布鲁顿酪氨酸激酶对破骨细胞增殖及分化作用的实验研究

目的 观察布鲁顿酪氨酸激酶（BTK）对破骨细胞增殖及分化的影响,探讨BTK对根尖周炎骨破坏的作用。方法 破骨前体细胞RAW264.7经100 ng·L ^-1核因子κB受体活化因子配体（RANKL）诱导5 d后,通过观察细胞形态、抗酒石酸酸性磷酸酶（TRAP）染色及实时荧光定量PCR（RT-qPCR）的方法,验证破骨细胞是否诱导成功。破骨细胞诱导成功后,转染BTK-小干扰RNA（siRNA）24 h,利用RT-qPCR检测TRAP mRNA的表达,采用CCK-8和TRAP酶活性检测法检测破骨细胞的增殖及分化情况。结果 RAW264.7细胞经RANKL诱导5 d后,可见大量体积较大,外观呈圆形、椭圆形,周围有不规则突起及TRAP染色阳性的多核破骨细胞。经BTK-siRNA转染24 h后,破骨细胞TRAP mRNA的表达水平显著降低（P<0.05）,其增殖及分化能力也明显受到抑制（P<0.05）。结论 抑制BTK表达,可以抑制破骨细胞的增殖及分化;BTK可作为抑制破骨细胞的新靶点。     

布鲁顿酪氨酸激酶对破骨细胞增殖及分化作用的实验研究

目的 观察布鲁顿酪氨酸激酶（BTK）对破骨细胞增殖及分化的影响,探讨BTK对根尖周炎骨破坏的作用。方法 破骨前体细胞RAW264.7经100 ng·L ^-1核因子κB受体活化因子配体（RANKL）诱导5 d后,通过观察细胞形态、抗酒石酸酸性磷酸酶（TRAP）染色及实时荧光定量PCR（RT-qPCR）的方法,验证破骨细胞是否诱导成功。破骨细胞诱导成功后,转染BTK-小干扰RNA（siRNA）24 h,利用RT-qPCR检测TRAP mRNA的表达,采用CCK-8和TRAP酶活性检测法检测破骨细胞的增殖及分化情况。结果 RAW264.7细胞经RANKL诱导5 d后,可见大量体积较大,外观呈圆形、椭圆形,周围有不规则突起及TRAP染色阳性的多核破骨细胞。经BTK-siRNA转染24 h后,破骨细胞TRAP mRNA的表达水平显著降低（P<0.05）,其增殖及分化能力也明显受到抑制（P<0.05）。结论 抑制BTK表达,可以抑制破骨细胞的增殖及分化;BTK可作为抑制破骨细胞的新靶点。     

低氧状态下骨细胞参与破骨细胞形成的作用机制研究

目的 探究低氧状态下骨细胞对破骨细胞形成的作用及其机制。方法 用去铁胺甲磺酸（DFO）刺激骨细胞系MLO-Y4建立体外低氧骨细胞培养体系。CCK-8实验检测DFO对MLO-Y4细胞增殖活性的影响;采用DFO处理后的MLO-Y4细胞培养液制备条件培养基诱导RAW264.7细胞分化,行抗酒石酸酸性磷酸酶（TRAP）染色检测;利用实时荧光定量聚合酶链反应、细胞免疫荧光和蛋白质印迹（Western blot）检测DFO作用下MLO-Y4表达低氧诱导因子（HIF）-1α与核因子κB受体活化因子配体（RANKL）的情况;检测HIF-1α siRNA转染对MLO-Y4表达HIF-1α和RANKL的影响。结果 100 μmol·L ^-1 DFO作用24 h时MLO-Y4增殖活性升高,之后随着时间延长细胞增殖活性下降（P<0.01）。加入可溶性RANKL后,低氧组可见破骨细胞形成明显增加（P<0.001）。100 μmol·L ^-1 DFO作用下,HIF-1α mRNA表达稳定,RANKL mRNA的表达随时间明显变化,24 h时最高（P<0.01）;HIF-1α、RANKL蛋白表达升高（P<0.01）。低氧状态下siHIF-1α转染可降低HIF-1α和RANKL的表达（P<0.01）,破骨细胞减少（P<0.01）。结论 低氧状态下MLO-Y4可通过上调HIF-1α的蛋白水平促进RANKL的生成,进而加速RAW264.7细胞向破骨细胞分化。     

低氧状态下骨细胞参与破骨细胞形成的作用机制研究

目的 探究低氧状态下骨细胞对破骨细胞形成的作用及其机制。方法 用去铁胺甲磺酸（DFO）刺激骨细胞系MLO-Y4建立体外低氧骨细胞培养体系。CCK-8实验检测DFO对MLO-Y4细胞增殖活性的影响;采用DFO处理后的MLO-Y4细胞培养液制备条件培养基诱导RAW264.7细胞分化,行抗酒石酸酸性磷酸酶（TRAP）染色检测;利用实时荧光定量聚合酶链反应、细胞免疫荧光和蛋白质印迹（Western blot）检测DFO作用下MLO-Y4表达低氧诱导因子（HIF）-1α与核因子κB受体活化因子配体（RANKL）的情况;检测HIF-1α siRNA转染对MLO-Y4表达HIF-1α和RANKL的影响。结果 100 μmol·L ^-1 DFO作用24 h时MLO-Y4增殖活性升高,之后随着时间延长细胞增殖活性下降（P<0.01）。加入可溶性RANKL后,低氧组可见破骨细胞形成明显增加（P<0.001）。100 μmol·L ^-1 DFO作用下,HIF-1α mRNA表达稳定,RANKL mRNA的表达随时间明显变化,24 h时最高（P<0.01）;HIF-1α、RANKL蛋白表达升高（P<0.01）。低氧状态下siHIF-1α转染可降低HIF-1α和RANKL的表达（P<0.01）,破骨细胞减少（P<0.01）。结论 低氧状态下MLO-Y4可通过上调HIF-1α的蛋白水平促进RANKL的生成,进而加速RAW264.7细胞向破骨细胞分化。     

IL-17联合IFN-γ体外诱导RAW264.7细胞增殖和凋亡的影响

目的研究白细胞介素-17(IL-17)联合干扰素-γ(IFN-γ)在小鼠破骨前体细胞系RAW264.7分化为成熟破骨细胞过程中,对细胞增殖和凋亡的影响。方法将核因子κB受体活化因子配体(RANKL)和巨噬细胞集落刺激因子(M-CSF)诱导小鼠破骨前体细胞系RAW264.7建立破骨细胞体外诱导分化研究模型,被诱导的RAW264.7细胞培养24 h后,分为IL-17组、IFN-γ组、IL-17+IFN-γ联合组(IL-17+IFN-γ等量组、IL-17固定+IFN-γ梯度组)进行干预。抗酒石酸酸性磷酸酶(TRAP)染色对破骨细胞成熟进行鉴定,利用CCK-8细胞增殖试验检测各组细胞增殖情况,AnnexinⅤ细胞凋亡实验评价各组细胞凋亡的差异,实时荧光定量PCR检测凋亡相关基因mRNA表达差异。结果 50 ng/m L IL-17与不同浓度的IFN-γ联合,随着IFN-γ浓度升高,IL-17对RAW264.7细胞生长抑制呈浓度依赖性。IL-17和IFN-γ的联合作用比单独应用IL-17,凋亡率更高;与单独应用IFN-γ相比,凋亡相关基因Fas L表达明显增加。结论试在RAW264.7细胞向破骨细胞的分化过程中,IL-17和IFN-γ联合能抑制破骨前体RAW264.7细胞向破骨细胞的增殖,并促进RAW264.7细胞的凋亡。     

白介素-17对破骨前体细胞RAW264.7增殖分化的影响

探讨白细胞介素-17对破骨前体细胞RAW264.7增殖分化功能的影响。方法：采用RANKL和M-CSF刺激破骨前体细胞系RAW264.7培养24小时后,将IL-17干预细胞培养。采用CCK-8试剂盒,及实时荧光定量PCR,分别评价破骨前体细胞增殖和特征性基因的表达差异。 结果：IL-17A促进小鼠RAW264.7细胞增殖,且呈时间、浓度依赖性;IL-17A浓度在50mg/ml时,特征性基因CathK,RANK和NAFTC1 mRNA表达升高,差异均有统计学意义（P<0.05）。 结论：IL-17A促进RAW264.7细胞增殖,促进破骨前体细胞分化。     

PMX205对致敏毒素C5a诱导RAW264.7向破骨细胞分化的影响

目的:探讨PMX205作用下,致敏毒素C5a对RAW264.7破骨细胞分化的影响.方法:采用MTT法检测不同浓度PMX205对RAW264.7细胞增殖水平的影响;在含核因子受体活化因子配体(RANKL)的破骨细胞诱导液条件下,实验分为3组:阴性对照组、C5a组和C5a+PMX205组,按上述条件培养5 d后,采用抗酒石酸酸性磷酸酶(TRAP)染色法检测各组破骨细胞分化水平;采用实时荧光定量聚合酶链反应(qRT-PCR)检测各组破骨细胞相关基因的表达水平.结果:MTT结果显示,PMX205对RAW264.7细胞的增殖水平无明显抑制作用.TRAP染色和qRT-PCR结果显示:10 ng/mL RANKL可成功诱导破骨细胞形成;与阴性对照组相比,1μmol/L C5 a组TRAP阳性多核细胞数目及破骨相关基因:c-fos、NFATc1和TRAF6均显著增多(P<0.01);而与C5a组相比,C5a+1μg/mL PMX205组TRAP阳性多核细胞数目及破骨相关基因的表达明显下调(P<0.01),但仍高于阴性对照组(P<0.01).结论:PMX205可通过C5a-C5aR轴抑制C5a诱导的RAW264.7破骨细胞分化.     

破骨细胞骨吸收上清液对成骨活动影响的实验研究

目的 研究破骨细胞骨吸收活动中的分泌产物对成骨细胞增殖、分化及成骨功能的影响,以了解活化的破骨细胞在体外对成骨细胞的影响.方法 诱导小鼠RAW264.7细胞为破骨细胞,用抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)染色和破骨细胞特异性基因检测鉴定.破骨细胞与牛骨磨片共培养,甲苯胺蓝染色.收集共培养上清液作用于小鼠MC3T3-E1细胞,用甲基噻唑基四唑法(methyl thiazolyl tetrazolium,MTT)法、酶联免疫吸附测定法、茜素红染色及反转录聚合酶链反应检测细胞的增殖、骨钙素水平、矿化及成骨特征基因的表达.结果 TRAP 染色、破骨细胞特异性基因检测及甲苯胺蓝染色表明RAW264.7细胞可分化为具有骨吸收能力的破骨细胞;破骨细胞骨吸收上清液作用后,MC3T3-E1细胞增殖受抑制(A值在0.062±0.004和0.405±0.033之间,P＜0.05);骨钙素水平增高[第10天上清液组的骨钙素质量浓度为(2.965±0.047) μg/L],钙化结节增多,碱性磷酸酶和Runt相关转录因子2的转录增强.结论 RAW264.7破骨细胞骨吸收上清液具有抑制成骨样细胞增殖、促进分化和钙化成骨的作用.     

压应力对小鼠单核细胞RAW264.7 DNAX活化蛋白12、抗酒石酸酸性磷酸酶表达的影响

目的 探讨压应力对小鼠单核细胞RAW264.7 DNAX活化蛋白12（DAP12）、抗酒石酸酸性磷酸酶（TRAP）表达的影响。方法 以小鼠单核细胞RAW264.7为研究对象,采用四点弯曲体外细胞加载装置加载压应力0、3、6、12 h,分别以逆转录聚合酶链反应（RT-PCR）、蛋白免疫印迹法检测DAP12、TRAP mRNA和DAP12蛋白的表达情况。结果 小鼠单核细胞RAW264.7经破骨细胞培养液培养后,体积变大,核数目增多,TRAP染色阳性。受压应力刺激后,DAP12、TRAP mRNA及DAP12蛋白表达随加力时间延长而增加（P<0.05）。结论 小鼠单核细胞RAW264.7经破骨细胞培养液培养后成功向破骨细胞转化,转化细胞受压应力刺激活化过程中存在DAP12高表达。     

人白细胞介素-10质粒转染抑制RANKL诱导的小鼠单核/巨噬细胞RAW264.7分化形成破骨细胞

目的:探讨人白细胞介素-10(human interlenkin-10,hIL-10)质粒转染后的表达产物对RANKL诱导的破骨细胞形成的影响。方法:将hIL-10及相应的空载体质粒在脂质体介导下转染293T细胞,获取含hIL-10蛋白的培养液;通过TRAP染色及骨吸收实验观察转染产物对RANKL诱导的小鼠单核/巨噬细胞RAW264.7分化形成破骨细胞的影响。结果:hIL-10质粒脂质体法瞬时转染293T及RAW264.7细胞获得成功,部分细胞发出绿色荧光,ELISA检测表明目的基因获得表达,RAW264.7细胞在RANKL诱导下可形成破骨细胞样细胞,在该培养体系中加入含hIL-10蛋白的培养液上清,TRAP染色阳性细胞及骨吸收陷窝面积显著减少(P<0.05)。结论:本实验所采用的hIL-10质粒可以成功转染293T及RAW264.7细胞,产生的hIL-10蛋白在体外具有抑制破骨细胞形成和骨吸收的生物学功能。     

Proliferation and differentiation of MC3T3-E1 cells on calcium phosphate/chitosan coatings

The incorporation of chitosan into electro-deposited calcium phosphate (CaP) coatings increases bone marrow stromal cell attachment. We hypothesized that such electrodeposited CaP/chitosan coatings can also enhance the proliferative ability and differentiation potential of osteoblasts. To verify this hypothesis, we cultured osteoblast-like MC3T3-E1 cells on these CaP coatings. It was found that MC3T3-E1 cells cultured on the electrodeposited CaP/chitosan coatings had cell proliferation rates higher than those on the electrodeposited CaP coatings. At the same time, both alkaline phosphatase activity and collagen expression were increased, and both bone sialoprotein and osteocalcin genes were up-regulated when MC3T3-E1 cells were cultured on the electrodeposited CaP/chitosan coatings. Additionally, within the range of selected chitosan concentrations in solution, no significant difference was found between the CaP/chitosan coatings. Our results suggest that the electrodeposited CaP/chitosan coatings are favorable to the proliferation and differentiation of MC3T3-E1 cells, which may endow them with great potential for future applications.     

钛表面RGD/仿生磷酸钙复合涂层对MC3T3-E1细胞黏附、增殖、分化的影响

目的:研究钛表面RGD/仿生磷酸钙复合涂层对MC3T3-E1细胞黏附、增殖和分化的影响。方法:1)两步法在钛表面制备仿生磷酸钙涂层,扫描电子显微镜(SEM)观察形态,X射线衍射(XRD)、傅立叶转换红外光谱(FTIR)检测表征;2)RGD固定至仿生磷酸钙涂层;3)实验分3组:纯钛组,钛/仿生磷酸钙涂层组,钛/RGD/仿生磷酸钙复合涂层组,细胞分别接种至3组材料表面。4)SEM观察细胞接种4 h和24 h的形态;5)结晶紫染色检测细胞接种4 h和8 h的黏附情况。6)MTT法检测细胞1、3、7 d的增殖活性;7)检测细胞3、7、14 d的ALP活性。结果:SEM细胞形态观察及结晶紫染色结果显示,RGD/仿生磷酸钙复合涂层组的细胞黏附效果优于对照组。MTT结果显示RGD/仿生磷酸钙复合涂层组的细胞增殖活性强于对照组。ALP检测结果显示RGD/仿生磷酸钙复合涂层组的成骨分化活性高于对照组。结论:钛表面RGD/仿生磷酸钙复合涂层对MC3T3-E1细胞的黏附、增殖、分化均有促进作用。     

电化学沉积钙磷涂层和钙磷-壳聚糖涂层的体外降解实验

目的 观察电化学沉积钙磷涂层和钙磷-壳聚糖涂层(electrodeposited calcium phosphate-chitosan coating,ECPC)的体外降解情况.方法 用电化学方法在钛合金表面制备钙磷涂层和ECPC涂层,将破骨细胞分别接种于两种涂层表面(细胞组);无细胞组不加细胞.测量第3、6天上清液的钙离子吸光度.结果 两种涂层表面均可形成破骨细胞吸收陷窝,原始培养液钙离子吸光度(0.183±0.002)均高于各组上清液(P<0.05);钙磷涂层和ECPC涂层第6天细胞组钙离子吸光度[(0.158±0.006)和(0.158±0.02)]高于无细胞组[(0.151±0.003)和(0.156±0.02),P<0.05].结论 钙磷涂层和ECPC涂层在体外均既可被破骨细胞吸收形成陷窝,也可在培养液中溶解.     

Electrolytic deposition of lithium into calcium phosphate coatings

ObjectivesLithium ions stimulate the Wnt signaling pathway and the authors previously demonstrated that lithium enhances the proliferation of tissue cultured human mesenchymal stem cells. The aim of this study was to prepare and characterize a calcium phosphate/lithium coating by means of electrolytic deposition. It was hypothesized that the hybrid coatings would enhance the proliferation of MG63 osteoblast-like cells in vitro.MethodsCalcium phosphate coatings were electrolytically deposited in electrolytes containing 0, 0.5 and 5 g/L lithium chloride, respectively. They were characterized by scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. The coating thickness, lithium content and release profile were also measured. The cell attachment and cell-doubling index of MG63 cells on these coatings were determined through a Cell Counting Kit-8.ResultsLithium inhibited calcium phosphate deposition in a dose-dependent manner. Both crystallinity and thickness of the coatings were reduced with increasing lithium concentration in the electrolyte. The incorporation of lithium was 2.2 and 5.5 μg/mg, respectively. The hybrid coatings demonstrated a burst lithium release within half an hour upon immersion into simulated physiological solution. Both attachment and early proliferation of MG63 cells on these hybrid coatings were enhanced.SignificanceThese results suggest that lithium can be effectively incorporated into calcium phosphate coatings. The incorporation of lithium interferes with calcium phosphate deposition; however, it enhances the biocompatibility of the coatings.     

Early bone apposition and 1‐year performance of the electrodeposited calcium phosphate coatings: an experimental study in rabbit femora

Objectives: The aim of this study was to investigate the early bone apposition and 1‐year performance of the electrodeposited calcium phosphate coatings with or without chitosan. Material and methods: Seventy‐two cylindrical implants with a length of 8 mm and a diameter of 3.3 mm were divided into three groups: electrodeposited calcium phosphate coated without chitosan, with chitosan, and an uncoated control. The implants adopted the special gap design and were inserted into the rabbit femora. After 2, 4, 26, and 52 weeks, the implants were retrieved and analyzed for bone formation, bone‐to‐implant contact, and coating degradation. Results: It was found that the coatings without chitosan had the highest bone contact at early time (P<0.05). The coatings with chitosan had the least bone formation within gaps after 2, 4, and 26 weeks of implantation (P<0.05). However, no difference was found among the three groups after 52 weeks. Both coatings showed degradation as early as 2 weeks post‐implantation. And after 52 weeks, most of the coatings had been degraded. There were no inflammatory reactions and hardly any osteoclasts around the implants and the coatings. The confocal laser scanning microscopy observation further demonstrated the different bone deposition characteristics. With scanning electron microscopy, no coatings could be found on both the implant surface and the bone interface. Conclusions: Bone apposition to both electrodeposited calcium phosphate coatings was different at early time but almost the same after 52 weeks. And both coatings showed early as well as a continued degradation in the rabbit femora. To cite this article:
Wang J, Sun C, Wang Y, Wang Y. Early bone apposition and 1‐year performance of the electrodeposited calcium phosphate coatings: an experimental study in rabbit femora.
Clin. Oral Impl. Res. 21 , 2010; 951–960.
doi: 10.1111/j.1600‐0501.2010.01935.x     

Preparation and characterization of electrodeposited calcium phosphate/chitosan coating on Ti6Al4V plates

Electrolytically deposited carbonate apatite coating demonstrates higher strength but weaker support for bone marrow stromal cell attachment than do biomimetically deposited coatings. It is hypothesized that the incorporation of chitosan will increase the biocompatibility of electrolytic coating while maintaining its original strength. To verify this hypothesis, we formed a hybrid calcium phosphate/chitosan coating through electrodeposition. We found that the incorporation of chitosan influenced calcium phosphate formation and crystallization. Moreover, coating thickness and surface roughness decreased with increasing chitosan concentration. Hybrid coating exhibited an increased dissolution rate in both acidic and neutral simulated physiologic solution, whereas no significant difference on adhesive strength was found between the hybrid and original coatings (P > 0.05). Most importantly, the calcium phosphate/chitosan coating proved to be a more favorable surface for goat bone marrow stromal cell attachment than an unincorporated coating (P < 0.01). Considering its economic and simple production, a hybrid calcium phosphate/chitosan coating is thought to be an attractive candidate for future applications.     

Nicotine stimulates osteoclast resorption in a porcine marrow cell model

BACKGROUND: Combustible tobacco use is generally linked with accelerated periodontal bone loss and diminished post-surgical wound healing; however, the pathogenesis of this process at the cellular level remains unclear. Nicotine is known to affect human gingival fibroblast orientation, attachment, and beta1 integrin expression, yet little is known about its effects on osteoclasts, the cells most responsible for bone resorption. The purpose of this study was to determine the effects of physiologically relevant nicotine levels on porcine osteoclast function as measured by resorption of calcium phosphate. METHODS: Pure nicotine was diluted in medium to the following concentrations: 0.03 microM, 0.15 microM, 0.30 microM, 0.60 microM, and 1.50 microM. Porcine osteoclasts were seeded onto calcium phosphate multi-test slides and incubated at 37 degrees C with half media changes every 24 hours. Cells received 0, 0.15, 0.30, 0.60, and 1.50 microM nicotine, or 25 nM parathyroid hormone (PTH). Osteoclast resorption was quantified by measuring the resorbed surface area of the calcium phosphate substrate. RESULTS: Osteoclast cultures resorbed bone slices and calcium phosphate substrate. All nicotine concentrations and PTH resulted in statistically significantly greater mean percent resorptions than the control group (P < 0.05). However, no statistical difference was found between the various nicotine doses or PTH. The number of osteoclasts increased in a linear relationship to the increasing nicotine concentrations; however, no correlation was found between osteoclast number and the amount of resorption. CONCLUSIONS: Nicotine is non-toxic to osteoclasts at the clinically relevant levels tested. Nicotine appears to stimulate osteoclast differentiation and resorption of calcium phosphate, the major component of bone. Nicotine-modulated osteoclast stimulation may, in part, explain the increased rapidity of periodontal bone loss and refractory disease incidence in smokers.