双氢青蒿素抑制核因子κB受体激动剂配体诱导RAW264.7细胞分化破骨细胞的研究

目的 探讨双氢青蒿素(DA)对核因子κB受体激动剂配体(RANKL)诱导RAW264.7细胞形成破骨细胞的影响。方法 通过细胞计数试剂盒(CCK-8)确定不同浓度(1、5、10、50、100、200 μmol/L)DA对RAW264.7细胞的生存影响。分别用50 ng/mL RANKL及50 ng/mL RANKL+5、10、50、100 μmol/L DA诱导RAW264.7细胞形成破骨细胞,共3 d。对形成的破骨细胞用抗酒石酸酸性磷酸酶(TRAP)染色并计数,TRAP染色阳性且细胞核数目＞3个认为是成熟的破骨细胞。50 ng/mL RANKL及50 ng/mL RANKL+10、100 μmol/L DA培养RAW264.7细胞24 h,用Trizol试剂提取总RNA,并使用荧光实时定量PCR检测破骨细胞分化相关基因NFATc1、c-fos及Cathepsin K的表达。结果 1、5、10、50、100 μmol/L DA对RAW264.7细胞毒性较小,细胞存活率均＞90%。TRAP染色显示,5、10、50、100 μmol/L DA可以减少RANKL诱导成熟破骨细胞的数目,并呈剂量依赖关系(F=139.156, P＜0.01)。实时荧光定量PCR结果显示,10、100 μmol/L DA具有下调破骨细胞分化关键基因NFATc1和c-fos表达的作用,且100 μmol/L抑制作用比10 μmol/L明显,但两种浓度DA对Cathepsin K的表达无明显影响。结论 DA对RAW 264.7细胞毒性较低,通过下调RAW 264.7细胞NFATc1和c-fos基因表达抑制RANKL诱导破骨细胞形成。DA可以作为治疗骨质破坏性疾病的潜在药物。     

人破骨细胞分化中重组结核杆菌热休克蛋白10的影响

背景:体外小鼠颅盖骨实验已证实了其结核杆菌热休克蛋白10的破骨效应。目的:在诱导破骨细胞分化的体外细胞培养系统中,研究重组结核杆菌热休克蛋白10对破骨细胞分化的影响及相关机制。方法:选用人巨噬细胞集落刺激因子依赖性附着性血液单个核细胞,实验分为:核因子κB受体活化因子配体+重组结核杆菌热休克蛋白10组、核因子κB受体活化因子配体组、重组结核杆菌热休克蛋白10组(1 mg/L)、阴性对照组(完全培养基),核因子κB受体活化因子配体+重组结核杆菌热休克蛋白10组、重组结核杆菌热休克蛋白10组中重组结核杆菌热休克蛋白10质量浓度为1 mg/L,在含有巨噬细胞集落刺激因子的a-MEM培养液重悬单核细胞,各组培养7,14,21 d后,观察所形成的抗酒石酸酸性磷酸酶阳性染色多核细胞的形态、数目、骨吸收面积;各组NFATc1、c-Fos基因及蛋白表达情况。结果与结论:阴性对照组无抗酒石酸酸性磷酸酶阳性多核破骨细胞分化生成,其余各组均有抗酒石酸酸性磷酸酶阳性多核破骨细胞分化生成,并在小牛骨磨片上形成吸收陷窝;重组结核杆菌热休克蛋白10组形成的破骨细胞分化细胞数目、吸收陷窝数目及陷窝面积显著低于核因子κB受体活化因子配体+重组结核杆菌热休克蛋白10组;阴性对照组NFATc1、c-Fos基因表达水平显著低于核因子κB受体活化因子配体+重组结核杆菌热休克蛋白10组和重组结核杆菌热休克蛋白10组,而重组结核杆菌热休克蛋白10组表达NFATc1、c-Fos蛋白,显著低于核因子κB受体活化因子配体+重组结核杆菌热休克蛋白10组。提示结核杆菌热休克蛋白10参与破骨细胞分化的形成,可能与诱导相关基因NFATc1、c-Fos表达上调有关。     

姜黄素治疗聚乙烯颗粒诱导骨溶解的体内实验

目的:探讨姜黄素对聚乙烯颗粒诱导骨溶解的治疗效果及作用机制,为防治假体无菌性松动提供新思路.方法:建立小鼠气囊植骨模型,随机分为:空白组、聚乙烯组、溶剂组、姜黄素组(姜黄素+溶剂).干预完成后杀死小鼠,取出背部气囊组织及其内植骨骨片.行抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色检测骨片中破骨细胞的数量.QPCR检测气囊组织中c-Fos,NFATc1的mRNA表达水平,Western印迹检测气囊组织中IκBa以及p-IκBa的蛋白含量.结果:TRAP染色发现,聚乙烯组和溶剂组骨片中染色的破骨细胞较多,而空白组和姜黄素组极少或没有染色的破骨细胞.QPCR检测基因表达情况:c-Fos,聚乙烯组＞溶剂组＞空白组＞姜黄素组,前两组与后两组相比,组间差异有统计学意义(P＜o.01);NFATc1,聚乙烯组＞溶剂组＞空白组＞姜黄素组,前两组与后两组相比,组间差异有统计学意义(P＜o.01).Western印迹检测IκBα蛋白相对灰度值:姜黄素组＞空白组＞溶剂组＞聚乙烯组,前两组与后两组相比,组间差异有统计学意义(P＜0.01);p-IκBa蛋白相对灰度值:聚乙烯组＞溶剂组＞空白组＞姜黄素组,前两组与后两组相比,组间差异有统计学意义(P＜0.o1).结论:在小鼠气囊植骨模型中,姜黄素可以通过抑制NF-κB的活化以及c-Fos,NFATc1的表达来抑制超高分子量聚乙烯(ultra-high molecular weight polyethylene,UHMWPE)诱导的骨溶解反应.     

骨水泥中的硫酸钡对破骨细胞的作用

目的验证骨水泥中的硫酸钡对破骨细胞形成及其生物学活性的影响。方法体外培养外周血单核细胞并加入巨噬细胞克隆集落刺激因子及核因子B激活因子配体诱导破骨细胞分化,实验组中分别加入含有或不含有硫酸钡的骨水泥颗粒。以抗酒石酸酸性磷酸酶阳性多核细胞及象牙磨片上虫蚀样骨吸收陷窝的形成昨作为检测破骨细胞形成及其骨吸收活性的检测指标,检验骨水泥中的硫酸钡对破骨细胞的影响。结果含或不含硫酸钡的骨水泥颗粒组抗酒石酸酸性磷酸酶阳性多核细胞形成均早于无骨水泥颗粒的对照组（4天vs6天）,而骨水泥颗粒中是否含有硫酸钡的两组间抗酒石酸酸性磷酸酶阳性多核细胞形成的时间无明显差异。各组间抗酒石酸酸性磷酸酶阳性多核细胞的数量无显著差异;含硫酸钡的骨水泥颗粒组象牙磨片上骨吸收陷窝的面积较不含硫酸钡的骨水泥颗粒组及阴性对照组均增大（28.26±4.98vs22.28±3.49vs14.58±2.82,〈0.05）。结论骨水泥颗粒中的硫酸钡能够促进破骨细胞分化并促进成熟破骨细胞的骨吸收活性。     

毛蕊花糖苷对类风湿关节炎小鼠骨侵蚀及JNK/ERK通路的影响

为探讨毛蕊花糖苷(verbascoside, VB)对类风湿关节炎(rheumatoid arthritis, RA)小鼠骨侵蚀的作用及其与JNK/细胞外信号调节激酶(extracellular signal-regulated kinase, ERK)通路的调控关系,将DBA/1J雌性小鼠随机分为正常组、胶原诱导的关节炎(collagen-induced arthritis, CIA)组、VB组和甲氨蝶呤(methotrexate, MTX)组,测量小鼠关节炎评分、后足肿胀、体质量和缩足阈值,观察踝关节病理变化;用ELISA检测血清中IL-6、TNF-α和抗牛胶原蛋白Ⅱ(collagenⅡ, CⅡ)特异性抗体水平;抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase, TRAP)染色观察踝关节破骨细胞变化;Western blotting检测关节组织中磷酸化JNK(phosphorylated JNK, p-JNK)、JNK、磷酸化ERK(phosphorylated ERK, p-ERK)、ERK蛋白表达。从C57BL/6小鼠中分离骨髓源性巨噬...     

巨噬细胞集落刺激因子/核因子κB受体激活物配体体外诱导培养高纯度破骨细胞:最佳剂量探讨

背景:巨噬细胞集落刺激因子(macrophage colony-stlimulating factor,M-CSF)/核因子κB受体激活物配体(receptor activatorof nuclear kappa B ligand,RANKL)两种细胞因子协同诱导骨髓干细胞形成破骨细胞是一种较新的,可以获取较高纯度和数量破骨细胞的诱导培养法,但尚缺乏统一的培养标准。目的:建立有效的M-CSF/RANKL诱导小鼠骨髓干细胞诱导分化破骨细胞的培养体系。方法:分离小鼠四肢骨获取骨髓干细胞。将小鼠骨髓干细胞在含有M-CSF的α-MEM培养基中培养24h,调整细胞浓度为107,108,109L-1。然后在培养基中同时加入10μg/LM-CSF和不同质量浓度(20,50,100μg/L)RANKL。行抗酒石酸酸性磷酸酶染色观察干细胞向破骨细胞的转变过程及细胞形态和染色情况,并对各组染色阳性的破骨细胞进行计数,比较不同诱导条件对破骨样细胞数量的影响。结果与结论:培养3d后可见少量破骨样细胞,胞质内含许多红色抗酒石酸酸性磷酸酶染色阳性颗粒,细胞内可见淡染的双核;培养6d后可见大量染色阳性破骨样细胞;培养9d后出现多核巨型染色阳性破骨样细胞,细胞明显增大,细胞核可达到3个以上。在固定细胞接种浓度条件下,RANKL质量浓度为100μg/L时诱导分化形成的破骨样细胞数量较另两质量浓度增多(P0.05);在固定RANKL质量浓度条件下,细胞浓度为108L-1时诱导分化形成的破骨样细胞数量较另两细胞接种浓度增多(P0.05);细胞接种浓度为108L-1,RANKL质量浓度为100μg/L时诱导分化形成的破骨样细胞数量高于其他条件组合(P0.05)。说明在M-CSF/RANKL诱导小鼠骨髓干细胞分化培养破骨细胞的体系中,RANKL最佳质量浓度为100μg/L,最佳细胞接种浓度为108L-1。     

外周血单个核细胞诱导培养破骨细胞

目的：从人外周血单个核细胞诱导培养获得高产量高纯度破骨细胞，为破骨细胞的体外研究提供丰富的细胞来源。 方法：从外周血分离单个核细胞贴壁培养，采用0.25％胰蛋白酶／0.02％EDTA联合消化，纯化后以RANKL和M-CSF加以诱导(A组)，并与未经消化之传统方法（B组）比较。结果：与B组相比，A组可获得(1 426±204)个破骨细胞（P<0.05），0.25％胰蛋白酶／0.02％EDTA联合消化可使破骨细胞纯化率达90％。诱导生成的破骨细胞TRAP染色阳性，功能试验显示具有噬骨能力。结论：联合消化结合RANKL/M-CSF诱导培养法可产生大量的破骨样细胞，方法简便而且经济实用。     

周期性张应变对破骨前体细胞和破骨细胞增殖和抗酒石酸酸性磷酸酶的影响

目的研究不同强度的力学载荷对破骨细胞及其前体细胞增殖、分化和功能的影响。方法以破骨诱导液培养RAW264.7破骨前体细胞,同时施加3 d的周期性张应变,然后培养4 d;另外一组RAW264.7细胞以破骨诱导液培养4 d,将其诱导为破骨细胞,再施加3 d的周期性张应变。结果在不同张应变下,两组细胞增殖活性的变化大致相同,但细胞抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatage,TRAP)活性和破骨细胞(TRAP阳性多核细胞)数量的变化明显不同。在2 500με的中等强度张应变下,第1组的TRAP活性降幅和破骨细胞数量减幅均最高,而后者TRAP活性降幅和破骨细胞数量减幅均最低。结论不同张应变对分化初期破骨前体细胞和已分化出破骨细胞的破骨前体细胞的破骨分化和功能状态的影响有明显差异。     

骨髓贴壁细胞向破骨细胞的分化

背景:一般认为采用贴壁分离法得到的小鼠骨髓中能够贴壁的细胞为间充质干细胞,并能分化成为成骨、成脂肪及成软骨细胞,其中贴壁的细胞是否有分化为破骨细胞的潜能尚不明确。目的:检测贴壁分离法得到的小鼠骨髓中能贴壁的细胞是否能够分化为破骨细胞。方法:采用贴壁法得到小鼠原代间充质干细胞,以及通过贴壁1-5 d后得到不同时间贴壁的骨髓细胞。原代间充质干细胞及不同时间贴壁的骨髓细胞分别用普通培养基,及含m-csf和RANKL培养基,培养9 d后进行碱性磷酸酶和抗酒石酸酸性磷酸酶染色。原代间充质干细胞传代后,第2代间充质干细胞分为4组,分别用普通培养基、含m-csf培养基、含RANKL培养基及含m-csf和RANKL的培养基培养,9 d后进行碱性磷酸酶及抗酒石酸酸性磷酸酶染色。结果与结论:贴壁法得到较均一的间充质干细胞,原代及传代贴壁细胞的联合诱导组抗酒石酸酸性磷酸酶染色均为阳性,说明原代和传代的贴壁骨髓细胞中均有可以分化为破骨细胞的细胞。不同时间贴壁的细胞诱导后碱性磷酸酶及抗酒石酸酸性磷酸酶染色有差异,说明不同时间贴壁的细胞存在分化差异。     

CD147抗体对破骨细胞中基质金属蛋白酶活性影响的体外研究

目的:建立人破骨细胞(Osteoclast,OCs)体外诱导分化模型,研究CD147单克隆抗体对OCs分化过程中基质金属蛋白酶-9(Matrix metalloproteinases 9,MMP-9)和基质金属蛋白酶-2(Matrix metalloproteinases 2,MMP-2)表达及活性的影响。方法:通过采集健康成年志愿者外周血所分离的单个核细胞贴壁培养,应用NF-κB配体激活因子(Receptor or Activator ofNF-KB Ligand,RANKL)与巨噬细胞集落刺激因子(Macrophage colony stimulating factor,M-CSF)诱导单个核细胞向OCs分化。本实验分为抗体组(RANKL+M-CSF+CD147单克隆抗体)与对照组(RANKL+M-CSF)。抗酒石酸酸性磷酸酶染色(Tartrate-resistant acid phosphatase,TRAP)与骨吸收实验检测鉴定OCs分化及活性情况,Real-Time PCR技术检测CD147、MMP-9和MMP-2 mRNA在破骨前体细胞(Osteoclast precursor cells,OPCs)中表达情况,明胶酶谱法检测细胞培养上清中MMP-9、MMP-2酶蛋白的活性变化情况。结果:①对照组经TRAP染色和骨吸收实验检测到破骨样细胞形成并具有骨吸收功能,而抗体组OCs分化及活性均受到抑制;②在24、48小时时相点上抗体组CD147、MMP-2及MMP-9 mRNA的相对表达量均低于相应的对照组(P<0.05),且MMP-2、MMP-9 mRNA的相对表达量与CD147 mRNA的表达量呈正相关。③明胶酶谱检测细胞培养上清,可见在24、48小时两时相点上抗体组OCs细胞中MMP-2、MMP-9酶原及活性酶的酶解量均较相应对照组明显降低(P<0.05)。结论:CD147单克隆抗体可以抑制OCs分化成熟过程中MMP-9及MMP-2的表达与活性;CD147对MMP-2、MMP-9活性的调节,可能是其对OCs活化调节的机制之一。     

Mitogen- and stress-activated protein kinase 1 activates osteoclastogenesis in vitro and affects bone destruction in vivo

Mitogen- and stress-activated protein kinase (MSK) 1 is an important regulator of immune response and mitogenic signaling. In this study, we report for the first time that MSK1 was activated by the osteoclast differentiation factor receptor activator of nuclear factor kappa B ligand (RANKL) in osteoclast precursor cells. Inhibition of upstream kinases ERK1/2 and p38, but not JNK, suppressed MSK activation upon RANKL stimulation. An MSK1 inhibitor efficiently prevented the induction of c-Fos and NFATc1 and CREB phosphorylation by RANKL. Inhibition of MSK1 also successfully blocked RANKL-induced osteoclastogenesis. MSK knockdown with small interfering RNA significantly inhibited osteoclast differentiation and bone resorption. MSK1 did not affect osteoclast survival. The induction of c-Fos and NFATc1 and the phosphorylation of CREB and ATF2 were also inhibited by MSK1 knockdown. Moreover, knockdown of MSK1 significantly blocked recruitment of c-Fos to the NFATc1 promoter upon RANKL stimulation. Therefore, NFATc1-inducible osteoclast-specific genes were downregulated by MSK1 blockade. NFATc1 retrovirus transduction almost completely rescued the differentiation defect of MSK1-silenced cells. In vivo knockdown of MSK1 reduced RANKL-induced bone resorption as well as osteoclast formation. Thus, our results suggested that MSK1 is an important novel molecule involved in RANKL signaling and osteoclast differentiation.     

Kaempferol Inhibits IL-1β-Stimulated,RANKL-mediated Osteoclastogenesis via Downregulation of MAPKs,c-Fos,and NFATc1

Kaempferol is one of the most common flavonoid that is present in a variety of vegetables and fruits and has effects on bone metabolism. The present study was performed to define the effects of kaempferol on interleukin (IL)-1β-stimulated receptor activator of NF-κB ligand (RANKL)-mediated osteoclast differentiation. Bone marrow cells were harvested from 6-week-old male imprinting control region mice, and the differentiation of osteoclasts from these cells was evaluated by tartrate-resistant acid phosphatase staining and resorption pit formation assay. Phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated p38, phosphorylated c-Jun amino-terminal kinase, NF-κB (p65), IκBα, c-Fos, and nuclear factor of activated T cells c1 (NFATc1) expressions were examined by Western blotting and quantitative RT-PCR. Kaempferol inhibits IL-1β-stimulated, RANKL-mediated osteoclast differentiation and also inhibits IL-1β-stimulated, RANKL-mediated phosphorylation of ERK 1/2, p38 and JNK MAP kinases, and expressions of c-Fos and NFATc1. These results indicate that kaempferol has an inhibitory role in the bone loss by preventing osteoclast formation and suggest that it might be a novel therapeutic agent for the treatment of inflammatory arthritis by managing bone destruction.     

Role of intercellular adhesion molecule-2 in osteoclastogenesis

Osteoclasts, multinucleated bone-resorbing cells, are specialized cells derived from the monocyte/macrophage lineage. Therefore, it is essential for mononuclear precursors to find a fusion partner during its differentiation. Our previous study showed an important role of cell communication via Mac-1 (CD11b/CD18) during osteoclastogenesis. However, the counter receptor of Mac-1 was still unknown. Flow cytometric analysis showed that bone marrow-derived mononuclear cells, used as osteoclast precursors, expressed intercellular adhesion molecule-1 and -2. Quantitative RT-PCR analysis revealed that expression level of ICAM-2 was higher than that of ICAM-1 in bone marrow cells. The osteoclastogenesis induced by receptor activator of NF-kappaB ligand (RANKL) was inhibited by anti-ICAM-2 neutralizing antibody but not by anti-ICAM-1 neutralizing antibody. The inhibitory effect of anti-ICAM-2 antibody on osteoclastogenesis was enhanced by simultaneous treatment of anti-CD11b neutralizing antibody. Furthermore, osteoclastogenesis induced by tumor necrosis factor α (TNFα) was also inhibited by anti-ICAM-2 neutralizing antibody. The involvement of lymphocytes in osteoclastogenesis was excluded, because anti-ICAM-2 antibody inhibited osteoclastogenesis using bone marrow-derived cells from immunodeficiency mice. Immunocytochemical staining demonstrated colocalization of ICAM-2 and Mac-1 during osteoclastogenesis; however, Mac-1 immunoreactivity was lost in differentiated multinucleated osteoclast. These results suggest the important role of ICAM-2/Mac-1 binding in osteoclastogenesis induced by either RANKL or TNFα.     

Inhibition of titanium particle-induced osteoclastogenesis through inactivation of NFATc1 by VIVIT peptide

Osteoclastogenesis induced by particulate wear debris is a major pathological factor contributing to periprosthetic osteolysis. Although the nuclear factor of activated T cells c1 (NFATc1) is known to be involved in osteoclast differentiation, its effect on osteoclastogenesis in response to wear particles remains unclear. In the present study, we investigated the role of NFATc1 in the regulation of osteoclast differentiation from bone marrow macrophages (BMMs) stimulated with titanium (Ti) particles. The results showed that Ti particles could stimulate BMMs to produce proinflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) and differentiate into multinucleated osteoclasts in the presence of receptor activator of nuclear factor-κB ligand (RANKL). NFATc1 was expressed in BMMs and multinucleated cells cultured with Ti particles and RANKL. Inactivation of NFATc1 by 11R-VIVIT peptide potently impeded the Ti particle-induced osteoclastogenesis. 11R-VIVIT peptide does not have toxic effect on BMMs. Based on these data, we conclude that inactivation of NFATc1 by VIVIT peptide would provide a promising therapeutic target for the treatment of periprosthetic osteolysis.     

Intercellular adhesion molecule-1 on synovial cells attenuated interleukin-6-induced inhibition of osteoclastogenesis induced by receptor activator for nuclear factor κB ligand

In a co-culture of osteoclast precursor cells and synovial cells, interleukin-6 (IL-6) induces osteoclast formation. In contrast, in a monoculture of osteoclast precursor cells, IL-6 directly suppresses receptor activator for nuclear factor κB ligand (RANKL)-induced differentiation of osteoclast precursor cells into osteoclasts. In the present study, we explored why the effect of IL-6 differed between the monoculture and the co-culture systems. In the monoculture, mouse osteoclast precursor cell line, RAW 264·7 (RAW) cells were cultured with soluble RANKL (sRANKL) for 24 h or 3 days. sRANKL increased both expression of osteoclastogenesis marker, tartrate-resistant acid phosphatase isoform 5b (TRAP5b) and nuclear factor of activated T cells cytoplasmic 1 (NFATc1), whereas the co-addition of IL-6 decreased them both in a dose-dependent manner. In the co-culture, RAW cells and human synovial cell line, SW982 cells were cultured with IL-6+soluble IL-6 receptor (sIL-6R) for 3 days. TRAP5b and NFATc1 expression reduced by IL-6 was increased by the addition of SW982 cells in a manner dependent upon the number of added cells. IL-6+sIL-6R treatment significantly induced RANKL production in SW982 cells, and anti-RANKL antibody inhibited IL-6+sIL-6R-induced osteoclastogenesis. SW982 cells expressed high levels of ICAM-1 originally, and ICAM-1 expression was increased significantly by IL-6+sIL-6R. Anti-ICAM-1 antibody suppressed IL-6-induced osteoclastogenesis. Finally, in the monoculture system, addition of sICAM-1 dose-dependently restored the expression of TRAP5b reduced by IL-6. Similar results were obtained when the formation of TRAP-positive multi-nuclear cells were examined using mouse bone marrow cells. In conclusion, IL-6 gave different results in the co-culture and monoculture systems because in the co-culture, ICAM-1 from the synovial cells restored osteoclastogenesis suppressed by IL-6.     

Mechanical stress directly suppresses osteoclast differentiation in RAW264.7 cells

Although it is known that mechanical stress to osteoblast and periodontal ligament cells suppresses osteoclast differentiation, little is known about the direct effect of mechanical stress on osteoclast differentiation. In this study, we examined the role of mechanical stress on osteoclast differentiation using murine pre-osteoclastic RAW264.7 cells treated with receptor activator of nuclear factor-kappaB ligand (RANKL). RAW cells were cultured with RANKL, and mechanical stress was applied for a given period. We counted the number of osteoclast cells which were tartrate-resistant acid phosphatase (TRAP)-positive and multinucleated (2 nuclei or more), and measured mRNA by RT-PCR. There was a decrease in the number of osteoclasts under mechanical stress compared with the number under no mechanical stress. The number of nuclei per osteoclast also decreased compared to the number of nuclei per osteoclast cultured with the application of mechanical stress. As the cells were cultured for a period of 1-7 days and/or for different periods of mechanical stress application, osteoclast differentiation decreased with mechanical stress and increased after removing mechanical stress. Expression of mRNA for the osteoclast-specific genes, TRAP, matrix metalloproteinase-9, cathepsin-K and calcitonin receptor, decreased with mechanical stress and was associated with the number of osteoclasts. Inducible nitric oxide synthase mRNA which inhibits osteoclast differentiation, increased with mechanical stress. In spite of the decrease in osteoclast number with mechanical stress, nuclear factor of activated T cell cytoplasmic 1 (NFATc1) and NFATc2 mRNA expression increased with mechanical stress. These findings indicate that mechanical stress directly suppresses osteoclast differentiation and increases NFATc1 and NFATc2 suggesting delayed differentiation.