Isolation of human osteoclasts formed in vitro: hormonal effects on the bone-resorbing activity of human osteoclasts

Osteoclasts are multinucleated cells that carry out bone resorption. Analysis of the direct effect of hormones on the bone-resorbing activity of human osteoclasts has been limited by difficulties in isolating these cells from the human skeleton. In this study, human osteoclasts formed from cultures of peripheral blood mononuclear precursors (PBMCs) on a Type-I collagen gel were isolated by collagenase treatment for investigating their resorptive activity. PBMCs were cultured in the presence of M-CSF, soluble RANKL, dexamethasone, and 1,25(OH)2D3. The isolated multinucleated cells expressed the osteoclast markers, TRAP, VNR, cathepsin K, calcitonin receptors and were capable of extensive lacunar resorption. Calcitonin inhibited the motility and resorptive activity of osteoclasts. RANKL significantly stimulated osteoclast resorption, but 1,25(OH)2D3, PTH, and OPG did not. These findings indicate that calcitonin and RANKL act directly on human osteoclasts to inhibit and stimulate osteoclast bone-resorbing activity, respectively, and that PTH, 1,25(OH)2D3, and OPG are more likely to influence osteoclast activity indirectly. This technique of human osteoclast isolation should permit the effects of cellular and hormonal/humoral factors on the bone-resorbing activity of mature human osteoclasts to be assessed independently of any effect such factors have on osteoclast formation. It should also make it possible to examine directly the resorptive activity and other characteristics of osteoclasts in specific bone disorders such as Paget's disease.     

Platelets increase while serum reduces the differentiation and activity of osteoclasts in vitro

Platelets modulate formation of osteoclasts and osteoblasts, but research with different preparations of platelets remains inconclusive. Here, we assessed whether serum components modulate the effect of platelet preparations. In murine bone marrow cultures, osteoclastogenesis was investigated in the presence of platelet‐released supernatant (PRS), serum containing PRS (SC‐PRS), and serum. Osteoclastogenesis was quantified by the numbers of tartrate‐resistant acid phosphatase (TRAP)‐positive multinucleated cells, TRAP activity and resorption assays. Also human osteoclastogenesis assays were performed. Viability and proliferation were tested by MTT and ³[H]thymidine incorporation assays, respectively. Osteoblastogenesis was assessed by histochemical staining for alkaline phosphatase‐of murine bone marrow cultures and human MG63 cells. We found PRS to increase the number of TRAP⁺ multinucleated cells in the early phase and TRAP activity in the later phase of osteoclastogenesis. SC‐PRS and serum decreased the number and activity of TRAP⁺ multinucleated cells. Both serum containing preparations reduced viability and proliferation of hematopoietic progenitors. PRS decreased the numbers of alkaline phosphatase‐positive colonies while SC‐PRS and serum increased osteoblastmarkers in MG63. Proliferation of MG63 was stimulated by all preparations. These results show that activated platelets support osteoclastogenesis, while platelet preparations that contain serum components decrease osteoclastogenesis and increase osteoblastogenesis in vitro, suggesting that serum components modulate the effects of platelets on osteoclastogenesis and osteoblastogenesis. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1561–1569, 2013     

体外培养破骨细胞的功能观察

观察破骨细胞在离体状态下的骨吸收功能。新鲜牛皮质骨经锯式切片机横切成５０μｍ厚０．５×０．５ｃｍ大小骨片。参照已建立的破骨细胞分离培养方法，从新生Ｗｉｓｔａｒ大鼠四肢长骨分离破骨细胞，培养于２．５ｃｍ细胞培养皿内或上述骨片上，相差倒置显微镜或扫描电镜观察，可见培养的细胞具有典型破骨细胞的形态特点，接种子骨片上的破骨细胞分别培养１、３、５、７天，扫描电镜观察，可见破骨细胞能在骨片表面形成吸收陷窝，其形态多样，深浅不一，边界清晰，底面粗糙，而且随培养时间延长，陷窝扩大，数量增多。结论，破骨细胞在体外培养条件下具有良好的骨吸收功能，可进行药物干预的研究。     

阿仑膦酸盐诱导破骨细胞调亡FAS基因的表达

本研究采用１０－８Ｍ１，２５（ＯＨ）２Ｄ３诱导ＳＤ鼠骨髓细胞形成破骨细胞，通过检测细胞上ＦＡＳ基因的表达，探讨骨吸收因子阿仑膦酸盐的作用机制。骨髓细胞培养６天即有多核巨细胞形成，加入阿仑膦酸盐致终浓度为１００μＭ，继续培养４８小时后，发现用药组破骨样细胞及圆形单核细胞的前体细胞呈现凋亡的形态学特征：胞浆收缩，核固缩等。ＦＡＳ抗原是与细胞凋亡密切相关的细胞表面蛋白，用抗ＦＡＳ抗体免疫组化方法，检测凋亡的破骨细胞及其前体细胞上ＦＡＳ基因的表达，结果呈阳性，而未凋亡细胞呈阴性，提示阿仑膦酸盐导致的破骨细胞及其前体细胞的凋亡，可能与细胞表面的ＦＡＳ基因表达相关     

Effect of retinoic acid on the resorptive activity of chick osteoclasts in vitro.

Mixed cell suspensions mechanically isolated from the long-bones of day 20 prehatch chicks were cultured for 24 h on bone and sperm whale dentine slices in the presence of 0, 10, 100, and 1000 nM retinoic acid (RA). Significant inhibitions in the numbers of discrete lacunae resorbed per dentine slice, and in the ratio of lacunae per tartrate-resistant acid phosphatase-positive multinuclear cell were observed with all concentrations of RA studied. Semi-automated, 3-dimensional analysis of 733 pits was performed on one series of experiments using a tandem scanning (confocal) microscope, interfaced to an image analyzing computer. The majority of lacunae were small and unilocular; the plan areas of 90% of control pits were below 500 microns 2. Small but statistically significant increases in lacunar areas, but not mean or maximum depths or volumes, were observed in the presence of 10 and 100 nM RA; however, these changes were much smaller than the magnitude of the decrease in pit numbers. Thus, the overall effect of RA in this system was inhibitory. Our findings contrast with the well known stimulatory action of retinoids on bone resorption both in vivo and in organ culture, but may parallel the inhibitory effects of prostaglandins observed in disaggregated osteoclast systems.     

依降钙素对破骨细胞的作用观察

目的 观察了国产降钙素-依降钙素对破骨细胞体外骨吸收功能的影响。并与进口降钙素-益钙宁的作用比较，方法 由10日龄幼兔四肢长骨机械分离破骨细胞于199培养液（含10%NCS 5?S），分别接种于象牙薄切片和培养板，置37℃，5%CO2培养箱中培养，细胞骨架观察采用荧光标记法（罗达明标记的鬼笔环肽），骨片吸收陷窝计数采用甲苯胺蓝染色法，益钙宁为阳性对照，等量培养液为阴性对照，结果 依降钙素组破骨细胞F-actin聚集，变短，骨片培养3d，吸收陷窝计数的结果显示，10^-10mol/L以上浓度依降钙素对破骨细胞吸收功能有明显抑制作用，并呈剂量相关性，10^-1mol/L时的抑制作用高达90%；依降钙素和益钙宁的抑制作用呈高度相关（r=0.99）。结论 依降钙素具有明显的抑制体外培养破骨细胞骨吸收活性的作用，与益钙宁的作用相仿。     

Statins prevent bisphosphonate-induced gamma,delta-T-cell proliferation and activation in vitro.

The acute phase response is the major adverse effect of intravenously administered N-BPs. In this study we show that N-BPs cause gamma,delta-T-cell activation and proliferation in vitro by an indirect mechanism through inhibition of FPP synthase, an effect that can be overcome by inhibiting HMG-CoA reductase with a statin. These studies clarify the probable initial cause of the acute phase response to N-BP drugs and suggest a possible way of preventing this phenomenon. INTRODUCTION: The acute phase response is the major adverse effect of intravenously administered nitrogen-containing bisphosphonate drugs (N-BPs), used in the treatment of metabolic bone diseases. This effect has recently been attributed to their action as non-peptide antigens and direct stimulation of gamma,delta-T-cells. However, because N-BPs are potent inhibitors of farnesyl diphosphate (FPP) synthase, they could cause indirect activation of gamma,delta-T-cells owing to the accumulation of intermediates upstream of FPP synthase in the mevalonate pathway, such as isopentenyl diphosphate/dimethylallyl diphosphate, which are known gamma,delta-T-cell agonists. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers and treated with N-BP, statin, or intermediates/inhibitors of the mevalonate pathway for 7 days in the presence of interleukin (IL)-2. Flow cytometric analysis of the T-cell-gated population was used to quantify the proportion of gamma,delta-T-cells in the CD3+ population. RESULTS AND CONCLUSIONS: The ability of N-BPs to stimulate proliferation of CD3+ gamma,delta-T-cells in human PBMC cultures matched the ability to inhibit FPP synthase. Gamma,delta-T-cell proliferation and activation (interferon gamma [IFNgamma] and TNFalpha release) was prevented by mevastatin or lovastatin, which inhibit HMG-CoA reductase upstream of FPP synthase and prevent the synthesis of isopentenyl diphosphate/dimethylallyl diphosphate. Desoxolovastatin, an analog of lovastatin incapable of inhibiting HMG-CoA reductase, did not overcome the stimulatory effect of N-BP. Furthermore, statins did not prevent the activation of gamma,delta-T-cells by a synthetic gamma,delta-T-cell agonist or by anti-CD3 antibody. Together, these observations show that N-BPs indirectly stimulate the proliferation and activation of gamma,delta-T-cells caused by inhibition of FPP synthase and intracellular accumulation of isopentenyl diphosphate/ dimethylallyl diphosphate in PBMCs. Because activation of gamma,delta-T-cells could be the initiating event in the acute phase response to bisphosphonate therapy, co-administration of a statin could be an effective approach to prevent this adverse effect.     

Carbonic anhydrase activity in isolated osteoclasts

Osteoclasts were isolated from the long bones of chicks by a nylon mesh filtering system. The cell purity, in terms of area of the slide occupied, was on the average 77.5% osteoclasts, 22% aggregated osteoblasts and matrix debris, and 1.5% individual blood and marrow cells. Viability, as measured by cytochalasin-blockable phagocytosis, of up to 99% was obtained. Electron microscopic examination revealed good retention of ultrastructural features. The presence of carbonic anhydrase and acid phosphatase in osteoclasts was verified by selective staining methods; the aggregates were positive for alkaline phosphatase. Carbonic anhydrase activity was 0.89 ± 0.13 × 10⁻⁴ micro Wilbur-Anderson units per osteoclast, and red blood cells had 0.12 ± 0.03 × 10⁻⁴ units/cell. Neither calcitonin nor parathyroid hormone influenced the activity of carbonic anhydrase. A review of other hormonal effects on carbonic anhydrase is provided.     

淫羊藿对体外培养破骨细胞的影响

目的 观察淫羊藿提取物对体外培养破骨细胞的影响。方法 以出生２ｄ的Ｗｉｓｔａｒ大鼠长骨为来源,培养已成熟生长的破骨细胞;以出生７￣９周的雄性Ｗｉｓｔａｒ大鼠骨髓为来源,在１．２５－（ＯＨ）２ＶｉｔＤ３作用下,培养出骨髓诱导而产生的破骨细胞。两种细胞培养过程中均分别加入２００μｇ／Ｌ的淫羊藿提取物。结果 皮质骨内层可培养出多核,体积大,抗酒石酸酸性磷酸酶（ＴＲＡＰ）染色阳性的破骨细胞,淫羊藿提取物对     

二甲双胍对破骨细胞体外分化的影响

目的 探讨二甲双胍对破骨细胞体外分化的影响及其可能机制.方法 采用RANKL诱导鼠巨噬细胞系Raw264.7细胞破骨分化模型,给予不同浓度的二甲双胍(400 μmol/L、800 μmol/L和1000μmol/L)和雷帕霉素(100 hmol/L)处理后,通过抗酒石酸酸性磷酸酶(tartrate-resistant Acid Phosphatase,TRAP)染色和破骨细胞骨架结构荧光染色观察破骨细胞数量,骨吸收培养板观察骨陷窝面积,RT-PCR技术检测破骨细胞特异性基因TRAP、组织蛋白酶K、降钙素受体和金属基质蛋白酶-9的表达,ELISA法检测肿瘤坏死因子-α(tumor necrosis factor,TNF-α)表达水平,Western-b1ot检测c-Fos蛋白以及哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin complex l,mTORC1)信号通路下游底物S6K1 Thr389、S6 Ser235/236、4EBP1 Thr37/46的表达及磷酸化水平.结果 二甲双胍和雷帕霉素均可使RANKL诱导的破骨细胞数量减少,抑制破骨细胞特异性基因的表达、抑制TNF-α、c-Fos蛋白以及mTORC1信号通路下游底物S6K1 Thr389、S6 Ser235/236、4E-BP1 Thr37/46的磷酸化,且二甲双胍的抑制作用具有浓度依赖性.结论 二甲双胍可抑制RANKL诱导的破骨前体细胞分化,其机制可能与抑制TNF-α和c-Fos蛋白的生成,以及抑制mTORC1信号通路激活有关.

Abstract：

Objective To investigate the effects of mefformin on the differentiation of osteoclastas well as relative mechanism.Methods Raw264.7 cells from the murine macrophage cell line was used.Receptor activator of NF-κB ligand (RANKL) was used to stimulate osteoclast differentiation from Raw264.7 cells.Osteoclast differentiation was assessed by tartrate-resistant acid phosphatase (TRAP) and actin fluorescence staining and counting the TRAP-positive cells after exposure to different concentrations of mefformin (0 μmol/L,400 μmol/L,800 μmol/L and 1000 μmol/L) or rapamicin (100 nmol/L) in the presence of 50 ng/ml RANKL for 5 days.Bone-resorbing activity was evaluated by BD BioCoatTM OsteologicTM Bone Cell Culture System.The expression of osteoclast-specific genes like TRAP,capthesin K,calcitonin receptor (CTR) and matrix metalloproteinase (MMP-9) was evaluated by RT-PCR.The expression of tumor necrosis factor-α(TNF-ct) S6K1Thr389,S6 Ser235/236,4E-BP1Thr37/46 and c-Fos protein was evaluated by ELISA kit and Western blot analysis,respectively.Results Mefformin dose-dependently inhibited RANKL-stimulated osteoclasts differentiation in Raw264.7 cell culture,as manifested by decrease of TRAP-positive multinucleated cells and pit erosion area,down-regulation of TRAP,cathepsin K,CTR and MMP-9 mRNA and reduction of TNF-α and c-Fos protein expression.Further study revealed that RANKL activated mTOR complex 1(mTORC1) signaling,while mefformin impaired RANKL-stimulated mTORC1 signaling.Rapamycin,an mTORCl-specific inhibitor and immunosuppressive macrolides could also prevent RANKL-induced osteoclast differentiation and bone resorption in vitro.Conclusion Mefformin inhibits osteoclastogenesis in vitro,which may due to reduction of TNF-α and c-Fos protein expression,and mTORC1 signaling is involved in this process.     

In vitro resorptive activity of isolated chick osteoclasts: effects of carbonic anhydrase inhibition

A potent inhibitor of carbonic anhydrase, 5-[3-hydroxybenzoyl]thiophene-2-sulfonamide (HTS), was shown to cause a 37% reduction in the area of resorption pits formed by isolated chick osteoclasts when used at a dose of 10(-7) M. HTS at doses of 10(-9) and 10(-7) M was also effective in reducing acid formation by the osteoclasts (14 and 36%, respectively). Additionally, the effect of HTS was found to be readily reversed by removing the agent, showing that it does not exert a toxic effect on the cells. This study indicates that the inhibitory effect of HTS on bone resorption is at the level of the acid-forming mechanism in osteoclasts and supports the view that carbonic anhydrase has a central role in the process.     

应用颅骨-破骨细胞联合培养体系研究先天性成骨不全破骨细胞骨吸收活性

目的 先天性成骨不全(OI)的主要临床表现为骨矿化过程不良,骨量丢失,骨骼畸形和骨折.但是其发病机理,尤其在其骨再建过程中成骨细胞(OB)及破骨细胞(OC)的功能改变尚不清楚.本实验以先天性成骨不全小鼠模型,oim/oim为基础,应用破骨细胞-颅骨联合培养体系研究OB和OC两种细胞在骨再建过程中的功能改变和相互作用.方法 本实验采用小鼠颅骨(CAL)组织培养模型.本模型采用颅骨组织培养,利用颅骨中成骨细胞可以从颅骨片游离出到培养皿及颅骨表面,从而支持培养皿及颅骨表面前体破骨细胞分化成为成熟破骨细胞,并吸收颅骨产生吸收陷窝.本实验中,共2组颅骨-破骨细胞联合培养体系:(1) 对照组(WT)颅骨与对照破骨细胞(WTCAL-WTOC);(2) OI颅骨与OI破骨细胞(OICAL-OIOC).联合培养颅骨及骨髓组织14日后,以TRAP免疫组化染色方法识别破骨细胞,ALP免疫组化染色方法识别成骨细胞,计算OC/OB.破骨细胞骨吸收活性以颅骨表面骨吸收陷窝占颅骨表面百分比并除以培养系统中的破骨细胞数表达.结果 第14日,OICAL-OIOC组的破骨细胞数低于WTCAL-WTOC组(92.50+23.18/mm2 对比 379.00+ 136.53/mm2,P<0.01); OICAL-OIOC组的OC/OB明显低于WTCAL-WTOC组(0.68+0.57对比1.65+0.67,P<0.01);OICAL-OIOC组OI破骨细胞的吸收能力高于WTCAL-WTOC组(27.76+22.81对比7.32+5.09,P<0.001).结论 oim/oim小鼠破骨细胞-颅骨培养体系中破骨细胞的数目明显减少,成骨细胞支持破骨细胞分化能力减低;但其破骨细胞骨吸收活性明显增强,以代偿成骨细胞功能,维持骨再建过程中成骨过程及骨吸收过程的平衡.     

体外培养的破骨细胞细胞化学测量观察

本文采用Spraque-Dawloy大鼠体外破骨细胞培养及新鲜长骨组织破骨细胞印片方洼研究两种材料中破骨细胞细胞化学的异同，检测了酸性磷酸酶(AcP)抗酒石酸酸性磷酸酶(TRACP)、β-酸性半乳糖苷酶、β-葡萄糖醛酸酶、乳酸脱氢酶、琥珀酸脱氢酶、NADH和NADPH脱氢酶．并使用Zeiss氏细胞扫描显微镜连接CD／MD20电脑图像分析系统，定量检测了以上酶的活性。结果表明，大鼠体外培养和新鲜组织印片的破骨细胞含有丰富的多种溶酶体酶和厌氧脱氢酶(线粒体酶)，新鲜组织印片的破骨细胞大多数脱氢酶的活性高于体外培养的破骨细胞。     

γ射线诱导下胱冬肽酶-3基因在犬胆管壁增殖平滑肌细胞凋亡中的表达及意义

目的：探讨在射线诱导下,胱冬肽酶-3(caspase-3)基因在犬胆管壁增殖平滑肌细胞凋亡中表达及意义。方法：将^103钯(^103Pd)放射性支架植入6只犬肝外胆管内,普通支架植入6只犬肝外胆管内,取出胆管标本,用RT—PCR方法检测γ射线诱导犬胆管壁凋亡的增殖平滑肌细胞中caspase-3基因的mRNA。结果:^103Pd支架组犬胆管组织中caspase-3基因表达较普通支架组明显,且caspase-3基因高表达组犬胆管出现明显增殖平滑肌细胞凋亡,而且犬肝外胆管无明显狭窄;caspase-3基因低表达组犬胆管未出现增殖平滑肌细胞明显凋亡,而且犬肝外胆管有明显狭窄。结论:caspase-3基因表达水平与细胞凋亡发生和对辐射敏感性有关,^103Pd放射性支架通过激活caspase-3基因,促进犬胆管增殖平滑肌细胞凋亡,从而抑制犬肝外胆管狭窄。     

Stretch injury causes calpain and caspase-3 activation and necrotic and apoptotic cell death in septo-hippocampal cell cultures

Traumatic brain injury (TBI) results in numerous central and systemic responses that complicate interpretation of the effects of the primary mechanical trauma. For this reason, several in vitro models of mechanical cell injury have recently been developed that allow more precise control over intra- and extracellular environments than is possible in vivo. Although we recently reported that calpain and caspase-3 proteases are activated after TBI in rats, the role of calpain and/or caspase-3 has not been examined in any in vitro model of mechanical cell injury. In this investigation, varying magnitudes of rapid mechanical cell stretch were used to examine processing of the cytoskeletal protein alpha-spectrin (280 kDa) to a signature 145-kDa fragment by calpain and to the apoptotic-linked 120-kDa fragment by caspase-3 in septo-hippocampal cell cultures. Additionally, effects of stretch injury on cell viability and morphology were assayed. One hour after injury, maximal release of cytosolic lactate dehydrogenase and nuclear propidium iodide uptake were associated with peak accumulations of the calpain-specific 145-kDa fragment to alpha-spectrin at each injury level. The acute period of calpain activation (1-6 h) was associated with subpopulations of nuclear morphological alterations that appeared necrotic (hyperchromatism) or apoptotic (condensed, shrunken nuclei). In contrast, caspase-3 processing of alpha-spectrin to the apoptotic-linked 120-kDa fragment was only detected 24 h after moderate, but not mild or severe injury. The period of caspase-3 activation was predominantly associated with nuclear shrinkage, fragmentation, and apoptotic body formation characteristic of apoptosis. Results of this study indicate that rapid mechanical stretch injury to septo-hippocampal cell cultures replicates several important biochemical and morphological alterations commonly observed in vivo brain injury, although important differences were also noted.     

Negative feedback loop in the Bim-caspase-3 axis regulating apoptosis and activity of osteoclasts.

Proapoptotic Bcl-2 family member Bim plays an essential role in the osteoclast apoptosis and is degraded through ubiquitin/proteasome pathways in a caspase-3-dependent manner. This negative feedback loop in the Bim-caspase-3 axis is important for regulating the survival and activity of osteoclasts. INTRODUCTION: Bim is a member of the proapoptotic Bcl-2 family and regulates the mitochondrial apoptosis pathway. Bim expression is post-translationally regulated in osteoclasts (OCs) through ubiquitin/proteasome pathways, and Bim is critical for their survival and activity. MATERIALS AND METHODS: Time-course of change in the expression of Bim in the course of OC apoptosis was examined, and the effect of various proteinase inhibitors on the degradation of Bim was analyzed. The role of caspase-3 and caspase-7 on Bim degradation was studied using RNA interference technique and caspase-3(-/-) mice. RESULTS: Bim was degraded after caspase-3 activation, which was suppressed by a caspase inhibitor and a proteasome inhibitor. Bim degradation was suppressed by gene knockdown of caspase-3 or in caspase-3(-/-) OCs but not by caspase-7 knockdown. OCs generated from caspase-3(-/-) bone marrow cells exhibited a shorter life span and higher bone-resorbing activity than normal OCs. Association of Bim with E3 ubiquitin ligase c-Cbl was suppressed by gene knockdown of caspase-3 or in caspase-3(-/-) OCs. Actin ring formation and cathepsin K expression were promoted in caspase-3(-/-) OCs. CONCLUSIONS: Caspase-3 negatively regulates Bim expression by stimulating its degradation through ubiquitin/proteasome pathways, thus creating a negative feedback loop in the Bim-caspase axis.     

高度糖基化终产物对破骨细胞酸性磷酸酶和抗酒石酸酸性磷酸酶活性的影响

目的：观察高度糖基化终产物（AGE）对破骨细胞酸性磷酸酶（ACP）和抗酒石酸酸性磷酸酶（TRACP）活性的影响。方法：用人血清白蛋白和葡萄糖恒温孵育制备人AGE蛋白，应用酶消化法从人髂骨松质骨分离培养破骨细胞。将AGE蛋白与培养的破骨细胞共同孵育，通过酶动力学法测定培养液ACP和TRACP活性，结果：低浓度AGE蛋白（50－200μg/ml）对破骨细胞ACP和TRACP活性无显著，而高浓度（500－1000μg/ml）则使ACP和TRACP活性显著增高，且ACP活性的增高主要来自TRACP活性的增高。结论：上述结果提示AGE蛋白可能促进破骨细胞的破骨功能。     

Polymethylmethacrylate particles stimulate bone resorption of mature osteoclasts in vitro

Background?Interaction between wear particle debris and the cells at the implant-bone interface is an important contributory factor to periprosthetic bone loss seen in arthroplasties.

Method?To investigate the effect of this particle-induced response on different stages of osteoclast maturation, polymethylmethacrylate (PMMA) particles were added to a murine osteoclastogenic bone marrow cell culture system at either day 0, day 4, or day 8 of culture, which represented PMMA particle stimulation of precursor osteoclasts, mature osteoclasts, or end-stage osteoclasts, respectively. The number of TRAP-posi-tive multinucleate cells (MNCs) and the degree of bone resorption in culture were measured

Results?Treatment of precursor osteoclasts with PMMA particles resulted in a statistically significant increase in TRAP-positive MNCs that persisted for 4 days, but there was no significant increase in bone resorption. Addition of particles to mature osteoclasts resulted in a significant increase in the number of TRAP-positive MNCs that lasted for 8 days, and also a significant increase in bone resorption. Treatment of end-stage osteoclasts with PMMA particles did not result in an increased number of TRAP-positive MNCs and there was no increase in bone resorption.

Interpretation?Treatment of mature osteoclasts with PMMA particles resulted in an elevated number of TRAP-positive cells. This persisted over a longer period of time than at the other stages of osteoclast development, and there was also a greater increase in bone resorption.