Mechanical properties and biocompatibility of melt processed,self-reinforced ultrahigh molecular weight polyethylene

The low efficiency of fabrication of ultrahigh molecular weight polyethylene (UHMWPE)-based artificial knee joint implants is a bottleneck problem because of its extremely high melt viscosity. We prepared melt processable UHMWPE (MP-UHMWPE) by addition of 9.8 wt% ultralow molecular weight polyethylene (ULMWPE) as a flow accelerator. More importantly, an intense shear flow was applied during injection molding of MP-UHMWPE, which on one hand, promoted the self-diffusion of UHMWPE chains, thus effectively reducing the structural defects; on the other hand, increased the overall crystallinity and induced the formation of self-reinforcing superstructure, i.e., interlocked shish-kebabs and oriented lamellae. Aside from the good biocompatibility, and the superior fatigue and wear resistance to the compression-molded UHMWPE, the injection-molded MP-UHMWPE exhibits a noteworthy enhancement in tensile properties and impact strength, where the yield strength increases to 46.3 ± 4.4 MPa with an increment of 128.0%, the ultimate tensile strength and Young's modulus rise remarkably up to 65.5 ± 5.0 MPa and 1248.7 ± 45.3 MPa, respectively, and the impact strength reaches 90.6 kJ/m². These results suggested such melt processed and self-reinforced UHMWPE parts hold a great application promise for use of knee joint implants, particularly for younger and more active patients. Our work sets up a new method to fabricate high-performance UHMWPE implants by tailoring the superstructure during thermoplastic processing.     

狂犬病病毒CTN-1v株原子力显微镜观察结果的初步分析

目的 利用原子力显微镜对狂犬病病毒进行观察.方法 超速离心制备狂犬病病毒CTN-1v株,采用磷钨酸负染透射电子显微镜进行观察,在此基础上进行原子力显微镜观察,采用轻敲模式在大气常温下扫描成像.结果 透射电子显微镜观察到狂犬病病毒的典型弹状病毒粒子,透射电镜提供病毒二维图像,可见刺突结构,原子力显微镜则呈现了狂犬病病毒三维图像,且可见病毒表面有凹凸不平的特征和边缘有齿轮状的突起,同时获得表面粗糙度等可以量化指标.两种方法最终得到相似的形态学结果.结论 利用原子力显微镜首次观察到狂犬病病毒的三维形态结构,与透射电镜观察相比,原子力显微镜是一种制样简单、观察直观的新型病毒形态学研究工具.

Abstract：

Objective To explore the application of atomic force microscopy( AFM ) on the research of morphology of the rabies viruses. Methods To prepare the rabies virus CTN-1v strains by ultracentrifugation, and observe it with transmission electron microscopy (TEM) which negatively stained by phosphotungstic acid. Then study the morphology of rabies virus with AFM based on the result of TEM. AFM image applies the tapping mode to rabies virus without any further treatment in air at room temperature. Results The TEM image is two-dimensional image which can be seen the classical bullet-shaped structure,and the spike structure can also be seen. The AFM image showed the rabies virus morphology with three-dimensional image which can shows the characteristics of the virus surface and edge. The rabies virus particle was successfully observed by TEM or AFM methods. Conclusion It's the first time to get the three-dimensional morphological structure of rabies virus by atomic force microscopy, compared with transmission electron microscopy, AFM is a new research tool for viral morphology study with the advantages of simple sample preparing and intuitionistic and visible interface for researchers.     

Molecular weights of individual proteins correlate with molecular volumes measured by atomic force microscopy

 Proteins are usually identified by their molecular weights, and atomic force microscopy (AFM) produces images of single molecules in three dimensions. We have used AFM to measure the molecular volumes of a number of proteins and to determine any correlation with their known molecular weights. We used native proteins (the TATA-binding protein Tbp, a fusion protein of glutathione-S-transferase and the renal potassium channel protein ROMK1, the immunoglobulins IgG and IgM, and the vasodilator-stimulated phosphoprotein VASP) and also denatured proteins (the red blood cell proteins actin, Band 3 and spectrin separated by SDS-gel electrophoresis and isolated from nitrocellulose). Proteins studied had molecular weights between 38 and 900 kDa and were imaged attached to a mica substrate. We found that molecular weight increased with an increasing molecular volume (correlation coefficient = 0.994). Thus, the molecular volumes measured with AFM compare well with the calculated volumes of the individual proteins. The degree of resolution achieved (lateral 5 nm, vertical 0.2 nm) depended upon the firm attachment of the proteins to the mica. This was aided by coating the mica with suitable detergent and by imaging using the AFM tapping mode which minimizes any lateral force applied to the protein. We conclude that single (native and denatured) proteins can be imaged by AFM in three dimensions and identified by their specific molecular volumes. This new approach permits detection of the number of monomers of a homomultimeric protein and study of single proteins under physiological conditions at the molecular level. Received: 14 February 1997 / Received after revision: 8 September 1997 / Accepted: 8 September 1997     

Immunolocalization of lamins and nuclear pore complex proteins by atomic force microscopy

The nuclear envelope functions as a selective barrier separating the nuclear from the cytosolic compartment. Nuclear pore complexes (NPCs) mediate nuclear import and export of macromolecules and, therefore, are potential regulators of gene expression. In this study we applied atomic force microscopy (AFM) to visualize the three dimensional (3D) structure of individual NPCs in the absence and presence of two different antibodies, one directed against a pore protein (gp62) and another directed against Xenopus lamin LIII, a component of the nuclear lamina, a filament meshwork localized on the nucleoplasmic side of the nuclear envelope (NE) adjacent to and interacting with NPCs. Using 12-nm gold-labelled secondary antibodies and transmission electron microscopy we could clearly localize the primary single anti-gp62 antibody on NPCs and the primary single anti-LIII antibody between NPCs. Using AFM, the secondary antibodies against anti-gp62 could be detected as particles 7 nm in height on the nucleoplasmic face of NPCs. The secondary antibodies against anti-LIII could be clearly identified between NPCs. The secondary antibodies, attached to a 12-nm colloidal gold particle and visualized on glass, revealed similar shapes and heights as found on NEs. According to the 3D images, the volume of a single gold particle conjugated with secondary antibodies was 10 203 nm³. This volume is equivalent to the volume of 38 IgG molecules associated with one individual gold particle. A similar volume of 11 987 nm³ was calculated from a model assuming that the 150-kDa IgG molecules perfectly cover the spherical gold particle. We conclude that AFM can be used for identifying antibodies or other macromolecules associated with biomembranes.     

Simultaneous processing of substrate-dependent monolayer cultures for scanning and transmission electron microscopy

Summary A reliable method is described for processing substrate-dependent cells raised on culture-grade plastic for both scanning (SEM) and transmission electron microscopy (TEM). This technique allows collection of specimens for TEM and SEM from the same culture dish or flask. In this way it is possible to study the surface morphology (SEM) and thin section ultrastructure of cells from contiguous regions of a culture. Specific regions of a culture can be selected and processed so that specimens retain orientation throughout mounting or embedment and sectioning. The method is applicable both to confluent cultures as well as isolated colonies.     

鼠疫耶尔森菌与表面抗体相互作用的原子力显微镜观测研究

目的 以原子力显微镜(atomic force microscopy,AFM)观察比较鼠疫耶尔森菌EV76株与正常兔血清以及兔抗F1抗体反应后微观形态变化,探讨以原子力显微镜为工具的鼠疫耶尔森菌的免疫检测方法.方法 用兔抗F1抗体及正常兔血清处理鼠疫耶尔森菌菌液,并与对照菌一起制样后在原子力显微镜下观察鼠疫耶尔森菌的表面结构的改变,并对其主要指标,包括Ra、Rq改变进行测量比较.结果 正常菌体组细胞呈椭圆形,两端钝圆,长为1.1～1.3 μm,宽为0.8～1.0 μm,阶高为0.04～0.06 μm,细胞形状规则,表面相对比较光滑;对照抗体及F1抗体加入组,细菌阶高均明显增高;F1抗体结合株菌体形状不规则,表面粗糙度明显增加.结论 获得原子力显微镜下的鼠疫耶尔森菌形态特征;表面抗体与鼠疫耶尔森菌结合后,对鼠疫耶尔森菌的表面超微结构有显著的影响,其中粗糙度可以作为原子力显微镜免疫检测的指标.     

Imaging the lamellipodium of migrating epithelial cells in vivo by atomic force microscopy

Cell locomotion originates at a specific region of the cell surface, the leading edge of a migrating cell. Various factors have been proposed to contribute to the propulsion of a cell over the substratum. Rapid turnover processes of cytoskeletal elements inside the cell and insertion of new plasma membrane at the leading edge of the cell permit the extension of a cell in a given direction. Our goal was to image in vivo plasma membrane turnover by means of atomic force microscopy (AFM) and to resolve dynamic processes at the nanometer level. As an experimental model we used migrating kidney cells derived from the Madin-Darby canine kidney (MDCK) cell line that was transformed by alkaline stress. These so-called MDCK-F cells exhibit spontaneous calcium-dependent oscillatory activity of plasma membrane potential associated with cell locomotion. We imaged cells during migration and observed dynamic invagination processes in the cell surface close to the leading edge, indicating internalization of plasma membrane. Invaginations were prevented by removal of calcium from the perfusate. During calcium reduction plasma membrane uncoupled from the underlying cytoskeleton and lipidic pores with diameters of about 30 nm could be disclosed and imaged. This study demonstrates that the AFM can readily trace dynamic physiological processes in vivo, emphasizing the potential role of calcium in maintaining plasma membrane integrity and function.     

Contribution of transmission electron microscopy to fine-needle aspiration biopsy diagnosis: Comparison of cytology and combined cytology and transmission electron microscopy with final histological diagnosis

This report evaluates 74 fine-needle aspiration biopsies processed for transmission electron microscopy with subsequent surgical procedure. The specificity of diagnosis obtained by cytology alone was compared to that obtained by cytology and electron microscopy, using histologic diagnosis as the gold standard. When cytology gave a diagnosis of malignancy but could not give tumor category or type, electron microscopy could correctly give both. When cytology could give tumor category but not type, electron microscopy correctly identified type in the majority of cases. When cytology gave tumor category and type, electron microscopy confirmed the diagnosis. Transmission electron microscopy is very helpful when the cytopathologist can diagnose malignancy but cannot give tumor category and/or type. When the cytopathologist is specific in his/her diagnosis, TEM is not as helpful. Diagn Cytopathol 1996;15: 282–287. © 1996 Wiley-Liss, Inc.     

Visualization of inositol 1,4,5-trisphosphate receptor by atomic force microscopy

Inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) acts as a ligand-gated channel that mediates neuronal signals by releasing Ca(2+) from the endoplasmic reticulum. The three-dimensional (3D) structure of tetrameric IP(3)R has been demonstrated by using electron microscopy (EM) with static specimens; however, the dynamic aspects of the IP(3)R structure have never been visualized in a native environment. Here we attempt to measure the surface topography of IP(3)R in solution using atomic force microscopy (AFM). AFM revealed large protrusions extending approximately 4.3 nm above a flat membrane prepared from Spodoptera frugiperda (Sf9) cells overexpressing mouse type 1 IP(3)R (Sf9-IP(3)R1). The average diameter of the large protrusions was approximately 32 nm. A specific antibody against a cytosolic epitope close to the IP(3)-binding site enabled us to gold-label the Sf9-IP(3)R1 membrane as confirmed by EM. AFM images of the gold-labeled membrane revealed 7.7-nm high protrusions with a diameter of approximately 30 nm, which should be IP(3)R1-antibody complexes. Authentic IP(3)R1 immuno-purified from mouse cerebella had approximately the same dimensions as those of the IP(3)R-like protrusions on the membrane. Altogether, these results suggest that the large protrusions on the Sf9-IP(3)R1 membrane correspond to the cytosolic domain of IP(3)R1. Our study provides the first 3D representation of individual IP(3)R1 particles in an aqueous solution.     

The molecular mass of adenovirus type 5 as determined by means of scanning transmission electron microscopy (STEM)

The mass of adenovirus type 5 was determined by means of computer-assisted scanning transmission electron microscopy (STEM). Arithmetic mean of 157 +/- 10(SD) X 10(6) daltons and mode between 160 and 170 X 10(6) daltons compare favourably with previously reported data. The advantages of the STEM-procedure over the physical and chemical techniques are: low amounts of purified virus particles are needed; visual control of the physical state of virus particles; no need to know the chemical composition or protein concentration of the virus sample.     

原子力显微镜在细胞与生物大分子超微结构和力学研究中的应用

作为微纳米科学理论与技术迅猛发展的代表,原子力显微镜(atomic force microscopy,AFM)在其25年的发展过程中极大地推进了生物学在微纳米尺度上的拓展,为微纳米生物学的诞生与发展提供了重要技术手段。本文在介绍AFM基本原理和检测模式的基础上,结合作者在该领域的研究成果和工作经验,从生物结构与形态学研究、表面物化性质表征、生物大分子的力学操纵三方面综述了AFM在细胞与生物大分子超微结构与生物力学特性研究中的具体应用,并重点探讨了AFM在细胞与生物大分子科学研究中亟待改进和解决的科学与技术问题,提出了一些探讨性的见解和建议。     

超高分子量聚乙烯在人工关节假体中的应用及评价方法

本文通过查阅人工关节领域的相关文献、技术标准、产品指导原则等对超高分子量聚乙烯在人工关节中的应用及评价方法进行了总结。在假体设计开发时,需综合考虑假体设计和影响产品临床使用的风险因素,全面评价产品的材料性能、锁定强度、疲劳性能、体外磨损、生物相容性等,以确保产品的安全有效性。     

Comparative studies of the same adenocarcinoma cells,macrophages, and mesothelial cells by light microscopy,scanning electron microscopy,and transmission electron microscopy

The identification of cells in body cavities of cancer patients is sometimes difficult to make. In order to make a definite cytological diagnosis, we observed the same cells by using light microscopy (LM)-scanning electron microscopy (SEM)-transmission electron microscopy (TEM). In this study we first stained cells by the Papanicolaou method after fixation in 1% glutaraldehyde for LM, and then attempted to observe them successively by SEM-TEM after fixation in 1% paraformaldehyde and 1.25% O^s0⁴. Our method and procedures in examining successively one and the same cells in body cavity fluids by using LM, SEM, and TEM ensured accurate discrimination among adenocarcinoma cells, mesothelial cells, and macrophages. The results of this study suggest that LM-SEM-TEM may be of diagnostic value in distinguishing among mesothelial cells, macrophages, and adenocarcinoma cells. This method also succeeded in disclosing differences between the ultrastructure of the cell surfaces, and those of the cytoplasm, and of the nuclei It is desirable that LM-SEM-TEM observation can be introduced into various aspects in order to obtain an improvement in the diagnosis by cytologic examination, the judgment of therapeutical effects, drug selection, and prognostic presumption. Diagn Cytopathol 1994; 11:333–342. © 1994 Wiley-Liss, Inc.     

神经元膜抗NMDAr1亚基IgG分子构象的原子力显微镜研究

目的　确定兔抗NMDAr1亚基 (N 甲基 D 天冬氨酸受体Nr1亚基 )IgG分子特征性构象。方法　应用原子力显微镜扫描生理状态下分布在云母表面的兔抗NMDAr1亚基蛋白IgG分子 ,并进行理论计算。结果　IgG分子为 136 .4 ×6 2 .8 × 2 6 .1 椭球形三亚基复合物。结论　原子力显微镜可以在生理状态下直观测定生物大分子纳米尺度介观结构。抗NMDAr1蛋白IgG分子的特征性构象 ,可以做为神经细胞膜表面NMDA受体 (N 甲基 D 天冬氨酸受体 )原子力显微镜观测的原位标记物     

基于原子力显微镜技术研究不同激活态下肌节超微结构与压弹性

目的搭建研究不同激活态下昆虫飞行肌纤维的超微结构与力学特性的系统,并以此开展近生理环境下的心肌生物力学研究,进而推动对心肌结构、力学特征和生理功能间关系的认识,为心肌病的基础研究和临床治疗提供更多的线索。方法采用轻敲模式的原子力显微镜技术研究僵直态、松弛态和活化态下昆虫飞行肌肌原纤维的超微精细结构;采用纳米压痕技术研究不同生理状态下肌纤维的定点弹性特性。结果肌原纤维在僵直态、松弛态和3种活化态的肌节长度分别为:(2.10±0.05)(、3.10±0.10)(、2.50±0.15)μm(2 mmol/L Ca2+)(,2.60±0.25)μm(5 mmol/L Ca2+)和(2.55±0.15)μm(10 mmol/L Ca2+),A带长度始终保持在1.50μm左右,而I带的长度则有较大的变化(0.7～1.6μm);力学实验发现,在同一生理状态,肌原纤维的弹性参数的大小满足Z线>M线>重叠区>I带;在不同活化状态下,钙离子浓度变化对Z线、M线和I带的压弹性影响的程度很接近。结论 5 mmol/L的Ca2+浓度是合适的肌纤维活化浓度,肌节长度的分布符合肌动蛋白与肌球蛋白在重叠区相对滑移的力学和空间结构模型;AFM是肌纤维超微结构与力学特性高分辨研究的潜力工具。     

Real-time monitoring of angiotensin II-induced contractile response and cytoskeleton remodeling in individual cells by atomic force microscopy

Physiological processes, occurring as a result of specific receptor stimulation, are generally assessed via molecular biology techniques and microscopic approaches with the involvement of specific molecular markers. The recent progress in experimental approaches, allowing the mechanical characterization of individual biological entities, now makes it possible to address cellular processes occurring in individual cells as a result of their stimulation by hormones. Here, we demonstrate that the atomic force microscope (AFM) can be used to mechanically probe individual cells following the activation of the angiotensin-1 receptor, a receptor well known for its role in cell homeostasis regulation. Our goal is to demonstrate that the measurement of cantilever deflection can be used to quantify in real time the mechanical and morphological cell activity associated with the activation of the receptor. By combining the AFM with time-lapse sequences of phase-contrast and confocal micrographs, we show that the angiotensin-1 receptor stimulation with 100 nM angiotensin II produces an actin-dependent contractile response with an amplitude of 262 ± 52 nm. We validated the mechanical origin of the responses by measuring the elastic modulus of the cell from indentation experiments performed at 30-s intervals. Additionally, nanoscaled height fluctuations of the cell membrane occurring after the initial contraction response could be attributed to an increased actin cytoskeleton activity and remodeling detected by confocal microscopy. Finally, by using inhibitors for specific elements of the angiotensin-1 receptor signaling pathways, we demonstrate that AFM real-time height monitoring allows a read out of the molecular processes responsible for the cell mechanical response.     

Aspiration cytology of neuroblastoma: light microscopy with transmission and scanning electron microscopic correlations

Fine-needle aspiration biopsies from three patients with neuroblastoma were studied by light microscopy, and the morphologic findings were correlated with those from transmission and scanning electron microscopy. Light microscopic examination of the aspiration smears from all three cases revealed small and large round cells with variable numbers of intertwining cytoplasmic processes. Transmission electron microscopy confirmed the light microscopic finding of cytoplasmic processes; in addition, it revealed the presence of other diagnostic morphologic features, including neurosecretory granules, microtubules, and synaptic cell junctions. Scanning electron microscopy demonstrated that the tumors were composed of a mixture of undifferentiated round cells and more differentiated cells with long cytoplasmic processes. The morphologic spectrum of these processes and their interrelationships with one another and with other cells could be studied in detail. These findings indicate that scanning electron microscopy may be used effectively in the morphologic evaluation and pathologic diagnosis of neuroblastoma.     

Chrysiasis: transmission electron microscopy, laser microprobe mass spectrometry and epipolarized light as adjuncts to diagnosis

Two patients receiving gold therapy for rheumatoid arthritis developed skin pigmentation, chrysiasis, which in one appeared 4 months after cessation of the therapy. The diagnosis was confirmed by transmission electron microscopy and mass spectrometry laser microprobe analysis of paraffin sections and its extent demonstrated by epipolarized light. The condition is poorly reported and clinically may be confused with silver and mercury impregnation. Tissue diagnosis requires ancillary methods and of these, transmission electron microscopy and laser microprobe mass spectrometry are excellent examples. The transmission electron microscopy findings differ from previous reports and raise doubts on the hypothesis on the role of the skin in gold excretion. Because of the renewed interest in crysotherapy and the latent period that can separate this from chrysiasis, an increase in chrysiasis and the need for its diagnosis can be anticipated.