唑来膦酸对体外破骨细胞性骨吸收影响的研究

目的：研究唑来膦酸（zoledronicacid,ZOL）对体外培养的破骨细胞骨（osteoclastic0C）吸收的抑制作用。方法：体外培养兔破骨细胞,通过抗酒石酸酸性磷酸酶（TRAP）染色破骨细胞计数、图像分析计算骨吸收陷窝面积、吖啶橙染色计算破骨细胞凋亡比率观察不同浓度唑来膦酸对细胞影响。结果：随着唑来膦酸浓度增高,TRAP阳性破骨细胞数量减少,骨吸收陷窝数量、陷窝面积亦减少;凋亡OC数目增多,凋亡率亦升高。结论：唑来膦酸通过抑制OC活性而直接抑制OC骨吸收功能,随其浓度增加而抑制作用增强。     

Neurokinin-1受体介导P物质对破骨细胞骨吸收功能的影响

目的 　了解P物质对体外培养大鼠破骨细胞骨吸收功能的影响和作用机制,探讨P物质在正畸牙槽骨改建中的调控机制。方法　 通过机械分离新生大鼠四肢长骨方法体外培养破骨细胞,细胞免疫组织化学染色观察NK1受体在大鼠破骨细胞内的表达情况;加入含不同浓度P物质（10-7-10-4 mol/L）和10-5 mol/L P物质受体拮抗剂（NK1受体拮抗剂）的培养液,观察P物质和NK1受体拮抗剂对破骨细胞骨吸收功能的影响。结果 免疫组化染色发现,大
鼠破骨细胞内NK1受体呈强阳性表达,着色位于细胞浆,胞核呈阴性;P物质明显增加破骨细胞的骨吸收陷窝面积（P<0.05）,但未见骨吸收陷窝数目明显增加（P>0.05）。NK1受体拮抗剂抑制了P物质对破骨细胞骨吸收功能的刺激
效应。结论 NK1受体可能介导P物质增强破骨细胞骨吸收功能,从而在正畸牙槽骨改建中发挥局部调节作用。     

白细胞介素-6对破骨细胞骨吸收效应及MMP-3表达影响的实验研究

目的：观察不同浓度IL－6对破骨细胞骨吸收的剂量效应及对破骨细胞基质金属蛋白酶－3表达的影响，以期进一步阐明IL－6介导基质金属蛋白酶在破骨细胞性骨吸收中的病理机制。方法：采用体外破骨细胞溶骨模型，通过原子吸收分光光度仪及免疫组化染色技术检测不同浓度IL－6对破骨细胞溶骨活性及其质金属蛋白酶－3表达的影响。结果：当IL－6＞10U／ml时，培养上清中Ca^2 浓度显著增加，牙本质片上骨吸收陷窝数目明显增多；当IL－6浓度为100U／ml、500U／ml时破骨细胞基质金属蛋白酶－3表达的阳性信号显著增强。结论：在IL－6作用下，破骨细胞表达基质金属蛋白酶－3。IL－6对破骨细胞具有激活作用，低浓度主要诱导破骨细胞形成，较高浓度刺激破骨细胞的溶骨活性。     

铜离子对牙各矿化成分破骨细胞性吸收的体外调控

目的研究铜离子对破骨细胞体外吸收人牙磨片功能的影响。方法体外分离、培养兔破骨细胞，与玻片和灭活人牙磨片共同培养，加入不同浓度的铜离子。抗酒石酸酸性磷酸酶染色鉴定玻片上的破骨细胞，显微摄影分析破骨细胞吸收造成的牙磨片上的吸收陷窝，原子吸收分光光度法测定溶出的钙，并将实验组上清液中钙离子浓度与对照组相比，定义为矿化组织吸收指数，以评价破骨细胞的功能。结果体外成功分离培养出多核的、抗酒石酸酸性磷酸酶染色（＋）的破骨细胞。破骨细胞吸收牙磨片时，首先在接近牙骨质或牙本质的部位开始形成吸收陷窝；破骨细胞在牙磨片上形成的吸收陷窝与骨片相比，数量较少，体积较小，多为正圆形；吸收深度较浅，常为大面积的浅吸收。（1×10^-14）～（1×10^-4）mol／L铜离子均能抑制矿化组织吸收，实验组矿化组织吸收指数均小于1。培养第3天，1×10^-10mol／L铜离子与对照组相比，其抑制效果差异有统计学意义（P〈0．05）。培养第7天，1×10^-4mol／L、（1×10^-10）～（1×10^-12）mol／L铜离子均能显著抑制矿化组织吸收（P〈0．05），但各浓度之间相比差异无统计学意义（P〉0．05）。结论一定浓度的铜离子可以抑制破骨细胞在牙磨片上的吸收。     

淫羊藿苷、黄芩苷和大黄素联合作用对体外骨吸收的影响

目的研究淫羊藿苷、黄芩苷和大黄素单独及联合作用对体外骨吸收的影响。方法将从新生兔长骨分离的细胞与牙本质磨片共培养,建立体外骨吸收模型,并加入白细胞介素(IL)-1β及淫羊藿苷0.01μg/ml、黄芩苷0.01μg/ml、大黄素10μg/ml或其等比组合进行干预,动态观察并测量吸收陷窝的数量和面积。结果分离的细胞与牙本质片共同培养24 h后,牙本质片上出现少量吸收陷窝。体外共培养10 d,加入IL-1β的阴性对照组的陷窝数量和面积大于空白对照组(P<0.05)。同时加入提取物的各组陷窝数量和面积均小于阴性对照组(P<0.05)。其中,淫羊藿苷组、黄芩苷组和大黄素组3组之间无显著差异(P>0.05),而组合组陷窝数量和面积均小于上述3组(P<0.01)。结论淫羊藿苷0.01μg/ml、黄芩苷0.01μg/ml、大黄素10μg/ml等比组合可抑制IL-1β诱导的骨吸收。     

阿伦膦酸盐在不同分化阶段对破骨细胞生成及骨吸收功能的影响

目的：研究阿伦膦酸盐作用在不同分化阶段对破骨细胞生成及骨吸收功能的影响。方法：体外分离骨髓单核细胞,用30μg/L M-CSF对其预诱导3d,然后将细胞分为A、B、C、D四组。A组（对照组）,用50μg/L M-CSF＋100μg/L RANKL进行破骨细胞诱导;B、C、D组除加入上述诱导因子外,在不同时间点加入0.1μmol/L阿伦膦酸盐（ALN）,加入时间分别为：B组第0～2天加入,C组第3～4天加入,D组第5～6天加入。检测每组细胞正式诱导第6天TRAP染色情况及牙本质磨片吸收陷窝情况。结果：各组细胞均有TRAP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A组TRAP阳性多核细胞数目、吸收陷窝数目及陷窝面积最高,D组次之,B组最差。结论：阿伦膦酸盐在破骨细胞分化阶段作用越早,对破骨细胞生成的抑制效应越大,所形成破骨细胞骨吸收能力越差。     

牙周炎牙槽骨吸收的基础研究—PGE2对破骨细胞性骨吸收的作用

本研究采用分离破骨细胞的体外培养方法,将分离的兔破骨细胞与牛骨片共同培养。相差显微镜观察前列腺素E_2.(Prostaglaodin E_2,PGE_2)对破骨细胞所形成的骨吸收陷窝数及形态学影响。并利用图象分析系统计算吸收陷窝的表面积。结果表明:PGE_2对体外分离的破骨细胞所形成的吸收陷窝数及吸收陷窝面积具有抑制作用。     

鼠破骨细胞培养过程中凋亡现象的观察研究

目的 观察研究鼠破骨细胞培养过程中的凋亡现象。方法 对机械分离法获得的鼠破骨细胞进行形态学、特异性耐酒石酸酸性磷酸酶(TRAP)染色及硬组织吸收实验鉴定。实验分为12h、24h、48h、72h组,分别在相应培养时间点行TRAP染色,计数每组阳性红染破骨细胞量。结果 机械分离法获得的鼠破骨细胞形态较大,TRAP染色见胞质呈酒红色不均匀沉淀,能形成不规则硬组织吸收陷窝。随着实验组培养时间的延长,TRAP阳性破骨细胞数量呈下降的趋势。在培养48h内,细胞数量减少不明显,当到达72h后,细胞数量明显减少。结论 鼠破骨细胞在培养过程中因细胞凋亡发生细胞数逐渐减少。     

Effects of 1,25 - （OH） 2 D3 on Stimulation of Osteoclast - like Cells Formationand Expression of ODF mRNA in Murine Marrow Cells in Vitro

目的：观察不同浓度的1，25-(OH)2D3对破骨细胞形成及对骨髓细胞ODFmRNA表达的影响：进一步阐明骨吸收刺激因子在正畸牙周组织改建中的作用。方法：应用不同浓度的1，25-(OH)2D3(0、10^-10、10^-8、10^-6mol／L)诱导大鼠骨髓细胞破骨样细胞的形成，采用体外破骨细胞溶骨模型，观察牙本质片上骨吸收陷窝数目，采用原位杂交技术检测骨髓基质细胞ODF的mRNA表达。结果：随着1，25-(OH)2D3浓度的增加，骨陷窝数明显增多，陷窝面积增大；ODF mRNA表达的阳性信号显著增强。结论：在体外，1，25-(OH)2D3可以调节破骨细胞的骨吸收活性，进而调节局部骨微环境的骨吸收及骨形成平衡的变化。     

大黄对破骨细胞性骨吸收作用的研究

从出生２４小时内的日本大耳白兔的四肢长骨中分离出破骨细胞，与牛骨磨片共同培养，培养液中加入大黄素，使终浓度为５×１０６ｍｏｌ／Ｌ和ｌ０５ｍｏｌ／Ｌ，另做空白对照。利用相差显微镜动态观察破骨细胞吸收骨的情况，连续观察７天。计数每个骨片上所形成的吸收陷窝数，并做统计学分析。结果表明：１０５ｍｏｌ／Ｌ的大黄可抑制破骨细胞性骨吸收，使破骨细胞在骨片上形成的吸收陷窝数减少（Ｐ＜０．０５）；而５×ｌ１０－６ｍｏｌ／Ｌ的大黄素虽可部分抑制破骨细胞性骨吸收，但对破骨细胞在骨片上形成的吸收陷窝数无明显影响（０．０５＜Ｐ＜０．２）。     

Polarization of osteoclasts on dental implant materials is similar to that observed on bone

ObjectivePolarized osteoclasts form sealing zones, (also called clear zones) detectable as actin rings, and ruffled borders to resorb bone. They secrete protons and catabolic enzymes, including tartrate-resistant acid phosphatase (TRAP), through the ruffled borders. We previously reported that polarized osteoclasts develop areas of TRAP activity (TRAP-marks) when cultured on dentin slices [11]. In this study, we examined how osteoclasts recognize dental implant materials.MethodsOsteoclasts obtained from murine co-cultures were cultured on implant materials such as titanium (Ti), alumina, zirconia, and sintered hydroxyapatite (sHA), in addition to dentin. Osteoclasts were also treated with reveromycin A (RM-A), which specifically acts on polarized osteoclasts and induces apoptosis. Polarization of osteoclasts cultured on implant materials was evaluated by measuring actin rings, TRAP-marks, and reveromycin A-induced apoptosis.ResultsOsteoclasts formed actin rings on all substrates examined. The formation of actin rings on Ti by osteoclasts was inhibited by the GRGDS peptide, but not by the GRGES peptide, suggesting an integrin-mediated polarization of osteoclasts on Ti. Calcitonin, an inhibitory hormone of osteoclast function, disrupted the actin rings that were preformed on Ti and sHA. Osteoclasts put TRAP-marks on sHA and dentin and formed resorption pits on dentin, but failed to form resorption pits on sHA. RM-A induced apoptosis in osteoclasts cultured on Ti and sHA; this was suppressed by calcitonin.ConclusionsThese results demonstrate that osteoclasts are able to polarize on dental implant materials similar to the polarization observed on bone.     

Decrease in the number and apoptosis of alveolar bone osteoclasts in estrogen-treated rats

BACKGROUND AND OBJECTIVE: Bone is a mineralized tissue that is under the influence of several systemic, local and environmental factors. Among systemic factors, estrogen is a hormone well known for its inhibitory function on bone resorption. As alveolar bone of young rats undergoes continuous and intense remodeling to accommodate the growing and erupting tooth, it is a suitable in vivo model for using to study the possible action of estrogen on bone. Thus, in an attempt to investigate the possibility that estrogen may induce the death of osteoclasts, we examined the alveolar bone of estrogen-treated rats. MATERIAL AND METHODS: Fifteen, 22-d-old female rats were divided into estrogen, sham and control groups. The estrogen group received estrogen and the sham group received corn oil used as the dilution vehicle. After 8 d, fragments containing alveolar bone were removed and processed for light microscopy and transmission electron microscopy. Sections were stained with hematoxylin and eosin and tartrate-resistant acid phosphatase (TRAP)-an osteoclast marker. Quantitative analysis of the number of TRAP-positive osteoclasts per mm of bone surface was carried out. For detecting apoptosis, sections were analyzed by the Terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling (TUNEL) method; TUNEL/TRAP combined methods were also used. RESULTS: The number of TRAP-positive osteoclasts per mm of bone surface was significantly reduced in the estrogen group compared with the sham and control groups. TRAP-positive osteoclasts exhibiting TUNEL-positive nuclei were observed only in the estrogen group. In addition, in the estrogen group the ultrastructural images revealed shrunken osteoclasts exhibiting nuclei with conspicuous and tortuous masses of condensed chromatin, typical of apoptosis. CONCLUSION: Our results reinforce the idea that estrogen inhibits bone resorption by promoting a reduction in the number of osteoclasts, thus indicating that this reduction may be, at least in part, a consequence of osteoclast apoptosis.     

厌氧菌荚膜对破骨细胞形成的影响

目的：观察３ 种厌氧菌荚膜对人骨髓长时间培养中破骨细胞形成和功能活化的影响。方法：采用合法堕胎５ 个月胎儿长干骨骨髓单个核细胞,在 Ｇ Ｍ－ Ｃ Ｓ Ｆ、马血清和荚膜存在下培养３ 周,观察破骨细胞的形成。结果：在所有培养中由单个核细胞融合形成的多核细胞多数具有破骨细胞特征：多核、细胞膜呈皱褶状、抗酒石酸盐酸性磷酸酶染色阳性,在牙本质片上可形成吸收陷窝等。牙龈卟啉菌荚膜组和中间型普里沃氏菌荚膜组中,抗酒石酸盐酸性磷酸酶染色阳性的多核和单个核细胞数和牙本质片上形成的吸收陷窝明显增多。牙髓卟啉菌荚膜组与对照组差异不明显。结论：厌氧菌荚膜可能参与了牙周病、尖周病后期的骨组织吸收。     

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目的    探讨正畸牙齿移动与肿瘤坏死因子α（TNF-α）表达的关系。方法    2008年9月至2009年1月于大连医科大学基础实验室,选取8周龄雄性SD大鼠18只,体重180～220 g。以大鼠上颌左侧牙弓为实验侧,右侧为对照侧。实验侧以大鼠上颌切牙为支抗牙,0.392 N（40 gf）力拉上颌第一磨牙向近中;对照侧不做任何处理。正畸加力第1、3、5、7、14、21天分别处死3只大鼠,取上颌双侧磨牙及周围组织制备切片,进行苏木素-伊红（HE）染色和抗酒石酸酸性磷酸酶（TRAP）染色,并采用免疫组化SABC法检测TNF-α变化（以第5天实验侧切片为阴性对照）。结果    正畸牙齿移动的骨改建过程中TNF-α水平随时间的增加呈先上升后下降的趋势。正常大鼠牙周组织中偶见TNF-α的表达,阴性对照未见TNF-α的表达。实验侧TNF-α的表达在加力第5天达到峰值,第21天接近对照侧的水平。结论    OC的形成分化和骨吸收的发生密切相关,是引发骨吸收的主要细胞;TNF-α是调控OC的形成和分化的重要因子,在正畸牙齿移动中发挥重要作用。     

Inhibitory effects of IL-12 on experimental tooth movement and root resorption in mice

ObjectiveInterleukin (IL)-12 is an important cytokine for innate and adaptive immunity. We previously reported that IL-12 inhibits tumour necrosis factor (TNF)-α-mediated osteoclast formation by inducing apoptosis. We also reported that TNF-α plays an important role in mechanical loading-induced osteoclast formation and bone resorption during orthodontic tooth movement. In this study, we investigated the effects of IL-12 on mechanical tooth movement in mice.DesignA Ni–Ti closed coil spring was inserted between the upper incisors and the upper left first molar in mice. IL-12 was injected locally adjacent to the first molar every other day during the experimental period, at doses varying from 0 to 1.5 μg/day. After 12 days, the animals were killed and their jaws were processed for histological evaluation using tartrate-resistant acid phosphatase (TRAP) and TdT-mediated dUTP-biotin nick end-labelling (TUNEL) staining, and measurements of the root resorption area.ResultsIn the IL-12-treated mice, tooth movement and root resorption appeared to be reduced. In TUNEL-stained sections, many apoptotic cells were recognized on the pressure side in the IL-12-treated mice.ConclusionsOur findings suggest that IL-12 inhibits not only mechanical tooth movement, but also root resorption during orthodontic tooth movement. These findings may arise through apoptosis induced by IL-12.     

In vitro osteoclast resorption of bone substitute biomaterials used for implant site augmentation: a pilot study

PURPOSE: This observational study examined the resorptive behavior of normal neonatal rabbit osteoclasts grown on slices of bovine cortical bone as compared to samples of commercially available bone substitute biomaterials. It also examined the surface characteristics of these materials. MATERIALS AND METHODS: The 11 materials tested fell into 3 groups: (1) bone-derived, including freeze-dried human rib block, human demineralized freeze-dried bone, and deproteinated bovine bone; (2) synthetic hydroxyapatites (HA); and (3) synthetic non-HA, including coated methacrylates and coated silica glass. After 4 days in culture, 1 group of samples of each material underwent scanning electron microscopy (SEM) to evaluate resorptive pitting versus controls, while another group underwent tartrate-resistant acid phosphatase staining and light microscopy to examine osteoclast numbers and morphology. The 2 bovine-derived HA materials also underwent immunohistochemical staining and surface chemistry analysis. RESULTS: While most of these materials supported osteoclast attachment, some spreading, and survival in culture, only the bone-derived materials, with the exception of sintered deproteinated bovine bone, showed large scalloped-edged resorption pits with trails and exposed collagen when examined by SEM, although not to the same extent as unprocessed natural bone material. The HA materials and the sintered deproteinated bovine bone showed evidence of etching with smaller pits but no evidence of resorptive trail formation. The non-HA materials showed no evidence of pit formation or trails. Under immunohistochemical staining, Bio-Oss appeared to be positive for type I collagen after osteoclast activity on its surface, while Osteograf/N showed no positive staining. Surface chemistry analysis revealed nitrogen present in Bio-Oss specimens (0.17% to 0.47%), while there was no nitrogen detected in the Osteograf/N (0.00%); the percent nitrogen observed in normal bovine bone controls was 6.01% to 9.25%. DISCUSSION: The bone-derived materials supported osteoclast activity on the material surface in a way that facilitated formation of the more complex resorption pits in vitro. Assuming the rate of pit formation observed in vitro mimics that observed in vivo, the quantity and type of osteoclastic remodeling seen on non-bone-derived materials--and perhaps sintered bone-derived materials--would be extremely slow to negligible. Physiologic removal of non-bone-derived bone substitutes in vivo may occur by methods other than osteoclast resorption. CONCLUSIONS: Allogenous and xenogenous bone-derived materials that undergo delayed physiologic resorption may be more appropriately used with a staged surgical approach when used in sites intended to support osseointegrated dental implants. The combination of collagen staining and the presence of nitrogen suggest that there may be residual protein in Bio-Oss.     

破骨细胞分化因子诱导小鼠骨髓细胞形成破骨细胞的实验研究

目的:探讨破骨细胞分化因子(ODF)和巨噬细胞集落刺激因子(M-CSF)联合应用进行体外诱导小鼠骨髓细胞形成破骨细胞(OC)的能力。方法:收集5～6周小鼠骨髓细胞,在含M-CSF(10 ng/ml)的α-MEM全培养液中培养24h,然后将悬浮生长的细胞接种到24孔培养板,并加入不同浓度的ODF和M-CSF。,通过观察抗酒石酸盐酸性磷酸酶(TRAP)染色的阳性细胞和能否在骨磨片上形成吸收陷窝来鉴定OC形成情况。结果:在只加入ODF或M-CSF一种细胞因子时,未见有TRAP阳性或CTR阳性细胞形成,同时加入ODF和M-CSF,可形成典型的OC。TRAP阳性多核细胞的数目和培养液中钙离子浓度的增加与ODF的浓度呈正相关。结论:小鼠骨髓来源的单核细胞在ODF和M-CSF共同作用下可形成具有骨吸收功能的OC,为体外研究OC的分化发育和功能调节提供了一种新的方法。     

白介素-23对破骨细胞分化及功能直接调节作用的实验研究

目的检测白介素-23（IL-23）是否对破骨细胞分化及功能有直接调节作用。方法在体外培养小鼠破骨细胞的过程中加入不同浓度的IL-23,观察破骨细胞形成数量及吸收陷窝的变化以及组织蛋白酶-K mRNA的表达变化。同时,以RAW264.7诱导破骨细胞,采用RT-PCR及FACS方法检测IL-23刺激下,RANK mRNA及蛋白的表达变化情况。结果 IL-23作用下,小鼠破骨细胞数量增加,形成的吸收陷窝增多,组织蛋白酶-K mRNA的表达增强。同时在IL-23刺激下,破骨前体细胞表面RANK mRNA及蛋白的表达增强。结论 IL-23可以通过调高破骨前体细胞表面RANK的表达,直接促进小鼠破骨细胞分化。