TOF-SIMS analysis of exhaled particles from patients with asthma and healthy controls.

Particles in exhaled air (PEx) may reflect the composition of respiratory tract lining fluid (RTLF); thus, there is a need to assess their potential as sources of biomarkers for respiratory diseases. In the present study, we compared PEx from patients with asthma and controls using time-of-flight-secondary ion mass spectrometry (TOF-SIMS) and multivariate analysis. Particles were collected using an instrument developed in-house. 15 nonsmoking subjects with physician-diagnosed asthma and 11 nonsmoking healthy controls performed 10 consecutive forced exhalations into the instrument. Particle concentrations were recorded and samples of particles collected on silicon plates were analysed by TOF-SIMS. Subjects with asthma exhaled significantly lower numbers of particles than controls (p=0.03) and the ratio of unsaturated to saturated phospholipids was significantly lower in samples from subjects with asthma (0.25 versus 0.35; p=0.036). Orthogonal partial least squares-discriminant analysis models showed good separation between both positive and negative spectra. Molecular ions from phosphatidylcholine and phosphatidylglycerol, and protein fragments were found to discriminate the groups. We conclude that analysis of PEx is a promising method to examine the composition of RTLF. In the present explorative study, we could discriminate between subjects with asthma and healthy controls based on TOF-SIMS spectra from PEx.

Free-form-fabricated commercially pure Ti and Ti6Al4V porous scaffolds support the growth of human embryonic stem cell-derived mesodermal progenitors.

Commercially-pure titanium (cp-Ti) and the titanium-aluminum-vanadium alloy (Ti6Al4V) are widely used as reconstructive implants for skeletal engineering applications, due to their good mechanical properties, biocompatibility and ability to integrate with the surrounding bone. Electron beam melting technology (EBM) allows the fabrication of customized implants with tailored mechanical properties and high potential in the clinical practice. In order to augment the interaction with the biological tissue, stem cells have recently been combined with metallic scaffolds for skeletal engineering applications. We previously demonstrated that human embryonic stem cell-derived mesodermal progenitors (hES-MPs) hold a great potential to provide a homogeneous and unlimited supply of cells for bone engineering applications. This study demonstrates the effect of EBM-fabricated cp-Ti and Ti6Al4V porous scaffolds on hES-MPs behavior, in terms of cell attachment, growth and osteogenic differentiation. Displaying different chemical composition but similar surface properties, EBM-fabricated cp-Ti and Ti6Al4V scaffolds supported cell attachment and growth, and did not seem to alter the expression of genes involved in osteogenic differentiation and affect the alkaline phosphatase activity. In conclusion, interfacing hES-MPs to EBM-fabricated scaffolds may represent an interesting strategy for design of third-generation biomaterials, with the potential to promote implant integration in clinical conditions characterized by poor bone quality.

Microbe-like inclusions in tree resins and implications for the fossil record of protists in amber.

During the past two decades, a plethora of fossil micro-organisms have been described from various Triassic to Miocene ambers. However, in addition to entrapped microbes, ambers commonly contain microscopic inclusions that sometimes resemble amoebae, ciliates, microfungi, and unicellular algae in size and shape, but do not provide further diagnostic features thereof. For a better assessment of the actual fossil record of unicellular eukaryotes in amber, we studied equivalent inclusions in modern resin of the Araucariaceae; this conifer family comprises important amber-producers in Earth history. Using time-of-flight secondary ion mass spectrometry (ToF-SIMS), we investigated the chemical nature of the inclusion matter and the resin matrix. Whereas the matrix, as expected, showed a more hydrocarbon/aromatic-dominated composition, the inclusions contain abundant salt ions and polar organics. However, the absence of signals characteristic for cellular biomass, namely distinctive proteinaceous amino acids and lipid moieties, indicates that the inclusions do not contain microbial cellular matter but salts and hydrophilic organic substances that probably derived from the plant itself. Rather than representing protists or their remains, these microbe-like inclusions, for which we propose the term 'pseudoinclusions', consist of compounds that are immiscible with the terpenoid resin matrix and were probably secreted in small amounts together with the actual resin by the plant tissue. Consequently, reports of protists from amber that are only based on the similarity of the overall shape and size to extant taxa, but do not provide relevant features at light-microscopical and ultrastructural level, cannot be accepted as unambiguous fossil evidence for these particular groups.

Material-tissue interfaces: the role of surface properties and processes.

The introduction of a foreign material into living tissue--intentionally as in biomedical applications (implants, protheses, drugs) or unintentionally as when minerals or fibers are inhaled--results in the creation of interfaces between the material and the surrounding tissue. This article identifies and discusses the possible role of material surface properties and molecular processes occurring at such interfaces. For kinetic and thermodynamic reasons, surfaces are different from the corresponding bulk of the material, and contain reactive (unsaturated) bonds, which in turn lead to the formation of surface reactive layers (e.g., surface oxides on metals) and adsorbed contamination layers. The encounter with the biological environment leads to further surface reactions modifying the surface, and to the adsorption of water, ions, and biomolecules, which are continuously exchanged. The exact nature of the dynamic, adsorbed water, ions, and biomolecule coating in turn influences the behavior of cells approaching the material surface, and hence the tissue response.

Surface spectroscopic characterization of titanium implants after separation from plastic-embedded tissue.

The method of plastic embedding of tissue and implant and subsequent separation of plastic and implant for preparing sections of tissue adjacent to solid metallic implants relies on a successful separation of the embedment and the implant. In this work, the surface of machined Ti implants has been analysed in order to investigate to what extent plastic remnants exist on the implant after separation. SEM and AES analyses show that at least 70% of the implant surface is free of plastic remnants to a proximity of 10 nm or less from the implant surface. The method is simple and suitable for both light and transmission electron microscopy of the interface tissue.

Accelerated oxide growth on titanium implants during autoclaving caused by fluorine contamination.

Titanium implants were occasionally found to be strongly discoloured after autoclaving. The discolouration is shown to be due to an accelerated growth of the surface oxide that covers the implants. Oxide thicknesses up to 650 A have been observed, i.e. more than ten times thicker than on normal implants. By applying surface sensitive spectroscopies (SIMS and XPS or ESCA) it is also shown that these oxide films contain considerable amounts of fluorine, alkali metals and silicon. Screening tests with alkali-halide solutions identify fluorine as the impurity responsible for the accelerated oxide growth. Discolouration after autoclaving can be observed for fluorine contaminations down to the ppm level. In those cases where discolouration was observed in the clinical situation, the source of fluorine was the textile cloths in which the titanium implant storage box had been wrapped during the autoclaving procedure. The cloths contained residual Na2SiF6 which had been used as an additive to the rinsing water used in the last step of the cloth laundry procedure. Since the biocompatibility of titanium implants is closely related to their surface oxides it is advisable to avoid all sources of fluorine in the implant preparation procedures.

Interactions between human whole blood and modified TiO2-surfaces: influence of surface topography and oxide thickness on leukocyte adhesion and activation.

An in vitro model (Nygren et al., J Lab Clin Med 129 (1997) 35-46) was used to investigate interactions between leukocytes and four modified TiO2-surfaces. Surface topography was measured using scanning electron microscopy and optical profilometry while Auger electron spectroscopy was used to determine surface composition and oxide thickness. The surfaces were either smooth or rough with either thin or thick oxides. All surfaces consisted of TiO2 covered by a carbonaceous layer. The surfaces were incubated with capillary blood for time periods of between 8 min and 32 h. Immunofluorescence techniques together with computer aided image analysis and chemiluminescence technique were used to detect cell adhesion, expression of adhesion receptors and the zymosan-stimulated respiratory burst response. Leukocyte adhesion to the surfaces increased during the first hours of blood-material contact and then decreased. Polymorphonuclear granulocytes were the dominating leukocytes on all surfaces followed by monocytes. Cells adhering to rough surfaces had higher normalized expression of adhesive receptors than cells on smooth surfaces. Maximum respiratory burst response occurred earlier on the smooth than on the rough surfaces. In conclusion, topography had a greater impact than oxide thickness on most cellular reactions investigated, but the latter often had a dampening effect on the responses.

Structure and surface of TiNi human implants.

The surface and the "bulk" structure of TiNi implants were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoemission spectroscopy (XPS), and scanning Auger microprobe analysis (AES). TiNi implants were compared with otherwise identically prepared non-implanted specimens, and sputter-cleaned and reoxidized samples. Non-implanted and implanted samples had essentially the same surface topography and microstructure. Ti, O, and C were the dominant elements detected on the surface. Trace amounts (approximately 1 at%) of Ni and Ca, N, Si, B, and S were also detected. Ti was present as TiO2 on the surface, while nickel was present in metallic form. A significant difference in Ni peak intensity was observed when retrieved or non-implanted control samples (a very low nickel content) were compared with sputter-cleaned and reoxidized samples (well-detected nickel). It is evident that the method of passivation is crucial for nickel loosening. No major changes occurred in the TiNi samples bulk structure or in the surface oxide during the implantation periods investigated.

Titanium with different oxides: in vitro studies of protein adsorption and contact activation.

Adsorption of albumin (HSA) and fibrinogen (Fib) from human blood plasma onto titanium surfaces with varying oxide properties was studied with an enzyme-linked immunosorbent assay. The intrinsic activation of blood coagulation (contact activation) was studied in vitro using a kallikrein-sensitive substrate. The sample surfaces were characterized with Fourier transform Raman spectroscopy. Auger electron spectroscopy and atomic force microscopy. Low Fib and high HSA adsorption was observed for all titanium samples except for the radio frequency plasma-treated and water-incubated samples, which adsorbed significantly lower amounts of both. Oxide thickness and carbon contamination showed no influence on protein adsorption or contact activation. Smooth samples with a surface roughness (Rrms) < 1 nm showed some correlation between surface wettability and adsorption of Fib and HSA, whereas rough surfaces (Rrms > 5 nm) did not. To varying degrees, all titanium surfaces indicated activation of the intrinsic pathway of coagulation as determined by their kallikrein formation in plasma.

Characterization of surface roughness in titanium dental implants measured with scanning tunnelling microscopy at atmospheric pressure.

Characterization of the surface topography of implant materials is important for understanding tissue response. We have measured, for the first time, the topography of titanium surfaces used in osseointegrated dental implants. Scanning tunnelling microscopy (STM) which provides 3D real space images was used. In addition to clinical samples, electropolished and anodically oxidized surfaces were also measured. Clinical samples are rather inhomogeneous in character showing grooves and steps with a maximum depth of 0.11 micron. Micropores with an average diameter of about 30 nm are also present. Electropolished samples are rather homogeneous and very smooth, showing steps of 1 to 5 nm in height. The measurements were performed under atmospheric conditions at a resolution in the subnanometer range.

Bone response to surface-modified titanium implants: studies on the early tissue response to machined and electropolished implants with different oxide thicknesses.

The bone formation around titanium implants with varied surface properties is investigated. Machined and electropolished samples with and without thick, anodically formed surface oxides were prepared, surface characterized and inserted in the cortical bone of rabbits (1, 3 and 6 weeks). Scanning electron microscopy, scanning Auger electron spectroscopy and atomic force microscopy revealed marked differences in oxide thickness, surface topography and roughness, but no significant differences in surface chemical composition, between the different groups of implants. Light microscopic morphology and morphometry showed that all implants were in contact with bone and had a large proportion of bone within the threads at 6 weeks. The smooth, electropolished implants, irrespective of anodic oxidation, were surrounded by less bone than the machined implants after 1 week. After 6 weeks the bone volume as well as the bone-implant contact were lower for the merely electropolished implants than for the other three groups. Our study shows that a high degree of bone contact and bone formation are achieved with titanium implants which are modified with respect to oxide thickness and surface topography. However, the result with the smooth (electropolished) implants indicates that a reduction of surface roughness, in the initial phase, decreases the rate of bone formation in rabbit cortical bone.

Bone response to surface modified titanium implants: studies on electropolished implants with different oxide thicknesses and morphology.

In a series of experimental studies, bone formation was analysed around systematically modified titanium implants. In the present study, machined, electropolished and anodically oxidized implants were prepared, surface characterized and inserted in the cortical bone of rabbits (7 wks and 12 wks). SEM, scanning Auger electron spectroscopy and atomic force microscopy revealed no differences in surface composition but marked differences in oxide thickness, surface topography and roughness. Light microscopic morphology and morphometry showed that all implants were in contact with bone, and had a large proportion of bone within the threads. The smooth, electropolished implants were surrounded by less bone than the machined implants with similar oxide thickness, (4-5 nm) and the anodically oxidized implants with thicker oxides (21 nm and 180 nm, respectively) after 7 wks. These studies show that a high degree of bone contact and bone formation can be achieved with titanium implants which are modified with respect to oxide thickness and surface topography. However, it appears that a reduction of surface roughness may influence the rate of bone formation in rabbit cortical bone.

Method for ultrastructural studies of the intact tissue-metal interface.

Samples were prepared for ultrastructural studies of the intact interface between metallic implants and tissue by transmission electron microscopy. The method is based on plastic embedding of implant and tissue and subsequent removal of the bulk metal by electrochemical dissolution (electropolishing), to facilitate preparation of ultrathin sections for transmission electron microscopy. Surface sensitive spectroscopy (Auger electron microscopy and X-ray photoemission spectroscopy) and transmission electron microscopy EDX results show that the method produces samples with an intact interface, containing the implant surface oxide and the adjacent tissue. Examples of application of the method on titanium, zirconium and aluminium implants in soft tissue are given.

Site-specific adhesion of Staphylococcus epidermidis (RP12) in Ti-Al-V metal systems.

Staphylococcus epidermidis (RP12) adhesion patterns were studied on the following titanium (Ti)-aluminium (Al)-vanadium (V) metal systems: (i) microfabricated samples consisting of Ti, Al and V islands deposited onto Ti or V substrata, (ii) pure Ti, Al and V metals, and (iii) medical grade Ti6Al4-V alloy. All of these surfaces were covered with their respective oxides formed upon exposure of the metals to air. Quantitative analysis of the number of cells bound per unit area indicates that S. epidermidis (RP12) exhibits greatest adhesion to pure V surfaces. When exposed to surfaces having controlled spatial variations in chemical composition on the 10 microns scale (microfabricated samples), the bacteria preferentially populate V islands versus Ti or Al substrata. In the case of the biphasic Ti6Al4V alloy, the bacteria tend to adhere to V-rich, mixed phase regions and phase boundaries. These findings demonstrate that enhanced and preferential adhesion of S. epidermidis (RP12) occurs on V surfaces in Ti-Al-V metal systems and suggest that bacterial interactions are influenced by surface oxide composition.

Osseointegration of fiber-reinforced composite implants: histological and ultrastructural observations.

OBJECTIVES: The aim of this study was to evaluate the bone tissue response to fiber-reinforced composite (FRC) in comparison with titanium (Ti) implants after 12 weeks of implantation in cancellous bone using histomorphometric and ultrastructural analysis. MATERIALS AND METHODS: Thirty grit-blasted cylindrical FRC implants with BisGMA-TEGDMA polymer matrix were fabricated and divided into three groups: (1) 60s light-cured FRC (FRC-L group), (2) 24h polymerized FRC (FRC group), and (3) bioactive glass FRC (FRC-BAG group). Titanium implants were used as a control group. The surface analyses were performed with scanning electron microscopy and 3D SEM. The bone-implant contact (BIC) and bone area (BA) were determined using histomorphometry and SEM. Transmission electron microscopy (TEM) was performed on Focused Ion Beam prepared samples of the intact bone-implant interface. RESULTS: The FRC, FRC-BAG and Ti implants were integrated into host bone. In contrast, FRC-L implants had a consistent fibrous capsule around the circumference of the entire implant separating the implant from direct bone contact. The highest values of BIC were obtained with FRC-BAG (58±11%) and Ti implants (54±13%), followed by FRC implants (48±10%), but no significant differences in BIC or BA were observed (p=0.07, p=0.06, respectively). TEM images showed a direct contact between nanocrystalline hydroxyapatite of bone and both FRC and FRC-BAG surfaces. CONCLUSION: Fiber-reinforced composite implants are capable of establishing a close bone contact comparable with the osseointegration of titanium implants having similar surface roughness.

Bone response to surface-modified titanium implants: studies on the early tissue response to implants with different surface characteristics.

In a series of experimental studies, the bone formation around systematically modified titanium implants is analyzed. In the present study, three different surface modifications were prepared and evaluated. Glow-discharge cleaning and oxidizing resulted in a highly stoichiometric TiO2 surface, while a glow-discharge treatment in nitrogen gas resulted in implants with essentially a surface of titanium nitride, covered with a very thin titanium oxide. Finally, hydrogen peroxide treatment of implants resulted in an almost stoichiometric TiO2, rich in hydroxyl groups on the surface. Machined commercially pure titanium implants served as controls. Scanning Auger Electron Spectroscopy, Scanning Electron Microscopy, and Atomic Force Microscopy revealed no significant differences in oxide thickness or surface roughness parameters, but differences in the surface chemical composition and apparent topography were observed. After surface preparation, the implants were inserted in cortical bone of rabbits and evaluated after 1, 3, and 6 weeks. Light microscopic evaluation of the tissue response showed that all implants were in contact with bone and had a large proportion of newly formed bone within the threads after 6 weeks. There were no morphological differences between the four groups. Our study shows that a high degree of bone contact and bone formation can be achieved with titanium implants of different surface composition and topography.

Ancient microbial activity recorded in fracture fillings from granitic rocks (Äspö Hard Rock Laboratory, Sweden).

Fracture minerals within the 1.8-Ga-old Äspö Diorite (Sweden) were investigated for fossil traces of subterranean microbial activity. To track the potential organic and inorganic biosignatures, an approach combining complementary analytical techniques of high lateral resolution was applied to drill core material obtained at -450 m depth in the Äspö Hard Rock Laboratory. This approach included polarization microscopy, time-of-flight secondary ion mass spectrometry (ToF-SIMS), confocal Raman microscopy, electron microprobe (EMP) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The fracture mineral succession, consisting of fluorite and low-temperature calcite, showed a thin (20-100 µm), dark amorphous layer lining the boundary between the two phases. Microscopic investigations of the amorphous layer revealed corrosion marks and, in places, branched tubular structures within the fluorite. Geochemical analysis showed significant accumulations of Si, Al, Mg, Fe and the light rare earth elements (REE) in the amorphous layer. In the same area, ToF-SIMS imaging revealed abundant, partly functionalized organic moieties, for example, C(x)H(y)⁺, C(x)H(y)N⁺, C(x)H(y)O⁺. The presence of such functionalized organic compounds was corroborated by Raman imaging showing bands characteristic of C-C, C-N and C-O bonds. According to its organic nature and the abundance of relatively unstable N- and O- heterocompounds, the organic-rich amorphous layer is interpreted to represent the remains of a microbial biofilm that established much later than the initial cooling of the Precambrian host rock. Indeed, δ¹³C, δ¹8O and 87Sr/86Sr isotope data of the fracture minerals and the host rock point to an association with a fracture reactivation event in the most recent geological past.

Analysis of hopanes and steranes in single oil-bearing fluid inclusions using time-of-flight secondary ion mass spectrometry (ToF-SIMS).

Steranes and hopanes are organic biomarkers used as indicators for the first appearance of eukaryotes and cyanobacteria on Earth. Oil-bearing fluid inclusions may provide a contamination-free source of Precambrian biomarkers, as the oil has been secluded from the environment since the formation of the inclusion. However, analysis of biomarkers in single oil-bearing fluid inclusions, which is often necessary due to the presence of different generations of inclusions, has not been possible due to the small size of most inclusions. Here, we have used time-of-flight secondary ion mass spectrometry (ToF-SIMS) to monitor in real time the opening of individual inclusions trapped in hydrothermal veins of fluorite and calcite and containing oil from Ordovician source rocks. Opening of the inclusions was performed by using a focused C(60)(+) ion beam and the in situ content was precisely analysed for C(27)-C(29) steranes and C(29)-C(32) hopanes using Bi(3)(+) as primary ions. The capacity to unambiguously detect these biomarkers in the picoliter amount of crude oil from a single, normal-sized (15-30 mum in diameter) inclusion makes the approach promising in the search of organic biomarkers for life's early evolution on Earth.