测PADI4、TRAP基因及其蛋白的表达水平。结果显示,低氧-M-CSF+RANKL组TRAP和PADI4 mRNA相对表达水平较常氧-M-CSF+RANKL组显著升高(P0.05)。低氧-M-CSF+RANKL组的TRAP阳性细胞数[(7.33±1.37)个/HP]显著高于常氧-M-CSF+RANKL组[(3.33±1.63)个/HP,P0.01],低氧-M-CSF+RANKL组较常氧-M-CSF+RANKL组TRAP活性显著增加(P0.001)。低氧-M-CSF+RANKL组骨吸收陷窝数[(107.00±12.42)个/片]较常氧-M-CSF+RANKL组[(77.40±8.79)个/片]显著增加(P0.05)。在低氧环境下,与对照组比较,M-CSF+RANKL+Cl-amidine组和M-CSF+RANKL+siRNA组TRAP mRNA表达水平、TRAP蛋白表达水平、TRAP阳性细胞数、TRAP活性、骨吸收陷窝数均显著降低(P0.05)。该研究提示,在低氧环境中PADI4可能是促进巨噬细胞向破骨细胞分化以及骨吸收的关键分子。     

人多发性骨髓瘤RPMI 8226细胞条件培养液诱导破骨前体细胞的分化成熟

目的: 探讨人多发性骨髓瘤RPMI 8226细胞条件培养液对破骨前体细胞RAW264.7的影响。方法: Western blotting检测可溶性核因子κB 受体激活剂配体(soluble receptor activator of NF-κB ligand, sRANKL)的蛋白表达。抗酒石酸酸性磷酸酶(tartrate－resistant acid phosphatase, TRAP)染色观察RAW264.7细胞的分化成熟情况。RT-PCR法检测RAW264.7细胞TRAP和cathepsin K mRNA的表达。结果: Western blotting法证实RPMI 8226细胞条件培养液中含有sRANKL。TRAP染色发现RPMI 8226细胞条件培养液能诱导RAW264.7细胞分化为TRAP阳性的多核成熟破骨细胞。人抑制性RANKL单克隆抗体拮抗30%RPMI 8226细胞条件培养液中sRANKL的作用,且具有剂量依赖性。30% RPMI 8226细胞条件培养液能刺激RAW264.7细胞上调TRAP和cathepsin K mRNA表达。结论: 人多发性骨髓瘤细胞RPMI 8226条件培养液中的sRANKL具有促RAW264.7破骨前体细胞分化成TRAP阳性的多核成熟破骨细胞的生物活性。人抑制性RANKL单克隆抗体阻断30%RPMI 8226细胞条件培养液中sRANKL的诱导分化作用,且具有浓度依赖性。     

子宫颈癌中CD147蛋白和MMP-9 mRNA的表达及意义

目的 探讨CD147蛋白、MMP-9 mRNA在子宫颈鳞状细胞癌中的表达及其与侵袭、转移的关系.方法 采用免疫组化MaxVision法、RT-PCR技术检测10例慢性子宫颈炎组织(对照组)和40例子宫颈鳞状细胞癌患者的外科手术切除标本组织中CD147蛋白、MMP-9 mRNA的表达,应用图像分析软件分别对CD147和MMP-9 mRNA的表达进行平均光密度和灰度分析.结果 CD147蛋白和MMP-9 mRNA的表达结果显示:子宫颈鳞状细胞癌组高于对照组慢性子宫颈炎组织(P值均＜0.01);有淋巴转移组高于无转移组(P＜0.01、P＜0.05);CD147蛋白在宫颈鳞状细胞癌低分化组表达与中分化组表达差异无显著性(P＞0.05),但MMP-9 mRNA表达为低分化组高于中分化组(P＜0.05).结论 CD147蛋白和MMP-9 mRNA在子宫颈鳞状细胞癌中呈高表达,它们之间的表达具有正相关性,二者可能共同参与了子宫颈鳞状细胞癌的侵袭和转移过程.     

Wnt/β-catenin信号途径抑制Raw264.7分化

目的 了解Wnt/β-catenin信号途径对小鼠单核/巨噬细胞Raw264.7的分化调控.方法 将细胞分为4组:阴性对照组(即空Raw264.7组)、实验对照组(即感染Ad-GFP组)、阳性对照组(即50 ng/mL RANKL诱导组)和实验组(即感染Ad-β-catenin组).分别给予上述4组处理因素后常规细胞培养3、6和9d提取细胞总RNA,荧光定量real-time (RT-PCR)检测核因子受体(RANK)、抗酒石酸酸性磷酸酶(TRAP)、组织蛋白酶K(Cathepsin K)及金属蛋白酶-9(MMP-9)mRNA的表达,再于同样处理因素培养6d通过酒石酸抗酸性磷酸酶(TRAP)染色观察Raw264.7的分化情况;Western blot检测TRAP、Cathepsin K蛋白的表达.结果 RT-PCR检测Ad-β-catenin组能下调RANK、TRAP、Cathepsin K和MMP-9 mRNA的表达;TRAP染色显示50 ng/mL RANKL诱导组能成功诱导Raw264.7成多核细胞,空白组和Ad-GFP组有个别多核细胞,Ad-β-catenin组为单核细胞;Western blot检测显示Ad-β-catenin组TRAP、Cathepsin K蛋白表达降低(P＜0.05).结论 Wnt/β-catenin信号途径能抑制RAW264.7分化成熟为破骨细胞.     

1, 25二羟基胆钙化醇诱导大鼠骨髓单核细胞向破骨细胞转化的机制

目的:探讨1,25二羟基胆钙化醇(1,25(OH)2D3)诱导大鼠骨髓单核细胞向破骨细胞转化时明胶酶表达及其参与骨陷窝形成机制。方法:分离乳鼠骨髓内细胞,诱导生成破骨样细胞。姬姆莎、抗酒石酸酸性磷酸酶(TRAP)染色鉴定。扫描电镜观察诱导出的细胞贴附于骨片上形成的骨陷窝,明胶酶谱检测细胞培养液中明胶酶表达水平。结果:单核细胞经1,25(OH)2D3诱导,第9日生成大量的破骨样细胞。姬姆莎染色显示出多核(≥3个),TRAP染色阳性,扫描电镜观察破骨细胞在骨片上培养时产生骨陷窝。1,25(OH)2D3组基质金属蛋白酶2(MMP-2)表达水平增加显著。结论:1,25(OH)2D3诱导单核细胞产生大量破骨细胞。参与骨陷窝形成的明胶酶是MMP-2。这可能是破骨细胞通过某些机制,促进其他非破骨细胞分泌有活性的MMP-2参与噬骨。     

下调骨细胞TGF-β/Smad4信号可抑制小鼠BMSCs成骨及破骨分化

目的检测骨细胞TGF-β/Smad4信号通路对骨髓间充质干细胞(BMSCs)成骨和破骨分化的作用,并初步探讨其相关机制。方法用条件性基因敲减Cre/loxp技术特异性敲减骨细胞Smad4,获得下调骨细胞TGF-β/Smad4信号通路的小鼠;体外分离骨细胞并与野生型小鼠骨髓间充质细胞(BMSCs)共培养;碱性磷酸酶(ALP)染色、茜素红(alizarin red)染色检测早期成骨分化和晚期钙盐沉积,酸性磷酸酶(TRAP)染色检测破骨细胞;real-time PCR检测成骨分化特异标志物Runx2、Osterix(OSX)、ALP、osteocalcin和破骨分化特异标志物RANKL和OPG的mRNA表达水平。Western blot检测成骨分化特异标志物Runx2和osteocalcin和破骨分化特异标志物RANK蛋白表达水平。结果下调骨细胞TGF-β/Smad4信号能够抑制BMSCs成骨转录因子Runx2、Osterix(P0.01)、成骨分化特异标志物ALP和osteocalcin(P0.01)以及破骨分化特异标志物RANK(P0.01)的表达;增加破骨分化抑制物OPG的表达(P0.05);而RANKL的表达无明显变化;最终下调了RANKL/OPG的比值(P0.05)。结论终末分化的骨细胞调控骨的代谢,下调其TGF-β/Smad4信号可抑制BMSCs成骨和破骨细胞的分化。     

低强度高频率振动对成骨细胞生物学特性的影响

目的　研究低强度高频率振动（low-magnitude high-frequency vibration, LMHFV）对成骨细胞生物学特性的影响。 方法　建立LMHFV加载MC3T3-E1细胞模型,观察不同频率LMHFV对MC3T3-E1细胞OPG/RANKL浓度比的影响,获得OPG/RANKL浓度比最高的频率（F）为后续研究频率;以0 Hz为对照,观察LMHFV对MC3T3-E1细胞碱性磷酸酶 (ALP)、骨钙素(OCN) mRNA和蛋白活性,及钙化结节形成的影响;LMHFV加载形成的条件培养液（CMF）孵育RAW264.7细胞,观察CMF对破骨细胞抗酒石酸酸性磷酸酶（TRAP）染色、多核破骨细胞形成、TRAP mRNA及蛋白活性的影响;观察LMHFV对MC3T3-E1细胞环氧化酶2(COX-2)蛋白水平的表达及COX-2抑制剂NS-398对LMHFV影响MC3T3-E1细胞分化的作用。 结果　30 Hz LMHFV获得OPG/RANKL浓度比最高,促进ALP、OCN mRNA及蛋白活性增加,增加钙化结节形成。30 Hz LMHFV形成的CM抑制RAW264.7细胞向多核破骨细胞分化,抑制TRAP mRNA及活性;LMHFV可诱导COX-2蛋白水平增加,NS-398能抑制LMHFV促进成骨细胞分化。 结论　30 Hz的LMHFV对MC3T3-E1细胞OPG/RANKL浓度比及成骨分化具有积极的影响,通过调控成骨细胞OPG/RANKL浓度比间接抑制骨吸收,COX-2通路参与了LMHFV对成骨细胞生物学特性的调节作用。     

活性维生素D3联合力学拉伸对成骨细胞MC3T3-E1增殖、分化及OPG和RANKL表达的影响

目的体外模拟成骨细胞在体内的生存环境,考察活性维生素D3(VD3)、力学拉伸以及两者联合对成骨细胞MC3T3-E1增殖、分化及破骨细胞抑制因子(OPG)和破骨细胞分化因子(RANKL)表达的影响。方法将10 nmol/L VD3、间断性力学拉伸以及两者联合作用于成骨细胞。流式细胞术检测细胞增殖。荧光探针试剂盒检测碱性磷酸酶(ALP)活性。实时定量PCR检测核心转录因子Runx2、OPG、RANKL mRNA水平,Western blotting检测其蛋白表达。结果 VD3抑制成骨细胞增殖,力学拉伸不能改变这种抑制效应。力学拉伸和VD3单独作用成骨细胞均能增加ALP活性及提高ALP、Runx2 mRNA水平,但当联合刺激后这些指标均降低,且成强度依赖性。力学拉伸增加OPG/RANKL比值,增强成骨作用,联合VD3后,OPG/RANKL比值下降。结论力学拉伸能有效诱导成骨分化,增加骨形成。VD3与力学拉伸联合抑制成骨细胞增殖和分化,并通过增加RANKL表达而影响骨重建。研究结果为骨质疏松及相关骨疾病的理论和治疗提供有意义的探索。     

活性维生素D3联合力学拉伸对成骨细胞MC3T3-E1增殖、分化及OPG和RANKL表达的影响（英文）

目的体外模拟成骨细胞在体内的生存环境,考察活性维生素D3(VD3)、力学拉伸以及两者联合对成骨细胞MC3T3-E1增殖、分化及破骨细胞抑制因子(OPG)和破骨细胞分化因子(RANKL)表达的影响。方法将10 nmol/L VD3、间断性力学拉伸以及两者联合作用于成骨细胞。流式细胞术检测细胞增殖。荧光探针试剂盒检测碱性磷酸酶(ALP)活性。实时定量PCR检测核心转录因子Runx2、OPG、RANKL mRNA水平,Western blotting检测其蛋白表达。结果 VD3抑制成骨细胞增殖,力学拉伸不能改变这种抑制效应。力学拉伸和VD3单独作用成骨细胞均能增加ALP活性及提高ALP、Runx2 mRNA水平,但当联合刺激后这些指标均降低,且成强度依赖性。力学拉伸增加OPG/RANKL比值,增强成骨作用,联合VD3后,OPG/RANKL比值下降。结论力学拉伸能有效诱导成骨分化,增加骨形成。VD3与力学拉伸联合抑制成骨细胞增殖和分化,并通过增加RANKL表达而影响骨重建。研究结果为骨质疏松及相关骨疾病的理论和治疗提供有意义的探索。     

Mac-1在破骨细胞分化中的作用

目的:探究巨噬细胞分化抗原1(Mac-1)分子在核因子κB受体活化因子配体(RANKL)诱导的破骨细胞分化中作用的分子机制。方法:取4周龄C57BL/6J小鼠脾细胞,用RANKL及巨噬细胞集落刺激因子(MCSF)诱导,同时用抗CD11b及抗CD18的特异性抗体进行处理,1周后对细胞核和细胞骨架进行染色;用抗CD11b抗体、干扰CD11b基因的慢病毒及其对照空载病毒处理RANKL诱导的破骨细胞,1周后对细胞进行免疫荧光染色;取4周龄C57BL/6J小鼠脾细胞用RANKL及M-CSF诱导,同时用抗CD11b抗体、干扰CD11b的慢病毒及其对照空载病毒分别进行处理,1周后提取总蛋白进行Western blot检测。结果:CD11b抗体组和双抗体组的多核细胞形成率低于对照组和CD18抗体组,双抗体组和CD11b抗体组之间多核细胞形成率的差异不具有统计学显著性;免疫荧光双标结果显示病毒组的CD11b、Syk及NFATc1表达强度均低于对照病毒组,CD11b抗体组的CD11b、Syk及NFATc1表达强度均低于对照组;Western blot结果显示,CD11b抗体组的CD11b、Syk、NFATc1、c-Fos及p-ERK/ERK水平均低于对照组,病毒组的CD11b、Syk、NFATc1、c-Fos及p-ERK/ERK水平均低于对照病毒组。结论:Mac-1分子中CD11b亚基对破骨细胞分化具有促进作用。该分子通过激活下游Syk通路,上调c-Fos,增加ERK活性,最终上调NFATc1而促进破骨细胞的分化。     

CD14- mononuclear stromal cells support (CD14+) monocyte-osteoclast differentiation in aneurysmal bone cyst

Aneurysmal bone cyst (ABC) is a benign osteolytic bone lesion in which there are blood-filled spaces separated by fibrous septa containing giant cells. The nature of the giant cells in this lesion and the mechanism of bone destruction in ABC is not certain. In this study, we have analysed several characteristics of mononuclear and multinucleated cells in the ABC and examined the cellular and molecular mechanisms of ABC osteolysis. The antigenic and functional phenotype of giant cells in ABC was determined by histochemistry/immunohistochemistry using antibodies to macrophage and osteoclast markers. Giant cells and CD14+ and CD14- mononuclear cells were isolated from ABC specimens and cultured on dentine slices and coverslips with receptor activator of nuclear factor κB ligand (RANKL)+/- macrophage-colony stimulating factor (M-CSF) and functional and cytochemical evidence of osteoclast differentiation sought. Giant cells in ABC expressed an osteoclast-like phenotype (CD51+, CD14-, cathepsin K+, TRAP+) and were capable of lacunar resorption, which was inhibited by zoledronate, calcitonin and osteoprotegerin (OPG). When cultured with RANKL±M-CSF, CD14+, but not CD14-, mononuclear cells differentiated into TRAP+ multinucleated cells that were capable of lacunar resorption. M-CSF was not necessary for osteoclast formation from CD14+ cell cultures. CD14- cells variably expressed RANKL, OPG and M-CSF but supported osteoclast differentiation. Our findings show that the giant cells in ABC express an osteoclast-like phenotype and are formed from CD14+ macrophage precursors. CD14- mononuclear stromal cells express osteoclastogenic factors and most likely interact with CD14+ cells to form osteoclast-like giant cells by a RANKL-dependent mechanism.     

Cell characterization of mononuclear and giant cells constituting pigmented villonodular synovitis

The aim of this study was to determine the histologic and cellular characteristics of 2 cell types, mononuclear cells (Mos) and multinuclear giant cells (GCs), that predominantly constitute pigmented villonodular synovitis (PVS). Synovial tissues examined in this study were obtained from 10 patients with PVS. Five methods were used for cell analysis: (1) enzyme-histochemistry for tartrate-resistant acid phosphatase (TRAP); (2) immunohistochemistry using antibodies for CD68, macrophage colony-stimulating factor (M-CSF), MIB-1, p53, p21, p16, and cathepsin-L (cath L); (3) TdT-mediated deoxyuridine triphosphate-biotin terminal end labeling (TUNEL) as a measure of apoptosis; (4) fluorescence-based polymerase chain reaction single-strand conformation polymorphism analyses (FPCR-SSCP) to detect p53 gene mutations; and (5) in situ hybridization using gene-specific oligoprobes for matrix metalloproteinase (MMP)-2, MMP-9, receptor activator of nuclear factor kappaB ligand (RANKL), and calcitonin receptor (CTR). Both Mos and GCs were shown to express the macrophage/histiocyte marker CD68. In GCs, TRAP and CTR, both of which are known as characteristic phenotype markers of osteoclasts, were expressed. M-CSF and RANKL, which are together essential for osteoclast differentiation, were expressed in both Mos and GCs. Mos were shown to express MIB-1, but GCs were not. Although proliferation-suppressor proteins p53, p21, and p16 were expressed in both Mos and GCs, little apoptotic phenomenon of lining Mos was detected by TUNEL. In our study, p53 gene mutations for exons 5, 7, and 8 in PVS synovial tissues were not detected by FPCR-SSCP analysis. Furthermore, both types of cells demonstrated the proteolytic enzymes MMP-2 and MMP-9 mRNA, and cath L protein. These results suggest that PVS has a hyperplastic property consisting of the CD68-positive monocytic cell lineage with differentiation of osteoclastic giant cells from monocyte and probably controlled against proliferation by wild-type p53, p21, and p16.     

LPS-stimulated human gingival fibroblasts inhibit the differentiation of monocytes into osteoclasts through the production of osteoprotegerin

Periodontitis is an inflammatory bone disease caused by Gram-negative anaerobic bacteria, but the precise mechanism of bone destruction remains unknown. Activated T lymphocytes secrete receptor activator of NF-kappaB ligand (RANKL) and support the differentiation of monocytes into mature osteoclasts. The purpose of this study was to examine the expression of RANKL and its inhibitor, osteoprotegerin (OPG), in inflamed gingival tissue and to clarify the role of human gingival fibroblasts (HGFs) in osteoclastogenesis regulated by RANKL. HGFs and gingival mononuclear cells (GMCs) were obtained from chronic periodontitis patients during routine periodontal surgery. Expression of OPG and RANKL mRNA in gingival tissue and HGFs was examined with RT-PCR. OPG production was measured using ELISA. Expression of RANKL, CD4, CD8 and CD69 on GMCs was determined by flow-cytometry using RANK-Fc fusion protein and the respective monoclonal antibodies. Osteoclastogenesis by RANKL was assayed by counting the number of tartarate-resistant acid phosphatase (TRAP)-positive cells after culturing human peripheral blood monocytes with recombinant human RANKL and macrophage-colony stimulating factor (M-CSF) for 10 days. OPG and RANKL mRNA were expressed in 80% (16/20) and 25% (5/20) of periodontitis lesions, respectively. OPG, but not RANKL, mRNA was expressed within HGFs. OPG mRNA expression and production by HGFs was augmented by LPS stimulation. All GMC samples expressed CD69, and two of five GMC samples expressed RANKL. The culture supernatant of LPS-stimulated gingival fibroblasts significantly reduced the number of TRAP positive cells generated by culturing monocytes with RANKL and M-CSF. The present study suggests that LPS-stimulated HGFs inhibit monocyte differentiation into osteoclasts through the production of OPG.     

LTB4 can directly stimulate human osteoclast formation from PBMC independent of RANKL

Leukotriene B4, as a kind of 5-lipoxygenase metabolite of arachidonic acid, is known to influence osteoclast formation and bone resorption. In order to determine whether Leukotriene B4 could directly stimulate human osteoclast differentiation and activation independent of RANKL (ODF), three different concentrations of Leukotriene B4 (10(-9)M, 10(-8)M, 10(-7)M) were added to the culture of CD14+ monocyte fraction of peripheral blood mononuclear cell (PBMC) in the presence of macrophage colony-stimulating factor (M-CSF). Under these conditions, Leukotriene B4 could induce multinucleated cells, which were positive for Tartrate-resistant acidic phosphatase (TRAP) staining and capable of bone resorption. Addition of osteoprotegerin (OPG) to PBMC cultures does not abrogate osteoclast formation induced by LTB4. Osteoclastogenesis induced by Leukotriene B4 were dose-dependently increased and weaker than that of RANKL. These results indicated that Leukotriene B4, elevated in many inflammatory diseases, is directly capable of inducing osteoclast formation by a RANKL-independent mechanism.     

Hydroxyapatite surface roughness: complex modulation of the osteoclastogenesis of human precursor cells

It is recognized that the surface roughness affects osteoblastic differentiation, but little information is available regarding its effect on osteoclastogenesis. With this work, the osteoclastogenic behaviour of human peripheral blood mononuclear cells (PBMCs), cultured isolated (1.5×10(6)cellscm(-2)) or co-cultured with human bone marrow cells (hBMCs; 10(3)cellscm(-2)), was assessed on surface-abraded hydroxyapatite disks with three different surface roughnesses (R(a) 0.0437-0.582 μm). Monocultures and co-cultures were performed for 21 days in the absence or presence of recombinant M-CSF and RANKL. Results showed that PBMCs supplemented with M-CSF and RANKL or co-cultured with hBMCs displayed typical osteoclastic features, i.e. multinucleated cells with actin rings, vitronectin and calcitonin receptors, gene expression of TRAP, cathepsin K, carbonic anhydrase 2, c-myc and c-src, TRAP activity and resorbing activity. The osteoclastogenic response increased with surface roughness in PBMCs cultured with M-CSF and RANKL but decreased in PBMCs co-cultured with hBMCs. However, co-cultures supplemented with the osteoclastogenic inducers displayed high and similar levels of osteoclast differentiation in the three tested surfaces. In conclusion, modulation of osteoclast differentiation by surface roughness seemed to be dependent on the mechanisms subjacent to the osteoclastogenic stimulus, i.e. the presence of soluble factors or direct cell-to-cell contacts between osteoblastic and osteoclastic cells.     

Proinflammatory cytokine (TNFalpha/IL-1alpha) induction of human osteoclast formation

TNFalpha and IL-1alpha are potent stimulators of bone resorption in vivo and in vitro. Recently, it has been demonstrated that these two cytokines directly induce osteoclastogenesis in mouse marrow cultures. This study determined whether TNFalpha (+/- IL-1alpha) is also capable of inducing human osteoclastogenesis. The CD14(+) monocyte fraction of human peripheral mononuclear cells was cultured with TNFalpha +/- IL-1alpha in the presence of M-CSF. TNFalpha induced the formation of multinucleated cells (MNCs) which were positive for TRAP, VNR and cathepsin K and showed evidence of resorption pit formation. IL-1alpha stimulated TNFalpha-induced lacunar resorption two- to four-fold. Osteoprotegerin, the decoy receptor for RANKL, did not inhibit this process. Anti-human IL-1alpha neutralizing antibodies significantly inhibited resorption without inhibiting the formation of TRAP(+)/VNR(+) MNCs. These results suggest that, in the presence of M-CSF, TNFalpha is sufficient for inducing human osteoclast differentiation from circulating precursors by a process which is distinct from the RANK/RANKL signalling pathway.     

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

Macrophages are commonly found within osteolytic secondary carcinomas in bone, but the manner in which these cells contribute to malignant bone resorption is uncertain. Macrophages isolated from primary breast carcinomas were co-cultured for up to 21 days with UMR 106 rat osteoblast-like cells on bone slices and glass coverslips in the presence and absence of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] and human macrophage colony stimulating factor (M-CSF). Cell cultures were then assessed for the presence of phenotypic markers of macrophage and osteoclast differentiation. Isolated cells were negative for osteoclast markers including tartrate-resistant acid phosphatase (TRAP), vitronectin receptor (VNR), and the ability to carry our lacunar bone resorption, but were positive for CD11b and CD14, macrophage markers which are not present on osteoclasts. In 21-day co-cultures of breast carcinoma tumour-associated macrophages (TAMs) and UMR 106 cells, incubated in the presence of 1,25(OH)₂D₃ and M-CSF, numerous TRAP- and VNR-positive multinucleated cells capable of extensive lacunar resorption were formed. Contact with UMR 106 cells and the presence of 1,25(OH)₂D₃ and M-CSF were absolute requirements for differentiation of human breast carcinoma TAMs into mature functional osteoclasts. TAM–osteoclast differentiation may represent an important cellular mechanism of osteolysis in metastatic skeletal carcinomas. © 1998 John Wiley & Sons, Ltd.     

Synovial fluid macrophages are capable of osteoclast formation and resorption

To determine whether synovial fluid (SF) macrophages isolated from the SF of osteoarthritis (OA), rheumatoid arthritis (RA) and pyrophosphate arthropathy (PPA) joints are capable of osteoclast formation, and to investigate the cellular and humoral factors required for this to occur, SF macrophages (CD14+) were isolated from the knee joint SF from patients with OA, RA and PPA and cultured for up to 14 days with macrophage-colony stimulating factor (M-CSF) and soluble receptor activator for nuclear factor-kappaB ligand (RANKL) or tumour-necrosis factor-alpha (TNFalpha) and interleukin-1alpha (IL-1alpha). Osteoclast differentiation was assessed by expression of tartrate-resistant acid phosphatase (TRAP) and vitronectin receptor (VNR), F-actin ring formation and lacunar resorption. Osteoclast formation and lacunar resorption was seen in RANKL-treated cultures of SF macrophages isolated from OA, RA and PPA joints with the largest amount of resorption noted in RA and PPA SF macrophage cultures. In TNFalpha/IL-1alpha-treated RA and PPA SF macrophage cultures, osteoclasts capable of lacunar resorption were also formed. Lacunar resorption was more extensive in RANKL than TNFalpha/IL-1alpha-treated cultures. These findings indicate that SF macrophages are capable of differentiating into mature osteoclasts capable of lacunar resorption. M-CSF in combination with RANKL or TNFalpha/IL-1alpha was required for osteoclast formation. As inflammatory synovial fluids contain an increase in the number of macrophages and an increase in the amounts of RANKL, TNFalpha and IL-1alpha, these findings suggest that one means whereby bone erosions may form in rheumatoid or crystal arthritis is by differentiation of synovial fluid macrophages into osteoclasts.     

Regulation of osteoclast differentiation and function by receptor activator of NFkB ligand and osteoprotegerin

The differentiation and functions of osteoclasts (OCs) are regulated by osteoblast-derived factors. Receptor activator of NFkB ligand (RANKL) is one of the key regulatory molecules in OC formation. Osteoprotegerin (OPG) is a novel secreted member of the TNF receptor superfamily that negatively regulates osteoclastogenesis and binds to RANKL. We examined the biological actions of macrophage-colony-stimulating factor (M-CSF), RANKL, and OPG on the differentiation of OCs isolated from cocultures of mouse osteoblastic cells and bone marrow cells. Preosteoclasts (pOCs) and OCs were characterized by their ultrastructure and the expression of OC markers such as tartrate-resistant acid phosphatase (TRAP) and vacuolar-type H(+)-ATPase. pOCs formed without any additives expressed TRAP, but showed little resorptive activity on cocultured dentine slices. TRAP-positive pOCs treated with M-CSF began to fuse with each other, but lacked a ruffled border (RB) and showed almost no resorptive activity. pOCs treated with RANKL became TRAP-positive multinucleated cells, which expressed intense vacuolar-type H(+)-ATPase along the RB membranes and exhibited prominent resorptive activity. Such effects of RANKL on pOCs were completely inhibited by the addition of OPG. OPG inhibited RB formation in mature OCs and reduced their resorptive activity, and also induced apoptosis of some OCs. These results suggest that 1) RANKL induces differentiation of functional OCs from pOCs, 2) M-CSF induces macrophage-like multinucleated cells, but not OCs, 3) OPG inhibits RB formation and resorptive activity in mature OCs, 4) OPG also induces apoptosis of OCs, and 5) RANKL and OPG are, therefore, important regulators of not only the terminal differentiation of OCs but also their resorptive function.     

Osteoclast formation and function in pigmented villonodular synovitis

Pigmented villonodular synovitis (PVNS) is a synovial tumour-like lesion that frequently causes osteolysis. PVNS contains numerous macrophages and osteoclast-like giant cells. In this study, we have analysed the cytochemical and functional characteristics of mononuclear and multinucleated cells in PVNS and determined the cellular and humoral mechanisms underlying giant cell formation and resorption in PVNS. Giant cells and CD14(+) and CD14(-) mononuclear cell populations were isolated from PVNS synovial tissue and cultured alone or in the presence and absence of the osteoclastogenic factors, RANKL and M-CSF. Osteoclast formation and activity was assessed by expression of TRAP and evidence of lacunar resorption. Giant cells in PVNS expressed an osteoclast-phenotype (CD51(+) , TRAP(+) , CD14(-) , HLA-DR(-) ) and were formed only in cultures of mononuclear cells that expressed the macrophage marker CD14. Osteoclast formation required RANKL and occurred in both the presence and absence of exogenous M-CSF. CD14(-) cells in PVNS expressed RANKL. Lacunar resorption by PVNS-derived giant cells was abolished by the addition of the bisphosphonate, zoledronate. Our findings indicate that osteoclasts form by a RANKL-dependent mechanism from CD14(+) mononuclear phagocytes in PVNS. Osteoclast formation occurred even in the absence of exogenous M-CSF, a finding which is in keeping with over-expression of M-CSF playing a pathogenic role in this condition. Anti-osteoclast resorptive treatment may be useful to control osteolysis in PVNS.