血浆蛋白对生物材料细菌粘附影响研究进展

防止生物材料细菌粘附是防治生物材料为中心感染(biomaterial centered infect,BCI)的重要环节.研究发现,人体血浆蛋白对生物材料细菌粘附有重要影响.因此,研究血浆蛋白与生物材料细菌粘附关系为防治BCI有重要的意义.本文综述了与生物材料细菌粘附相关的血浆蛋白、血浆蛋白对生物材料细菌粘附影响的有关机制及如何提高血浆蛋白抗细菌粘附作用的展望.     

Characterization of plasma proteins adsorbed onto biomaterials. By MALDI-TOFMS

The analysis of plasma proteins adsorbed onto a polyurethane (PU) biomaterial was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). This article marks the first study on MALDI-TOFMS analysis of multiple proteins adsorbed from plasma, in vitro, onto the surface of a biomaterial to easily enable their characterization. Plasma standards from three different hosts were placed in contact with non-porous PU, a model biomaterial. Following the use of washing protocols developed in our laboratory, the biomaterial was analyzed, directly, with MALDI-TOFMS. Proteins with molecular weights (Mr) ranging from ca. 6.5 to 150 kDa were observed in the mass spectra and characterized upon comparison with proteins of known Mr. The proteins observed were tentatively identified as those known to adsorb onto PU, both in vitro and in vivo. In an attempt to model in vivo sorption, the PU biomaterial was exposed to freshly collected canine plasma, in vitro, for different lengths of time. Corresponding MALDI-TOFMS spectra displayed increasing protein signal for a number of different proteins with increasing times of exposure to plasma. This method provided qualitative and semi-quantitative analysis of the proteins adsorbed onto the biomaterial surface.     

Micronucleus formation detected by live-cell imaging

Although micronuclei (MNi) have been extensively used to evaluate genotoxic effects and chromosome instability, the most basic issue regarding their formation was not completely addressed until recently, due to limitations of traditional experimental methods. The development of live-cell imaging, combined with genetically engineered chromosome labelling techniques makes it possible to investigate the origin of a micronucleus in a single cell in a real-time and high-throughput manner. Here, we review all the available studies on the origins of MNi in live cells and discuss novel findings based on this recently emerged methodology. Some unsolved questions on MNi formation and limitations of live-cell imaging in the investigation of MNi have also been discussed.     

Characterization of surface microphase structures of poly(urethane urea) biomaterials by nanoscale indentation with AFM

Atomic force microscopy utilizing both tapping mode and force mode imaging is used to visualize the separated microphases in poly(urethane urea) films under ambient and aqueous conditions. The topography of the PUU surface changed upon hydration with the formation of nanometer-sized features on the surface. The surface becomes enriched in hard domains with hydration time and this enrichment is irreversible after dehydration. Force mode measurements were used to quantify mechanical properties as both indentation and modulus measurements. Analysis of the modulus during indentation reveals the three-dimensional nature of the structures, with the surface being covered by a 2-20-nm-thick soft segment overlayer under ambient conditions, while hydration leads to the loss of this overlayer. The force measurements also reveal the presence of regions having modulus values between those of the hard and soft phases and located spatially near the interface between the hard and soft domains. However, such regions with intermediate modulus were only rarely seen following hydration. Calculation of the Young's modulus from the compression data shows that hydration increases the modulus of the PUU surface by both enrichment of the amount of hard domain present and increasing the modulus of the individual hard and soft phases themselves. Direct visualization of the distribution of these different domains on the surface by nanoscale measurements provides an important path to characterizing the relationships between the surface properties of these materials and subsequent performance in biomedical applications.     

Modulating bone cells response onto starch-based biomaterials by surface plasma treatment and protein adsorption

The effect of oxygen-based radio frequency glow discharge (rfGD) on the surface of different starch-based biomaterials (SBB) and the influence of proteins adsorption on modulating bone-cells behavior was studied. Bovine serum albumin, fibronectin and vitronectin were used in single and complex protein systems. RfGD-treated surfaces showed to increase in hydrophilicity and surface energy when compared to non-modified SBB. Biodegradable polymeric blends of cornstarch with cellulose acetate (SCA; 50/50wt%), ethylene vinyl alcohol (SEVA-C; 50/50wt%) and polycaprolactone (SPCL; 30/70wt%) were studied. SCA and SCA reinforced with 10% hydroxyapatite (HA) showed the highest degree of modification as result of the rfGD treatment. Protein and control solutions were used to incubate with the characterized SBB and, following this, MG63 osteoblast-like osteosarcoma cells were seeded over the surfaces. Cell adhesion and proliferation onto SCA was found to be enhanced for non-treated surfaces and on SCA+10%HA no alteration was brought up by the plasma modification. Onto SCA surfaces, BSA, FN and VN single solutions improved cell adhesion, and this same effect was found upscaled for ternary systems. In addition, plasma treated SEVA-C directed an increase in both adhesion and proliferation comparing to non-treated surfaces. Even though adhesion onto treated and untreated SPCL was quite similar, plasma modification clearly promoted MG63 cells proliferation. Regarding MG63 cells morphology it was shown that onto SEVA-C surfaces the variation of cell shape was primarily defined by the protein system, while onto SPCL it was mainly affected by the plasma treatment.     

Intracranial abnormalities detected by three-dimensional magnetic resonance imaging in Prader-Willi syndrome

The neuropathologic abnormalities associated with Prader-Willi syndrome (PWS) are largely unknown. PWS is due to the loss of several paternally expressed genes in chromosome 15q11-q13 region. Several of the imprinted genes in the 15q11-q13 region are normally expressed in the brain and thought to be necessary for neuronal growth and development. Thus, we hypothesized that we would find abnormalities in gray and white matter growth in individuals with PWS. We evaluated three-dimensional (3-D) MRI scans of 20 individuals with PWS, aged three months to 39 years, and compared them to 3-D MRI scans of 21 normal weight sibling controls and 16 individuals with early-onset morbid obesity (EMO) of unknown etiology. The interpreters of the scans were blinded to the diagnosis of the subjects. Intracranial abnormalities in individuals with PWS included ventriculomegaly (100% of individuals), decreased volume of brain tissue in the parietal-occipital lobe (50%), sylvian fissure polymicrogyria (60%), and incomplete insular closure (65%). None of the EMO or normal weight control subjects had any of these findings. We found multiple morphologic brain abnormalities in subjects with PWS suggesting that the loss of paternally expressed genes in chromosome 15q11-q13 region may result in abnormalities of neuronal development. The specific mechanisms underlying these neuropathological abnormalities and their correlation with the clinical phenotype remain to be elucidated.     

Monitoring health by values of acute phase proteins

A systemic acute phase reaction may develop during infection and inflammation, due to the action of peripherally liberated proinflammatory cytokines. Hepatic metabolism changes, and negative and positive acute phase proteins (APPs) can be measured in the blood: the APPs therefore represent appropriate analytes to assess health. While they are non-specific markers, their levels change with biological effects and this can be used to assess nutritional deficits and reactive processes, especially when positive and negative acute phase variables are combined in an index. Unfortunately, at present, no comprehensive, easy-to-use and cheap system is available to assess various acute phase proteins in serum or blood samples. Protein micro-array technology may satisfy this need; it will permit simultaneous analysis of numerous analytes in the same small volume sample and enable integration of information derived from systemic reactivity and nutrition with disease-specific variables. Applying such technology may help to address health problems in many countries.     

Beyond the lateral resolution limit by phase imaging

We present a theory to extend the classical Abbe resolution limit by introducing a spatially varying phase into the illumination beam of a phase imaging system. It allows measuring lateral and axial distance differences between point sources to a higher accuracy than intensity imaging alone. Various proposals for experimental realization are debated. Concretely, the phase of point scatterers' interference is experimentally visualized by high numerical aperture (NA = 0.93) digital holographic microscopy combined with angular scanning. Proof-of-principle measurements are presented by using sub-wavelength nanometric holes on an opaque metallic film. In this manner, Rayleighs classical two-point resolution condition can be rebuilt. With different illumination phases, enhanced bandpass information content is demonstrated, and its spatial resolution is theoretically shown to be potentially signal-to-noise ratio limited.     

Micro-Raman analysis and AFM imaging of Acidithiobacillus ferrooxidans biofilm grown on uranium ore

Acidithiobacillus ferrooxidans biofilm grown on uranium ore substrate was analyzed by a micro-Raman spectrometer and an atomic force microscope (AFM). The bacterium employed for this study, A. ferrooxidans BM1, was isolated from a uranium mine (Jaduguda, India). Micro-Raman analysis revealed the different constituents of molecular fragments present in microbial cells and in secreted extracellular polymeric substances (EPSs). AFM images clearly revealed bacterial cells surrounded by EPS. From Raman spectral data, the composition of EPS from A. ferrooxidans BM1 appeared to be similar to that of EPS secreted in a different Pseudomonas bacterium.     

Aldosterone receptor sites on plasma membrane of human vascular endothelium detected by a mechanical nanosensor

The mineralocorticoid hormone aldosterone acts on target cells of kidney, colon, and the cardiovascular system through genomic and nongenomic pathways. Although the classical intracellular mineralocorticoid receptor plays a key role in mediating both pathways, it is unclear whether there are specific aldosterone receptors located on the cell surface. To search for such sites in vascular endothelium, we used an atomic force microscope (AFM) which measures unbinding forces based on single molecular recognition between an aldosterone-loaded AFM tip and the cell membrane. Aldosterone was tethered covalently via linker molecules to an AFM tip. Human endothelial cells (EA.hy926) were grown in culture and studied in buffer at 37°C. Using the aldosterone-functionalized AFM tip as a mechanical nanoscale indenter, unbinding forces could be measured at randomly chosen sites of the plasma membrane. Sites with strong interactions between AFM tip and cell surface could be identified exhibiting unbinding forces of about 65 pN. The binding probability between the aldosterone-loaded tip and the cell surface at selected membrane sites was 53 ± 7.2%. Addition of an excess supply of aldosterone to the bath solution blocked the binding of the aldosterone-loaded tip to the cell surface. The binding probability was reduced to 8.0 ± 1.8% when an excess supply of aldosterone was added to the bath. However, it was not influenced by the addition of spironolactone or dexamethasone. We conclude that aldosterone receptor sites exist on the cell surface of vascular endothelial cells distinct from the classical mineralocorticoid receptors and insensitive to glucocorticoids. Binding of aldosterone to these receptors initiates an intracellular signaling cascade that precedes the classical genomic response and most likely participates in the control of vascular resistance.     

Plasma lithography--thin-film patterning of polymeric biomaterials by RF plasma polymerization I: Surface preparation and analysis

Plasma lithography, combining plasma deposition with photolithography, is described as a versatile method to manufacture all-polymeric substrates with thin-film patterns for applications in biomedical engineering. Patterns of a hydrophobic fluorocarbon plasma polymer with feature sizes between 5 and 100 microm were deposited on a base substrate in a lift-off process: an intermediate tetraglyme plasma polymer layer provides non-fouling properties to the base substrate. Careful analysis of critical process parameters identified the narrow window of process conditions that led to the formation of functional surface patterns. High pattern fidelity, aspect ratios, and resolution of the patterns are demonstrated by atomic force microscopy. Electron spectroscopy for chemical analysis (ESCA) and secondary ion mass spectroscopy (SIMS) were used to characterize the surfaces, showing good retention of the original chemical structure of the pattern components throughout the process. SIMS imaging was used for specific chemical imaging of the components. Potential applications for the patterned polymer films, e.g., for studying cell behavior in vitro in dependence of shape and size of adhering cells, are discussed.     

Nonfouling biomaterials based on polyethylene oxide-containing amphiphilic triblock copolymers as surface modifying additives: adsorption of proteins from human plasma to copolymer/polyurethane blends

Three polyethylene oxide-polyurethane-polyethylene oxide (PEO-PU-PEO) block copolymers of variable PEO block size (MW 550, 2000, and 5000) were used to modify the surface of a conventional segmented polyurethane (PU) with the objective of inhibiting interactions with proteins. The surface-active copolymers were blended with the PU by solution methods. Protein adsorption from human plasma to the modified materials was investigated using radiolabeling and immunoblotting methods. From the radiolabeling experiments, it was found that fibrinogen adsorption from plasma to all of the modified surfaces was much lower than to the unmodified PU matrix. For blends of low copolymer content, resistance to adsorption was greatest on the copolymer 1 (PEO550)-modified materials, and increased with increasing copolymer content for all three blend types. At high copolymer content inhibition of adsorption was very strong and independent of PEO block size. The immunoblotting experiments showed that on materials of high copolymer content (20 wt %), the proteins investigated (fibrinogen, albumin, complement C3, and apolipoprotein A-I) were undetectable. At low copolymer content (< or = 5 wt %), the blends of copolymer 1, with the shortest PEO block, exhibited greater protein resistance than those of copolymers 2 and 3 (PEO blocks of MW 2000 and 5000, respectively), and resistance decreased with decreasing protein size. Evidence of complement activation was seen for the blends of low copolymer content. Adsorption of C3 and complement activation decreased with increasing content of the copolymers. It was concluded that surface density of PEO is more important than chain length for protein resistance in contact with plasma.     

Association of surface IgM with two membrane proteins on murine B lymphocytes detected by chemical crosslinking

Surface proteins of intact murine B lymphocytes were crosslinked by the bifunctional reagent, 4,4'-diphenyldiazoniumdisulphidfluoroborate and then radiolabelled with 125I. After solubilization of the cells, Ig-containing complexes (Ig and protein(s) covalently crosslinked to Ig) were isolated and analyzed by two-dimensional SDS polyacrylamide gel electrophoresis (2D-SDS-PAGE). Ig-containing complexes were separated in the first dimension by cylindrical SDS gels. Subsequently, the disulphide bridges of the isolated surface Ig molecules and of the crosslinking reagent were cleaved, and the products electrophoresed in the second dimension on SDS slab gels. In addition to the Ig polypeptide chains, two proteins with mol. wts of 46,000 and 56,000 could be identified. In order to answer the question whether these proteins are associated with IgM and/or IgD Ig-complexes were separated with regard to their isotype and analyzed separately by 2D-SDS-PAGE. It was found that both proteins are associated with subunits of IgM. In the case of IgD, no associated structures could be demonstrated by the method used.     

Human IgE and IgG antibodies to mosquito proteins detected by the immunoblot technique

Immunoblot technique was used in this study to detect IgE and IgG antibodies in human sera against mosquito antigens. Mosquito proteins were separated on SDS-polyacrylamide gels and transferred electrophoretically to nitrocellulose papers. After incubation with sera from different individuals, the precipitated bands were analyzed with enzyme-labeled goat anti-human immunoglobulins. Distinct patterns of antigen were recognized by IgE and IgG antibodies.     

Silicone rubber-hydrogel composites as polymeric biomaterials III. An investigation of phase distribution by scanning electron microscopy

The structure of silicone rubber-hydrogel composite materials was investigated by scanning electron microscopy (SEM) and light microscopy. The polymer phases in these materials composed of the polysiloxane matrix and very small particles of lightly cross-linked poly(2-hydroxyethylmethacrylate) or poly(2-hydroxyethylmethacrylate-co-methacrylic acid) were visualized using both methods. The distribution of polymer phases was studied by SEM of fracture surfaces of the materials. The results are discussed in relation to the transport properties of the materials.     

X射线相位衬度成像的研究进展

在临床医学和材料科学等领域,基于吸收衬度的X射线成像技术是一种非常重要的诊断工具.然而,对于生物医学软组织、聚合物或纤维材料等,由于它们对X射线的弱吸收,这种传统X射线成像技术的应用受到了限制.相位衬度成像是目前X射线成像领域的最新前沿技术和研究热点之一,它能检测对X射线弱吸收的轻元素物质,空间分辨率可达微米甚至亚微米量级,与传统X射线吸收成像技术相比具有独特的优势,并且在医学、生物学、材料科学等领域上获得了成功.介绍了X射线相位衬度成像的原理、成像特点和应用情况.