钛颗粒负荷对成骨细胞释放细胞因子的影响

假体周围的溶骨性因子是造成无菌性松动的主要原因之一。前期的研究表明,磨损颗粒抑制成骨细胞分化和矿化能力,为探索不同直径的磨损颗粒对成骨细胞释放细胞因子的影响,我们采用双夹心抗体ELISA、RT-PCR法分析3种直径钛颗粒对兔成骨细胞表达IL-6I、L-10和破骨细胞分化因子(ODF)的影响。结果显示:0.9μm钛颗粒刺激成骨细胞产生大量促骨吸收因子(IL-6、ODF)的同时快速而短暂地释放抑骨吸收因子(IL-10);2.7μm和6.9μm钛颗粒,尤其是后者主要是刺激成骨细胞缓慢而强烈地产生促骨吸收因子。提示:磨屑颗粒作用成骨细胞的生物学反应是双相的,且反应程度因颗粒大小和作用时间的不同而又有所不同。可见如何抑制假体周围溶骨性因子的产生以及人工材料的优化选择是提高人工假体寿命的关键。     

钛颗粒负荷对成骨细胞白细胞介素6生成的影响

磨损碎屑颗粒所诱发的无菌性松动是人工关节置换术后最常见的并发症.研究表明,假体-骨界面的骨组织细胞吞噬磨屑颗粒后,以自分泌或旁分泌的方式释放细胞间介质因子,而引起假体旁骨改建紊乱,导致假体松动.为了探明不同直径的磨屑颗粒对细胞释放细胞间介质因子的影响,本研究采用3种不同直径(mean sizeφ0.9 μm、φ2.7μm、φ6.9 μm)的钛颗粒,在浓度分别为0.25wt%,0.1wt%,0.05wt%下与兔成骨细胞共孵育4、8、12、24、36、48 h后,检测各组细胞白介素6(IL-6)分泌的变化情况.结果表明,3种钛颗粒均能刺激成骨细胞分泌IL-6因子,但其上调作用因不同直径,不同负荷浓度,不同负荷时间而有所不同.随着浓度增加和时间的推移,φ2.7μm和φ6.9 μm钛颗粒对IL-6生成的上调作用持续升高,而φ 0.9 μm钛颗粒的上调作用却呈先升高后快速下降的双相变化过程.结果提示磨屑颗粒能明显刺激细胞释放细胞间介质因子IL-6,而颗粒直径大小不同,上调IL-6的程度有所不同,使得细胞间介质因子影响假体旁骨改建紊乱的机制有所不同,这种由细胞因子介导的信号传递机制若进一步深入研究必将有助于人工关节材质的优化选择及临床预防用药指导.     

钛颗粒负荷对切应力作用下骨髓间质干细胞F-actin表达和DNA含量的影响

假体磨损碎屑颗粒是引起假体一骨界面无菌性炎症和骨溶解而致全关节置换术失败的主要原因之一。磨屑颗粒所诱发的骨溶解须有周围骨组织中成骨细胞分泌足够的骨基质以弥补丢失的骨量，而成骨细胞正常的数量和质量有赖于其来源骨髓祖细胞—骨髓问质干细胞的正常增殖分化能力的维持。为了探讨磨屑钛颗粒对大鼠骨髓间质干细胞(Rat MSCs，rMSCs)产生细胞毒性的可能细胞分子机制，选用健康3月龄SD雄性大鼠，采用Percoll等密度梯度离心法分离获取rMSCs，经体外传代纯化培养后，与不同直径、不同负荷浓度、不同负荷作用时间的钛颗粒悬液共孵育，再采用精准的流室系统对钛颗粒负荷的rMSCs施加一定的流体剪切应力(Fluid shear stress，FSS)后立刻固定细胞，经免疫荧光抗体染色，结合激光共聚焦显微镜技术和图像分析软件定性定量分析rMSCs F—actin表达和DNA含量的变化情况。同时设置相应的未经钛颗粒孵育的rMSCs细胞为对照组细胞。结果显示，切应力作用可上调rMSCs胞内F—actin的表达。亚微(Submieron)直径(0．9μm)的钛颗粒负荷对rMSCs F—actin表达和DNA含量的抑制作用最为显著，并伴有凋亡小体出现；直径为2．7μm的钛颗粒负荷产生的抑制作用略为减弱，而较大直径(6．9μm)的抑制效应最弱。相同条件下，钛颗粒负荷对F—actin的抑制效应有一定的时间和浓度依赖性：以0．1wt％浓度对F—actin表达和DNA含量的抑制效应最为明显，亦有凋亡小体的出现；随着浓度的降低，抑制作用亦减弱，以0．01wt％浓度最弱；随着作用时间的延长，F—actin表达和DNA含量逐渐降低，至实验中的32h达到最低值。提示：磨屑颗粒对rMSCs活力的抑制作用是假体无菌性松动的可能分子机制，对其具体细胞分子机制进行深入研究，必将有助于有效防治假体松动药物的研发应用以及人工关节材料的优化设计，从而为全关节置换术患者真正带来福音。     

磨损颗粒与人工关节无菌性松动

人工关节无菌性松动是影响人工关节长期使用和妨碍人工关节发展的最重要的并发症。许多研究表明,人工关节无菌性松动与假体磨损有着密切关系。磨损颗粒的产生及其所诱发的一系列生物反应是导致假体周围骨溶解及假体无菌性松动的重要因素,其中巨噬细胞、成纤维细胞释放的骨吸收介质和成骨细胞的功能抑制可能是假体无菌性松动发生的重要机制。本文就近年来有关磨损颗粒与人工关节无菌性松动机制的一些研究进展进行综述     

磨损颗粒与人工关节无菌性松动

人工关节无菌性松动是影响人工关节长期使用和妨碍人工关节发展的最重要的并发症。许多研究表明,人工关节无菌性松动与假体磨损有着密切关系。员颗粒的产生及其所诱发的一系列生物反应是导致假体周围骨溶解及本无菌性松动的重要因素,其中巨噬细胞,成纤维细胞释放的骨吸收介质和成骨的功能抑制可能是假体无菌性松动性的重要机制。本语文就近年来有关磨损颗粒与人工关节无菌性松动机制的一些研究进展进行综述。     

CRTER杂志“骨科植入物研究”栏目关于“脊柱外科植入物”的热点选题

<正>○RANK/RANKL在假体松动中的分子机制○TFE抑制TNF-α分泌的机制以及关节假体无菌性松动防治○假体松动应力遮挡引起的骨质重新塑形○钛颗粒刺激巨噬细胞而释放的肿瘤坏死因     

CRTER杂志“骨科植入物研究”栏目关于“假体松动作用机制”的热点选题

<正>○RANK/RANKL在假体松动中的分子机制○TFE抑制TNF-α分泌的机制以及关节假体无菌性松动防治○假体松动应力遮挡引起的骨质重新塑形○钛颗粒刺激巨噬细胞而释放的肿瘤坏死因子对人工关节松动的作用     

人工关节磨屑的提取及表征

背景：磨屑引起生物学反应从而导致人工关节的无菌性松动是假体失效的主要原因,因此获取人工关节磨屑研究其生物学反应对提高人工关节寿命有重要意义。 目的：获取纳米级人工关节磨损磨屑,进一步研究生物学反应。 方法：通过酸消化法和酶降解法获取人工关节磨屑,比较其分离效果,并获取不同尺寸的粒子,观察纳米粒子的形态。 结论与结论：①全髋关节模拟试验机运转循环次数对超高相对分子质量聚乙烯磨屑大小、形貌有一定的影响。②磨屑粒径分布范围很广,大到100 μm以上,小到小于1 μm。③从消化方法上来看,酶消化和酸消化法分离超高相对分子质量聚乙烯磨屑有效可行。     

磨损颗粒引起人工关节无菌性松动的研究与进展

背景：研究显示磨损颗粒诱导的假体周围骨溶解是导致人工假体无菌性松动的最主要原因。 目的：对磨损颗粒引起人工关节假体无菌性松动在分子生物学方面的研究现状及新进展作一综述。 方法：应用计算机检索CNKI和Pubmed数据库中1991年1月至2012年3月关于人工关节磨损颗粒的文章,在标题和摘要中以“关节置换,磨损颗粒,假体松动”或“arthroplasty,wear particles,loosening of the prosthesis”为检索词进行检索。最终选择30篇文献进行综述。 结果与结论：假体无菌性松动是人工关节置换后手术失败的最主要原因,在此过程中磨损颗粒起到关键性作用。通过对细胞因子及信号传导途径研究的综述,为预防及药物治疗假体无菌性松动提供理论依据。然而,假体的无菌性松动受多种因素的影响,所以预防应从整体着眼,全面深入研究,为假体的松动防治提供方向。     

滚动式人工膝关节设计

针对人工关节中超高分子量聚乙烯(UHMWPE)磨粒导致的骨吸收、骨溶解及由此引起的假体远期松动问题,提出一种滚动式膝关节假体设计。这种膝关节假体利用金属—陶瓷滚动摩擦副代替UHMWPE-金属滑动摩擦副,通过降低膝关节伸屈运动时的摩擦阻力和假体—骨界面间的应力,从而减少磨屑产生,降低假体远期松动的可能性。     

Stimulative and regulative functions of osteoblasts loaded under the titanium particles on osteoclasts

Our previous studies on the function of the osteoblasts (OBs) have shown that worn titanium particles decrease osteoblast function and promot secretion of bone resorption cytokines of OBs surrounding the synovium-like interface membrane of loosening implants. The current study was aimed to test the hypothesis that osteoclasts (OCs) bone absorption function is induced by conditioned media (CM) prepared from OBs loaded in the presence or absence of titanium particles (with three mean diameters 6.9 microm, 2.7 microm, and 0.9 microm, respectively). The effects of CM on OCs function were examined using a combination of the morphological characteristics tests, i.e., TRAP dyeing, scanning electron microscopy, F-actin immunofluorescence protocol for confocal microscopy, bone resorption lacunae assay, osteoclastic calcium tracking, with biochemical evaluation, i.e., C-terminal cross-linked telopeptides of type I collagen evaluated with ABC-ELISA method. The results showed that CM from 0.9 microm titanium particles could induce osteoclastic differentiation and formation, could partially influence the survival of the OCs; while CM of 2.7 microm and 6.9 microm titanium particles, especially the latter, could obviously augmented osteoclastic activity, survival, or differentiation. The stimulation of osteoclast function may be due to a parallel increase in the intracellular free calcium concentration. The present study provides strong support for the hypothesis that osteoclastic activity, survival, or differentiation are very important in the development of aseptic loosening. The development of therapeutic interventions to reduce osteoclastic function and optimization of biomaterials may be useful approaches for improving the performance of orthopaedic implants.     

Hydroxy apatite microspheres enhance gap junctional intercellular communication of human osteoblasts composed of connexin 43 and 45

The aseptic loosening of artificial joints with associated periprosthetic bone resorption may be partly due to the suppression of osteoblast function to form new bone by wear debris from the joint. To assess the effect of wear debris on osteoblasts, effects of model wear debris on gap junctional intercellular communication (GJIC) of normal human osteoblasts were estimated. The GJIC activity of the osteoblasts after a 1-day incubation with the microspheres was similar to that of normal osteoblasts. However, hydroxy apatite particles, which have been reported to enhance the differentiation of osteoblasts in contact with them, enhanced the GJIC function of the osteoblasts. From RT-PCR studies, not only connexin 43 but also connexin 45 is suggested to play a role in the GJIC of the osteoblasts in an early stage of coculture with the microspheres, although it is still unclear how these connexins work and are regulated in the GJIC and differentiation. However, this study suggests that there is a relationship between the early levels of GJIC and the differentiation of the cells. Therefore, estimating the effect of biomaterials, even in the microsphere form, on the GJIC of model cells, with which the biomaterials may be in contact in vivo, can provide important information about their biocompatibility.     

Phagocytosis of wear debris by osteoblasts affects differentiation and local factor production in a manner dependent on particle composition

Wear debris is considered to be one of the main factors responsible for aseptic loosening of orthopaedic endoprostheses. Whereas the response of cells in the monocytic lineage to foreign materials has been extensively studied, little is known about cells at the bone formation site. In the present study, we examined the hypothesis that the response of osteoblasts to wear debris depends on the chemical composition of the particles. We produced particles from commercially pure titanium (cpTi), Ti-6Al-4V (Ti-A), and cobalt-chrome (CoCr) and obtained ultrahigh molecular weight polyethylene (UHMWPE; GUR 4150) particles from a commercial source. The equivalent circle diameters of the particles were comparable: 1.0 +/- 0.96 microm for UHMWPE; 0.84 +/- 0.12 microm for cpTi; 1.35 +/- 0.09 microm for Ti-A, and 1.21 +/- 0.16 microm for CoCr. Confluent primary human osteoblasts and MG63 osteoblast-like cells were incubated in the presence of particles for 24 h. Harvested cultures were examined by transmission electron microscopy to determine if the cells had phagocytosed the particles. Particles were found intracellularly, primarily in the cytosol, in both the primary osteoblasts and MG63 cells. The chemical composition of the particles inside the cells was confirmed by energy-dispersive X-ray analysis. Morphologically, both cell types had extensive ruffled cell membranes, less-developed endoplasmic reticulum, swollen mitochondria, and vacuolic inclusions compared with untreated cells. CpTi, Ti-A, and CoCr particles were also added to cultures of MG63 cells to assess their effect on proliferation (cell number) and differentiation (alkaline phosphatase activity), and PGE2 production. All three types of particles had effects on the cells. The effect on cell number was dependent on the chemical composition of the particles; Ti-A and CoCr caused a dose-dependent increase, while cpTi particles had a biphasic effect with a maximal increase in cell number observed at the 1:10 dilution. Alkaline phosphatase specific activity was also affected and cpTi was more inhibitory than Ti-A or CoCr. PGE2 production was increased by all particles, but the magnitude of the effect was particle-dependent: CoCr > cpTi > Ti-A. This study demonstrates clearly that human osteoblast-like cells and MG63 cells can phagocytose small UHMWPE, CoCr, Ti-A, and cpTi particles. Phagocytosis of the particles is correlated with changes in morphology, and analysis of MG63 response shows that cell proliferation, differentiation, and prostanoid production are affected. This may have negative effects on bone formation adjacent to an orthopaedic implant and may initiate or contribute to the cellular events that cause aseptic loosening by inhibiting bone formation. The effects on alkaline phosphatase and PGE2 release are dependent on the chemical composition of the particles, suggesting that both the type and concentration of wear debris at an implant site may be important in determining clinical outcome.     

Abriebpartikel

The aseptic prosthetic loosening of hip and knee prosthesis is the most important cause of implant insufficiency. Bone loss as a result of the biological effect of wear particles is the main cause of such loosening. Wear particles develop their biological activity along different cellular pathways, above all via macrophages, foreign body giant cells as well as fibroblasts of the periprosthetic membrane. These cells induce particle-dependent bone resorption by means of proinflammatory cytokines, such as IL-1beta, TNF-alpha, IL-6 and PGE2. These factors induce the activation of osteoclasts as well as the suppression of osteoblasts. Neutrophil granulocytes and lymphocytes do not play an important role in the process of aseptic loosening. The different wear particles, such as ultra-high molecular weight polyethylene, metal particles, ceramic particles and polymethylmethacrylate can be morphologically recognized very easily. From the clinical point of view, the differentiation between acute or chronic implant infection and particle induced prosthetic loosening is very important, with the histomorphological differential diagnosis between septic and aseptic loosening and their combination being the key clinicopathological factor.     

The potential role of human osteoblasts for periprosthetic osteolysis following exposure to wear particles

Aseptic loosening in total hip replacement is mainly caused by wear particles inducing inflammation and osteolysis. Wear can be a consequence of micromotions at the interface between implant and bone cement. Due to complex cellular interactions, different mediators (e.g. cytokines, proteinases) are released, which can promote osteolytic processes in the periprosthetic tissue followed by loosening of the implant. Furthermore, a reduced matrix synthesis and an induced apoptosis rate can be observed. The purpose of this study was to evaluate to what extent human primary osteoblasts exposed to wear particles are involved in the osteolysis. The viability, the secretion of collagen and collagenases and the variety of released cytokines after particle exposure was examined. Therefore, human osteoblasts were incubated with particles experimentally generated in the interface between hip stems with rough and smooth surface finishings as well as different material compositions (Ti-6Al-7Nb, Co-28Cr-6Mo and 316L) and bone cement mantle made of Palacos R containing zirconium oxide particles. Commercially pure titanium particles, titanium oxide, polymethylmethacrylate and particulate zirconium oxide were used as references. The results revealed distinct effects on the cytokine release of human osteoblasts towards particulate debris. Thereby, human osteoblasts released increased levels of interleukine (IL)-6 and IL-8 after treatment with metallic wear particles. The expression of VEGF was slightly induced by all particle entities at lower concentrations. Apoptotic rates were enhanced for osteoblasts exposed to all the tested particles. Furthermore, the de novo synthesis of type 1 collagen was reduced and the expression of the matrix metalloproteinase (MMP)-1 was considerably increased. However, wear particles of Co-28Cr-6Mo stems seemed to be more aggressive, whereas particles derived from stainless steel stems caused less adverse cellular reaction. Among the reference particles, which caused less altered reactions in the metabolism of osteoblasts in general, ZrO2 can be assumed as the material with the smallest cell biological effects.     

Effect of size and dose on bone resorption activity of macrophages by in vitro clinically relevant ultra high molecular weight polyethylene particles

Polyethylene wear debris generated at the bearing surfaces of total artificial hip joints is thought to play an important role in the periprosthetic osteolysis and ultimately the aseptic loosening of these prostheses. The macrophage is believed to be central to this process by releasing various cytokines and other mediators of osteolysis upon phagocytosis of the polyethylene wear debris. This study evaluated the in vitro bone resorption response of C3H murine peritoneal macrophages to clinically relevant GUR 1120 polyethylene particles. Macrophages were co-cultured in vitro with GUR 1120 particles with a mean size of 0.24, 0.45, 1.71, and 7.62, and GUR 1120 polyethylene resin with a mean size of 88 microm at various particle volume (microm)(3): macrophage ratios (0.1:1; 1:1; 10:1; and 100:1). The conditioned supernatants were incubated with (45)calcium radio-labeled mouse calvariae, and bone resorption was measured as (45)calcium release. The results showed that the 0.24 microm particles stimulated the macrophages to generate bone resorbing activity at a ratio of 10(microm)(3) per macrophage. The 0.45 and 1.71 microm particles were active at a ratio of 100( microm)(3) per macrophage, and the 7.62 and 88 microm particles were inactive at all the doses tested. The co-culture supernatants were also assayed for TNF-alpha, IL-1beta, IL-6, and PGE(2). The results followed the same trend for particle size and volume dose to that observed for the bone resorbing activity. This study has demonstrated, for the first time, the importance of size and dose of clinically relevant polyethylene particles on the osteolytic response of macrophages in vitro.     

Quantitative analysis of ultrahigh molecular weight polyethylene (UHMWPE) wear debris associated with total knee replacements

The size and morphology of particulate wear debris retrieved from tissues around 18 failed total knee replacements (TKR) were characterized. Interfacial membranes from nine cemented and nine uncemented TKR were harvested from below the tibial components during revision surgery. Wear debris were extracted using papain and potassium hydroxide digestion. Ultrahigh molecular weight polyethylene (UHMWPE) particles from around cemented or uncemented TKR were similar in size and morphology. The mean size was 1.7 +/- 0. 7 microm with a range of 0.1-18 microm. Thirty-six percent of the particles were less than 1 microm and 90% were less than 3 microm. Morphologically the particles were predominantly spherical with occasional fibrillar attachments and flakes. Particles from TKR were greater than threefold larger than previously characterized particles from total hip replacements, which were 0.5 microm in mean size. Differences in joint conformity and wear patterns between the hip and knee articulations may explain the disparity in size of the wear debris. Since particle size represents an important variable influencing the magnitude of the biological response, it is possible that in vivo the larger TKR debris results in a diminished mediator release, which in turn may account for the lower incidence of osteolysis and aseptic loosening in some designs of TKR.     

Fabrication of submicron titanium-alloy particles for biological response studies.

Biologic response to generated wear particles and subsequent aseptic loosening is a critical factor limiting the long-term survival of total hip replacements. To better understand the sequence of events leading to aseptic loosening and the role of the individual material components, fabricating metal particles similar to those present clinically is very important. We describe a simple milling technique to generate significant amounts of fine titanium-alloy (TiAlV) debris. A TiAlV rod was milled against a TiAlV plate in distilled water supplemented with antibiotics. The resulting debris were sedimented in alcohol and the fine debris were separated. Scanning electron microscopy analysis and particle size analysis demonstrated that the mean size of particles was 1.1 +/- 0.9 microm (range 0.2-4.2 microm). Sixty-two percent were smaller than 1.0 microm, and 85% were smaller than 2.0 microm. The particles generated had varying shapes, including angular or shard-like shapes with jagged and irregular outlines.     

Histomorphometric analysis of the intramedullary bone response to titanium particles in wild-type and IL-1R1 knock-out mice: a preliminary study

Aseptic loosening of implants following total joint arthroplasty remains a major cause of implant failure. Particulate debris generated primarily from wear results in inflammatory mediated periprosthetic osteolysis. Titanium is a commonly utilized metal in joint arthroplasty and titanium debris induces the production of the pro-inflammatory cytokine IL-1. To further elucidate the role of IL-1, this study examined the response of murine femora to the presence of titanium particles following implantation of an intramedullary rod in mice lacking the receptor for IL-1. We hypothesized that the inflammatory effects of wear debris on bone would be mitigated in IL-1R1 deficient mice with a resultant decrease in resorption. Femora receiving titanium particles demonstrated a marked inflammatory response in wild-type mice with increased endocortical resorption, periprosthetic membrane formation, and significant histomorphometric changes. Femora exposed to titanium particles in the knockout mice also demonstrated osteolysis with irregular deposition of trabecular bone and increased cortical porosity. The persistence of inflammation and osteolysis, despite the lack of functional IL-1R1, suggests a multi-factorial role for IL-1 in the proinflammatory cascade resulting from wear debris. This intramedullary murine model provides the ability to evaluate and quantify the proinflammatory cascade in an in vivo model approximating prosthesis failure.