淫羊藿苷防治人工关节无菌性松动的实验研究
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摘要
研究背景
自从人工关节置换术应用于临床以来,它逐渐成为恢复关节功能的主要方法,并取得了良好的效果。然而随着该技术的广泛推广,假体使用年限的延长,术后的并发症随之增多,其中假体无菌性松动是人工关节置换术后期最常见的并发症。造成假体松动的因素主要包括两大类,一类为机械因素,即假体周围的应力遮挡,早期松动,假体微动,关节液压力,手术医师固定方法不正确等；另一类为生物学因素,即假体使用过程中产生的磨损颗粒及其引发的一系列生物化学反应所造成的骨溶解。目前主流的观点是“微粒病”的学说。该学说认为,人工关节长期磨损后产生了大量的磨损微粒,磨损微粒刺激假体周围界膜组织中的多种细胞,如单核-巨噬细胞、成骨细胞、成纤维细胞产生溶骨性因子,这些溶骨性因子可刺激破骨细胞活化和增殖,产生骨溶解和骨吸收,假体与周围骨床之间出现空隙,假体松动,而松动的人工关节可使磨损进一步加剧,从而形成了由磨损-松动-磨损的恶性循环。
磨损颗粒包括聚乙烯颗粒、骨水泥颗粒、陶瓷颗粒和金属颗粒等。对聚乙烯颗粒和骨水泥颗粒,已有较多的研究。钛为人工关节假体和骨折内固定常见的材料,股骨柄与骨水泥间及与骨界面间微动,股骨柄与骨质磨擦,微孔涂层与骨间微动及螺钉与臼杯间微动,是钛颗粒产生的主要来源,因此钛颗粒是人工关节置换术后常见的金属颗粒之一。随着新一代金属对金属人工假体的推广运用,金属颗粒对假体周围骨溶解的作用也逐渐引起了人们的重视。
关于钛颗粒对骨代谢的影响,目前研究的焦点主要集中在破骨细胞。研究认为,钛颗粒可以激活破骨细胞引起骨溶解。然而骨代谢是破骨细胞和成骨细胞共同作用的结果。成骨细胞作为具有成骨潜能的主要功能细胞,能够合成和分泌Ⅰ型胶原蛋白和多种非胶原蛋白,分泌类骨质和碱性磷酸酶等促矿化物质,从而促进新骨形成。磨损微粒在激活破骨细胞活性的同时,不可避免的和周围大量的成骨细胞紧密接触,是否会对成骨细胞的生物学行为造成影响?目前还没有深入地研究。
磨损颗粒刺激假体周围界膜组织中的细胞释放大量的溶骨性因子,它们在体内共同构成了一个复杂的网络,可单独或联合作用参与假体周围骨溶解的代谢过程。在众多炎性因子中,IL-6是引起细胞因了级联反应、诱导破骨细胞活化和增殖、刺激炎性细胞向假体周围聚集、引起假体周围骨溶解和骨重建紊乱的重要的溶骨性因子之一。它主要由成骨细胞合成,在原发性骨质疏松发病机制中所起的作用主要是刺激原始破骨细胞分化成成熟破骨细胞,增加骨小梁中破骨细胞的数量,并促进破骨细胞分化、成熟和活性增强,还通过增加胶原酶释放,促进骨基质降解,使骨吸收增加,骨密度下降。IL-6作为磨损颗粒刺激产生的一种重要的炎性介质,参与了假体周围骨溶解的病理过程。在全髋关节置换术后发生假体松动的患者的关节液中,IL-6的水平升高,因此可以将IL-6水平升高视为人工假体周围骨溶解的生物标志。
近年来发现,成骨细胞可通过OPG/RANKLRANK系统调节破骨细胞的增殖、分化和成熟。RANK作为一种Ⅰ型跨膜蛋白高度表达于破骨前体细胞的表面。RANKL由成骨细胞分泌,它可结合到RANK上,启动细胞内的信号传导通路,激活破骨细胞,促进其分化增殖并抑制其调亡。同时,成骨细胞可分泌OPG,它作为一种诱骗受体,可以竞争性的与RANKL结合,阻断RANKL与破骨细胞表面的RANK结合,抑制破骨细胞的分化成熟。成骨细胞合成RANKL/OPG比例的变化对于破骨细胞的分化增殖具有重要的意义,当RANKL/OPG的比例上调,破骨细胞的数量和活性将增加,RANKL/OPG的比例下降时破骨细胞的数量和活性将降低。因此,假体周围骨重建和骨量的稳定还依赖于OPG和RANKL之间表达的平衡。
目前针对骨量减少的药物治疗主要是抑制骨溶解和骨吸收,包括抑制溶骨性因子和抑制破骨细胞等。但骨溶解是多种因素联合作用的结果,涉及到多种途径,单一抑制某种因素的药物很难完全控制。抑制破骨细胞的药物如降钙素在使用一段时间后会失效,二磷酸盐则因为长期使用会产生各种严重的毒副作用及昂贵的价格限制了它的使用。因此还需要进一步地探索。
假体无菌性松动是假体周围骨形成和骨吸收失衡的结果。骨形成的减少对假体无菌性松动也具有重要的意义。应用药物促进松动假体周围骨形成,使假体得到良好的生物学固定,从而治疗人工关节无菌性松动可能是另一种有效途径,但目前相关的研究较少。淫羊藿苷是一种“强骨”中药,它可以诱导骨髓间充质干细胞向成骨细胞分化,增加钙盐含量,促进骨钙素分泌,增加钙化结节数量,同时上调Runx-2、bFGF-1和Osterix等成骨相关基因的表达,对骨形成具有促进作用。在临床上,淫羊藿苷能促进骨折愈合和骨质疏松患者骨量增加；并且它来源广泛,价格低廉,制剂方便,副作用小,安全性高,具有传统药物不可比拟的优点,因此目前已广泛用于治疗骨质疏松。人工关节无菌性松动的病理过程和骨质疏松类似,都存在骨形成减少。对松动假体周围的成骨细胞,淫羊藿苷是否也具有促进骨形成,提供骨保护的作用?本研究通过体外实验观察淫羊藿苷对钛颗粒刺激下成骨细胞的影响,为进一步动物研究探讨淫羊藿苷防治人工关节无菌性松动提供实验依据。
目的
l、研究钛颗粒对成骨细胞增殖、表型和形态结构的影响。
2、研究淫羊藿苷对钛颗粒刺激下成骨细胞增殖和分化的影响。
3、研究淫羊藿苷对钛颗粒刺激下成骨细胞表达溶骨性因子IL-6的影响。
4、研究淫羊藿苷对钛颗粒刺激下成骨细胞OPG、RANKI,mRNA表达的影响。方法
1、多次酶消化法体外分离新生大鼠颅骨成骨细胞,进行原代培养。碱性磷酸酶染色进行细胞鉴定。配制钛颗粒培养基,鲎试验排除钛颗粒内毒素水平超标。取生长良好的第3代成骨细胞,分别向细胞中加入浓度为0.01、0.05、0.1、0.5、
1mg/ml的钛颗粒(n=6)。培养7d后用CCK-8法检测OD值,比较不例浓度的钛颗粒对成骨细胞增殖能力的影响。ELISA法检测各组细胞ALP和Ⅰ型胶原的表达(n=6),观察钛颗粒对成骨细胞分化表型的影响。对细胞进行FITC-phalloidin和PI双重荧光染色,激光共聚焦显微镜下观察成骨细胞吞噬钛颗粒后形态结构的改变。
2、体外用钛颗粒作用于大鼠成骨细胞,并分别加入10-5、10-6、10-7、10-8、10-9、10-10mol/L的淫羊藿苷进行干预(n=6)。于混合培养7d后用CCK-8法检测各组细胞增殖活力,观察淫羊藿苷对钛颗粒刺激下成骨细胞增殖活力的影响,筛选出最佳药物浓度。以最佳药物浓度的淫羊藿苷干预钛颗粒刺激下的成骨细胞,分别于培养第3d、7d、14d用ELISA法检测各组细胞ALP活性和Ⅰ型胶原的表达(n=6),于培养第21d用茜素红法进行钙化结节染色。
3、将体外培养的大鼠成骨细胞分成五组：第一组对照组成骨细胞,第二组钛颗粒组成骨细胞+钛颗粒,第三组成骨细胞+钛颗粒+10-10mol/L淫羊藿苷,第四组成骨细胞+钛颗粒+10曲mol/L淫羊藿苷,第五组成骨细胞+钛颗粒+10-8mol/L淫羊藿苷(n=6)。分别施加不同干预措施,于培养7d后吸取上清,用ELISA法检测各组上清液中IL-6的含量。
4、将体外培养的大鼠成骨细胞分成四组：第一组对照组成骨细胞,第二组钛颗粒组成骨细胞+钛颗粒,第三组成骨细胞+淫羊藿苷,第四组成骨细胞+钛颗粒+淫羊藿苷(n=6)。分别施加不同干预措施,于培养7d后用RT-PCR技术检测各组细胞OPG/RANKLmRNA的表达及RANKL/OPG比值的变化。
5、应用SPSS18.0软件对实验数据进行统计分析,实验资料以均数±标准差(x±s)表示,主效应与交互效应采用两因素析因分析,若存在交互效应,则单独效应采用单因素方差分析,先做方差齐性检验,若方差齐,则组间比较采用LSD检验,若方差不齐,则采用Tamhane检验。所有检验均采用双侧检验,检验水准为α=0.05,当P<0.05时差异有统计学意义。
结果
1、各组成骨细胞增殖活力的差异有统计学意义(F=517.480,P=0.000)。不同浓度的钛颗粒均能抑制成骨细胞增殖,与对照组相比有显著差异(P<0.05)。浓度为1mg/ml的钛颗粒对成骨细胞增殖活力的抑制作用要大于浓度为0.01、0.05、0.1、0.5、1mg/ml的钛颗粒(P<0.05)。浓度为0.01mg/ml、0.05mg/ml的钛颗粒对成骨细胞增殖活力的影响无显著差异(P=0.240)。各组成骨细胞ALP活性和Ⅰ型胶原表达的差异有统计学意义(ALP：F=828.024,P=0.000；Ⅰ型胶原：F=2258.835,p=0.000)。不同浓度的钛颗粒均能明显抑制成骨细胞ALP活性和Ⅰ型胶原表达,与对照组相比有统计学差异(P<0.05)。浓度为1mg/ml的钛颗粒对成骨细胞ALP活性和Ⅰ型胶原表达的抑制作用要大于浓度为0.01、0.05、0.1、0.5n、1mg/ml的钛颗粒(P<0.05)。浓度为0.01mg/ml、0.05mg/ml的钛颗粒对成骨细胞Ⅰ型胶原表达的影响无显著差异(P=0.240)。激光共聚焦显微镜下可见成骨细胞吞噬钛颗粒后皱缩变形,伪足收缩变短,微丝排列紊乱。2、各组成骨细胞的增殖能力有显著差异(F=829.652,P=0.000)。浓度为10-10-10-6mol/L的淫羊藿苷能抑制钛颗粒介导的成骨细胞增殖能力下降,与钛颗粒组相比有显著差异(P<0.05)。10-8mol/L为淫羊藿苷的最佳作用浓度,与其它各浓度淫羊藿苷组相比有显著差异(P<0.05)。当淫羊藿苷浓度上升至10-5mol/L时,对成骨细胞的增殖有抑制作用(P<0.05)。析因分析结果显示,不同组间ALP浓度有显著差异(F分组=445.548,P=0.000),不同时间点也有显著差异(F时间=194.242,P=0.000),且存在交互效应(F分组×时间=81.439,P=0.000)。单因素方差分析显示,同一时间点不同组间差异有统计学意义(3d：F=18.699,P=0.000;7d:F=129.110,P=0.000;14d:F=459.210, P=0.000),相同组不同时间点间差异也有统计字蒽义(control:F=130.968,P=0.000；Ti:F=23.070,P=0.000;ICA: F=232.660,P=0.000；Ti+ICA:F=43.024,P=0.000)。不同组间COL-I浓度有显著差异(F分组=1843.514,P=0.000),不同时间点也有显著差异(F时间=341.296,P=0.000),且存在交互效应(F分组×州=250.979,P=0.000)。单因素方差分析显示,同一时间点不同组间差异有统计学意义(3d：F=86.658,P=0.000；7d：F=923.149,P=0.000；14d：F=1293.625,P=0.000),相同组不同时间点间差异也有统计学意义(control:F=268.229,P=0.000；Ti:F=206.053,P=0.000；ICA:F=519.974, P=0.000；Ti+ICA:F=120.072,P=0.000)。两两比较结果显示,钛颗粒组成骨细胞的ALP活性和Ⅰ型胶原表达在3d、7d、14d时均低于同时期的对照组(P<0.05),钛颗粒+淫羊藿苷组成骨细胞的ALP活性和Ⅰ型胶原表达在7d、14d时均高于同时期的钛颗粒组(P<0.05)。淫羊藿苷的作用存在时间-效应关系,干预第7d时,钛颗粒+淫羊藿苷组成骨细胞的ALP活性和Ⅰ型胶原浓度最高,与3d、14d相比有显著差异(P<0.05)。钙化结节染色显示淫羊藿苷干预组的成骨细胞钙化结节数和分布面积明显高于钛颗粒组。3、各组成骨细胞IL-6表达的差异有统计学意义(F=7202.778,P=0.000)。与对照组相比,钛颗粒明显促进了成骨细胞IL-6的表达(P<0.05)。不同浓度的淫羊藿苷对钛颗粒介导的IL-6的表达均有抑制作用,与钛颗粒组相比有统计学差异妒<0.05)。浓度为10吨mol/L的淫羊藿苷对IL-6表达的抑制作用要大于浓度为10-10、10-9mol/L的淫羊藿苷(P<0.05)。4、各组成骨细胞OPG mRNA、RANKLmRNA的表达量及RANKL/OPG比值的差异有统计学意义(OPG:F=4277.834P=0.000；RANKL:F=5599.081P=0.000； RANKL/OPG:F=1334.919P=0.000)。与对照组相比,钛颗粒组成骨细胞OPGmRNA表达下降,RANKL mRNA表达上升,RANKL/OPG的比值上升,差异有统计学意义(P<0.05)。淫羊藿苷干预后,成骨细胞OPG mRNA的表达上升,RANKL mRNA的表达下降,RANKL/OPG的比值下降,与钛颗粒组相比有显著差异(P<0.05)。
结论
1、钛颗粒可以抑制体外培养的大鼠成骨细胞的增殖和分化成熟,降低成骨能力。成骨细胞可吞噬钛颗粒并且细胞形态和骨架发生改变。
2、淫羊藿苷能够逆转钛颗粒对成骨细胞增殖、分化成熟和矿化能力的抑制作用,维持细胞表型,促进骨形成。
3、淫羊藿苷可以抑制钛颗粒介导的成骨细胞分泌溶骨性因子IL-6,抑制骨溶解。
4、淫羊藿苷可以通过调控钛颗粒介导的成骨细胞OPG、RANKL mRNA的表达,抑制破骨细胞的分化成熟。
Background
Since arthroplasty used clinically, it has become the main method of reconstructing joint function and achieved good results. However, with expansion of application, increasing number of operation and extension of service life,-the postoperation complications also increased. Among them, prosthesis aseptic loosening is the most common complication on the later period of artificial joint replacement. The factors which cause prosthetic loosening are mainly divided into two categories:one is mechanical factors, that is stress shielding around prosthesis, early loosening, prosthesis micromove, joint hydraulic pressure, loosed fixation of prosthesis, etc. Another is biological factors, which refers to that wear particles are generated in utilization and osteolysis is caused by a series of related biochemical reactions. At present, in the mainstream view was proposed the concept of "particles disease". It proposed that artificial joints produce a lot of wear particles, the wear particles stimulate monocytes-macrophages, osteoblasts, fibroblasts to produce osteolytic factors. Thereby it activates periprosthetic osteoclasts, produces bone resorption and bone dissolution. Then the gap appears between the prosthesis and the surrounding bone, so that the prosthesis loses support, and eventually lead to artificial joints aseptic loosening. The loosening artificial joints make further exacerbated wear. Thus formed a vicious cycle from wear to loosen.
Wear particles include polyethylene particles, cement particles, ceramic particles and metal particles. There are many researches about polyethylene particles and bone cement particles. Titanium is common material for artificial joint prostheses and fracture internal fixation. Fretting between femoral stem and cement and bone interface, friction between femoral stem and bone, fretting between micro-porous coating, fretting between screws and acetabular cup, are the main source of titanium particles. So titanium particles is one of common metal particles after artificial joint replacement surgery. With the spread application of the new generation of metal-on-metal prosthesis, the periprosthetic osteolysis role of metal particles has gradually attracted people's attention.
When it refers to the effect of titanium particles on bone metabolism, the present study focused on osteoclasts. Studies suggest that the titanium particles can cause osteolysis by activation of osteoclasts. However, bone metabolism balance is a result of osteoclasts and osteoblasts joint action. As the main functional cells with osteogenic potential, osteoblasts are capable of synthesizing and secreting a variety of type I collagen and non-collagen protein, secreting mineralized substance as osteoid mineralization substances and alkaline phosphatase, thus promoting new bone formation. At the same time wear particles activate osteoclasts activity, inevitably closely contact with large amount of osteoblasts surrounding around. There is no in-depth study about whether it will effect the biological behavior of osteoblasts.
Wear particles stimulate cells of membrane arround the prosthesis to release large amounts of osteolytic factors, which constitute a complex network inbody, and are involved in the bone resolved metabolic process around prosthesis alone or together. IL-6is mainly synthesized by osteoblasts, its primary role in the pathogenesis of osteoporosis is stimulates the original osteoclasts to differentiate into mature osteoclasts, increases the number of osteoclasts in trabecular bone, promote osteoclast differentiation, maturation, and by increasing the release of collagenase to promote bone matrix degradation, so that to increase bone resorptionand decrease BMD. IL-6as an important inflammatory mediator produced by stimulating wear particles, it involves in the pathological process of dissolution of bone around the prosthesis. In the synovial; fluid of the the patients who occurred prosthesis loosening after total hip arthroplasty, the levels of IL-6elevated, and therefore the increasing level of IL-6can be considered as the biomarker of bone dissolved around atificial prosthesis.
Receptor activator of nuclear factor-KB belongs to type I transmembrane protein, it is one of the TNF family members. RANK is highly expressed on the surface of the osteoclast precursor cells. Receptor activator of nuclear factor-KB factor ligand binds to RANK expressed on the surface of osteoclast precursor cells and mature osteoclasts, promote the differentiation of osteoclasts and inhibit its apoptosis. Osteoprotegerin as a decoy receptor, competitively binding with RANKL, and thereby blocking the binding of RANKL to RANK on osteoclasts surface, inhibitting osteoclast maturation. The change of RANKL/OPG ratio is essential for oosteoclasts production. In general, when the RANKL/OPG ratio upregulates, the number of osteoclasts and their activity will increase, when the RANKL/OPG ratio downregulates, the number of osteoclasts and their activity will decrease. Therefore, the bony remodeling and the stability of bone mass around prothesis depended upon the balance between OPG and RANKL.
At present, the treatment for joint prosthesis aseptic loosening is inhibitting osteolysis and bone resorption, including the control of osteolytic factors and osteoclasts. However, osteolysis is resulted from the co-effects of various factors through various methods. It is difficult to take complete control of prosthesis loosening by medicine which inhibits solo osteolytic factor. Some medicine which inhibit osteoclasts, such as calcitonin, would be ineffective after taking for a period of time, while the long-term utilization of bisphosphonate will lead to various serious toxic and side effects and the expensive price limits its application. Therefore, it is necessary to conduct further research.
Prosthesis aseptic loosening is caused by the imbalance between bone formation and bone resorption. The reduction of bone formation also has important influence on prosthesis aseptic loosening. It may be another effective method to conduct treatment of joint prosthesis aseptic loosening by applying medicines to prompt bone formation around loosening prosthesis and make prosthesis biologically fixed. However, few related researches are at present. Icariin is a kind of "bone reinforcement" Chinese medicine, which can induced bone marrow mesenchymal stem cells to differentiate into osteoblasts, improve ALP activity, increase calcium salt content, prompt osteocalcin secretion, increase the number of calcified nodules and upregulate the expression of factors related to bone-formation, such as Runx-2, bFGF-1and Osterix etc. Icariin can prompt the recovery of bone fracture and increase bone mass of patients of osteoporosis. And it has a wide range of source, low cost, easy preparation, small side effects, high safety and incomparable advantages of traditional medicines, therefore it has been widely used for the treatment of osteoporosis. The pathological process of joint prosthesis aseptic loosening is similar to osteoporosis, both of them have decreased bone formation. For those osteoblasts around the loose prosthesis, whether icariin can promote bone formation, provide protective effect? In this study we observe the effects of icariin on osteoblasts stimulated by titanium particles in vitro, so as to provide experimental foundations for further animal experiment.
Ojectives
1、Study the effect of titanium particles on proliferation, phenotype and morphology of osteoblasts.
2、Study the effect of icarrin on proliferation, differenciation of osteoblasts stimulated by titanium particles.
3、Study the effect of icarrin on osteolytic factor IL-6secretion of osteoblasts stimulated by titanium particles.
4、Study the effect of icarrin on OPG, RANKL mRNA expression of osteoblasts stimulated by titanium particles.
Methods
1、Calvarial osteoblasts of newborn rats were seperated in vitro by repeated enzyme digestion, and received primary culture. Alkaline phosphatase staining was carried out to identify them. Titanium culture medium was prepared and limulus assay was performed to exclude excessive endotoxin level. Titanium particles with concentrations of0.01,0.05,0.1,0.5as well as1mg/ml were respectively added to the third generation osteoblasts well grown(n=6). After cultured for seven days, OD was detected by CCK-8method to compare the influence of different titanium particles concentrations on osteoblasts proliferation. ALP activity and type I collagen expression were detected by ELISA to observe the effect of titanium particles on differentiation and phenotype of osteoblasts(n=6). The cells received double fluorescence staining with FITC-phalloidin and PI to observe cellular morphology variation of osteoblasts which swallowed titanium particles under laser scanning confocal microscope.
2、Titanium particles were used to treat rats osteoblasts, and icariin with concentrations of10-5,10-6，10-7,10-8,10-9,10-10mol/L were added for interference respectively(n=6). The effect of icariin on proliferation of osteoblasts stimulated with titanium particles was observed after cultured for seven days, and the optimum drug concentration was screened out. Icariin with optimum drug concentration was added to intervene osteoblasts stimulated with titanium particles(n=6), ELISA method was carried out to detected ALP activity and type I collagen expression on the3rd7th、14th day after cultur, calcified nodules were stained by alizarin S red stain method on the21th day.
3、The rats osteoblasts cultured in vitro were divided into five groups:the first group, the control group, osteoblasts; the second group, the tatinium particlesgroup, osteoblasts plus titanium particles; the third group, osteoblasts plus titanium particles plus10-10mol/L icariin; the fourth group, osteoblasts plus titanium particles plus10-9mol/L icariin; the fifth group osteoblasts plus titanium particles plus10-8mol/L icariin(n=6). They received differern intervene measures. After cultured for seven days, supernatant was extracted, and ELISA method was performed to detect IL-6concentration in the supernatant of each group.
4、The rats osteoblasts cultured in vitro were divided into four groups:the first group, the control group,osteoblasts; the second group, titanium particles group, osteoblasts plus titanium particles; the third group, osteoblasts plus icariin; the fourth group, osteoblasts plus titanium particles plus icariin(n=6). Each group received differern intervene measures. After cultured for seven days, RT-PCR was carried out to detect relative expression of OPG and RANKL mRNA and RANKL/OPG ratio variation of each group.
Results
1、The differences of osteoblasts proliferation activity within each group was statistically significant (F=517.480, P=0.000). Different concentrations of titanium particles could inhibit the proliferation of osteoblasts, which had a significant difference (P<0.05) compared with the control group. The inhibition effect of titanium particles with concentration of lmg/ml on osteoblast proliferation activity was greater than those of titanium particles with concentration of0.01,0.05,0.1,0.5,1mg/ml (P<0.05). The effect of of titanium particles on osteoblast proliferation activity showed no significantly difference between concentration of0.01mg/ml and0.05mg/ml (P=0.240). The differences of osteoblasts ALP activity and type I collagen expression within each group was statistically significant (ALP:F=828.024, P=0.000; type I collagen:F=2258.835, P=0.000). Different concentrations of titanium particles could significantly inhibit ALP activity and type I collagen expression of osteoblasts, there was significant difference (P<0.05) compared with the control group. The inhibition effect of titanium particles with concentration of lmg/ml on osteoblast ALP activity and type I collagen expression was greater than those of titanium particles with concentration of0.01,0.05,0.1,0.5,1mg/ml (P<0.05). The effect of of titanium particles on osteoblast ALP activity and type I collagen expression showed no significantly difference between concentration of0.01mg/ml and0.05mg/ml (P=0.240). Laser scanning confocal microscope showed cellular shrinking, microfilaments distortion, pseudopodia contraction of osteoblasts swallowed with titanium particles.
2、The differences of osteoblasts proliferation activity within each group was statistically significant (F=829.652, P=0.000). Icarrin with concentrations of10-10-10-6mol/L could inhibit decreased proliferation ability of osteoblasts stimulated by titanium particles, which showed significant difference compared with the titanium particles group (P<0.05).10-8mol/L was icarrin's optimum drug concentration, which had significant differences compared with other various concentrations of icariin groups (P<0.05). When the concentration of icariin rise to10-5mol/L, it inhibitted the proliferation of osteoblasts (P<0.05). Factorial analysis showed that there was significant differences of ALP concentration within different groups (Fgroup=445.548, P=0.000), so were different time points (Ftime=194.242, P=0.000), and there existed interaction (Fgroup×time=81.439, P=0.000). One-way ANOVA analysis showed that, there were significant differences within different groups of the same time point (3d:F=18.699, P=0.000;7d:F=129.110, P=0.000;14d:F=459.210, P=0.000), so were different time point of the same group (control: F=130.968, P=0.000; Ti:F=23.070, P=0.000; ICA:F=232.660, P=0.000; Ti+ICA: F=43.024, P=0.000). There were significant differences of type I collagen expression within different groups (Fgroup=1843.514, P=0.000), so were different time points (Ftime=341.296, P=0.000), and there existed interaction (Fgroup×time=250.979, P=0.000). One-way ANOVA analysis showed that, there were significant differences within different groups of the same time point (3d:F=86.658, P=0.000;7d: F=923.149, P=0.000;14d:F=1293.625, P=0.000), so were different time point of the same group (control:F=268.229, P=0.000; Ti:F=206.053, P=0.000; ICA: F=519.974, P=0.000; Ti+ICA:F=120.072, P=0.000). Pairwise comparisons showed that, ALP activity and type I collagen expression of osteoblasts in titanium particles group on3d,7d,14d are lower than that of control group during the same period (P<0.05). while ALP activity and type I collagen expression of osteoblasts in titanium particles+icarrin groups on7d,14d.are higher than that of titanium particles groups during the same period (P<0.05). The role of icariin had a time-effect relationship,, ALP activity and type I collagen expression of titanium particles+icarrin reached the highest on7d,which had significant difference compared with that of3d and7d (P<0.05). Alizarin red stain showed that the amount of cacified nodules and distribution area of osteoblast were higher in icarrin intervene group than in titanium particles group.
3The differences of IL-6expression of osteoblasts within each group was statistically significant (F=7202.778, P=0.000). Compared with the control group, titanium particles obviously promoted the osteoblasts'expression of IL-6(P<0.05). Different concentrations of icariin could significantly inhibit IL-6expression mediated by titanium particles, which was significant different compared with the titanium particles group(P<0.05). The inhibition effect of icarrin with concentration of108mol/L on IL-6expression was greater than those of icarrin with concentrations of10-10、10-9mol/L(P<0.05).
4、The differences of OPG mRNA、RANKL mRNA expression of osteoblasts and RANKL/OPG ratio within each group were statistically significant (OPG:F=4277.834P=0.000; RANKL:F=5599.081P=0.000; RANKL/OPG: F=1334.919P=0.000). Compared with the control group, OPG mRNA expression decreased, RANKL mRNA expression increased, and RANKL/OPG ratio rised in titanium particles group, which showed statistic difference (P<0.05). After icarrin intervention, OPG mRNA expression rised, while RANKL mRNA expression decreased, and RANKL/OPG ratio declined, which showed statistic difference compared with the titanium particles group (P<0.05)
Conclusions
1-, Titanium particles can inhibit the proliferation and differenciation ability of cultured rat osteoblasts in vitro, decreased bone formation. Titanium particles can be phagocytosed by osteoblasts and make changes in cell morphology and cytoskeleton.
2、Icariin can reverse the inhibition of effect of titanium particles on osteoblasts proliferation, differentiation, maturation and mineralization, maintain cell phenotype and promote bone formation.
3、Icariin can inhibit titanium particles-mediated osteoblasts'secretion of osteolytic factor IL-6, thus inhibit osteolysis.
4、Icariin can regulate osteoblasts OPG, RANKL mRNA expression mediated by titanium particles, thus inhibit differenciation and maturation of osteoclasts..
自从人工关节置换术应用于临床以来,它逐渐成为恢复关节功能的主要方法,并取得了良好的效果。然而随着该技术的广泛推广,假体使用年限的延长,术后的并发症随之增多,其中假体无菌性松动是人工关节置换术后期最常见的并发症。造成假体松动的因素主要包括两大类,一类为机械因素,即假体周围的应力遮挡,早期松动,假体微动,关节液压力,手术医师固定方法不正确等；另一类为生物学因素,即假体使用过程中产生的磨损颗粒及其引发的一系列生物化学反应所造成的骨溶解。目前主流的观点是“微粒病”的学说。该学说认为,人工关节长期磨损后产生了大量的磨损微粒,磨损微粒刺激假体周围界膜组织中的多种细胞,如单核-巨噬细胞、成骨细胞、成纤维细胞产生溶骨性因子,这些溶骨性因子可刺激破骨细胞活化和增殖,产生骨溶解和骨吸收,假体与周围骨床之间出现空隙,假体松动,而松动的人工关节可使磨损进一步加剧,从而形成了由磨损-松动-磨损的恶性循环。
磨损颗粒包括聚乙烯颗粒、骨水泥颗粒、陶瓷颗粒和金属颗粒等。对聚乙烯颗粒和骨水泥颗粒,已有较多的研究。钛为人工关节假体和骨折内固定常见的材料,股骨柄与骨水泥间及与骨界面间微动,股骨柄与骨质磨擦,微孔涂层与骨间微动及螺钉与臼杯间微动,是钛颗粒产生的主要来源,因此钛颗粒是人工关节置换术后常见的金属颗粒之一。随着新一代金属对金属人工假体的推广运用,金属颗粒对假体周围骨溶解的作用也逐渐引起了人们的重视。
关于钛颗粒对骨代谢的影响,目前研究的焦点主要集中在破骨细胞。研究认为,钛颗粒可以激活破骨细胞引起骨溶解。然而骨代谢是破骨细胞和成骨细胞共同作用的结果。成骨细胞作为具有成骨潜能的主要功能细胞,能够合成和分泌Ⅰ型胶原蛋白和多种非胶原蛋白,分泌类骨质和碱性磷酸酶等促矿化物质,从而促进新骨形成。磨损微粒在激活破骨细胞活性的同时,不可避免的和周围大量的成骨细胞紧密接触,是否会对成骨细胞的生物学行为造成影响?目前还没有深入地研究。
磨损颗粒刺激假体周围界膜组织中的细胞释放大量的溶骨性因子,它们在体内共同构成了一个复杂的网络,可单独或联合作用参与假体周围骨溶解的代谢过程。在众多炎性因子中,IL-6是引起细胞因了级联反应、诱导破骨细胞活化和增殖、刺激炎性细胞向假体周围聚集、引起假体周围骨溶解和骨重建紊乱的重要的溶骨性因子之一。它主要由成骨细胞合成,在原发性骨质疏松发病机制中所起的作用主要是刺激原始破骨细胞分化成成熟破骨细胞,增加骨小梁中破骨细胞的数量,并促进破骨细胞分化、成熟和活性增强,还通过增加胶原酶释放,促进骨基质降解,使骨吸收增加,骨密度下降。IL-6作为磨损颗粒刺激产生的一种重要的炎性介质,参与了假体周围骨溶解的病理过程。在全髋关节置换术后发生假体松动的患者的关节液中,IL-6的水平升高,因此可以将IL-6水平升高视为人工假体周围骨溶解的生物标志。
近年来发现,成骨细胞可通过OPG/RANKLRANK系统调节破骨细胞的增殖、分化和成熟。RANK作为一种Ⅰ型跨膜蛋白高度表达于破骨前体细胞的表面。RANKL由成骨细胞分泌,它可结合到RANK上,启动细胞内的信号传导通路,激活破骨细胞,促进其分化增殖并抑制其调亡。同时,成骨细胞可分泌OPG,它作为一种诱骗受体,可以竞争性的与RANKL结合,阻断RANKL与破骨细胞表面的RANK结合,抑制破骨细胞的分化成熟。成骨细胞合成RANKL/OPG比例的变化对于破骨细胞的分化增殖具有重要的意义,当RANKL/OPG的比例上调,破骨细胞的数量和活性将增加,RANKL/OPG的比例下降时破骨细胞的数量和活性将降低。因此,假体周围骨重建和骨量的稳定还依赖于OPG和RANKL之间表达的平衡。
目前针对骨量减少的药物治疗主要是抑制骨溶解和骨吸收,包括抑制溶骨性因子和抑制破骨细胞等。但骨溶解是多种因素联合作用的结果,涉及到多种途径,单一抑制某种因素的药物很难完全控制。抑制破骨细胞的药物如降钙素在使用一段时间后会失效,二磷酸盐则因为长期使用会产生各种严重的毒副作用及昂贵的价格限制了它的使用。因此还需要进一步地探索。
假体无菌性松动是假体周围骨形成和骨吸收失衡的结果。骨形成的减少对假体无菌性松动也具有重要的意义。应用药物促进松动假体周围骨形成,使假体得到良好的生物学固定,从而治疗人工关节无菌性松动可能是另一种有效途径,但目前相关的研究较少。淫羊藿苷是一种“强骨”中药,它可以诱导骨髓间充质干细胞向成骨细胞分化,增加钙盐含量,促进骨钙素分泌,增加钙化结节数量,同时上调Runx-2、bFGF-1和Osterix等成骨相关基因的表达,对骨形成具有促进作用。在临床上,淫羊藿苷能促进骨折愈合和骨质疏松患者骨量增加；并且它来源广泛,价格低廉,制剂方便,副作用小,安全性高,具有传统药物不可比拟的优点,因此目前已广泛用于治疗骨质疏松。人工关节无菌性松动的病理过程和骨质疏松类似,都存在骨形成减少。对松动假体周围的成骨细胞,淫羊藿苷是否也具有促进骨形成,提供骨保护的作用?本研究通过体外实验观察淫羊藿苷对钛颗粒刺激下成骨细胞的影响,为进一步动物研究探讨淫羊藿苷防治人工关节无菌性松动提供实验依据。
目的
l、研究钛颗粒对成骨细胞增殖、表型和形态结构的影响。
2、研究淫羊藿苷对钛颗粒刺激下成骨细胞增殖和分化的影响。
3、研究淫羊藿苷对钛颗粒刺激下成骨细胞表达溶骨性因子IL-6的影响。
4、研究淫羊藿苷对钛颗粒刺激下成骨细胞OPG、RANKI,mRNA表达的影响。方法
1、多次酶消化法体外分离新生大鼠颅骨成骨细胞,进行原代培养。碱性磷酸酶染色进行细胞鉴定。配制钛颗粒培养基,鲎试验排除钛颗粒内毒素水平超标。取生长良好的第3代成骨细胞,分别向细胞中加入浓度为0.01、0.05、0.1、0.5、
1mg/ml的钛颗粒(n=6)。培养7d后用CCK-8法检测OD值,比较不例浓度的钛颗粒对成骨细胞增殖能力的影响。ELISA法检测各组细胞ALP和Ⅰ型胶原的表达(n=6),观察钛颗粒对成骨细胞分化表型的影响。对细胞进行FITC-phalloidin和PI双重荧光染色,激光共聚焦显微镜下观察成骨细胞吞噬钛颗粒后形态结构的改变。
2、体外用钛颗粒作用于大鼠成骨细胞,并分别加入10-5、10-6、10-7、10-8、10-9、10-10mol/L的淫羊藿苷进行干预(n=6)。于混合培养7d后用CCK-8法检测各组细胞增殖活力,观察淫羊藿苷对钛颗粒刺激下成骨细胞增殖活力的影响,筛选出最佳药物浓度。以最佳药物浓度的淫羊藿苷干预钛颗粒刺激下的成骨细胞,分别于培养第3d、7d、14d用ELISA法检测各组细胞ALP活性和Ⅰ型胶原的表达(n=6),于培养第21d用茜素红法进行钙化结节染色。
3、将体外培养的大鼠成骨细胞分成五组：第一组对照组成骨细胞,第二组钛颗粒组成骨细胞+钛颗粒,第三组成骨细胞+钛颗粒+10-10mol/L淫羊藿苷,第四组成骨细胞+钛颗粒+10曲mol/L淫羊藿苷,第五组成骨细胞+钛颗粒+10-8mol/L淫羊藿苷(n=6)。分别施加不同干预措施,于培养7d后吸取上清,用ELISA法检测各组上清液中IL-6的含量。
4、将体外培养的大鼠成骨细胞分成四组：第一组对照组成骨细胞,第二组钛颗粒组成骨细胞+钛颗粒,第三组成骨细胞+淫羊藿苷,第四组成骨细胞+钛颗粒+淫羊藿苷(n=6)。分别施加不同干预措施,于培养7d后用RT-PCR技术检测各组细胞OPG/RANKLmRNA的表达及RANKL/OPG比值的变化。
5、应用SPSS18.0软件对实验数据进行统计分析,实验资料以均数±标准差(x±s)表示,主效应与交互效应采用两因素析因分析,若存在交互效应,则单独效应采用单因素方差分析,先做方差齐性检验,若方差齐,则组间比较采用LSD检验,若方差不齐,则采用Tamhane检验。所有检验均采用双侧检验,检验水准为α=0.05,当P<0.05时差异有统计学意义。
结果
1、各组成骨细胞增殖活力的差异有统计学意义(F=517.480,P=0.000)。不同浓度的钛颗粒均能抑制成骨细胞增殖,与对照组相比有显著差异(P<0.05)。浓度为1mg/ml的钛颗粒对成骨细胞增殖活力的抑制作用要大于浓度为0.01、0.05、0.1、0.5、1mg/ml的钛颗粒(P<0.05)。浓度为0.01mg/ml、0.05mg/ml的钛颗粒对成骨细胞增殖活力的影响无显著差异(P=0.240)。各组成骨细胞ALP活性和Ⅰ型胶原表达的差异有统计学意义(ALP：F=828.024,P=0.000；Ⅰ型胶原：F=2258.835,p=0.000)。不同浓度的钛颗粒均能明显抑制成骨细胞ALP活性和Ⅰ型胶原表达,与对照组相比有统计学差异(P<0.05)。浓度为1mg/ml的钛颗粒对成骨细胞ALP活性和Ⅰ型胶原表达的抑制作用要大于浓度为0.01、0.05、0.1、0.5n、1mg/ml的钛颗粒(P<0.05)。浓度为0.01mg/ml、0.05mg/ml的钛颗粒对成骨细胞Ⅰ型胶原表达的影响无显著差异(P=0.240)。激光共聚焦显微镜下可见成骨细胞吞噬钛颗粒后皱缩变形,伪足收缩变短,微丝排列紊乱。2、各组成骨细胞的增殖能力有显著差异(F=829.652,P=0.000)。浓度为10-10-10-6mol/L的淫羊藿苷能抑制钛颗粒介导的成骨细胞增殖能力下降,与钛颗粒组相比有显著差异(P<0.05)。10-8mol/L为淫羊藿苷的最佳作用浓度,与其它各浓度淫羊藿苷组相比有显著差异(P<0.05)。当淫羊藿苷浓度上升至10-5mol/L时,对成骨细胞的增殖有抑制作用(P<0.05)。析因分析结果显示,不同组间ALP浓度有显著差异(F分组=445.548,P=0.000),不同时间点也有显著差异(F时间=194.242,P=0.000),且存在交互效应(F分组×时间=81.439,P=0.000)。单因素方差分析显示,同一时间点不同组间差异有统计学意义(3d：F=18.699,P=0.000;7d:F=129.110,P=0.000;14d:F=459.210, P=0.000),相同组不同时间点间差异也有统计字蒽义(control:F=130.968,P=0.000；Ti:F=23.070,P=0.000;ICA: F=232.660,P=0.000；Ti+ICA:F=43.024,P=0.000)。不同组间COL-I浓度有显著差异(F分组=1843.514,P=0.000),不同时间点也有显著差异(F时间=341.296,P=0.000),且存在交互效应(F分组×州=250.979,P=0.000)。单因素方差分析显示,同一时间点不同组间差异有统计学意义(3d：F=86.658,P=0.000；7d：F=923.149,P=0.000；14d：F=1293.625,P=0.000),相同组不同时间点间差异也有统计学意义(control:F=268.229,P=0.000；Ti:F=206.053,P=0.000；ICA:F=519.974, P=0.000；Ti+ICA:F=120.072,P=0.000)。两两比较结果显示,钛颗粒组成骨细胞的ALP活性和Ⅰ型胶原表达在3d、7d、14d时均低于同时期的对照组(P<0.05),钛颗粒+淫羊藿苷组成骨细胞的ALP活性和Ⅰ型胶原表达在7d、14d时均高于同时期的钛颗粒组(P<0.05)。淫羊藿苷的作用存在时间-效应关系,干预第7d时,钛颗粒+淫羊藿苷组成骨细胞的ALP活性和Ⅰ型胶原浓度最高,与3d、14d相比有显著差异(P<0.05)。钙化结节染色显示淫羊藿苷干预组的成骨细胞钙化结节数和分布面积明显高于钛颗粒组。3、各组成骨细胞IL-6表达的差异有统计学意义(F=7202.778,P=0.000)。与对照组相比,钛颗粒明显促进了成骨细胞IL-6的表达(P<0.05)。不同浓度的淫羊藿苷对钛颗粒介导的IL-6的表达均有抑制作用,与钛颗粒组相比有统计学差异妒<0.05)。浓度为10吨mol/L的淫羊藿苷对IL-6表达的抑制作用要大于浓度为10-10、10-9mol/L的淫羊藿苷(P<0.05)。4、各组成骨细胞OPG mRNA、RANKLmRNA的表达量及RANKL/OPG比值的差异有统计学意义(OPG:F=4277.834P=0.000；RANKL:F=5599.081P=0.000； RANKL/OPG:F=1334.919P=0.000)。与对照组相比,钛颗粒组成骨细胞OPGmRNA表达下降,RANKL mRNA表达上升,RANKL/OPG的比值上升,差异有统计学意义(P<0.05)。淫羊藿苷干预后,成骨细胞OPG mRNA的表达上升,RANKL mRNA的表达下降,RANKL/OPG的比值下降,与钛颗粒组相比有显著差异(P<0.05)。
结论
1、钛颗粒可以抑制体外培养的大鼠成骨细胞的增殖和分化成熟,降低成骨能力。成骨细胞可吞噬钛颗粒并且细胞形态和骨架发生改变。
2、淫羊藿苷能够逆转钛颗粒对成骨细胞增殖、分化成熟和矿化能力的抑制作用,维持细胞表型,促进骨形成。
3、淫羊藿苷可以抑制钛颗粒介导的成骨细胞分泌溶骨性因子IL-6,抑制骨溶解。
4、淫羊藿苷可以通过调控钛颗粒介导的成骨细胞OPG、RANKL mRNA的表达,抑制破骨细胞的分化成熟。
Background
Since arthroplasty used clinically, it has become the main method of reconstructing joint function and achieved good results. However, with expansion of application, increasing number of operation and extension of service life,-the postoperation complications also increased. Among them, prosthesis aseptic loosening is the most common complication on the later period of artificial joint replacement. The factors which cause prosthetic loosening are mainly divided into two categories:one is mechanical factors, that is stress shielding around prosthesis, early loosening, prosthesis micromove, joint hydraulic pressure, loosed fixation of prosthesis, etc. Another is biological factors, which refers to that wear particles are generated in utilization and osteolysis is caused by a series of related biochemical reactions. At present, in the mainstream view was proposed the concept of "particles disease". It proposed that artificial joints produce a lot of wear particles, the wear particles stimulate monocytes-macrophages, osteoblasts, fibroblasts to produce osteolytic factors. Thereby it activates periprosthetic osteoclasts, produces bone resorption and bone dissolution. Then the gap appears between the prosthesis and the surrounding bone, so that the prosthesis loses support, and eventually lead to artificial joints aseptic loosening. The loosening artificial joints make further exacerbated wear. Thus formed a vicious cycle from wear to loosen.
Wear particles include polyethylene particles, cement particles, ceramic particles and metal particles. There are many researches about polyethylene particles and bone cement particles. Titanium is common material for artificial joint prostheses and fracture internal fixation. Fretting between femoral stem and cement and bone interface, friction between femoral stem and bone, fretting between micro-porous coating, fretting between screws and acetabular cup, are the main source of titanium particles. So titanium particles is one of common metal particles after artificial joint replacement surgery. With the spread application of the new generation of metal-on-metal prosthesis, the periprosthetic osteolysis role of metal particles has gradually attracted people's attention.
When it refers to the effect of titanium particles on bone metabolism, the present study focused on osteoclasts. Studies suggest that the titanium particles can cause osteolysis by activation of osteoclasts. However, bone metabolism balance is a result of osteoclasts and osteoblasts joint action. As the main functional cells with osteogenic potential, osteoblasts are capable of synthesizing and secreting a variety of type I collagen and non-collagen protein, secreting mineralized substance as osteoid mineralization substances and alkaline phosphatase, thus promoting new bone formation. At the same time wear particles activate osteoclasts activity, inevitably closely contact with large amount of osteoblasts surrounding around. There is no in-depth study about whether it will effect the biological behavior of osteoblasts.
Wear particles stimulate cells of membrane arround the prosthesis to release large amounts of osteolytic factors, which constitute a complex network inbody, and are involved in the bone resolved metabolic process around prosthesis alone or together. IL-6is mainly synthesized by osteoblasts, its primary role in the pathogenesis of osteoporosis is stimulates the original osteoclasts to differentiate into mature osteoclasts, increases the number of osteoclasts in trabecular bone, promote osteoclast differentiation, maturation, and by increasing the release of collagenase to promote bone matrix degradation, so that to increase bone resorptionand decrease BMD. IL-6as an important inflammatory mediator produced by stimulating wear particles, it involves in the pathological process of dissolution of bone around the prosthesis. In the synovial; fluid of the the patients who occurred prosthesis loosening after total hip arthroplasty, the levels of IL-6elevated, and therefore the increasing level of IL-6can be considered as the biomarker of bone dissolved around atificial prosthesis.
Receptor activator of nuclear factor-KB belongs to type I transmembrane protein, it is one of the TNF family members. RANK is highly expressed on the surface of the osteoclast precursor cells. Receptor activator of nuclear factor-KB factor ligand binds to RANK expressed on the surface of osteoclast precursor cells and mature osteoclasts, promote the differentiation of osteoclasts and inhibit its apoptosis. Osteoprotegerin as a decoy receptor, competitively binding with RANKL, and thereby blocking the binding of RANKL to RANK on osteoclasts surface, inhibitting osteoclast maturation. The change of RANKL/OPG ratio is essential for oosteoclasts production. In general, when the RANKL/OPG ratio upregulates, the number of osteoclasts and their activity will increase, when the RANKL/OPG ratio downregulates, the number of osteoclasts and their activity will decrease. Therefore, the bony remodeling and the stability of bone mass around prothesis depended upon the balance between OPG and RANKL.
At present, the treatment for joint prosthesis aseptic loosening is inhibitting osteolysis and bone resorption, including the control of osteolytic factors and osteoclasts. However, osteolysis is resulted from the co-effects of various factors through various methods. It is difficult to take complete control of prosthesis loosening by medicine which inhibits solo osteolytic factor. Some medicine which inhibit osteoclasts, such as calcitonin, would be ineffective after taking for a period of time, while the long-term utilization of bisphosphonate will lead to various serious toxic and side effects and the expensive price limits its application. Therefore, it is necessary to conduct further research.
Prosthesis aseptic loosening is caused by the imbalance between bone formation and bone resorption. The reduction of bone formation also has important influence on prosthesis aseptic loosening. It may be another effective method to conduct treatment of joint prosthesis aseptic loosening by applying medicines to prompt bone formation around loosening prosthesis and make prosthesis biologically fixed. However, few related researches are at present. Icariin is a kind of "bone reinforcement" Chinese medicine, which can induced bone marrow mesenchymal stem cells to differentiate into osteoblasts, improve ALP activity, increase calcium salt content, prompt osteocalcin secretion, increase the number of calcified nodules and upregulate the expression of factors related to bone-formation, such as Runx-2, bFGF-1and Osterix etc. Icariin can prompt the recovery of bone fracture and increase bone mass of patients of osteoporosis. And it has a wide range of source, low cost, easy preparation, small side effects, high safety and incomparable advantages of traditional medicines, therefore it has been widely used for the treatment of osteoporosis. The pathological process of joint prosthesis aseptic loosening is similar to osteoporosis, both of them have decreased bone formation. For those osteoblasts around the loose prosthesis, whether icariin can promote bone formation, provide protective effect? In this study we observe the effects of icariin on osteoblasts stimulated by titanium particles in vitro, so as to provide experimental foundations for further animal experiment.
Ojectives
1、Study the effect of titanium particles on proliferation, phenotype and morphology of osteoblasts.
2、Study the effect of icarrin on proliferation, differenciation of osteoblasts stimulated by titanium particles.
3、Study the effect of icarrin on osteolytic factor IL-6secretion of osteoblasts stimulated by titanium particles.
4、Study the effect of icarrin on OPG, RANKL mRNA expression of osteoblasts stimulated by titanium particles.
Methods
1、Calvarial osteoblasts of newborn rats were seperated in vitro by repeated enzyme digestion, and received primary culture. Alkaline phosphatase staining was carried out to identify them. Titanium culture medium was prepared and limulus assay was performed to exclude excessive endotoxin level. Titanium particles with concentrations of0.01,0.05,0.1,0.5as well as1mg/ml were respectively added to the third generation osteoblasts well grown(n=6). After cultured for seven days, OD was detected by CCK-8method to compare the influence of different titanium particles concentrations on osteoblasts proliferation. ALP activity and type I collagen expression were detected by ELISA to observe the effect of titanium particles on differentiation and phenotype of osteoblasts(n=6). The cells received double fluorescence staining with FITC-phalloidin and PI to observe cellular morphology variation of osteoblasts which swallowed titanium particles under laser scanning confocal microscope.
2、Titanium particles were used to treat rats osteoblasts, and icariin with concentrations of10-5,10-6，10-7,10-8,10-9,10-10mol/L were added for interference respectively(n=6). The effect of icariin on proliferation of osteoblasts stimulated with titanium particles was observed after cultured for seven days, and the optimum drug concentration was screened out. Icariin with optimum drug concentration was added to intervene osteoblasts stimulated with titanium particles(n=6), ELISA method was carried out to detected ALP activity and type I collagen expression on the3rd7th、14th day after cultur, calcified nodules were stained by alizarin S red stain method on the21th day.
3、The rats osteoblasts cultured in vitro were divided into five groups:the first group, the control group, osteoblasts; the second group, the tatinium particlesgroup, osteoblasts plus titanium particles; the third group, osteoblasts plus titanium particles plus10-10mol/L icariin; the fourth group, osteoblasts plus titanium particles plus10-9mol/L icariin; the fifth group osteoblasts plus titanium particles plus10-8mol/L icariin(n=6). They received differern intervene measures. After cultured for seven days, supernatant was extracted, and ELISA method was performed to detect IL-6concentration in the supernatant of each group.
4、The rats osteoblasts cultured in vitro were divided into four groups:the first group, the control group,osteoblasts; the second group, titanium particles group, osteoblasts plus titanium particles; the third group, osteoblasts plus icariin; the fourth group, osteoblasts plus titanium particles plus icariin(n=6). Each group received differern intervene measures. After cultured for seven days, RT-PCR was carried out to detect relative expression of OPG and RANKL mRNA and RANKL/OPG ratio variation of each group.
Results
1、The differences of osteoblasts proliferation activity within each group was statistically significant (F=517.480, P=0.000). Different concentrations of titanium particles could inhibit the proliferation of osteoblasts, which had a significant difference (P<0.05) compared with the control group. The inhibition effect of titanium particles with concentration of lmg/ml on osteoblast proliferation activity was greater than those of titanium particles with concentration of0.01,0.05,0.1,0.5,1mg/ml (P<0.05). The effect of of titanium particles on osteoblast proliferation activity showed no significantly difference between concentration of0.01mg/ml and0.05mg/ml (P=0.240). The differences of osteoblasts ALP activity and type I collagen expression within each group was statistically significant (ALP:F=828.024, P=0.000; type I collagen:F=2258.835, P=0.000). Different concentrations of titanium particles could significantly inhibit ALP activity and type I collagen expression of osteoblasts, there was significant difference (P<0.05) compared with the control group. The inhibition effect of titanium particles with concentration of lmg/ml on osteoblast ALP activity and type I collagen expression was greater than those of titanium particles with concentration of0.01,0.05,0.1,0.5,1mg/ml (P<0.05). The effect of of titanium particles on osteoblast ALP activity and type I collagen expression showed no significantly difference between concentration of0.01mg/ml and0.05mg/ml (P=0.240). Laser scanning confocal microscope showed cellular shrinking, microfilaments distortion, pseudopodia contraction of osteoblasts swallowed with titanium particles.
2、The differences of osteoblasts proliferation activity within each group was statistically significant (F=829.652, P=0.000). Icarrin with concentrations of10-10-10-6mol/L could inhibit decreased proliferation ability of osteoblasts stimulated by titanium particles, which showed significant difference compared with the titanium particles group (P<0.05).10-8mol/L was icarrin's optimum drug concentration, which had significant differences compared with other various concentrations of icariin groups (P<0.05). When the concentration of icariin rise to10-5mol/L, it inhibitted the proliferation of osteoblasts (P<0.05). Factorial analysis showed that there was significant differences of ALP concentration within different groups (Fgroup=445.548, P=0.000), so were different time points (Ftime=194.242, P=0.000), and there existed interaction (Fgroup×time=81.439, P=0.000). One-way ANOVA analysis showed that, there were significant differences within different groups of the same time point (3d:F=18.699, P=0.000;7d:F=129.110, P=0.000;14d:F=459.210, P=0.000), so were different time point of the same group (control: F=130.968, P=0.000; Ti:F=23.070, P=0.000; ICA:F=232.660, P=0.000; Ti+ICA: F=43.024, P=0.000). There were significant differences of type I collagen expression within different groups (Fgroup=1843.514, P=0.000), so were different time points (Ftime=341.296, P=0.000), and there existed interaction (Fgroup×time=250.979, P=0.000). One-way ANOVA analysis showed that, there were significant differences within different groups of the same time point (3d:F=86.658, P=0.000;7d: F=923.149, P=0.000;14d:F=1293.625, P=0.000), so were different time point of the same group (control:F=268.229, P=0.000; Ti:F=206.053, P=0.000; ICA: F=519.974, P=0.000; Ti+ICA:F=120.072, P=0.000). Pairwise comparisons showed that, ALP activity and type I collagen expression of osteoblasts in titanium particles group on3d,7d,14d are lower than that of control group during the same period (P<0.05). while ALP activity and type I collagen expression of osteoblasts in titanium particles+icarrin groups on7d,14d.are higher than that of titanium particles groups during the same period (P<0.05). The role of icariin had a time-effect relationship,, ALP activity and type I collagen expression of titanium particles+icarrin reached the highest on7d,which had significant difference compared with that of3d and7d (P<0.05). Alizarin red stain showed that the amount of cacified nodules and distribution area of osteoblast were higher in icarrin intervene group than in titanium particles group.
3The differences of IL-6expression of osteoblasts within each group was statistically significant (F=7202.778, P=0.000). Compared with the control group, titanium particles obviously promoted the osteoblasts'expression of IL-6(P<0.05). Different concentrations of icariin could significantly inhibit IL-6expression mediated by titanium particles, which was significant different compared with the titanium particles group(P<0.05). The inhibition effect of icarrin with concentration of108mol/L on IL-6expression was greater than those of icarrin with concentrations of10-10、10-9mol/L(P<0.05).
4、The differences of OPG mRNA、RANKL mRNA expression of osteoblasts and RANKL/OPG ratio within each group were statistically significant (OPG:F=4277.834P=0.000; RANKL:F=5599.081P=0.000; RANKL/OPG: F=1334.919P=0.000). Compared with the control group, OPG mRNA expression decreased, RANKL mRNA expression increased, and RANKL/OPG ratio rised in titanium particles group, which showed statistic difference (P<0.05). After icarrin intervention, OPG mRNA expression rised, while RANKL mRNA expression decreased, and RANKL/OPG ratio declined, which showed statistic difference compared with the titanium particles group (P<0.05)
Conclusions
1-, Titanium particles can inhibit the proliferation and differenciation ability of cultured rat osteoblasts in vitro, decreased bone formation. Titanium particles can be phagocytosed by osteoblasts and make changes in cell morphology and cytoskeleton.
2、Icariin can reverse the inhibition of effect of titanium particles on osteoblasts proliferation, differentiation, maturation and mineralization, maintain cell phenotype and promote bone formation.
3、Icariin can inhibit titanium particles-mediated osteoblasts'secretion of osteolytic factor IL-6, thus inhibit osteolysis.
4、Icariin can regulate osteoblasts OPG, RANKL mRNA expression mediated by titanium particles, thus inhibit differenciation and maturation of osteoclasts..
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