Atomic force microscopic studies on the structure of bovine femoral cortical bone at the collagen fibril-mineral level

The structure of cortical bone at the collagen-mineral level was investigated by means of atomic force microscopy. Surfaces of the specimens treated with collagenase and ethylenediaminetetraacetic acid (EDTA) were examined. Images of blob-like objects observed in intact specimen became clearly outlined after collagenase treatment; the sizes of the blob decreased, suggesting that each blob had been fragmented by the collagenase treatment. Following EDTA treatment of an intact specimen, an image of thread-like objects appeared; the thread was partly constructed by trains of blobs and the other parts of the threads had a periodic pattern along its longer axis. The period was almost equal to the collagen D-period of the Hodge–Petruska model, indicating that the threads are collagen fibrils and that the blobs are related to the mineral phase in bone. It was concluded that minerals were deposited on and along collagen fibrils. A decorated collagen fibril model for the spatial relationship between mineral and collagen fibril was proposed. According to our model, the mineral inside the collagen fibril is about one forth of the extrafibrillar mineral.     

In situ observation of fluoride-ion-induced hydroxyapatite-collagen detachment on bone fracture surfaces by atomic force microscopy

The topography of freshly fractured bovine and human bone surfaces was determined by the use of atomic force microscopy (AFM). Fracture surfaces from both kinds of samples exhibited complex landscapes formed by hydroxyapatite mineral platelets with lateral dimensions ranging from ～90?nm × 60?nm to ～20?nm × 20?nm. Novel AFM techniques were used to study these fracture surfaces during various chemical treatments. Significant topographical changes were observed following exposure to aqueous solutions of ethylenediaminetetraacetic acid (EDTA) or highly concentrated sodium fluoride (NaF). Both treatments resulted in the apparent loss of the hydroxyapatite mineral platelets on a timescale of a few seconds. Collagen fibrils situated beneath the overlying mineral platelets were clearly exposed and could be resolved with high spatial resolution in the acquired AFM images. Time-dependent mass loss experiments revealed that the applied agents (NaF or EDTA) had very different resulting effects. Despite the fact that the two treatments exhibited nearly identical results following examination by AFM, bulk bone samples treated with EDTA exhibited a ～70% mass loss after 72?h, whereas for the NaF-treated samples, the mass loss was only of the order of ～10%. These results support those obtained from previous mechanical testing experiments, suggesting that enhanced formation of superficial fluoroapatite dramatically weakens the protein-hydroxyapatite interfaces. Additionally, we discovered that treatment with aqueous solutions of NaF resulted in the effective extraction of noncollagenous proteins from bone powder.     

Intact free prostate-specific antigen and free and total human glandular kallikrein 2. Elimination of assay interference by enzymatic digestion of antibodies to F(ab')2 fragments

Various blood constituents can interfere with immunoassays, usually by binding the Fc portion of antibodies. Our previously developed assays for intact free prostate-specific antigen (PSA), free human kallikrein 2 (hK2), and total hK2 frequently yielded falsely high results despite including an excess of scavenger antibodies. We investigated whether this interference could be eliminated by replacing monoclonal capture or tracer antibodies with F(ab')2 or recombinant Fab fragments. Female heparin plasma samples (n = 1092), which should have negligible PSA and hK2, and male samples (n = 957) were analyzed to identify samples manifesting interference, which then were used to optimize protocols for the immunoassays. We compared original assays (monoclonal antibodies) versus optimized assays (F(ab')2 fragments: denatured mouse IgG added as scavenger) using another set of EDTA plasma (n = 113), heparin plasma (n = 160), and serum samples (n = 171). With the original assays, the frequency of falsely elevated hK2 and intact free PSA was 15 and 13%, respectively. The optimized assays eliminated 70-85% of these falsely elevated results and importantly reduced the magnitude in the remainder. F(ab')2 fragmentation was the most important factor in reducing interference. The optimized intact free PSA, free hK2, and total hK2 assays manifested high accuracy close to the lower limit of detection.     

民用飞机噪声适航审定中伪纯音影响的去除方法

在民用飞机噪声适航合格审中计算有效感觉噪声级时,通过引入纯音修正因子来考虑噪声频谱不规则性对人主观感觉的影响,但布置在地面上的传声器测量的噪声频谱中含有地面影响导致的伪纯音,使得纯音修正因子可能被高估,需要加以识别并去除。该文提出了一种识别和去除地面影响引入的伪纯音对纯音修正因子计算影响的飞行试验方法和分析方法。在适航噪声测量点旁布置倒置传声器,得到飞机直达噪声,可靠地识别实测适航噪声频谱中的伪纯音,基于"CCAR-36部"及其附件中方法的改进方法,去除伪纯音对纯音修正因子计算的影响。通过某型民机噪声适航审定飞行试验对该方法进行了验证,结果表明该方法满足"CCAR-36部"等规章要求,易于试验和分析实现,能准确去除由地面影响引入的伪纯音影响,提高了有效感觉噪声级的计算准确度。     

Effects of deproteinization and ashing on site-specific properties of cortical bone

Buffered sodium hypochlorite (NaOCl) solution was used to remove selectively the collagen phase from bovine cortical bone. Changes in the mechanical behaviour and material properties were studied over a wide range of resolution (from 5 m to 3 mm) using an integrated combination of experimental techniques. Optical microscopy indicated that timed immersion in NaOCl results in cortical bone specimens that consist of a mineralized tissue core surrounded by a layer of deproteinized or anorganic bone. With increased NaOCl treatment, the mechanical behaviour in three-point flexure of the intact specimens became increasingly characteristic of a brittle ceramic material. Localized material properties were evaluated using histology, scanning electron microscopy and microhardness testing. The site-specific properties and the mineralization of the cores were not significantly affected by the treatment; however, the interactions and structural framework of the hydroxyapatite crystallites within the anorganic material were compromised. This destruction of crystallite interlocking was not observed in samples in which the organic phase was removed by ashing at 800°C. The ashed samples maintained microhardness values three times those of the bleached samples. Because of its damaging effects on cortical bone structural integrity, the NaOCl treatment did not provide a reasonable means of studying, as a function of the phasic mass fraction, incremental changes in bone mechanical behaviour or the relative roles of collagen and mineral within the structural hierarchy.     

A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry

Recent advances in phosphopeptide enrichment prior to mass spectrometric analysis show genuine promise for characterization of phosphoproteomes. Tandem mass spectrometry of phosphopeptide ions, using collision-activated dissociation (CAD), often produces product ions dominated by the neutral loss of phosphoric acid. Here we describe a novel method, termed Pseudo MS(n), for phosphopeptide ion dissociation in quadrupole ion trap mass spectrometers. The method induces collisional activation of product ions, those resulting from neutral loss(es) of phosphoric acid, following activation of the precursor ion. Thus, the principal neutral loss product ions are converted into a variety of structurally informative species. Since product ions from both the original precursor activation and all subsequent neutral loss product activations are simultaneously stored, the method generates a "composite" spectrum containing fragments derived from multiple precursors. In comparison to analysis by conventional MS/MS (CAD), Pseudo MS(n) shows improved phosphopeptide ion dissociation for 7 out of 10 synthetic phosphopeptides, as judged by an automated search algorithm (TurboSEQUEST). A similar overall improvement was observed upon application of Pseudo MS(n) to peptides generated by enzymatic digestion of a single phosphoprotein. Finally, when applied to a complex phosphopeptide mixture, several phosphopeptides mis-assigned by TurboSEQUEST under the conventional CAD approach were successfully identified after analysis by Pseudo MS(n).     

Analysis of trace levels of sulfonamide and tetracycline antimicrobials in groundwater and surface water using solid-phase extraction and liquid chromatography/mass spectrometry

A method has been developed for the trace analysis of two classes of antimicrobials consisting of six sulfonamides (SAs) and five tetracyclines (TCs), which commonly are used for veterinary purposes and agricultural feed additives and are suspected to leach into ground and surface water. The method used solid-phase extraction and liquid chromatography/mass spectrometry (LC/MS) with positive ion electrospray. The unique combination of a metal chelation agent (Na2EDTA) with a macroporous copolymer resulted in quantitative recoveries by solid-phase extraction (mean recovery, 98 +/- 12%) at submicrogramper-liter concentrations. An ammonium formate/formic acid buffer with a methanol/water gradient was used to separate the antimicrobials and to optimize the signal intensity. Mass spectral fragmentation and ionization characteristics were determined for each class of compounds for unequivocal identification. For all SAs, a characteristic m/z 156 ion representing the sulfanilyl fragment was identified. TCs exhibited neutral losses of 17 amu resulting from the loss of ammonia and 35 amu from the subsequent loss of water. Unusual matrix effects were seen only for TCs in this first survey of groundwater and surface water samples from sites around the United States, requiring that TCs be quantitated using the method of standard additions.     

Effects of Addition of Mannitol Crystals on the Porosity and Dissolution Rates of a Calcium Phosphate Cement

The bone defect repair functions of calcium phosphate cement (CPC) are related to its osteoconductivity and its gradual replacement by new bone. Adding mannitol to CPC may enhance its bone repair potential by increasing CPCs macroporosity and dissolution rate. The objective of the study was to assess microporosity and macroporosity and dissolution rates for CPC mixed with mannitol. Three groups of CPC discs were prepared by combining an equimolar mixture of tetracalcium phosphate and anhydrous dicalcium phosphate with (0 %, 10 %, or 50 %) mass fraction (hereafter expressed as mass %) of mannitol. Macroporosity and microporosity of the samples were calculated from volume and mass measurements of the discs. Discs were then placed in a pH 3.0 demineralizing solution simulating acidified physiological solution, and dissolution rates were measured by a previously described constant-composition titration method. Pure CPC exhibited no macropores and microporosity (mean ± s.d.; n = 5) of (46.8 ± 0.8) % volume fraction (hereafter expressed as vol %). Adding 10 mass % mannitol resulted in 15.6 ± 3.9 vol % macroporosity and 39.4 ± 1.8 vol % microporosity, and adding 50 mass % mannitol produced 54.7 ± 0.8 vol % macroporosity and 21.1 ± 0.4 vol % microporosity. The dissolution rates (mean ± s.d.; n = 5) of CPC with (0, 10, and 50) mass % mannitol incorporation were (30.6 ± 3.4, 44.8 ± 10.2, and 54.7 ± 3.6, respectively) μg · cm(-2) · min(-1), or (0.018 ± 0.002, 0.032 ± 0.007, and 0.072 ± 0.005, respectively) μL · cm(-2) · min(-1). Adding either 10 mass % or 50 mass % mannitol into CPC significantly (p < 0.05) increased CPC dissolution rates. Adding mannitol readily increased macroporosity and dissolution rate of CPC, which may enhance the capacity of CPC to be osteoconductive.     

纳滤处理模拟电镀废水

分别以硫酸铜、氯化铜、硝酸铜溶液为模拟电镀废水进行了纳滤实验,所采用纳滤膜为Osmonics的DK、DL膜和日东电工的NTR-7450膜.实验表明,三种膜通量大小顺序为DL>DK>NTR-7450,随原料液浓度的增大膜通量降低,而膜对铜离子的截留率有升高的趋势;纳滤膜对各盐的截留率大小排序为硫酸盐>氯盐>硝酸盐.实验还考察了添加其它离子对纳滤膜通量和截留率的影响.结果表明,加入CaCl2后,对DL膜性能影响不显著;加入Na2SO4后,DL膜的通量变化不明显,截留率略有升高;加入EDTA能明显提高DL膜通量和对铜离子的截留率.     

Systematic Assessment of Synthesized Tri-magnesium Phosphate Powders(Amorphous,Semi-crystalline and Crystalline) and Cements for Ceramic Bone Cement Applications

Magnesium phosphate cements have come under investigation in recent years for use as an alternative to calcium phosphate cements for bone void repair applications.Evidence indicates that magnesium phosphate cements obtain higher initial strengths after cement reaction and resorption in more clinically appropriate time frames than commercially available calcium phosphate cements.In this study,amorphous,partially amorphous and crystalline tri-magnesium phosphate powders were synthesized via an aqueous precipitation reaction with subsequent thermal treatment,and characterized using techniques such as X-ray diffraction and Fourier transform infrared spectroscopy.These materials were assessed for their functionality in cementing reaction with a 3.0 mol/L,pH 7.0 ammonium phosphate solution,including setting time and pH evolution in phosphate buffered saline solution.Results indicated that the amorphous and semi-crystalline tri-magnesium phosphate powders were highly reactive with the setting solution but resulted in mechanically weak cements,while the crystalline tri-magnesium phosphate powder reacted efficiently with the cement solution and were mechanically strong following the cement reaction.X-ray diffraction and scanning electron microscopy analyses indicated significant changes in the phase composition and morphology of the cements following incubation in phosphate buffered saline.These were perceived to be detrimental to the integrity of the amorphous and semi-crystalline tri-magnesium phosphate derived cements but not to those created with fully crystalline tri-magnesium phosphate.The crystalline tri-magnesium phosphate material resulted in the most functional cement as this embodiment displayed the most clinically relevant setting time as well as the highest mechanical resilience and neutral pH during incubation in saline solution rendering them potentially viable as bone void fillers.     

In vivo evaluation of bone tissue behavior on ion implanted surfaces

The ion implantation process offers several unique advantages over other surfaces modifications techniques, in regard to ion release and material mechanical characteristics. The aim of this study was to evaluate the in vivo bone tissue response to ion implanted surfaces. Untreated and nitrogen-ion-implanted stainless steel implants were inserted in the tibia diaphysis (cortical bone) and proximal tibia epiphysis (trabecular bone) of 12 New Zealand White rabbits. The animals were divided into three groups of four animals each, which were maintained for 4, 12 and 24 weeks according to internationally accepted and standardized procedures. At sacrifice, the implants were retrieved with surrounding bone and fixed in 4% neutral buffered formaldehyde and embedded in polymethylmethacrylate (PMMA). The samples were reducted in slices and stained with hematoxylin-eosin, light-green, fuchsin acid and giemsa solution for histological evaluation; fluorescent markers were also used to assess bone apposition. Histomorphometric evaluation was used to determine the extent of bone-material contact. Results from histological and morphometrical analyses revealed active remodeling of bone around both types of implants (control and ion implanted). However, faster bone deposition was observed around the treated material (12 weeks). Both materials reached similar endpoints, as no significant differences between them were evident at 24 weeks. The results demonstrate that ion implanted stainless steel has similar, or slightly enhanced, biological compatibility in contact with bone compared to untreated material; thus it may be a useful material in biomedical applications where reduced ion release or enhanced mechanical properties (as provided by ion implantation) are required. © 2001 Kluwer Academic Publishers     

Removal of hazardous metals from MSW fly ash—An evaluation of ash leaching methods

Incineration is a commonly applied management method for municipal solid waste (MSW). However, significant amounts of potentially hazardous metal species are present in the resulting ash, and these may be leached into the environment. A common idea for cleaning the ash is to use enhanced leaching with strong mineral acids. However, due to the alkalinity of the ash, large amounts of acid are needed and this is a drawback. Therefore, this work was undertaken in order to investigate some alternative leaching media (EDTA, ammonium nitrate, ammonium chloride and a number of organic acids) and to compare them with the usual mineral acids and water.All leaching methods gave a significant increase in ash specific surface area due to removal of soluble bulk (matrix) compounds, such as CaCO₃ and alkali metal chlorides. The use of mineral acids and EDTA mobilised many elements, especially Cu, Zn and Pb, whereas the organic acids generally were not very effective as leaching agents for metals. Leaching using NH₄NO₃ was especially effective for the release of Cu. The results show that washing of MSW filter ash with alternative leaching agents is a possible way to remove hazardous metals from MSW fly ash.     

锌镀层硅酸盐钝化膜的耐腐蚀性能

为了考察锌镀层硅酸盐钝化膜的耐腐蚀性能,通过中性盐雾试验、3%CuSO4点滴试验对比研究了锌镀层、硅酸盐钝化膜及低铬酸盐钝化膜的耐腐蚀性能,并用极化曲线电化学测试方法研究了硅酸盐钝化膜的电化学性能。结果表明:硅酸盐钝化膜明显提高了锌镀层的耐腐蚀性能,其耐蚀性优于低铬酸盐钝化膜;硅酸盐钝化膜也明显提高了锌镀层的自腐蚀电位,有效地控制了其腐蚀的电化学过程,属阳极控制型。锌镀层硅酸盐钝化膜具有较高的耐蚀性能。     

Promoting learning, memory, and transfer in a time-constrained, high hazard environment

Two methods of metacognitive reflection for promoting compliance with an aviation safety rule were tested in a transfer design. Two groups of pilots (n = 10) conducted a simulated flight entailing a search for a target on the ground. During this flight, only 35% of the pilots stayed above an altitude of 500 ft, the minimum allowed by relevant regulations. Following the flight, one group completed a self-explanation questionnaire, in which they explained their actions during the initial flight and what they would do in future flights. The other group completed a relapse-prevention questionnaire, in which they identified the circumstances leading to safety lapses and their future avoidance. A third group (n = 10) formed a rest control; they conducted a familiarization flight without a ground target or debriefing. One week later, all pilots conducted a series of test flights with the same or different ground targets as the initial flight. The self-explanation group showed 100% compliance when the ground target remained the same, but less so (<70%) when the ground target was different. The relapse-prevention group and control groups both showed low levels of compliance across all test flights (<30%). The results are discussed from theoretical and applied perspectives.     

Multisource-sputtered ErBa2Cu3O7−x films on single-crystal and buffered crystalline substrates

High-quality superconducting films of erbium-barium-copper-oxide have been produced in a multisource sputtering system on a variety of substrates, including buffered sapphire and silicon wafers as well as various single-crystal materials. Fine-grained polycrystalline films with narrow (<4 K) resistive transitions have been grown on a number of different substrates. The use of erbium in the 12–3 compound leads to improved film morphology and to more forgiving conditions for establishing of the superconducting phase. Sputtered neutral mass spectroscopy (SNMS) yields useful information about the compositional profile of the films.     

FTMS structure elucidation of natural products: application to muraymycin antibiotics using ESI multi-CHEF SORI-CID FTMS(n), the top-down/bottom-up approach,and HPLC ESI capillary-skimmer CID FTMS

The molecular formulas for the structures and substructures of muraymycin antibiotics A1 (C52H90N14O19, MW 1214) and B1 (C49H83N11O18, MW 1113) were determined using electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS). The muraymycin A1 and B1 structures were elucidated by utilizing capillary-skimmer fragmentation with up to five stages of mass spectrometry (MS5). Multi-CHEF, a multiple ion isolation method, was used at each stage of MS(n) to isolate a parent ion and up to four reference ions, for exact-mass calibration. The parent ions were fragmented by SORI-CID and the product ions internally calibrated with average absolute mass errors less than 1 ppm at each stage in the fragmentation processes. Using the top-down/bottom-up approach, the molecular formulas for the antibiotics were determined by summing the elemental formulas of the neutral losses, obtained by measuring the mass differences (<500 Da) between the genetically related sequential parent ion masses in the MS(n) spectra, with the unique elemental formula of the lowest parent ion mass (<500 Da). The structures of 12 additional compounds in the muraymycin complex were elucidated using HPLC ESI capillary-skimmer CID FTMS by correlating their fragmentation patterns with those of muraymycins A1 and B1. Sequential neutral losses of an aminosugar, a valine, a uridine, and an ester fatty acid from the muraymycin parent ions provided diagnostic fragments for characterization.     

Heavy metal contaminants removal by soil washing

The feasibility of soil washing for decontaminating a silty sand spiked with cadmium, chromium, lead, and zinc was evaluated in laboratory-scale batch and column experiments. Soil samples were subjected to chelant extraction using a solution of disodium salt of ethylenediaminetetraacetic acid (Na(2)EDTA), sodium metabisulfite (Na(2)S(2)O(5)) solution (an inexpensive reducing reagent), and a solution containing a mixture of the two reagents. Batch and column washing of the contaminated soil with deionized water (DI water) revealed that approximately 70% of the cadmium in the sample is weakly bound and readily mobilized in aqueous solution at neutral pH, followed by approximately 25%-30% of zinc, approximately 20%-25% chromium, and only approximately 10% of lead. Of the washing reagents tested, Na(2)EDTA solutions were generally more effective than Na(2)S(2)O(5) for removing heavy metals from the soil samples. Na(2)EDTA preferentially extracted lead over zinc and cadmium but exhibited little impact on chromium removal. Cadmium and, especially zinc, removal by a 0.01-M Na(2)EDTA solution were enhanced considerably by inclusion of 0.1 M Na(2)S(2)O(5), suggesting that a mixture of the two reagents may provide an economically optimum solution for certain contaminated soils.     

Effects of hypergravity on immunologic function

The purpose of this study was to compare the effects of hypergravity exposure (2g) with those of exposure to space flight in the Cosmos 2044 flight. To do so, rats were centrifuged continuously for 14 days. Two different experiments were carried out on tissue obtained from the centrifuged rats. In the first experiment, rat bone marrow cells were examined for their response to recombinant murine colony stimulating factor-granulocyte/monocyte (GM-CSF). In the second experiment, rat spleen and bone marrow cells were stained in with a variety of antibodies directed against cell surface antigenic markers. These cells were preserved and analyzed on a flow cytometer. The results of the studies indicated that bone marrow cells from centrifuged rats showed no significant change in response to GM-CSF as compared to bone marrow cells from control rats. Spleen cells from flown rats showed some statistically significant changes in leukocytes subset distribution, but no differences that appeared to be of biological significance. These results indicate that hypergravity did not greatly affect the same immunological parameters affected by space flight in the Cosmos 2044 mission.     

Comparison between infrared and Raman spectroscopic analysis of maturing rabbit cortical bone

The molecular composition of the organic and inorganic matrices of bone undergoes alterations during maturation. The aim of this study was to compare Fourier transform infrared (FT-IR) and near-infrared (NIR) Raman microspectroscopy techniques for characterization of the composition of growing and developing bone from young to skeletally mature rabbits. Moreover, the specificity and differences of the techniques for determining bone composition were clarified. The humeri of female New Zealand White rabbits, with age range from young to skeletally mature animals (four age groups, n = 7 per group), were studied. Spectral peak areas, intensities, and ratios related to organic and inorganic matrices of bone were analyzed and compared between the age groups and between FT-IR and Raman microspectroscopic techniques. Specifically, the degree of mineralization, type-B carbonate substitution, crystallinity of hydroxyapatite (HA), mineral content, and collagen maturity were examined. Significant changes during maturation were observed in various compositional parameters with one or both techniques. Overall, the compositional parameters calculated from the Raman spectra correlated with analogous parameters calculated from the IR spectra. Collagen cross-linking (XLR), as determined through peak fitting and directly from the IR spectra, were highly correlated. The mineral/matrix ratio in the Raman spectra was evaluated with multiple different peaks representing the organic matrix. The results showed high correlation with each other. After comparison with the bone mineral density (BMD) values from micro-computed tomography (micro-CT) imaging measurements and crystal size from XRD measurements, it is suggested that Raman microspectroscopy is more sensitive than FT-IR microspectroscopy for the inorganic matrix of the bone. In the literature, similar spectroscopic parameters obtained with FT-IR and NIR Raman microspectroscopic techniques are often compared. According to the present results, however, caution is required when performing this kind of comparison.     

Simple identification of a cross-linked hemoglobin by tandem mass spectrometry in human serum

Hemoglobin-based oxygen therapeutics are prepared by reaction of hemoglobin with cross-linking molecules and are utilized as blood substitutes. They can be used as doping agents to increase the oxygen-carrying capacity of hemoglobin. We have compared a glutaraldehyde-polymerized bovine hemoglobin (Oxyglobin, Biopure Corp.) with natural bovine hemoglobin by mass spectrometry in order to detect specific fragment ions of the cross-linked protein for further potential applications in doping control of human blood samples. HCl acid (6 N) hydrolysis was performed in parallel on both proteins. Hydrolysates were then analyzed by direct infusion electrospray mass spectrometry (ESIMS) using a triple quadrupole mass spectrometer. Confirmation and precision were obtained by LC-ESIMS(n) experiments performed on an ion trap mass spectrometer. Chromatographic and mass spectrometry data allowed detection of two potential Oxyglobin-specific ions--m/z 299 and 399--that were shown to lose a 159 u neutral fragment under collision-induced dissociation conditions. Thus, monitoring of constant neutral loss of 159 u on acid hydrolysates of human serum samples spiked with different amounts of Oxyglobin has proved to be an efficient screening method to specifically detect and identify Oxyglobin. LC-MS of the spiked serum sample hydrolysates enabled detection of Oxyglobin at a detection limit of 4 g x L(-1).