不同质量浓度阿仑膦酸钠干预牙槽骨吸收模型兔牙周组织破骨细胞分化因子和骨保护素的表达

背景：有研究表明阿仑膦酸钠能够防治骨质疏松症,但其应用到口腔牙周组织干预牙槽骨吸收较为罕见。 目的：建立兔牙槽骨吸收模型,观察不同质量浓度阿仑膦酸钠干预下炎症牙周组织破骨细胞分化因子和骨保护素的表达。 方法：将大耳白兔建立牙槽骨吸收模型,建模成功后随机分成5组,胶原+0.5 g/L阿仑膦酸钠组、胶原+1 g/L阿仑膦酸钠组、胶原+2 g/L阿仑膦酸钠组、胶原组、对照组。各组分别于用药后2和4周用免疫组织化学方法检测破骨细胞分化因子和骨保护素表达情况,并进行药效评价。 结果与结论：破骨细胞分化因子和骨保护素阳性细胞在5组成骨细胞、破骨细胞、成纤维细胞中均有表达,胞浆呈棕色或棕褐色颗粒状着色。对照组和胶原组牙槽嵴吸收区破骨细胞分化因子和骨保护素比率明显大于其他3组(P < 0.05),胶原+1 g/L阿仑膦酸钠组在用药后2和4周均高于胶原+0.5 g/L阿仑膦酸钠组(P < 0.05)。结果证实,阿仑膦酸钠能够降低牙槽骨破骨细胞分化因子和骨保护素的比率,应用含1 g/L 阿仑膦酸钠胶原海绵载体更适合抑制牙槽骨吸收。     

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。     

大鼠破骨细胞体外分离培养和鉴定

采用机械分离方法，从新生大骨长管骨分离破骨细胞获得成功。分离的破骨细胞具有如下特性：１．在体外迅速贴附于盖玻片或骨片；２．多核（一般为３￣１０个细胞核）；３．伪足运动活跃，降钙素或降钙素基因相关肽可以抑制其伪足运动；４．酸性磷酸酶染色强阳性，酸性醋酸酯酶弱阳性，ＰＡＳ中等阳性；５．细胞悬液与骨磨片共同培养，可以形成骨吸收陷窝。由此可见，本研究分离的细胞，符合破骨细胞的公认特性，故本法分离培养破骨细     

双膦酸盐对破骨细胞分化中组织蛋白酶K及骨吸收功能的影响

背景:有研究表明双膦酸盐可抑制破骨细胞的骨吸收功能,但对其骨吸收功能关键细胞因子组织蛋白酶K是否产生作用,至今少有报道。目的:观察双膦酸盐对破骨细胞分化中组织蛋白酶K及骨吸收功能影响。方法:用小鼠单核巨噬细胞RAW264.7诱导培养破骨细胞。实验分2组:对照组加入质量浓度100μg/L核因子κB受体活化因子配体进行诱导至收获细胞,双膦酸盐组在对照组的基础上加入10-7 mol/L阿仑膦酸盐处理至收获细胞。培养第7天检测各组破骨细胞生成和骨吸收功能,培养72 h免疫荧光检测两组组织蛋白酶K表达差异,Western blot检测组织蛋白酶K蛋白表达情况。结果与结论:两组均有抗酒石酸酸性磷酸酶阳性多核破骨细胞生成,并在牙本质磨片上形成吸收陷窝;但对照组抗酒石酸酸性磷酸酶阳性多核细胞数目、吸收陷窝数目及陷窝面积均大于双膦酸盐组(P0.01)。免疫荧光检测组织蛋白酶K表达对照组强于双膦酸盐组(P0.01);Western blot检测组织蛋白酶K表达双膦酸盐组低于对照组(P0.01)。结果证实,双膦酸盐通过抑制组织蛋白酶K因子的表达,阻碍破骨细胞的骨吸收功能。     

破骨细胞TRPV5通道对体外降解生物珊瑚人工骨的影响

背景：生物珊瑚人工骨是一种修复长段骨缺损的良好替代材料,但其在体内的降解吸收速率与新生骨的生长速率不够匹配,因而其在临床上的应用受到限制。目的：探讨破骨细胞TRPV5通道对生物珊瑚人工骨降解的影响。方法：取生长状态良好的小鼠单核巨噬细胞株RAW264.7,接种于生物珊瑚人工骨片上,加入破骨细胞诱导分化培养液,观察有破骨细胞出现后,分别加入含0,50,500,5 000μmol/L钌红(TRPV5通道抑制剂)的破骨细胞诱导分化培养液。培养一定时间后,激光共聚焦显微镜下观察TRPV5通道蛋白表达,扫描电镜下观察生物珊瑚人工骨片上的吸收陷窝,应用Western Blot检测细胞TRPV5通道蛋白表达。结果与结论：(1)激光共聚焦显微镜：TRPV5表达于破骨细胞的胞浆和胞膜,随着钌红浓度的增加,破骨细胞上TRPV5表达减少;(2)扫描电镜：0 nmol/L钌红组骨片上可见大片连续的骨吸收陷窝,其他浓度钌红组骨片上的骨吸收陷窝减少,并散在分布,并且随着钌红浓度的升高,骨陷窝面积逐渐减少;(3)Western Blot检测：与0 nmol/L钌红组比较,其他浓度钌红组TRPV5通道蛋白表达明显降低...     

双膦酸盐对破骨细胞分化及抗酒石酸酸性磷酸酶的影响

背景:抗酒石酸酸性磷酸酶是破骨细胞分化及骨吸收功能的特异性标志酶,是破骨细胞分化成熟的标志。目的:观察双膦酸盐对破骨细胞分化及骨吸收功能相关因子抗酒石酸酸性磷酸酶的影响。方法:小鼠单核巨噬细胞RAW264.7诱导培养破骨细胞。实验分2组:对照组开始时加入质量浓度100μg/L核因子kB受体活化因子配体进行诱导至收获细胞,双膦酸盐组在对照组的基础上加入10-7 mol/L阿仑膦酸盐处理至收获细胞。培养第7天检测各组破骨细胞生成和骨吸收功能,免疫荧光检测两组抗酒石酸酸性磷酸酶表达的差异,Western blot检测抗酒石酸酸性磷酸酶蛋白表达情况。结果与结论:各组细胞均有抗酒石酸酸性磷酸酶阳性多核破骨细胞生成,并在牙本质磨片上形成吸收陷窝;但对照组抗酒石酸酸性磷酸酶阳性多核细胞数目、吸收陷窝数目及陷窝面积均大于双膦酸盐组(P0.01)。免疫荧光检测显示,对照组抗酒石酸酸性磷酸酶表达均强于双膦酸盐组(P0.01)。Western blot检测显示,双膦酸盐组抗酒石酸酸性磷酸酶蛋白的表达低于对照组(P0.01)。说明双膦酸盐通过抑制抗酒石酸酸性磷酸酶蛋白的表达,阻碍破骨细胞分化生成及骨吸收功能。     

空泡型质子泵阻断剂Baf. A1抑制破骨样细胞体外骨吸收研究

目的 从骨吸收机制比较骨巨细胞瘤中的破骨样细胞和破骨细胞,明确参与破骨样细胞骨吸收的质子泵类型及其在骨吸收中的作用,进一步探讨破骨样细胞的特征和来源。方法 采用骨片与破骨样细胞体外共同培养的方法,观察破骨样细胞形成的骨吸收陷窝数和陷窝面积,比较线粒体型质子泵Ｆ－ＡＴＰａｓｅ阻断剂Ｏｌｉｇｍｙｃｉｎｅ和空泡型质子泵阻断剂Ｂａｆｌｏｍｙｃｉｎｅ Ａ１对破骨样骨吸收的影响。结果 只有Ｂａｆ．Ａ１明显阻断     

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。     

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。     

构建创伤性股骨头塌陷坏死大鼠模型及阿仑膦酸钠预防的机制

背景：临床随访研究表明阿仑膦酸钠对于预防股骨头坏死塌陷有效,但尚缺乏其预防塌陷作用的机制研究。 目的：分析阿仑膦酸钠预防股骨头坏死塌陷的效果及其作用机制。 方法：将45只SD大鼠随机分成3组,每组15只。安慰剂组在建立股骨头坏死模型后给予生理盐水治疗;阿仑膦酸钠组建立股骨头坏死模型后给予药物阿仑膦酸钠治疗;假手术组给予同样剂量的生理盐水治疗。造模后5周处死大鼠,取造模侧股骨标本分别行大体标本观察,X射线、Micro-CT及组织学检测。 结果与结论：大体标本观察安慰剂组股骨头明显塌陷畸形,阿仑膦酸钠组股骨头轻度变形。股骨头高度与宽度的比值假手术组>阿仑膦酸钠组>安慰剂组,差异均有显著性意义。Micro-CT扫描结果显示阿仑膦酸钠组骨小梁平均数量多于安慰剂组,少于假手术组,差异均有显著性意义。阿仑膦酸钠组骨小梁平均厚度小于安慰剂组,但和假手术组比差异无显著性意义。阿仑膦酸钠组骨小梁平均间距小于安慰剂组,但大于假手术组,差异均有显著性意义。阿仑膦酸钠组股骨头骨组织体积、骨表面积、骨矿盐密度均大于安慰剂组,小于假手术组,差异均有显著性意义。组织学检测结果显示,阿仑膦酸钠组存在明显的死骨,破骨细胞明显受到抑制,破骨细胞数量较安慰剂组明显减少,成骨细胞和新生血管也受到了一定程度的抑制。结果表明阿仑膦酸钠可通过全面抑制破骨细胞、成骨细胞及血管新生而抑制骨坏死的修复反应,减慢坏死骨的吸收,保存骨量及股骨头形态,对大鼠创伤性股骨头坏死早期塌陷具有一定的预防作用。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

破骨细胞释放凋亡小体微小RNA-30a对成骨活性的影响

目的 探讨破骨细胞凋亡释放凋亡小体介导成骨活性的作用.方法 通过小鼠(n=10)骨髓单核细胞体外诱导破骨细胞,用抗酒石酸酸性磷酸酶(TRAP)染色和细胞骨架F-actin与DAPI双标免疫荧光鉴定破骨细胞,破骨细胞与小鼠成骨细胞MC-3T3E1共培养体系,DNA片段化ELISA分析破骨细胞凋亡,凋亡小体标志物检测,骨形...     

葛根素在体外对成骨细胞增殖和分化的影响

背景：成骨细胞是骨代谢平衡过程中的关键功能细胞,植物雌激素对成骨细胞的增殖和分化有重要影响,葛根素作为植物雌激素的一种,在体外以较大范围浓度对成骨细胞功能的影响仍少见报道。 目的：观察葛根素在体外对大鼠成骨细胞增殖和分化功能的影响。 方法：取新生Wistar大鼠的颅盖骨,对成骨细胞进行分离、培养、纯化及鉴定。将培养的成骨细胞随机分为对照组、10-3~10-10 mol/L不同浓度葛根素组,观察不同浓度葛根素对体外培养的成骨细胞增殖和碱性磷酸酶活性表达的影响。 结果与结论：细胞经葛根素处理后10-5~10-9 mol/L组成骨细胞增殖活性较对照组明显增加(P < 0.05),第3天增殖最快(P < 0.01),第4天开始下降;诱导第4天,各组碱性磷酸酶活性与对照组相比,差异均有显著性意义(P < 0.01),其中以10-6 mol/L组最显著(P < 0.01)。然而葛根素10-3 mol/L组成骨细胞增殖活性、碱性磷酸酶活性表达较对照组均减少(P < 0.05)。提示葛根素对成骨细胞的影响存在剂量依赖性,并且具有双向性,即在低浓度(10-5~10-8 mol/L)下刺激骨形成;在高浓度(10-3~10-4 mol/L)下抑制骨形成。     

Absence of complement factor H alters bone architecture and dynamics

Complement system is an important arm of the immune system that promotes inflammation. Complement Factor H (FH) is a critical regulator of the alternative complement pathway. Its absence causes pathology in different organs resulting in diseases such as age related macular degeneration and dense deposit disease. Recent studies suggest that the complement system plays a role in bone development and homeostasis. To determine the role of FH in bone architecture, we studied the FH knockout (fh-/-) mice. 3D reconstructions of femur from 16 week old fh-/- mice reveal significant changes, such as decreased BV/TV (4.5%, p?<?0.02), trabecular number (22%, p?<?0.01), tissue mineral density (16%, p?<?0.04), and increased marrow area (16% p?<?0.01), compared to their wild type (WT) counterparts. Kidney function and histology remained normal indicating that bone changes occurred prior to kidney dysfunction. Next we examined cultured osteoblasts and osteoclasts isolated from bone marrow. FH is expressed ubiquitously in the osteoblasts and in the cytoplasm of osteoclasts. The changes caused by absence of FH include: increase in number of osteoblasts (362%) and osteoclasts (342%), increase in RNA (180%) and protein expression of cathepsin K and increased osteoclast function (pit formation, 233%). Actin rearrangement in both osteoblasts and osteoclasts was altered, with a loss of integrity of the F-actin ring at the periphery of the osteoclasts. For the first time our studies demonstrate a direct role of FH in the maintenance of bone structure and function and is highlighted as a promising therapeutic target in bone diseases.     

Gene disruption of the calcium channel Orai1 results in inhibition of osteoclast and osteoblast differentiation and impairs skeletal development

Calcium signaling plays a central role in the regulation of bone cells, although uncertainty remains with regard to the channels involved. In previous studies, we determined that the calcium channel Orai1 was required for the formation of multinucleated osteoclasts in vitro. To define the skeletal functions of calcium release-activated calcium currents, we compared the mice with targeted deletion of the calcium channel Orai1 to wild-type littermate controls, and examined differentiation and function of osteoblast and osteoclast precursors in vitro with and without Orai1 inhibition. Consistent with in vitro findings, Orai1(-/-) mice lacked multinucleated osteoclasts. Yet, they did not develop osteopetrosis. Mononuclear cells expressing osteoclast products were found in Orai1(-/-) mice, and in vitro studies showed significantly reduced, but not absent, mineral resorption by the mononuclear osteoclast-like cells that form in culture from peripheral blood monocytic cells when Orai1 is inhibited. More prominent in Orai1(-/-) mice was a decrease in bone with retention of fetal cartilage. Micro-computed tomography showed reduced cortical ossification and thinned trabeculae in Orai1(-/-) animals compared with controls; bone deposition was markedly decreased in the knockout mice. This suggested a previously unrecognized role for Orai1 within osteoblasts. Analysis of osteoblasts and precursors in Orai1(-/-) and control mice showed a significant decrease in alkaline phosphatase-expressing osteoblasts. In vitro studies confirmed that inhibiting Orai1 activity impaired differentiation and function of human osteoblasts, supporting a critical function for Orai1 in osteoblasts, in addition to its role as a regulator of osteoclast formation.     

Endothelial nitric oxide synthase gene-deficient mice demonstrate marked retardation in postnatal bone formation, reduced bone volume, and defects in osteoblast maturation and activity

Nitric oxide (NO) has been implicated in the local regulation of bone metabolism. However, the contribution made by specific NO synthase (NOS) enzymes is unclear. Here we show that endothelial NOS gene knockout mice (eNOS-/-) have marked abnormalities in bone formation. Histomorphometric analysis of eNOS-/- femurs showed bone volume and bone formation rate was reduced by up to 45% (P: < 0.01) and 52% (P: < 0.01), respectively. These abnormalities were prevalent in young (6 to 9 weeks old) adults but by 12 to 18 weeks bone phenotype was restored toward wild-type. Dual energy X-ray absorptiometry analysis confirmed the age-related bone abnormalities revealing significant reductions in femoral (P: < 0.05) and spinal bone mineral densities (P: < 0.01) at 8 weeks that were normalized at 12 weeks. Reduction in bone formation and volume was not related to increased osteoclast numbers or activity but rather to dysfunctional osteoblasts. Osteoblast numbers and mineralizing activity were reduced in eNOS-/- mice. In vitro, osteoblasts from calvarial explants showed retarded proliferation and differentiation (alkaline phosphatase activity and mineral deposition) that could be restored by exogenous administration of a NO donor. These cells were also unresponsive to 17ss-estradiol and had an attenuated chemotactic response to transforming growth factor-beta. In conclusion, eNOS is involved in the postnatal regulation of bone mass and lack of eNOS gene results in reduced bone formation and volume and this is related to impaired osteoblast function.     

Constitutive c-fos expression in osteoblastic MC3T3-E1 cells stimulates osteoclast maturation and osteoclastic bone resorption.

The effect of culture supernatants of c-fos-transfected MC3T3-E1 osteoblastic cells on osteoclastic bone resorption was studied. Human c-fos cDNA was integrated in the expression vector pH8, and the cells were transfected using the calcium phosphate precipitation technique. Osteoclastic bone resorption was quantified by the pit formation assay, and the osteoclast maturation from precursor was assessed by the generation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNC). The culture supernatants of MC3T3-E1 transfectants constitutively expressing c-fos gene enhanced osteoclast-like MNC formation from haematopoietic blast cells compared with those of control transfectants (P < 0.01). The culture supernatants also promoted osteoclastic bone resorption: the pit number, 118.7 +/- 38.5, was significantly higher than 19.0 +/- 10.1 of the control (P < 0.05). The absorption area, 12,394 +/- 3145 mm2, was significantly larger than 1646 +/- 314 mm2 of the control (P < 0.05). The culture supernatants also promoted bone resorption by purified chick osteoclasts (P < 0.05). The results show that constitutive expression of c-fos gene in osteoblastic MC3T3-E1 cells stimulates osteoclast maturation and osteoclastic bone resorption by releasing humoral mediator(s).     

The role of nacreous factors in preventing osteoporotic bone loss through both osteoblast activation and osteoclast inactivation

Excessive bone resorption by osteoclasts relative to bone formation by osteoblasts results in the development of osteoporosis. Anti-osteoporotic agents that are able both to inhibit bone resorption and to stimulate bone formation are not available. We now show that water-soluble nacreous factors prepared from the pearl oyster Pteria martensii prevent osteoporotic bone loss associated with estrogen deficiency in mice mainly through osteoclast inactivation. Nacreous factors stimulated osteoblast biomineralization in?vitro in association with activation of signaling by c-Jun NH(2)-terminal kinase (JNK) and Fos-related antigen-1 (Fra-1). They also suppressed both osteoclast formation by blocking up-regulation of nuclear factor of activated T cells cytoplasmic 1 (NFATc1) as well as bone pit formation mediated by mature osteoclasts, likely by disrupting the actin ring of these cells. Our findings thus show that the components of a natural material have beneficial effects on bone remodeling that are mediated through regulation of both osteoblast and osteoclast function. They may thus provide a basis for the development of biomimetic bone material as well as anti-osteoporotic agents.     

唑来膦酸对大鼠成骨细胞护骨素及肿瘤坏死因子α mRNA表达的影响

背景：唑来膦酸属于第3代双瞵酸盐类药物,在临床上被广泛应用于治疗骨吸收增加类疾病,其对破骨细胞的作用及影响已取得了共识,而对于成骨细胞的影响尚有争议。 目的：观察唑来膦酸对大鼠成骨细胞增殖、分化和护骨素及肿瘤坏死因子α mRNA表达的影响。 方法：体外培养新生24 h内SD大鼠颅盖骨来源的成骨细胞,用10^-5~10^-9 mol/L唑来膦酸进行干预,分别于干预后第3,5,7天采用MTT法来测吸光度值,以检测唑来膦酸对大鼠成骨细胞增殖的影响;硝基苯基质动力学法和RT-PCR在干预后72 h,测量细胞碱性磷酸酶活性和护骨素及肿瘤坏死因子α mRNA的表达。 结果与结论：较高浓度(10^-5 mol/L)的唑来膦酸明显抑制成骨细胞增殖, 10^-7~10^-9 mol/L唑来膦酸不影响成骨细胞的分化。10^-5~10^-9 mol/L唑来膦酸能使成骨细胞护骨素mRNA表达增加,肿瘤坏死因子α mRNA表达下降。说明唑来膦酸在低浓度时不影响成骨细胞的增殖及分化,其可通过调节成骨细胞护骨素、肿瘤坏死因子α mRNA的表达,来发挥其抗骨吸收的作用。