Optimization and comparison of two different 3D culture methods to prepare cell aggregates as a bioink for organ printing

The ultimate goal of tissue engineering is to design and fabricate functional human tissues that are similar to natural cells and are capable of regeneration. Preparation of cell aggregates is one of the important steps in 3D tissue engineering technology, particularly in organ printing. Two simple methods, hanging drop (HD) and conical tube (CT) were utilized to prepare cell aggregates. The size and viability of the aggregates obtained at different initial cell densities and pre-culture duration were compared. The proliferative ability of the cell aggregates and their ability to spread in culture plates were also investigated. In both methods, the optimum average size of the aggregates was less than 500 μm. CT aggregates were smaller than HD aggregates. 5,000 cells per drop HD aggregates showed a marked ability to attach and spread on the culture surface. The proliferative ability reduced when the initial cell density was increased. Comparing these methods, we found that the HD method having better size controlling ability as well as enhanced ability to maintain higher rates of viability, spreading, and proliferation. In conclusion, smaller HD aggregates might be a suitable choice as building blocks for making bioink particles in bioprinting technique.     

Solvent vapour lock: an extreme case of the problems caused by lignified and suberized cell walls during resin infiltration

To determine the cause of floating tissue pieces and poor resin infiltration, sugarcane stem tissue was monitored during processing for microscopy. Bubbles formed within cells at the early stages of solvent replacement (the timing dependent on the fixation used), and increased in volume to fill the cells as resin concentration increased. It is concluded that bubbles form by solvent cavitation (formation of a solvent vapour phase) as hydraulic pressure decreases within the cells, because the thick, lignified and suberized walls are freely permeable to the solvent but not to the resin molecules. The pressure difference across the walls that would be required to release the vapour lock cannot be achieved by vacuum infiltration. In more flexible, thinner-walled, suberized and lignified tissues in maize roots, the lowering of hydrostatic pressure in the cells results in distortion by cytorrhysis (cell collapse). The analogy is drawn between the cavitation and cytorrhysis of cells during resin infiltration and the same processes which occur in living plant cells during water stress. Cavitation and cytorrhysis did not occur in sugarcane or maize tissues processed by anhydrous freeze substitution.     

Electron microscopy of mammalian cells in the absence of fixing,freezing, dehydration,or specimen coating

Human osteoblast-like (bone-forming) cells were imaged using environmental scanning electron microscopy (ESEM). The cells were hydrated, unfrozen, and uncoated. Specimens were cooled to 3 degrees C and imaged in water vapor, with partial pressures varying from saturated conditions to a humidity of approximately 50%, relative to pure water. The ESEM images show the presence of cell nuclei, nucleoli, and cytoplasmic membranes. Comparisons between chemically fixed and unfixed specimens (neither dried nor coated) show that cell morphologies are similar in both cases. These results are compared with a fixed, dried, carbon-coated specimen. Thermodynamic and kinetic arguments are used to show that humidities significantly lower than 100% correspond to metastable states suitable for stabilizing hydrated biological tissues and cells. The ability to perform observations with minimal specimen preparation is potentially useful for studying interactions between mammalian cells and biomaterials that are developed for tissue engineering. The methods employed are equally applicable to the study of specimens in the biological, materials, and physical sciences where careful control over specimen stability is required.     

Freeze-substitution: the addition of water to polar solvents enhances the retention of structure and acts at temperatures around –60°C

High‐pressure freezing followed by freeze substitution and plastic embedding is becoming a more widely used method for TEM sample preparation. Here, we have investigated the influence of solvents, fixative concentrations and water content in the substitution medium on the sample quality of high‐pressure frozen, freeze‐substituted and plastic embedded mammalian cell culture monolayers. We found that the visibility of structural details was optimal with acetone and that extraction increased with both increasing and decreasing solvent polarity. Interestingly, the addition of water to polar solvents increased the sample quality, while being destructive when added to apolar solvents. The positive effect of water addition is saturable in acetone and ethanol at 5%(v/v), but even addition of up to 20% water has no negative effect on the sample structure. Therefore, a medium based on acetone containing fixatives and 5% water is most optimal for the substitution of mammalian cell cultures. In addition, our results suggest that the presence of water is critical for the retention of structure at temperatures around –60°C.     

一种含磺酸酯基亲和性变化树脂的合成及其热分解性能的研究--磺酸酯种类及其取代基对树脂热分解性能的影响

合成了一类可以热分解产生磺酸的三元树脂,并系统考察了不同磺酸酯的热分解特性及其对树脂水溶解性变化的影响.三元树脂由乙烯基磺酸酯、甲基丙烯酸甲酯(MMA)与甲基丙烯酸(MAA)按不同的配比共聚而成.通过DSC、TG、IR等分析发现,三元树脂的稳定性和热分解温度主要依赖磺酸酯的种类和取代基,适当调整分子结构中磺酸酯以及羧酸的含量,热处理后的树脂薄膜可以在中性水中完全溶解.由于这类树脂的热解可以导致水不溶向水溶的转换,因而有可能在构筑中性水处理,甚至,"免处理"的环保型热敏成像材料中得到应用.     

Single- and two-photon spectral imaging of intrinsic fluorescence of transformed human hepatocytes

Autofluorescence (AF) originating from the cytoplasmic region of mammalian cells has been thoroughly investigated; however, AF from plasma membranes of viable intact cells is less well known, and has been mentioned only in a few older publications. Herein, we report results describing single- and two-photon spectral properties of a strong yellowish-green AF confined to the plasma-membrane region of transformed human hepatocytes (HepG2) grown in vitro as small three-dimensional aggregates or as monolayers. The excitation-emission characteristics of the membrane AF indicate that it may originate from a flavin derivative. Furthermore, the AF was closely associated with the plasma membranes of HepG2 cells, and its presence and intensity were dependent on cell metabolic state, membrane integrity and presence of reducing agents. This AF could be detected both in live intact cells and in formaldehyde-fixed cells.     

The microstructure of petroleum vacuum residue films for bituminous concrete: a microscopy approach

Selected carbon-rich refinery residues ('binders') mixed with mineral particles can form composite materials ('bituminous concrete') with bulk mechanical properties comparable to those of cement concrete. The microstructural mechanism underlying the remarkable composite properties has been related to the appearance of a rigid percolating network consisting of asphaltenes and mineral particles [Wilbrink M. et al. (2005) Rigidity percolation in dispersions with a structured visco-elastic matrix. Phys. Rev. E71, 031402]. In this paper, we explore the microstructure of thin binder films of varying thickness with a number of microscopic characterization techniques, and attempt to relate the observed microstructure to the distinctive mechanical behaviour. Two binders, only one of which has been proven to be suitable for bituminous concrete were investigated, and their microstructure compared. Both binders show the formation of asphaltene aggregates. The binder suitable for bituminous concrete is distinguished by the fact that the asphaltenes show a stronger tendency towards such aggregation, due to a higher concentration and less stabilization in the maltene phase. They also show a clear affinity to other species (such as waxes) and may act as nucleation sites for crystals and aggregates of those species.     

Enhancement of Lubrication Properties of Nano-oil by Controlling the Amount of Fullerene Nanoparticle Additives

In this article, the tribological properties of fullerene nanoparticles-added mineral oil were investigated as a function of volume concentration of fullerene nanoparticle additives (e.g., 0.01, 0.05, 0.1, and 0.5 vol.%). The lubrication tests were performed at the disk-on-disk type tester under the various normal forces and fullerene volume concentrations. Tribological properties were evaluated by measuring the friction surface temperature and friction coefficient, and then interpreted in terms of Stribeck curves. At the same time the friction surfaces tested were evaluated by observing their SEM images, surface roughness, and AFM images. The results showed that the nano-oil containing the higher volume concentration of fullerene nanoparticles resulted in the lower friction coefficient and less wear in the fixed plate, indicating that the increase of fullerene nanoparticle additives improved the lubrication properties of regular mineral oil.     

The structure and tribological behavior of nanocomposite carbide coating on steel 45

It is shown that fullerene black interacts with the steel 45 surface and forms a nanocomposite coating including orthorhombic iron carbide Fe₃C with crystallites sized 100–150 nm and dispersed spots of fullerene black with particles sized 40–50 nm. Tribotests show the coefficient of friction of the composite coating to be three-four times lower and the wear resistance about two times higher than these parameters for original annealed steel 45. It is proved that in friction, frictional transfer occurs of hard high-disperse wear products of the coating carbide base and fullerene black to the counterbody, which encourages the decrease in the friction of the pair with one of its elements coated with Fe₃C-fullerene black. The causes of the improved tribological behavior of the carbide coating are discussed.     

样品制备对化合物NMR结构解析的影响

同所有的化学分析测试方法一样,样品制备是核磁共振分析检测中的一个不可忽视的环节。本文根据实验室多年来开展NMR结构分析研究的体会,就样品制备对核磁共振实验的影响进行分析,重点探讨溶剂选择、样品浓度、溶解水及辅助试剂等几个问题,并通过具体实例说明样品制备环节的合理设计可能在改善核磁共振实验效果和提高谱图质量方面起到关键作用。     

Freeze-fracture organization of hair cell synapses in the sensory epithelium of guinea pig organ of Corti

The fine structure of both the afferent and efferent hair cell synapses in the sensory epithelium of guinea pig organ of Corti was examined by freeze-fracture electron microscopy. In the afferent synapse, barlike aggregates of intramembrane particles (IMPs) of about 10 nm in diameter were seen on the P-face of the afferent presynaptic membrane directly beneath the presynaptic dense projection which is located in the active zone of the presynaptic membrane. Small and large depressions have been seen on the presynaptic membrane. The former were observed in the proximity of the barlike aggregates, while the latter were observed some distance from the aggregate. In outer hair cells, IMPs of about 10 nm in diameter were seen on the P-face of the afferent postsynaptic membrane at a density of 3,000/μm². In the efferent synapse, many aggregates composed of from several to tens of large IMPs of 13 nm in diameter were observed on the presynaptic membrane. These aggregates were localized to small membrane depressions, which tended to be deeper as particle number per aggregate increased. Dense populations of IMPs of about 9 nm in diameter were observed on the P-face of the efferent postsynaptic membrane at a density of 4,000/μm². A fenestrated subsynaptic cistern completely covers the efferent postsynaptic membrane. Moreover, the subsynaptic cistern spans several efferent postsynaptic membranes when efferent synapses are gathered in a group. In the afferent and efferent synapses of hair cells, specializations of the synaptic membranes were represented by marked aggregates characteristic of IMPs. In the efferent synapse, IMP movement inside the synaptic membrane was proposed in relationship to retrival of synaptic vesicle membrane. Structural relationship between the subsynaptic cistern and efferent postsynaptic membrane was revealed.     

The study on the atomic force microscopy base nanoscale electrical discharge machining

This study proposes an innovative atomic force microscopy (AFM) based nanoscale electrical discharge machining (AFM-based nanoEDM) system which combines an AFM with a self-produced metallic probe and a high-voltage generator to create an atmospheric environment AFM-based nanoEDM system and a deionized water (DI water) environment AFM-based nanoEDM system. This study combines wire-cut processing and electrochemical tip sharpening techniques on a 40-μm thick stainless steel sheet to produce a high conductive AFM probes, the production can withstand high voltage and large current. The tip radius of these probes is approximately 40 nm. A probe test was executed on the AFM using probes to obtain nanoscales morphology of Si wafer surface. The silicon wafer was as a specimen to carry out AFM-base nanoEDM process in atmospheric and DI water environments by AFM-based nanoEDM system. After experiments, the results show that the atmospheric and DI water environment AFM-based nanoEDM systems operate smoothly. From experimental results, it can be found that the electric discharge depth of the silicon wafer at atmospheric environments is a mere 14.54 nm. In a DI water environment, the depth of electric discharge of the silicon wafer can reach 25.4 nm. This indicates that the EDM ability of DI water environment AFM-based nanoEDM system is higher than that of atmospheric environment AFM-based nanoEDM system. After multiple nanoEDM process, the tips become blunt. After applying electrochemical tip sharpening techniques, the tip radius can return to approximately 40 nm. Therefore, AFM probes produced in this study can be reused.     

Effect of Sand Concentration in the Medium and Travel Distance and Speed on the Slurry Wear Response of a Zinc-Based Alloy Alumina Particle Composite

Slurry wear characteristics of a zinc-based alloy composite containing 10 wt.% alumina particles have been analyzed by sample rotation technique in this study. The slurry comprised 0.4 g sodium chloride and 0.5 cm³ concentrated sulphuric acid dissolved in one litre of water plus varying concentration (up to 60 wt.%) of sand particles. The traversal distance was varied in the range of 15–500 km while test speeds were maintained at 4.71 and 7.02 m/s. It was observed that the wear response of the composite was influenced by parameters such as traversal speed and distance and the content of the sand particles in the environment. Wear rate reduced with increasing speed when tests were conducted in the liquid-only medium while the trend tended to reverse in the presence of the suspended sand particles in the medium. Further, the wear rate increased initially with distance, attained a peak and decreased thereafter at longer traversal distances. Testing the samples in the environment without sand led to higher wear rate when compared with the ones in the media containing sand. Further, within the liquid plus sand media, intermediate sand content (in the test environment) caused maximum wear rate. In this case, the maximum sand content exhibited minimum wear rate. The wear behaviour of the composite under varying conditions has further been substantiated through the characteristic features of the affected surfaces and subsurface regions. Operating wear mechanisms have been observed to be corrosion assisted erosion and abrasion. The predominance of one mechanism over the other(s) actually controlled the wear response of the composite in specific conditions of wear.     

可溶滤膜富集-石墨炉原子吸收法测定钯

本文提出一种基于将钯(Ⅱ)与1-亚硝基-2-萘酚-3,6-二磺酸钠形成阴离子螯合物,再与十四烷基苄基胺阳离子表面活性剂缔合,此离子缔合物放收集在o.3μm硝酸纤维素膜上,用小体积的浓硫酸溶解滤膜和缔合物后于石墨炉原子吸收法测定ng/L级钯的新方法。在2.0~108ng钯/5mL范围内线性良好,富集倍数超过40倍,在10倍预富集时,检测限为12ng/L,常见离子不干扰,用于富集和测定来源不同的河水和海水中钯(Ⅱ),加标回收率90.6%~100.4%。     

静磁场对固液界面表面电荷性质的影响

考虑磁场对固液界面表面电荷性质的影响与微纳流体系统的流体阻力相关,本文采用原子力显微镜(AFM)研究了静磁场对去离子水黏度以及高硼硅玻璃-去离子水界面表面电荷性质的影响,并分析了静磁场对去离子水性质影响的机理。研究结果表明,将去离子水静置于磁场强度为0~0.6T的静磁场下30min时,去离子水的黏度随磁场强度的增加而减小,而高硼硅玻璃-磁化水界面的表面电荷密度随磁场强度的增加而增加;静磁场对去离子水性质影响的机理是磁场引起的去离子水内氢键以及氢氧键的断裂。研究结果同时表明,磁场可以有效地改变固液界面的表面电荷性质。本文的研究结果为利用磁场有效地控制微纳流体系统的流体阻力提供了可能。     

Silver enhancement of gold probes (5–40 nm): Single and double labeling of antigenic sites on cell surfaces imaged with backscattered electrons

Silver enhancement of immunogold-labeled cells was carried out to increase the applicability of colloidal gold probes for visualization in the backscatter electron imaging (BEI) mode of a scanning electron microscope. Optimum conditions were established for single particle discrimination and differential counting of labeling density at low magnifications. Red blood cells double-labeled with 15 + 40 nm and 5 + 20 nm gold probes were silver-enhanced for 6 min and 20 min, respectively, at which times both pairs of labels increased to about 25 + 50 nm. The gold probes still appeared spherical after enhancement and were easily discriminated. Cells were also single-labeled with the above probes and enhanced accordingly. The present method enables visualization of individual particles of any probe size, labeling one, or simultaneously two, antigenic sites on cell surfaces. The silver enhancement procedure thereby allows cells to be labeled with small probes with increased labeling efficiency.     

Intermediate aggregates resulting in the interaction of bile salt with liposomes studied by transmission electron microscopy and light scattering techniques

The interaction of sodium cholate (SC) with phosphatidylcholine liposomes was studied by means of transmission electron microscopy (TEM), changes in the mean particle size (quasielastic light scattering, QELS) and in the static light scattering (SLS) of the system during liposome solubilization. A good correlation was found between the TEM diameter of particles and the mean hydrodynamic diameter (HD) determined by QELS. The intermediate aggregates resulting in this interaction were dependent on the SC concentration in the system. Thus, an initial vesicle growth occurred when the SC concentration in the system was 13.79 mol%. Additional SC amounts (41.17 mol% SC) led to the formation of the largest vesicles (HD 410 nm). Increasing SC amounts led to a slight fall in the vesicle diameter and in the SLS of the system. Thus, for 47.08 mol% SC, TEM images still showed the presence of vesicles albeit with traces of smaller structures and signs of vesicle fusion. When SC concentration exceeded 48 mol% an abrupt decrease in SLS occurred, the size curve starting to show a bimodal distribution. Thus, for 50 mol% SC a sharp distribution curve appeared at 52 nm indicating the formation of small particles and TEM images showed clear signs of vesicle disintegration with formation of tubular structures. The subsequent self organization of these tubular structures (54 mol% SC) led to the formation of open multilayered structures in coexistence with small particles. A gradual increase in the number of these small particles (mixed micelles) led to the complete solubilization of liposomes.     

Acetonitrile as a substitute for ethanol/propylene oxide in tissue processing for transmission electron microscopy: comparison of fine structure and lipid solubility in mouse liver, kidney, and intestine.

Tissue processing for transmission electron microscopy (TEM) is commonly accomplished using ethanol (EtOH) as a dehydrating solvent and propylene oxide (PO) as a transition fluid. Both solvents have some undesirable properties: EtOH solubilizes lipids; PO is highly flammable, volatile, toxic, and potentially carcinogenic. Their replacement by a compound devoid of these characteristics is therefore desirable. Acetonitrile (AN) appears to be such a solvent. It is freely miscible with water, alcohols, acetone, and epoxy resins; it does not interfere with epoxy polymerization; and the resulting cured resins have excellent cutting quality and beam stability. AN is also an excellent dehydrating agent whose use does not necessitate modification of current techniques. Most importantly, the low solubility of phospholipids (PL) in AN limits the loss of membrane lipids and, hence, leads to a better preservation of tissue features.     

Speed measurement of ink droplet by using edge detection techniques

Measurement techniques for the jetting speed and the diameter of an ink droplet are discussed. For the experiment, nano silver ink with nanoparticles of approximately 10 nm diameter dissolved in DI (de-ionised) water (20 wt%) was used as the jetting material. For analysing ink droplet from a CCD (charge coupled device) camera image, the edge detection technique is used where a region of interest (ROI) is defined by a line in the direction of the ink jet. Then, the droplet speed is calculated by varying trigger delay time of strobe LED (light emitting diode) light which is synchronized with respect to the firing signal for jetting. Finally, the measured speed results using the edge detection are compared with the results from particle analysis based on the binary image.     

Investigation of micro-phase separation of the additive,cyclotriphosphazene, in lubricant films of hard disk media

The micro-phase separation of the additive, cyclotriphosphazene (X-1P), in perfluoropolyether (PFPE) lubricant films on hard disk media was studied. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) indicated that the small spots appearing on the disk surface consisted predominantly of X-1P. Observation using an atomic force microscope (AFM) revealed the micro-phase separation process to be the sudden, continuous appearance of new spots some time after coating the film. Some spots grew over previous ones, while some spots dissolved. Finally, they stopped growing and the number of spots became saturated. The solubility of X-1P in the lubricant film increased in the order of ZDIAC, ZDOL2000, ZDOL4000 and Z03, and that in bulk lubricant increased in the order of Z03, ZDOL4000, ZDOL2000 and ZDIAC. The order of solubility of X-1P in the film did not correspond to that in the bulk.