金属离子与金属颗粒生物学活性实验研究
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摘要
研究背景和目的
人工关节置换术是治疗骨关节疾病的有效方法,能有效达到解除关节疼痛、重建关节活动功能的目的。但全髋关节置换术的致命缺憾是有限的假体使用寿命,目前人工关节的使用寿命多在10-15年之间。假体无菌性松动(aseptic loosening)是导致人工关节使用寿命较短的主要原因。假体无菌性松动主要有以下两大主要因素引起：一为机械性因素,即假体的材质、设计、配伍、安装、撞击等因素引起假体无菌性松动；另一为生物性因素,即假体无菌性松动是由人工关节磨损颗粒刺激炎性细胞(单核巨噬细胞)分泌大量溶骨性因子包括肿瘤坏死因子-α (TNF-α)、白介素-1β (IL-1β),白介素-6(IL-6)等,这些溶骨性因子能刺激破骨细胞产生骨溶解,最终导致假体无菌性松动。目前一般认为假体机械磨损产生的磨损颗粒可诱导假体周围组织细胞产生一系列生物学反应,直接或间接激活破骨细胞并导致假体周围骨溶解是人工关节晚期松动的主要原因。
金属假体以其良好的刚度和强度及其良好的抗磨损性在全髋置换中获得广泛的运用,但金属假体周围骨溶解仍是导致假体中远期失败的一个重要原因,而破骨细胞(OC,osteoclast)在假体周围骨溶解中起了重要的作用,许多研究已证明金属假体周围存在大量金属离子,以钻和铬离子较为常见。
骨是一个代谢活跃的器官,通过骨重建而获得持续的更新,骨重建对于调节和维持骨骼的完整性非常重要。在这个过程中,破骨细胞吸收旧骨,成骨细胞(OB,osteoblast)形成新骨,因此骨重建需要骨吸收和骨形成两种过程的精密协调。OB来源于骨髓间充质干细胞,负责骨基质的合成、分泌和矿化；OC来源于造血干细胞中的单核前体细胞,是一种大的多核细胞。在骨髓微环境中,破骨前体细胞分化为成熟的OC,并行使骨吸收功能。正常情况下OB和OC数量和功能通过各种因子和信号通路维持稳定的水平,从而使它们所介导的骨形成与骨吸收处于平衡状态。在病理条件下这种平衡被打破,导致骨骼结构和功能的异常,从而有相应的临床表现如假体周围骨丢失。重建是一个复杂的过程,受到各种细胞因子、激素及信号通路的调控,OB和OC之间的相互调节有助于骨重建过程中骨吸收和骨形成两过程之间的偶联。
核因子-K B受体活化因子(RANK, receptor activator of nuclear factor-κB)属于I型跨膜蛋白,是TNF家族成员之一。RANK高度表达于破骨前体细胞的表面。核因子-K B受体活化因子配体(RANKL, receptor activator of nuclear factor-κ B ligand)结合到破骨前体细胞和成熟OC表面表达的RANK上,促进OC的分化活化并抑制其凋亡。骨保护素(OPG, osteoprotegerin)作为一种诱骗受体,可以竞争性的与RANK结合,从而封闭RANKL与OC表面的RANK结合,抑制OC的分化成熟。RANKL/OPG的比例的变化对于OC产生至关重要,一般来说,当RANKL/OPG的比例上调OC的数量和活性将增加,RANKL/OPG的比例下降时OC的数量和活性将降低。总之,正常的骨重建和骨量的稳定依赖于OPG和RANKL这间的平衡。
TNF-α是炎性细胞释放的一种重要的炎性介质,它能刺激炎性细胞分泌IL-1和IL-6,而这两者又能引起巨噬细胞和破骨细胞的趋化作用,向界膜内聚集。TNF-α还能刺激破骨细胞释放前列腺素E(PGE),引起破骨细胞性骨吸收。近年有研究表明TNF-α能促进人成骨细胞骨保护素配体(OPGL)的表达,而骨保护素配体能促进破骨细胞分化成熟并增强其功能活性,从而进一步引起假体松动发生。
细胞凋亡(apoptosis)是由基因控制的一种细胞的生理性、程序性死亡。当刺激因素作用于细胞后,通过启动相关基因转录及蛋白质合成而导致细胞内各种蛋白酶及内源性核酸内切酶等的活化,从而引起细胞的一系列具有特征性的生化及形态学改变。近年来研究表明,无菌性松动的假体周围界膜组织中存在单核巨噬细胞凋亡的现象。单核巨噬细胞的凋亡会导致更多单核巨噬细胞的聚集由此引起级联反应导致假体周围骨丢失。
金属颗粒和金属离子均能诱导假体周围骨溶解。但金属颗粒与金属离子在无菌性松动及骨溶解中活性比较至今无相关研究。本课题旨在通过体外试验研究,对金属离子与金属颗粒在假体无菌性松动中的活性进行比较,为临床骨科材料的选取提供参考。
目的
1、通过真空球磨法制备符合实验要求的金属颗粒及金属离子。
2、观察金属磨损颗粒对破骨细胞体外分化的影响。
3、观察金属离子对小鼠成骨细胞骨保护素及核因子-κ B受体活化因子配体表
达的影响。
4、金属离子与金属颗粒对单核巨噬细胞影响的活性比较。
材料与方法
1、真空球磨法制备金属颗粒及其物理、生物学活性检测
通过真空球磨法体外制备常用人工关节金属磨损颗粒,同时对磨损颗粒进行元素分析、扫描电镜观察、激光粒度仪检测、并与RAw264.7细胞共培养检测细胞毒性。
2、观察金属磨损颗粒对破骨细胞体外分化的影响
体外培养小鼠破骨前体细胞(RAw264.7),分别用高浓度(10mg/ml)、中浓度(1.0mg/ml)、低浓度(0.1mg/ml)金属磨损颗粒悬液进行干预。RANKL诱导培养7天后,通过抗酒石酸酸性磷酸酶(TRAP)染色观察TRAP阳性多核细胞形成的变化并进行计数。
3、观察金属离子对小鼠成骨细胞骨保护素及核因子-K B受体活化因子配体表达的影响。
体外培养小鼠成骨细胞MC3T3E1根据培养基不同分为两组,A实验组使用含10mg/LCoCl2及150mg/L CrCl3溶液的培养基,B对照组使用不含金属离子的培养基。RT-PCR检测各组RANKL、OPG mRNA的表达；ELISA法检测上清液RANKL、 OPG蛋白的表达。计算RANKL/OPG mRNA和蛋白表达比值变化。
4、金属离子与金属颗粒对单核巨噬细胞影响的活性比较
从30名健康志愿者分别抽取5mL外周血；离心后吸取单个核细胞,将细胞进行体外培养。实验分九组：(1)Fe3＋组、(2)Ti3＋组、(3)Cr3＋组、(4)Co2＋组、(5)铁颗粒组、(6)铬颗粒组、(7)钴颗粒组、(8)钛颗粒组、(9)生理盐水组；用Fe3+、Ti3+、Cr3+、Co2+、铁颗粒、钛颗粒、铬颗粒、钴颗粒分别进行干预,空白组加入生理盐水作为对照。分别在6h、12h、24h、36h、48h用MTT方法检测九组细胞活性；在6h、12h、24h、36h、48h运用ELISA试剂盒检测九组细胞上清液中TNF-α的表达；流式细胞仪检测细胞凋亡情况。
结果
1、通过真空球磨法所取得金属颗粒的中位径2.193μm,体积平均径3.108μm；面积平均径2.119μ m；98%的颗粒直径在6.167μ m以下。与人工关节界膜组织内提取的颗粒直径相仿,且生物活性相仿,在试验浓度(通常低于lmg/ml)没有显示细胞毒性。
2、培养7d后,在200×视野下观察破骨细胞计数,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对照组(P<0.05),中、高浓度组破骨细胞数均显著高于低浓度组(P<0.05),高浓度组破骨细胞数亦显著高于中浓度组(P<0.05)。
3、随时间增加,实验组RANKL、 OPGmRNA表达量明显增加,培养24、48h与对照组比较,差异有统计学意义(P<0.05)。实验组组内培养48h时RANKL/OPG mRNA比值较24h明显升高,差异有统计学意义(P<0.05)；对照组则轻度下降,差异无统计学意义(P>0.05)。培养24、48h时,实验组RANKL蛋白表达量较对照组明显增多分别为对照组的61.6倍及13.8倍,差异有统计学意义(P<0.05)：OPG蛋白表达量较对照组增长32.1%及17.8%,差异有统计学意义(P<0.05)。培养24、48h实验组RANKL/OPG mRNA的比值分别为0.860、1.232,较对照组0.695、0.688明显增加,差异有统计学意义(P<0.05)
4、(1)单核细胞与金属颗粒共培养6h、12h、24h、36h、48h后TNF-α水平。经统计学分析24h、36h各颗粒组TNF-α量高于对照组(p<0.05)；
(2)钴、铬颗粒诱导细胞分泌TNF-α作用比铁、钛颗粒更强(p<0.05),钴离子诱导细胞分泌TNF-α作用较铬、铁、钛离子更强(p<0.05)；钴、铬离子组肿瘤坏死因子水平于24h到达峰值,36h又下降。
(3)各实验组金属颗粒均能诱导人单核细胞产生凋亡,且随时间发展细胞凋亡率逐渐升高,经统计学分析钴、铬、铁颗粒每个时间点实验组细胞凋亡率均高于空白对照组(p<0.05)；
(4)金属离子能诱导人单核细胞产生凋亡,且随时间发展细胞凋亡率逐渐升高,经统计学分析钴、铬诱导细胞凋亡作用强于铁、钛颗粒；铁、钴离子诱导细胞凋亡作用强于铬、钛颗粒；
(5)钛颗粒和钛离子组诱导人单核细胞分泌TNF-α与细胞凋亡明显低于铁、铬、钴颗粒和离子组；
结论
1、实验证实真空球磨法体外制备人工关节金属磨损颗粒简单方便,能大量制备人工关节金属磨损颗粒可重复性好,可用于相关体外实验。
2、RAW264.7是一种较好的破骨前体细胞模型。RANKL诱导RAW264.7形成破骨细胞方法简便易行、可重复性好。传代超过20代的RAW264.7仍然保持了在RANKL的诱导下分化成为破骨细胞的能力。金属磨损颗粒和RAW264.7细胞共培养后,可以促进RANKL诱导RAW264.7形成破骨细胞的数量增加。
3、Co2+、Cr3+共同作用可刺激成骨细胞RANKL、OPGmRNA表达的增加,以及RANKL/OPG mRNA比值的增加,并显著促进RANKL蛋白的分泌,从而影响破骨细胞前体细胞向破骨细胞分化与成熟,促进破骨细胞骨性吸收。
4、金属颗粒与金属离子能诱导人单核细胞凋亡,随时间细胞凋亡率逐步升高；金属颗粒与金属离子能通过刺激单核细胞释放TNF-α引发假体周围炎性反应,并且通过诱发单核细胞凋亡参与这一病理过程从而导致假体松动；不同的金属材料金属颗粒及离子生物学活性不同,钛金属材料生物学活性明显低于铁、钻、铬材料。钴、铬颗粒生物学活性高于离子：铁颗粒和铁离子生物学活性无明显差异
Background
Joint replacements as an effective strategy in treating bone and joint diseases, provides pleasing results in releasing pain and increase the motion range of joints. However, popularize joint replacements are somehow limited by relatively short lifespan of prosthesis. According to those long-term follow-up data, aseptic loosening plays a key role in this process. Aseptic loosening of the components are induced by biological factors and mechanical factors including material design compatibility and installation of the prosthesis. Fragments of wear debris may cause an inflammatory reaction involving IL1β(interleukin-1β) IL-6(interleukin-6) and TNF-α (TNF-α, tumor necrosis factor-a) with bone absorption which can cause loosening. This phenomenon is known as osteolysis. It is generally believed that the wear particles generated by the mechanical wear of the prosthesis can induce periprosthetic tissue cells to produce a series of biological reactions activating the osteoclasts causing periprosthetic osteolysis which is the main reason for long-term loosening of the artificial joints. With the technology development of material selection and process design wear and tear caused by mechanical loosening has been well controlled.
The femoral prosthesis is commonly made of metal which is widely used in total hip arthroplasty because of its appropeiate stiffness and rigidity. However, osteolysis surrounding the host bone following metal prosthesis implantation made its application controversial. Osteolcast (OC) plays a significant role in osteolysis surrounding prosthesis. Numerous researches have evidenced the metal ion, Mainaly cobalt and chrome, located surrounding the femoral prosthesis.
Bone is an active metabolic organ. Bone remodeling is important for regulating and maintaining skeletal integrity. In this process, OC absorbed the old bones meanwhile osteoblasts (OB, osteoblast) form the new bone. Therefore, bone reconstruction need these two processes precisely coordinate with each other. In the bone marrow microenvironment osteoclast differentiation of progenitor cells into mature OC, and bone resorption.Number of OB and OC under normal conditions and by a variety of factors and signaling to maintain stable levels, so that bone formation and bone resorption mediated by them in a State of balance.This balance is broken in pathological conditions, resulting in abnormal structure and function of bones, thus the corresponding clinical manifestations such as periprosthetic bone loss.Redevelopment is a complex process, subject to various cytokines, hormones and signaling pathways of regulation, mutual regulation between the OB and OC help bone resorption and bone formation in the bone remodeling process coupling between the two processes.
RANK (RANK, Nuclear factor-B receptor activation factor) is type I transmembrane protein which belongs to TNF family. RANK highly expresse on the surface of osteoclast precursor. Nuclear factor Kb ligand receptor activation factor (RANKL, receptor activator of nuclear factor-K b ligand) binding to RANK not only promoting the differentiation of OC but also inhibiting the apoptosis of OC. OPG (OPG, osteoprotegerin) secreted by osteoblast can competitive binding to RANK against RANKL. RANKL/OPG rate is essential for OC. Ehance in the proportion of RANKL/OPG lead to the increase in the number of OC and its activity. Decrease in the proportion of RANKL/OPG will reduce the number of OC and its activity. In short, the normal bone remodeling and bone mass stability depends on the rate of RANKL/OPG.
TNF-a is a kind of important inflammatory mediators which can can stimulate the inflammatory cells secrete IL-land IL-6lead to the macrophages and osteoclasts gathering at the place where inflammatory reaction take place. TNF-a can also stimulate osteoclast release PGE (PGE, prostaglandinE) which can induce osteoclastic bone resorption. In recent years, research has shown that TNF-α can promote human osteoblasts of osteoprotegerin ligand (OPGL) expression and osteoprotegerin physical distribution activity of osteoclast differentiation and maturation and increase its functionality, which further lead to implant loosening.
Apoptosis is the process of programmed cell death controlled by genes. Stimulunts affect cells by initiating gene transcription and protein synthesis causing intracellular protease and activating endogenous DNA endonuclease. In recent years, studies have shown that around the aseptic loosening prosthesis exists the phenomenon of mononuclear macrophage apoptosis. Apoptosis of mononuclear macrophage will lead to more gathering of mononuclear macrophage causing a cascade reaction resulted in periprosthetic bone loss.
Both metal particles and metal ions can induce Periprosthetic osteolysis leading to the loosening of prosthesis. But there is no relevant research comparing the effect between metal particles and metal ions on aseptic loosening and osteolysis. This study aim at compare the effect between metal particles and metal ions on aseptic loosening and osteolysis through vitro experiment providing reference for the choice of prosthesis materials.
Purpose
1. Prepare metal debris and metal ions which meet the requirements of the experiment.
2. Observe the effect of metal debris on the differentiation of osteoclast in vitro.
3. Observe the effect of metal ions on the expression RANKL and OPG secreted by osteoblast.
4. Compare the biological activity difference between metal ions and metal debris on mononuclear macrophage.
Method
1. Prepare metal debris through the vacuum ball milling method. Analyse the elements of the metal debris. Observe its character through electron microscope. Measure its diameter by laser particle size analyzer. Detect its cytotoxicity by coculture with RAW264.7cell line.
2. Treating RAW264.7cells which induced by RANKL with high concentration (10mg/ml) medium concentration (1.0mg/ml) low concentration (0.1mg/ml) metal debris respectively. Take count of TRAP (TRAP, tartrate-resistant acid phosphatase) stain positive multinucleate cells after7days culturing.
3. In vitro culture of MC3T3-E1cell line. Divide it into two groups according to its medium. The experimental group including10mg/L C0Cl2and150mg/L CrC13in the culture medium defined as group A. The control group with normal culture medium defined as group B. Detect RANKL and OPG mRNA by RT-PCR. Detect RANKL and OPG protein by ELASIA.
4. Mononuclear cells obtained from peripheral blood of30healthy volunteers. Coculture mononuclear cells with iron particles cobalt particles chromium particles titanium particles Iron ions chromium ions cobalt ions titanium ions and physiological saline respectively. Assess activity of the cells by MTT at6h12h24h36h48h respectively. Assess the expression of TNF-α protein by ELASIA at6h12h24h36h48h respectively.
Results
1. Median diameter volume mean diameter and surface mean diameter of metal particles obtained by vacuum ball milling is2.193μm3.018μm and2.119μm respectively. Diameter of98%particles is less than6.167μm. The diameter and biological activity in our experiment is in common with particles extracted from artificial joint interface tissue.In the testing concentration, typically less than1mg/ml, showed no cytotoxicity.
2. The results show that as the wear particles suspension concentration increased RANKL induces formation of osteoclast cells increased. A number of osteoclast in the high concentration medium concentration low concentration group was higher than that in control group (P<0.05). A number of osteoclast in the high concentration medium concentration group was higher than that in low concentration group (P<0.05). A number of osteoclast in the high concentration group was higher than that in medium concentration group (P<0.05).
3. The expression of RANKL OPG mRNA was increased with time in the experimental group. The expression of RANKL OPG mRNA was higher than that in the control group at2448h. Rate of RANKL/OPG mRNA is0.8601.232at2448h in the experimental group higher than that0.6950.688in the control group(P<0.05). Rate of RANKL/OPG protein at2448h in the experimental group was higher than that in the control group (P<0.05)
4. The expression of TNF-a was higher in iron particles cobalt particles chromium particles titanium particles group at2436h than that in the control group (P<0.05). The expression of TNF-a was higher in cobalt particles chromium particles group than that in the iron particles titanium particles group (P<0.05). The expression of TNF-a was higher in cobalt ions group than that in the iron ions chromium ions titanium ions group. Iron particles cobalt particles chromium particles titanium particles can induce mononuclear cells to apoptpsis. Iron ions chromium ions cobalt ions titanium ions can induce mononuclear cells to apoptpsis. The ability of inducing mononuclear cells to apoptosis for cobalt particles chromium particles was greater than iron particles titanium particles. The ability of inducing mononuclear cells to apoptosis for iron ions cobalt ions was greater than chromium ions titanium ions.
Conclusion
1. Metal debris prepared by vacuum ball milling method not only simple and convenient but also has good reproducibility and productive thus can be used for vitro experiment.
2. RAW264.7is a good osteoclast precursor cells model. RANKL induced RAW264.7to form osteoclasts is simple also has good repeatability. The number of osteoclase has increased after coculture with metal debris.
3. Co2+, Cr3+interaction can stimulate osteogenesis cells increase expression of RANKL and OPGmRNA as well as the increase in the ratio of RANKL/OPG mRNA and greatly promote the secretion of the protein RANKL thus affecting the osteoclast precursor cells differentiate into osteoclasts and enhance the ability of bone absorption.
4. Iron particles cobalt particles chromium particles titanium particles Iron ions chromium ions cobalt ions titanium ions can induce human mononuclear cells to apoptosis and its rate gradually increased over time.Metal particles and metal ions through stimulating mononuclear cells causing inflammatory reaction around the prosthesis and by inducing apoptosis of mononuclear cells lead to implant loosening. The biological activity of titanium was significantly lower than iron cobalt and chromium.
人工关节置换术是治疗骨关节疾病的有效方法,能有效达到解除关节疼痛、重建关节活动功能的目的。但全髋关节置换术的致命缺憾是有限的假体使用寿命,目前人工关节的使用寿命多在10-15年之间。假体无菌性松动(aseptic loosening)是导致人工关节使用寿命较短的主要原因。假体无菌性松动主要有以下两大主要因素引起：一为机械性因素,即假体的材质、设计、配伍、安装、撞击等因素引起假体无菌性松动；另一为生物性因素,即假体无菌性松动是由人工关节磨损颗粒刺激炎性细胞(单核巨噬细胞)分泌大量溶骨性因子包括肿瘤坏死因子-α (TNF-α)、白介素-1β (IL-1β),白介素-6(IL-6)等,这些溶骨性因子能刺激破骨细胞产生骨溶解,最终导致假体无菌性松动。目前一般认为假体机械磨损产生的磨损颗粒可诱导假体周围组织细胞产生一系列生物学反应,直接或间接激活破骨细胞并导致假体周围骨溶解是人工关节晚期松动的主要原因。
金属假体以其良好的刚度和强度及其良好的抗磨损性在全髋置换中获得广泛的运用,但金属假体周围骨溶解仍是导致假体中远期失败的一个重要原因,而破骨细胞(OC,osteoclast)在假体周围骨溶解中起了重要的作用,许多研究已证明金属假体周围存在大量金属离子,以钻和铬离子较为常见。
骨是一个代谢活跃的器官,通过骨重建而获得持续的更新,骨重建对于调节和维持骨骼的完整性非常重要。在这个过程中,破骨细胞吸收旧骨,成骨细胞(OB,osteoblast)形成新骨,因此骨重建需要骨吸收和骨形成两种过程的精密协调。OB来源于骨髓间充质干细胞,负责骨基质的合成、分泌和矿化；OC来源于造血干细胞中的单核前体细胞,是一种大的多核细胞。在骨髓微环境中,破骨前体细胞分化为成熟的OC,并行使骨吸收功能。正常情况下OB和OC数量和功能通过各种因子和信号通路维持稳定的水平,从而使它们所介导的骨形成与骨吸收处于平衡状态。在病理条件下这种平衡被打破,导致骨骼结构和功能的异常,从而有相应的临床表现如假体周围骨丢失。重建是一个复杂的过程,受到各种细胞因子、激素及信号通路的调控,OB和OC之间的相互调节有助于骨重建过程中骨吸收和骨形成两过程之间的偶联。
核因子-K B受体活化因子(RANK, receptor activator of nuclear factor-κB)属于I型跨膜蛋白,是TNF家族成员之一。RANK高度表达于破骨前体细胞的表面。核因子-K B受体活化因子配体(RANKL, receptor activator of nuclear factor-κ B ligand)结合到破骨前体细胞和成熟OC表面表达的RANK上,促进OC的分化活化并抑制其凋亡。骨保护素(OPG, osteoprotegerin)作为一种诱骗受体,可以竞争性的与RANK结合,从而封闭RANKL与OC表面的RANK结合,抑制OC的分化成熟。RANKL/OPG的比例的变化对于OC产生至关重要,一般来说,当RANKL/OPG的比例上调OC的数量和活性将增加,RANKL/OPG的比例下降时OC的数量和活性将降低。总之,正常的骨重建和骨量的稳定依赖于OPG和RANKL这间的平衡。
TNF-α是炎性细胞释放的一种重要的炎性介质,它能刺激炎性细胞分泌IL-1和IL-6,而这两者又能引起巨噬细胞和破骨细胞的趋化作用,向界膜内聚集。TNF-α还能刺激破骨细胞释放前列腺素E(PGE),引起破骨细胞性骨吸收。近年有研究表明TNF-α能促进人成骨细胞骨保护素配体(OPGL)的表达,而骨保护素配体能促进破骨细胞分化成熟并增强其功能活性,从而进一步引起假体松动发生。
细胞凋亡(apoptosis)是由基因控制的一种细胞的生理性、程序性死亡。当刺激因素作用于细胞后,通过启动相关基因转录及蛋白质合成而导致细胞内各种蛋白酶及内源性核酸内切酶等的活化,从而引起细胞的一系列具有特征性的生化及形态学改变。近年来研究表明,无菌性松动的假体周围界膜组织中存在单核巨噬细胞凋亡的现象。单核巨噬细胞的凋亡会导致更多单核巨噬细胞的聚集由此引起级联反应导致假体周围骨丢失。
金属颗粒和金属离子均能诱导假体周围骨溶解。但金属颗粒与金属离子在无菌性松动及骨溶解中活性比较至今无相关研究。本课题旨在通过体外试验研究,对金属离子与金属颗粒在假体无菌性松动中的活性进行比较,为临床骨科材料的选取提供参考。
目的
1、通过真空球磨法制备符合实验要求的金属颗粒及金属离子。
2、观察金属磨损颗粒对破骨细胞体外分化的影响。
3、观察金属离子对小鼠成骨细胞骨保护素及核因子-κ B受体活化因子配体表
达的影响。
4、金属离子与金属颗粒对单核巨噬细胞影响的活性比较。
材料与方法
1、真空球磨法制备金属颗粒及其物理、生物学活性检测
通过真空球磨法体外制备常用人工关节金属磨损颗粒,同时对磨损颗粒进行元素分析、扫描电镜观察、激光粒度仪检测、并与RAw264.7细胞共培养检测细胞毒性。
2、观察金属磨损颗粒对破骨细胞体外分化的影响
体外培养小鼠破骨前体细胞(RAw264.7),分别用高浓度(10mg/ml)、中浓度(1.0mg/ml)、低浓度(0.1mg/ml)金属磨损颗粒悬液进行干预。RANKL诱导培养7天后,通过抗酒石酸酸性磷酸酶(TRAP)染色观察TRAP阳性多核细胞形成的变化并进行计数。
3、观察金属离子对小鼠成骨细胞骨保护素及核因子-K B受体活化因子配体表达的影响。
体外培养小鼠成骨细胞MC3T3E1根据培养基不同分为两组,A实验组使用含10mg/LCoCl2及150mg/L CrCl3溶液的培养基,B对照组使用不含金属离子的培养基。RT-PCR检测各组RANKL、OPG mRNA的表达；ELISA法检测上清液RANKL、 OPG蛋白的表达。计算RANKL/OPG mRNA和蛋白表达比值变化。
4、金属离子与金属颗粒对单核巨噬细胞影响的活性比较
从30名健康志愿者分别抽取5mL外周血；离心后吸取单个核细胞,将细胞进行体外培养。实验分九组：(1)Fe3＋组、(2)Ti3＋组、(3)Cr3＋组、(4)Co2＋组、(5)铁颗粒组、(6)铬颗粒组、(7)钴颗粒组、(8)钛颗粒组、(9)生理盐水组；用Fe3+、Ti3+、Cr3+、Co2+、铁颗粒、钛颗粒、铬颗粒、钴颗粒分别进行干预,空白组加入生理盐水作为对照。分别在6h、12h、24h、36h、48h用MTT方法检测九组细胞活性；在6h、12h、24h、36h、48h运用ELISA试剂盒检测九组细胞上清液中TNF-α的表达；流式细胞仪检测细胞凋亡情况。
结果
1、通过真空球磨法所取得金属颗粒的中位径2.193μm,体积平均径3.108μm；面积平均径2.119μ m；98%的颗粒直径在6.167μ m以下。与人工关节界膜组织内提取的颗粒直径相仿,且生物活性相仿,在试验浓度(通常低于lmg/ml)没有显示细胞毒性。
2、培养7d后,在200×视野下观察破骨细胞计数,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对照组(P<0.05),中、高浓度组破骨细胞数均显著高于低浓度组(P<0.05),高浓度组破骨细胞数亦显著高于中浓度组(P<0.05)。
3、随时间增加,实验组RANKL、 OPGmRNA表达量明显增加,培养24、48h与对照组比较,差异有统计学意义(P<0.05)。实验组组内培养48h时RANKL/OPG mRNA比值较24h明显升高,差异有统计学意义(P<0.05)；对照组则轻度下降,差异无统计学意义(P>0.05)。培养24、48h时,实验组RANKL蛋白表达量较对照组明显增多分别为对照组的61.6倍及13.8倍,差异有统计学意义(P<0.05)：OPG蛋白表达量较对照组增长32.1%及17.8%,差异有统计学意义(P<0.05)。培养24、48h实验组RANKL/OPG mRNA的比值分别为0.860、1.232,较对照组0.695、0.688明显增加,差异有统计学意义(P<0.05)
4、(1)单核细胞与金属颗粒共培养6h、12h、24h、36h、48h后TNF-α水平。经统计学分析24h、36h各颗粒组TNF-α量高于对照组(p<0.05)；
(2)钴、铬颗粒诱导细胞分泌TNF-α作用比铁、钛颗粒更强(p<0.05),钴离子诱导细胞分泌TNF-α作用较铬、铁、钛离子更强(p<0.05)；钴、铬离子组肿瘤坏死因子水平于24h到达峰值,36h又下降。
(3)各实验组金属颗粒均能诱导人单核细胞产生凋亡,且随时间发展细胞凋亡率逐渐升高,经统计学分析钴、铬、铁颗粒每个时间点实验组细胞凋亡率均高于空白对照组(p<0.05)；
(4)金属离子能诱导人单核细胞产生凋亡,且随时间发展细胞凋亡率逐渐升高,经统计学分析钴、铬诱导细胞凋亡作用强于铁、钛颗粒；铁、钴离子诱导细胞凋亡作用强于铬、钛颗粒；
(5)钛颗粒和钛离子组诱导人单核细胞分泌TNF-α与细胞凋亡明显低于铁、铬、钴颗粒和离子组；
结论
1、实验证实真空球磨法体外制备人工关节金属磨损颗粒简单方便,能大量制备人工关节金属磨损颗粒可重复性好,可用于相关体外实验。
2、RAW264.7是一种较好的破骨前体细胞模型。RANKL诱导RAW264.7形成破骨细胞方法简便易行、可重复性好。传代超过20代的RAW264.7仍然保持了在RANKL的诱导下分化成为破骨细胞的能力。金属磨损颗粒和RAW264.7细胞共培养后,可以促进RANKL诱导RAW264.7形成破骨细胞的数量增加。
3、Co2+、Cr3+共同作用可刺激成骨细胞RANKL、OPGmRNA表达的增加,以及RANKL/OPG mRNA比值的增加,并显著促进RANKL蛋白的分泌,从而影响破骨细胞前体细胞向破骨细胞分化与成熟,促进破骨细胞骨性吸收。
4、金属颗粒与金属离子能诱导人单核细胞凋亡,随时间细胞凋亡率逐步升高；金属颗粒与金属离子能通过刺激单核细胞释放TNF-α引发假体周围炎性反应,并且通过诱发单核细胞凋亡参与这一病理过程从而导致假体松动；不同的金属材料金属颗粒及离子生物学活性不同,钛金属材料生物学活性明显低于铁、钻、铬材料。钴、铬颗粒生物学活性高于离子：铁颗粒和铁离子生物学活性无明显差异
Background
Joint replacements as an effective strategy in treating bone and joint diseases, provides pleasing results in releasing pain and increase the motion range of joints. However, popularize joint replacements are somehow limited by relatively short lifespan of prosthesis. According to those long-term follow-up data, aseptic loosening plays a key role in this process. Aseptic loosening of the components are induced by biological factors and mechanical factors including material design compatibility and installation of the prosthesis. Fragments of wear debris may cause an inflammatory reaction involving IL1β(interleukin-1β) IL-6(interleukin-6) and TNF-α (TNF-α, tumor necrosis factor-a) with bone absorption which can cause loosening. This phenomenon is known as osteolysis. It is generally believed that the wear particles generated by the mechanical wear of the prosthesis can induce periprosthetic tissue cells to produce a series of biological reactions activating the osteoclasts causing periprosthetic osteolysis which is the main reason for long-term loosening of the artificial joints. With the technology development of material selection and process design wear and tear caused by mechanical loosening has been well controlled.
The femoral prosthesis is commonly made of metal which is widely used in total hip arthroplasty because of its appropeiate stiffness and rigidity. However, osteolysis surrounding the host bone following metal prosthesis implantation made its application controversial. Osteolcast (OC) plays a significant role in osteolysis surrounding prosthesis. Numerous researches have evidenced the metal ion, Mainaly cobalt and chrome, located surrounding the femoral prosthesis.
Bone is an active metabolic organ. Bone remodeling is important for regulating and maintaining skeletal integrity. In this process, OC absorbed the old bones meanwhile osteoblasts (OB, osteoblast) form the new bone. Therefore, bone reconstruction need these two processes precisely coordinate with each other. In the bone marrow microenvironment osteoclast differentiation of progenitor cells into mature OC, and bone resorption.Number of OB and OC under normal conditions and by a variety of factors and signaling to maintain stable levels, so that bone formation and bone resorption mediated by them in a State of balance.This balance is broken in pathological conditions, resulting in abnormal structure and function of bones, thus the corresponding clinical manifestations such as periprosthetic bone loss.Redevelopment is a complex process, subject to various cytokines, hormones and signaling pathways of regulation, mutual regulation between the OB and OC help bone resorption and bone formation in the bone remodeling process coupling between the two processes.
RANK (RANK, Nuclear factor-B receptor activation factor) is type I transmembrane protein which belongs to TNF family. RANK highly expresse on the surface of osteoclast precursor. Nuclear factor Kb ligand receptor activation factor (RANKL, receptor activator of nuclear factor-K b ligand) binding to RANK not only promoting the differentiation of OC but also inhibiting the apoptosis of OC. OPG (OPG, osteoprotegerin) secreted by osteoblast can competitive binding to RANK against RANKL. RANKL/OPG rate is essential for OC. Ehance in the proportion of RANKL/OPG lead to the increase in the number of OC and its activity. Decrease in the proportion of RANKL/OPG will reduce the number of OC and its activity. In short, the normal bone remodeling and bone mass stability depends on the rate of RANKL/OPG.
TNF-a is a kind of important inflammatory mediators which can can stimulate the inflammatory cells secrete IL-land IL-6lead to the macrophages and osteoclasts gathering at the place where inflammatory reaction take place. TNF-a can also stimulate osteoclast release PGE (PGE, prostaglandinE) which can induce osteoclastic bone resorption. In recent years, research has shown that TNF-α can promote human osteoblasts of osteoprotegerin ligand (OPGL) expression and osteoprotegerin physical distribution activity of osteoclast differentiation and maturation and increase its functionality, which further lead to implant loosening.
Apoptosis is the process of programmed cell death controlled by genes. Stimulunts affect cells by initiating gene transcription and protein synthesis causing intracellular protease and activating endogenous DNA endonuclease. In recent years, studies have shown that around the aseptic loosening prosthesis exists the phenomenon of mononuclear macrophage apoptosis. Apoptosis of mononuclear macrophage will lead to more gathering of mononuclear macrophage causing a cascade reaction resulted in periprosthetic bone loss.
Both metal particles and metal ions can induce Periprosthetic osteolysis leading to the loosening of prosthesis. But there is no relevant research comparing the effect between metal particles and metal ions on aseptic loosening and osteolysis. This study aim at compare the effect between metal particles and metal ions on aseptic loosening and osteolysis through vitro experiment providing reference for the choice of prosthesis materials.
Purpose
1. Prepare metal debris and metal ions which meet the requirements of the experiment.
2. Observe the effect of metal debris on the differentiation of osteoclast in vitro.
3. Observe the effect of metal ions on the expression RANKL and OPG secreted by osteoblast.
4. Compare the biological activity difference between metal ions and metal debris on mononuclear macrophage.
Method
1. Prepare metal debris through the vacuum ball milling method. Analyse the elements of the metal debris. Observe its character through electron microscope. Measure its diameter by laser particle size analyzer. Detect its cytotoxicity by coculture with RAW264.7cell line.
2. Treating RAW264.7cells which induced by RANKL with high concentration (10mg/ml) medium concentration (1.0mg/ml) low concentration (0.1mg/ml) metal debris respectively. Take count of TRAP (TRAP, tartrate-resistant acid phosphatase) stain positive multinucleate cells after7days culturing.
3. In vitro culture of MC3T3-E1cell line. Divide it into two groups according to its medium. The experimental group including10mg/L C0Cl2and150mg/L CrC13in the culture medium defined as group A. The control group with normal culture medium defined as group B. Detect RANKL and OPG mRNA by RT-PCR. Detect RANKL and OPG protein by ELASIA.
4. Mononuclear cells obtained from peripheral blood of30healthy volunteers. Coculture mononuclear cells with iron particles cobalt particles chromium particles titanium particles Iron ions chromium ions cobalt ions titanium ions and physiological saline respectively. Assess activity of the cells by MTT at6h12h24h36h48h respectively. Assess the expression of TNF-α protein by ELASIA at6h12h24h36h48h respectively.
Results
1. Median diameter volume mean diameter and surface mean diameter of metal particles obtained by vacuum ball milling is2.193μm3.018μm and2.119μm respectively. Diameter of98%particles is less than6.167μm. The diameter and biological activity in our experiment is in common with particles extracted from artificial joint interface tissue.In the testing concentration, typically less than1mg/ml, showed no cytotoxicity.
2. The results show that as the wear particles suspension concentration increased RANKL induces formation of osteoclast cells increased. A number of osteoclast in the high concentration medium concentration low concentration group was higher than that in control group (P<0.05). A number of osteoclast in the high concentration medium concentration group was higher than that in low concentration group (P<0.05). A number of osteoclast in the high concentration group was higher than that in medium concentration group (P<0.05).
3. The expression of RANKL OPG mRNA was increased with time in the experimental group. The expression of RANKL OPG mRNA was higher than that in the control group at2448h. Rate of RANKL/OPG mRNA is0.8601.232at2448h in the experimental group higher than that0.6950.688in the control group(P<0.05). Rate of RANKL/OPG protein at2448h in the experimental group was higher than that in the control group (P<0.05)
4. The expression of TNF-a was higher in iron particles cobalt particles chromium particles titanium particles group at2436h than that in the control group (P<0.05). The expression of TNF-a was higher in cobalt particles chromium particles group than that in the iron particles titanium particles group (P<0.05). The expression of TNF-a was higher in cobalt ions group than that in the iron ions chromium ions titanium ions group. Iron particles cobalt particles chromium particles titanium particles can induce mononuclear cells to apoptpsis. Iron ions chromium ions cobalt ions titanium ions can induce mononuclear cells to apoptpsis. The ability of inducing mononuclear cells to apoptosis for cobalt particles chromium particles was greater than iron particles titanium particles. The ability of inducing mononuclear cells to apoptosis for iron ions cobalt ions was greater than chromium ions titanium ions.
Conclusion
1. Metal debris prepared by vacuum ball milling method not only simple and convenient but also has good reproducibility and productive thus can be used for vitro experiment.
2. RAW264.7is a good osteoclast precursor cells model. RANKL induced RAW264.7to form osteoclasts is simple also has good repeatability. The number of osteoclase has increased after coculture with metal debris.
3. Co2+, Cr3+interaction can stimulate osteogenesis cells increase expression of RANKL and OPGmRNA as well as the increase in the ratio of RANKL/OPG mRNA and greatly promote the secretion of the protein RANKL thus affecting the osteoclast precursor cells differentiate into osteoclasts and enhance the ability of bone absorption.
4. Iron particles cobalt particles chromium particles titanium particles Iron ions chromium ions cobalt ions titanium ions can induce human mononuclear cells to apoptosis and its rate gradually increased over time.Metal particles and metal ions through stimulating mononuclear cells causing inflammatory reaction around the prosthesis and by inducing apoptosis of mononuclear cells lead to implant loosening. The biological activity of titanium was significantly lower than iron cobalt and chromium.
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