不同氧环境中破骨细胞的发育分化和功能变化研究

目的 本实验拟观察不同氧浓度下破骨细胞诱导过程中的分化发育,寻找破骨细胞体外培养的适宜氧浓度,为骨转换平衡的修复提供依据.方法 取野生型C57B/L小鼠(2个月龄左右,雄性)骨髓进行破骨细胞的诱导培养.用RANKL(10ng/ml)和M-CSF(10ng/ml)联合的诱导方案,将小鼠骨髓中单核-巨噬细胞系体外诱导为破骨细胞样细胞.将原代破骨细胞置于20%O2、7%O2、2%O2下诱导培养,MOCP5不同氧浓度下普通培养.用MTT法检测MOCP5的增殖变化,用抗酒石酸酸性磷酸酶(TRAP)染色检测破骨细胞形成的变化,并进行TRAP阳性细胞计数,用象牙骨片骨吸收陷窝甲苯胺蓝染色检测破骨细胞骨吸收活性的变化.结果 骨髓中单核-巨噬细胞体外经RANKL和M-CSF联合诱导可分化为多核的破骨细胞样细胞,在诱导第3天细胞开始融合,第5天TRAP染色强阳性,第8天可见象牙骨片上形成圆形、椭圆形、腊肠形等多种形态的骨吸收陷窝.MTT检测显示MOCP5在20%O2培养时一直处于增殖状态,7%O2条件下由增殖期进入平台期,2%O2时增殖缓慢且没有规律.20%O2、7%O2、2%O2培养下形成的TRAP阳性破骨细胞数分别为22±5.97、34±2.97、7±1.39(P<0.05),原代诱导的破骨细胞在20%O2、7%O2、2%O2形成的骨吸收陷窝面积(μm2)分别为3892.28±1642.78、5356.7±1655.6、2573±994.48(P<0.05).结论 体外RANKL和M-CSF联合可将骨髓单核-巨噬细胞诱导成多核的破骨细胞样细胞作为破骨细胞的研究模型.常氧条件下破骨细胞的TRAP阳性细胞数和骨吸收活性均低于7%O2.7%O2培养下的破骨细胞更接近于体内生理状态的破骨细胞.     

骨桥蛋白对Ⅰ型神经纤维瘤病小鼠破骨细胞功能的影响

Ⅰ型神经纤维瘤病( NFI)患者有明显的骨质疏松和骨量减少[1-2],我们报道了神经纤维瘤病破骨细胞功能增强是引起骨改变的机制之一[3].骨桥蛋白(OPN)具有细胞因子和基质蛋白两种性质,能影响骨代谢平衡,造成骨质疏松.我们应用Nf1基因杂合型小鼠模型,研究外源性骨桥蛋白对其破骨细胞生物学功能的影响,并与同源野生型小鼠破骨细胞功能进行统计学比较.     

辛伐他汀抑制甲状旁腺素相关肽诱导的破骨细胞生成和功能

目的:探讨辛伐他汀对骨髓来源的破骨细胞形成和功能的影响。方法:取Balb/C雄性小鼠双侧股骨和胫骨的骨髓,以不含血清的α-MEM培养液洗涤并收集骨髓细胞,再将细胞重新悬浮于含100 m l/L胎牛血清的α-MEM培养液中,细胞计数后,配成1.5×109/L的细胞悬液,同时加入甲状旁腺素相关肽(PTHrP)和不同剂量的辛伐他汀(10-7、10-6、10-5mol/L)于24孔培养板进行培养,并设阳性对照(只加PTHrP)和阴性对照(PTHrP和辛伐他汀都不加)组,每组均有1孔放置骨磨片1片,培养6 d后;去除上清,抗酒石酸(TRAP)染色检测培养板底部破骨细胞形成;骨磨片用甲苯胺蓝染色,电镜检测骨磨片的吸收陷窝。结果:小鼠骨髓细胞在PTHrP的诱导下获得大量的TRAP染色阳性的破骨细胞,骨磨片有吸收陷窝形成;用辛伐他汀(10-7、10-6mol/L)和PTHrP共同培养下TRAP染色阳性的破骨细胞形成数量均明显减少(P<0.01),辛伐他汀在10-5mol/L时则无TRAP染色阳性的破骨细胞形成;辛伐他汀在10-7mol/L时骨磨片有吸收陷窝的形成但少于阳性对照组(P<0.01),在10-6、10-5mol/L时骨磨片则无吸收陷窝的形成。结论:辛伐他汀对小鼠骨髓来源的破骨细胞的形成有着明显的抑制作用,并且呈剂量依赖关系。     

共育体系中成骨细胞和破骨细胞生物学特性观察

目的建立成骨细胞和破骨细胞的体外共育体系,观察在此体系中成骨细胞和破骨细胞生物学特性的变化,探讨成骨细胞和破骨细胞间的相互作用。方法取髂骨松质骨,Ⅱ型胶原酶消化,分次获得成骨细胞和破骨细胞。建立培养上清相通但二者互不接触的成骨细胞-破骨细胞共育模型。以细胞增殖(MTT法)、碱性磷酸酶(ALP)活性代表成骨细胞的成骨活性,以抗酒石酸酸性磷酸酶(TRAP)活性、骨吸收陷窝面积代表破骨细胞的破骨能力,检测共育对成骨细胞和破骨细胞生物学特性的影响。结果成骨细胞呈饱满的梭形,ALP染色阳性;破骨细胞呈多核,TRAP染色阳性,可以吸收骨质形成骨陷窝。当成骨细胞与破骨细胞共育后,其MTT法OD值(0.60±0.08)较单独培养时(0.36±0.03)明显提高(P=0.000);其ALP活性(23.37±2.48)u/mg较单独培养时(18.33±0.34)u/mg明显提高(P=0.000)。破骨细胞与成骨细胞共育后,形成骨吸收陷窝的平均面积犤(6.55±0.34)×10-2犦μm2较单独培养时犤(5.15±0.17)×10-2犦μm2明显增大(P=0.000)。结论共育体系中成骨细胞和破骨细胞的功能相互促进,为骨组织代谢的体外研究提供了可靠的模型。     

Transwell小室内建立小鼠成骨-破骨细胞共培养体系

目的 :Transwell小室内建立体外小鼠成骨-破骨细胞共培养体系,并检测体系对成骨及破骨细胞活性的影响。方法:体外培育小鼠成骨细胞MC3T3-E1和小鼠单核巨噬细胞RAW264.7,RANKL诱导小鼠单核巨噬细胞RAW264.7分化为成熟破骨细胞后,于Transwell小室内建立成骨-破骨细胞共培养体系。通过CCK-8实验、茜素红染色、TRAP染色检测细胞的成骨、破骨活性。采用PCR、Western Blot方法检测成骨细胞MC3T3-E1中OPG、ALP、RANKL、TGF-b1的基因表达以及RANKL的蛋白表达,检测破骨细胞RANK、NF-κB的基因表达和蛋白表达。结果 :小鼠成骨细胞MC3T3-E1和小鼠破骨细胞可在Transwell小室内建立共培养体系;共培养体系影响小鼠成骨细胞与破骨细胞的分化活性,镜下可见成骨细胞分化增多,破骨细胞分化稍减少。共培养体系中成骨细胞基因OPG(0.65±0.08)、ALP(0.16±0.01)较单独培养OPG(1.00±0.08)、ALP(1.01±0.16)表达下降,而TGF-b1(4.42±0.21)、RANKL(4.12±1.04)较单独培养组TGF-b1(1.00±0.10)、RANKL(1.00±0.09)表达上升;破骨细胞相关RANK(0.63±0.06)、NF-κB(0.64±0.08)基因表达较单独培养组的RANK(1.00±0.08)、NF-κB(1.00±0.09)下降,差异均有统计学意义。同时共培养组的OPG(0.43±0.05)、NF-κB(0.59±0.05)的蛋白表达较单独培养组的OPG(0.84±0.06)、NF-κb(1.13±0.03)减少;共培养组RANKL(0.54±0.03)的蛋白表达则较单独培养组的RANKL(0.31±0.03)增加,差异有统计学意义,均与基因表达变化趋势一致。结论:小鼠成骨细胞MC3T3-E1和小鼠破骨细胞可在Transwell小室内建立共培养体系,共培养体系中成骨细胞活性高于破骨细胞活性。     

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.     

不同浓度金属磨损颗粒对破骨细胞体外分化的影响

[目的]观察不同浓度金属磨损颗粒对RAW 264.7在体外分化成破骨细胞的影响,明确浓度与破骨细胞分化数量的关系.[方法]真空球磨法制备人工关节磨损颗粒:RANKL诱导RAW 264.7体外分化成破骨细胞,通过TRAP染色,电镜扫描检测骨片吸收陷窝来鉴定破骨细胞;不同浓度人工关节磨损颗粒混悬液作用RAW 264.7,并用RANKL诱导后第7 d,TRAP染色后,光镜下计数破骨细胞数量.[结果]不同浓度磨损颗粒作用于RAW 264.7 7 d后,显微镜下计数破骨细胞数量,结果显示随着磨损颗粒混悬液浓度增加,RANKL诱导生成的破骨细胞增多,低、中、高浓度3组破骨细胞数均显著高于空白对照组(P<0.05),中、高浓度组破骨细胞数均显著高于低浓组(P<0.05),高浓度组破骨细胞数亦显著高于中浓组(P<0.05).[结论](1)RAW 264.7是一种较好的破骨前体细胞模型,RAW 264.7诱导形成破骨细胞的方法简便易行,所获得破骨细胞均一性好;(2)人工关节金属磨损颗粒为RAW264.7细胞向具有骨质吸收功能的破骨细胞转化发挥正向作用,而且与混悬液的浓度有量效关系.     

阿伦膦酸钠对骨髓生成破骨细胞及骨吸收作用的影响

[目的]研究阿伦膦酸钠对体外培养的小鼠骨髓生成破骨细胞及其骨吸收作用的影响。[方法]收集小鼠骨髓细胞于含有10^-8mol／L的1，25二羟基维生素D3[1，25-（OH）2D3]的α-MEM完全培养基中体外培养，设置不同浓度的阿伦膦酸钠给药，并于培养的第6、9、12d观察记录抗酒石酸酸性磷酸酶（tartrate-resistant acidphosphatase，TRAP）阳性多核巨细胞[破骨样细胞（osteoclast-like cell，OLC）]生成数，反映破骨细胞生成情况。记数培养12d骨磨片上骨吸收陷窝数及吸收面积，反映骨吸收情况。[结果]随着阿伦膦酸钠浓度的增高，TRAP阳性的细胞数减少，骨吸收陷窝数及面积均减少。[结论]阿伦膦酸钠可抑制骨髓细胞体外培养中破骨样细胞的形成，体外可抑制破骨细胞骨吸收作用。     

磨损颗粒诱导骨溶解的两种动物模型形态学比较研究

目的建立两种磨损颗粒诱导骨溶解模型,比较炎症水平、骨溶解情况以及破骨细胞计数等指标,为骨溶解相关实验动物模型的选择提供依据。方法分别采用小鼠颅骨模型、小鼠气囊植骨模型制备两组磨损颗粒诱导骨溶解模型。通过HE染色法进行形态学观察;利用扫描电子显微镜观察骨溶解情况;采用TRAP染色法进行破骨细胞计数。结果小鼠气囊植骨模型组骨吸收面积为(385.30±5.11)nm2,明显高于小鼠颅骨模型组的(288.60±4.89)nm2(P0.05);小鼠气囊植骨模型组破骨细胞数目为(16.75±0.53)个,明显高于小鼠颅骨模型组的(9.55±0.48)个(P0.05)。结论两种动物模型均构建成功;小鼠气囊植骨模型在炎症水平、骨溶解情况及破骨细胞计数等方面均优于小鼠颅骨模型,因此在模拟磨损颗粒诱导的骨溶解病理过程中小鼠气囊植骨模型模拟度更高。     

小鼠成骨细胞和破骨细胞共培养模型的建立

目的 建立小鼠成骨细胞和破骨细胞共培养模型,为探讨成骨细胞和破骨细胞之间的相互调控作用奠定基础.方法 利用24 h内新生小鼠颅骨和4～8周龄成年小鼠四肢长骨分别分离、培养成骨细胞和破骨细胞,采用间接接触的培养模式将接种了骨髓单核细胞的玻片或骨片置入提前24 h接种了成骨细胞的培养皿中进行共培养.共培养一定时间后进行破骨细胞抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色鉴定和骨吸收功能检测,并进一步采用RT-PCR对破骨细胞标志酶基因基质金属蛋白酶.9(MMP-9)、TRAP 和组织蛋白酶K (Cathepsin K ) 的表达进行检测.结果 共培养5天后可见TRAP( )多核细胞形成,13天TRAP( )多核细胞数目达到高峰;骨吸收陷窝在共培养7天后开始出现,随着培养时间的延长,陷窝面积呈增加趋势;破骨细胞标志基因TRAP 在共培养3天时开始表达,而MMP-9和 Cathepsin K则在共培养5天后表达.结论 共培养体系中成骨细胞对破骨细胞调控作用显著,诱导的破骨细胞具有噬骨能力,该共培养模型可用于成骨细胞和破骨细胞之间的相互调控作用研究.     

The R740S mutation in the V‐ATPase a3 subunit increases lysosomal pH,impairs NFATc1 translocation,and decreases in vitro osteoclastogenesis

Vacuolar H⁺‐ATPase (V‐ATPase), a multisubunit enzyme located at the ruffled border and in lysosomes of osteoclasts, is necessary for bone resorption. We previously showed that heterozygous mice with an R740S mutation in the a3 subunit of V‐ATPase (+/R740S) have mild osteopetrosis resulting from an ～90% reduction in proton translocation across osteoclast membranes. Here we show that lysosomal pH is also higher in +/R740S compared with wild‐type (+/+) osteoclasts. Both osteoclast number and size were decreased in cultures of +/R740S compared with +/+ bone marrow cells, with concomitant decreased expression of key osteoclast markers (TRAP, cathepsin K, OSCAR, DC‐STAMP, and NFATc1), suggesting that low lysosomal pH plays an important role in osteoclastogenesis. To elucidate the molecular mechanism of this inhibition, NFATc1 activation was assessed. NFATc1 nuclear translocation was significantly reduced in +/R740S compared with +/+ cells; however, this was not because of impaired enzymatic activity of calcineurin, the phosphatase responsible for NFATc1 dephosphorylation. Protein and RNA expression levels of regulator of calcineurin 1 (RCAN1), an endogenous inhibitor of NFATc1 activation and a protein degraded in lysosomes, were not significantly different between +/R740S and +/+ osteoclasts, but the RCAN1/NFATc1 ratio was significantly higher in +/R740S versus +/+ cells. The lysosomal inhibitor chloroquine significantly increased RCAN1 accumulation in +/+ cells, consistent with the hypothesis that higher lysosomal pH impairs RCAN1 degradation, leading to a higher RCAN1/NFATc1 ratio and consequently NFATc1 inhibition. Our data indicate that increased lysosomal pH in osteoclasts leads to decreased NFATc1 signaling and nuclear translocation, resulting in a cell autonomous impairment of osteoclastogenesis in vitro. © 2013 American Society for Bone and Mineral Research     

Tartrate-resistant acid phosphate activity as osteoclastic marker: Sensitivity of cytochemical assessment and serum assay in comparison with standardized osteoclast histomorphometry

Tartrate-resistant acid phosphatase (TRAP) activity is regarded as an important cytochemical marker of osteoclasts; its concentration in serum is utilized as a biochemical marker of osteoclast function and degree of bone resorption. This study was carried out to assess the sensitivity of TRAP activity both as a cytochemical marker in histological sections and as a biochemical marker in serum in comparison with the standardized histomorphometric variables of osteoclasts. To this end we investigated 24 patients (21 women, 3 men; 60±17 years of age) affected with various metabolic bone diseases. Osteoclast surface (OcS/BS) and osteoclast number (OcN/BS) were evaluated by standardized histomorphometry in iliac crest biopsies. On the basis of TRAP cytochemical activity, TRAP-positive osteoclast surface (TRAP+OcS/BS) and number (TRAP+OcN/BS) were measured. TRAP-positive cells adjacent to bone and showing one nucleus or no nuclei at all in the plane of section were included in the counts as osteoclasts. Serum TRAP activity was determined by spectrophotometric assay. Values of OcS/BS and OcN/BS were much lower than those of TRAP+OcS/BS (–50%) and TRAP+OcN/BS (–60%), respectively. Correlations between OcS/BS and TRAP+OcS/BS, and between OcN/BS and TRAP+OcN/BS, were highly significant. Serum TRAP was significantly correlated with OcS/BS, OcN/BS, and TRAP+OcN/BS. These correlations, however, were rather low. Moreover, serum TRAP did not correlate with TRAP+OcS/BS. From these results, the conclusion can be drawn that while TRAP activity is confirmed as a valid cytochemical marker for identification of osteoclasts, serum TRAP activity is an osteoclastic marker of weak sensitivity. This may be due to known factors, such as synthesis of the enzyme not being unique to osteoclasts, enzyme instability, and the presence of inhibitors in serum. Mononucleated osteoclasts do not significantly influence the serum enzyme levels.     

Down-regulation of osteoclast differentiation by daidzein via caspase 3.

Phytoestrogens are plant-derived compounds with estrogen-like activity. Phytoestrogen-rich diets may prevent postmenopausal osteoporosis and these molecules maintain bone mass in ovariectomized animals. We compared the effects of the isoflavone daidzein, which has no action on tyrosine kinases, and 17beta-estradiol on the development and activity of osteoclasts in vitro. Nonadherent porcine bone marrow cells were cultured on dentine slices or on culture slides in the presence of 10-8 M of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], with or without 10(-8) M of daidzein, 10(-8) M of 17beta-estradiol for 9-11 days. Multinucleated tartrate-resistant acid phosphatase-positive (TRAP+) cells that resorbed bone (osteoclasts) developed in the presence of 1,25(OH)2D3. The number of osteoclasts formed in response to 1,25(OH)2D3 was reduced by 58 +/- 8% by daidzein and 52 +/- 5% by estrogen (p < 0.01); these effects were reversed by 10-6 M of ICI 182,780. The area resorbed by mature osteoclasts was reduced by 39 +/- 5% by daidzein and 42 +/- 6% by estradiol (p < 0.01). Both compounds also inhibited the 1,25(OH)2D3-induced differentiation of osteoclast progenitors (mononucleated TRAP+ cells), 53 +/- 8% by daidzein and 50 +/- 7% by estradiol (p < 0.05). Moreover, daidzein and estradiol promoted caspase-8 and caspase-3 cleavage and DNA fragmentation of monocytic bone marrow cells. Caspase-3 cleavage was reversed by 10-8 M of ICI 182,780. Both compounds up-regulated the expression of nuclear estrogen receptors ER-alpha and ER-beta. Thus, daidzein, at the same concentration as 17beta-estradiol, inhibits osteoclast differentiation and activity. This may be caused by, at least in part, greater apoptosis of osteoclast progenitors mediated by ERs.     

Tetracyclines induce apoptosis in osteoclasts

Chemically modified tetracyclines (CMTs) are thought to inhibit bone resorption through inhibition of matrix metalloproteinases. Here we report that some tetracyclines also induce apoptosis in rabbit osteoclasts and inhibit differentiation and activity of osteoclasts in murine osteoblast/marrow cocultures. Apoptosis of mature rabbit osteoclasts increased from 5.5 +/- 1.4% (mean +/- SD) in control cultures to 44.9 +/- 6.3% (p < 0.001) and 18.9 +/- 4.0% (p < 0.005) with CMT-3 and doxycycline (10 microg/mL), respectively. CMT-2 or CMT-5 did not alter osteoclast viability even at 25 microg/mL. In murine osteoblast/marrow cocultures over 11 days, CMT-3 and doxycycline (5 microg/mL) reduced the formation of mature osteoclasts and inhibited resorption to 21 +/- 9% (p < 0.01) and 49 +/- 4% (p < 0.01) of untreated cultures. Induction of osteoclast apoptosis is an additional property of tetracyclines that may contribute to their ability to inhibit bone resorption.     

紧密连接蛋白2缺失对小鼠胆汁及胆固醇结石形成的影响分析

目的 旨在探究紧密连接蛋白2（Cldn2）在胆汁分泌中的相关作用。方法 实验将受试小鼠分为Cldn2^-/-基因敲除组（Cldn2^-/-组）与Cldn2^+/+野生型组（Cldn2^+/+组），进行4周致石饮食，两组小鼠分别获得肝脏及胆管组织，通过组织学、生物化学、电生理学分析等分析两组小鼠肝脏和胆管内水及相关离子渗透压梯度运动差异；评价两组小鼠胆结石的形成情况。结果 Cldn2 ^+/+组小鼠的胆汁流速为[（58.0±4.4）μL/ （min·kg）]，约为Cldn2^-/-组[29.0±3.3 μL/（min·kg）]的2倍，Cldn2^-/-组胆汁中总胆固醇、磷脂、总胆汁酸及总胆红素均显著高于Cldn2+/+组（P＜0.01）。当将Cldn2^+/+组的肝内胆管单位（IBDU）置于低渗缓冲液中时，腔内空间立即增加且持续至90 s；相反，当IBDU置于高渗缓冲液中时，Cldn2^+/+组腔内空间立即降低并且持续收缩至90 s；Cldn2^-/-组具有相同的趋势，但变化幅度显著低于Cldn2^+/+组，表现为90 s低渗时[（1.20±0.02）% vs（1.34±0.02）%，P=0.035]，高渗时[（0.67±0.01）% vs（ 0.82±0.01）%，P=0.025]。与Cldn2^+/+组比，Cldn2^-/-组Na⁺浓度降低，但K⁺浓度升高，跨上皮电传导速度显著降低。致石饮食后所有Cldn2^-/-组肉眼均可见明显的胆结石形成，结石的主要成分是胆固醇（＞98%），而Cldn2^+/+组中仅1 只小鼠有结石形成。结论 Cldn2 可能通过调控胆汁中的水分、胆固醇含量以及离子浓度，进而影响胆汁流速和成分，该蛋白的缺失会导致胆固醇结石发生的风险增加。     

Lysosomal Ca2+ Signaling is Essential for Osteoclastogenesis and Bone Remodeling

Lysosomal Ca²⁺ emerges as a critical component of receptor‐evoked Ca²⁺ signaling and plays a crucial role in many lysosomal and physiological functions. Lysosomal Ca²⁺ release is mediated by the transient receptor potential (TRP) family member TRPML1, mutations that cause the lysosomal storage disease mucolipidosis type 4. Lysosomes play a key role in osteoclast function. However, nothing is known about the role of lysosomal Ca²⁺ signaling in osteoclastogenesis and bone metabolism. In this study, we addressed this knowledge gap by studying the role of lysosomal Ca²⁺ signaling in osteoclastogenesis, osteoclast and osteoblast functions, and bone homeostasis in vivo. We manipulated lysosomal Ca²⁺ signaling by acute knockdown of TRPML1, deletion of TRPML1 in mice, pharmacological inhibition of lysosomal Ca²⁺ influx, and depletion of lysosomal Ca²⁺ storage using the TRPML agonist ML‐SA1. We found that knockdown and deletion of TRPML1, although it did not have an apparent effect on osteoblast differentiation and bone formation, markedly attenuated osteoclast function, RANKL‐induced cytosolic Ca²⁺ oscillations, inhibited activation of NFATc1 and osteoclastogenesis‐controlling genes, suppressed the formation of tartrate‐resistant acid phosphatase (TRAP)‐positive multinucleated cells (MNCs), and markedly reduced the differentiation of bone marrow–derived macrophages into osteoclasts. Moreover, deletion of TRPML1 resulted in enlarged lysosomes, inhibition of lysosomal secretion, and attenuated the resorptive activity of mature osteoclasts. Notably, depletion of lysosomal Ca²⁺ with ML‐SA1 similarly abrogated RANKL‐induced Ca²⁺ oscillations and MNC formation. Deletion of TRPML1 in mice reduced the TRAP‐positive bone surfaces and impaired bone remodeling, resulting in prominent osteopetrosis. These findings demonstrate the essential role of lysosomal Ca²⁺ signaling in osteoclast differentiation and mature osteoclast function, which play key roles in bone homeostasis. © 2016 American Society for Bone and Mineral Research.     

NF-kappaB p50 and p52 expression is not required for RANK-expressing osteoclast progenitor formation but is essential for RANK- and cytokine-mediated osteoclastogenesis.

Expression of RANKL by stromal cells and of RANK and both NF-kappaB p50 and p52 by osteoclast precursors is essential for osteoclast formation. To examine further the role of RANKL, RANK, and NF-KB signaling in this process, we used NF-kappaB p50-/- ;p52-/- double knockout (dKO) and wild-type (WT) mice. Osteoclasts formed in cocultures of WT osteoblasts with splenocytes from WT mice but not from dKO mice, a finding unchanged by addition of RANKL and macrophage colony-stimulating factor (M-CSF). NF-kappaB dKO splenocytes formed more colony-forming unit granulocyte macrophage (CFU-GM) colonies than WT cells, but no osteoclasts were formed from dKO CFU-GM colonies. RANKL increased the number of CFU-GM colonies twofold in WT cultures but not in dKO cultures. Fluorescence-activated cell sorting (FACS) analysis of splenocytes from NF-kappaB dKO mice revealed a two-to threefold increase in the percentage of CD11b (Mac-1) and RANK double-positive cells compared with WT controls. Treatment of NF-kappaB dKO splenocytes with interleukin (IL)-1, TNF-alpha, M-CSF, GM-CSF, and IL-6 plus soluble IL-6 receptor did not rescue the osteoclast defect. No increase in apoptosis was observed in cells of the osteoclast lineage in NF-kappaB dKO or p50-/-;p52+/- (3/4KO) mice. Thus, NF-kappaB p50 and p52 expression is not required for formation of RANK-expressing osteoclast progenitors but is essential for RANK-expressing osteoclast precursors to differentiate into TRAP+ osteoclasts in response to RANKL and other osteoclastogenic cytokines.     

BM210955对破骨细胞骨吸收的抑制作用

细胞水平研究国产双磷酸盐药物－ＢＭ２１０９５５的骨吸收抑制作用。由１日龄ＳＤ大鼠四肢长骨分离破骨细胞（ＯＣ）并接种于牛皮质骨薄切片上,在不同浓度ＢＭ２１０９５５作用下培养,定时取骨片作ＴＲＡＰ免疫组化染色,计数阳性多核细胞后,经超声去除细胞后作甲苯胺蓝染色,光镜下作吸收陷窝计数,数据以ｘ±ｓ表示,并与对照比较作ｔ检验。结果显示：①ＢＭ２１０９５５能降低体外培养ＯＣ的数目,１０－８ｍｏｌ／Ｌ组的ＴＲＡＰ阳性多核细胞较对照组减少７３％,差异具有非常显著性,Ｐ＜０．０１。②ＢＭ２１０９５５抑制ＯＣ的陷窝形成能力,１０－１０、１０－８ｍｏｌ／Ｌ组的抑制率分别为７６．３２％和８７．９９％,均与对照有明显差异,Ｐ＜０．０１。③上述结果与剂量有关,剂量越高,抑制效应越明显     

Osteoclast polarization is not required for degradation of bone matrix in rachitic FGF23 transgenic mice

Hypophosphatemic transgenic (tg) mice overexpressing FGF23 in osteoblasts display disorganized growth plates and reduced bone mineral density characteristic of rickets/osteomalacia. These FGF23 tg mice were used as an in vivo model to examine the relation between osteoclast polarization, secretion of proteolytic enzymes and resorptive activity. Tg mice had increased mRNA expression levels of the osteoblast differentiation marker Runx2 and mineralization-promoting proteins alkaline phosphatase and bone sialoprotein in the long bones compared to wild type (wt) mice. In contrast, expression of alpha1(I) collagen, osteocalcin, dentin matrix protein 1 and osteopontin was unchanged, indicating selective activation of osteoblasts promoting mineralization. The number of osteoclasts was unchanged in tg compared to wt mice, as determined by histomorphometry, serum levels of TRAP 5b activity as well as mRNA expression levels of TRAP and cathepsin K. However, tg mice displayed elevated serum concentrations of C-terminal telopeptide of collagen I (CTX) indicative of increased bone matrix degradation. The majority of osteoclasts in FGF23 tg mice lacked ultrastructural morphological signs of proper polarization. However, they secreted both cathepsin K and MMP-9 at levels comparable to osteoclasts with ruffled borders. Mineralization of bone matrix thus appears essential for inducing osteoclast polarization but not for secretion of osteoclast proteases. Finally, release of CTX by freshly isolated osteoclasts was increased on demineralized compared to mineralized bovine bone slices, indicating that the mineral component limits collagen degradation. We conclude that ruffled borders are implicated in acidification and subsequent demineralization of the bone matrix, however not required for matrix degradation. The data collectively provide evidence that osteoclasts, despite absence of ruffled borders, effectively participate in the degradation of hypomineralized bone matrix in rachitic FGF23 tg mice.     

Characterization of the Bone Phenotype in ClC-7-Deficient Mice

Mice deficient in the chloride channel ClC-7, which is likely involved in acidification of the resorption lacuna, display severe osteopetrosis. To fully characterize the osteopetrotic phenotype, the phenotypes of osteoclasts and osteoblasts were evaluated. ClC-7^−/− mice and their corresponding wild-type littermates were killed at 4–5 weeks of age. Biochemical markers of bone resorption (CTX-I), osteoclast number (TRAP5b), and osteoblast activity (ALP) were evaluated in serum. Splenocytes were differentiated into osteoclasts using M-CSF and RANKL. Mature osteoclasts were seeded on calcified or decalcified bone slices, and CTX-I, Ca²⁺, and TRAP were measured. Acidification rates in membrane vesicles from bone cells were measured using acridine orange. Osteoblastogenesis and nodule formation in vitro were investigated using calvarial osteoblasts. ClC-7^−/− osteoclasts were unable to resorb calcified bone in vitro. However, osteoclasts were able to degrade decalcified bone. Acid influx in bone membrane vesicles was reduced by 70% in ClC-7^−/− mice. Serum ALP was increased by 30% and TRAP5b was increased by 250% in ClC-7^−/− mice, whereas the CTX/TRAP5b ratio was reduced to 50% of the wild-type level. Finally, evaluation of calvarial ClC-7^−/− osteoblasts showed normal osteoblastogenesis. In summary, we present evidence supporting a pivotal role for ClC-7 in acidification of the resorption lacuna and evidence indicating that bone formation and bone resorption are no longer balanced in ClC-7^−/− mice.