Surface characterization, in vitro and in vivo biocompatibility of Mg-0.3Sr-0.3Ca for temporary cardiovascular implant.

Magnesium-based alloys are attractive candidate materials for medical applications. Our earlier work showed that the ternary Mg-0.3Sr-0.3Ca alloy exhibits slower degradation rates than both binary Mg-Sr and Mg-Ca alloys. The ternary alloy immersed in simulated body fluid (SBF) forms a compact surface layer of corrosion products that we hypothesized to be a Sr-substituted hydroxyapatite (HA). The main objectives of the current work are to understand the bio-degradation mechanism of Mg-0.3Sr-0.3Ca, to identify the exact nature of its protective layer and to evaluate the in vitro and in vivo biocompatibility of the alloy for cardiovascular applications. To better simulate the physiological environment, the alloy was immersed in SBF which was daily refreshed. Raman spectroscopy and X-Ray photoelectron spectroscopy (XPS) confirmed the formation of a thin, Sr-substituted HA layer at the interface between the alloy and the corrosion products. In vitro biocompatibility evaluated via indirect cytotoxicity assays using HUVECs showed no toxicity effect and ions extracted from Mg-0.3Sr-0.3Ca in fact increased the viability of HUVECs after one week. In vivo tests were performed by implanting a tubular Mg-0.3Sr-0.3Ca stent along with a WE43 control stent into the right and left femoral artery of a dog. Post implantation and histological analyses showed no thrombosis in the artery with Mg-0.3Sr-0.3Ca stent after 5weeks of implantation while the artery implanted with WE43 stent was extensively occluded and thrombosed. Microscopic observation of the Mg-0.3Sr-0.3Ca implant-tissue interface confirmed the in situ formation of Sr-substituted HA on the surface during in vivo test. These results show that the interfacial layer protects the surface of the Mg-0.3Sr-0.3Ca alloy both in vitro and in vivo, and is the key factor in the bio-corrosion resistance of the alloy.

Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: an ultrastructural, compositional and comparative analysis with mouse bone.

Bone cell culture systems are essential tools for the study of the molecular mechanisms regulating extracellular matrix mineralization. MC3T3-E1 osteoblast cell cultures are the most commonly used in vitro model of bone matrix mineralization. Despite the widespread use of this cell line to study biomineralization, there is as yet no systematic characterization of the mineral phase produced in these cultures. Here we provide a comprehensive, multi-technique biophysical characterization of this cell culture mineral and extracellular matrix, and compare it to mouse bone and synthetic apatite mineral standards, to determine the suitability of MC3T3-E1 cultures for biomineralization studies. Elemental compositional analysis by energy-dispersive X-ray spectroscopy (EDS) showed calcium and phosphorus, and trace amounts of sodium and magnesium, in both biological samples. X-ray diffraction (XRD) on resin-embedded intact cultures demonstrated that similar to 1-month-old mouse bone, apatite crystals grew with preferential orientations along the (100), (101) and (111) mineral planes indicative of guided biogenic growth as opposed to dystrophic calcification. XRD of crystals isolated from the cultures revealed that the mineral phase was poorly crystalline hydroxyapatite with 10 to 20nm-sized nanocrystallites. Consistent with the XRD observations, electron diffraction patterns indicated that culture mineral had low crystallinity typical of biological apatites. Fourier-transform infrared spectroscopy (FTIR) confirmed apatitic carbonate and phosphate within the biological samples. With all techniques utilized, cell culture mineral and mouse bone mineral were remarkably similar. Scanning (SEM) and transmission (TEM) electron microscopy showed that the cultures had a dense fibrillar collagen matrix with small, 100nm-sized, collagen fibril-associated mineralization foci which coalesced to form larger mineral aggregates, and where mineralized sites showed the accumulation of the mineral-binding protein osteopontin. Light microscopy, confocal microscopy and three-dimensional reconstructions showed that some cells had dendritic processes and became embedded within the mineral in an osteocyte-like manner. In conclusion, we have documented characteristics of the mineral and matrix phases of MC3T3-E1 osteoblast cultures, and have determined that the structural and compositional properties of the mineral are highly similar to that of mouse bone.

A mineralogical characterization of biogenic calcium carbonates precipitated by heterotrophic bacteria isolated from cryophilic polar regions.

Precipitation of calcium carbonate (CaCO3(s) ) can be driven by microbial activity. Here, a systematic approach is used to identify the morphological and mineralogical characteristics of CaCO3(s) precipitated during the heterotrophic growth of micro-organisms isolated from polar environments. Focus was placed on establishing mineralogical features that are common in bioliths formed during heterotrophic activity, while in parallel identifying features that are specific to bioliths precipitated by certain microbial phylotypes. Twenty microbial isolates that precipitated macroscopic CaCO3(s) when grown on B4 media supplemented with calcium acetate or calcium citrate were identified. A multimethod approach, including scanning electron microscopy, high-resolution transmission electron microscopy, and micro-X-ray diffraction (µ-XRD), was used to characterize CaCO3(s) precipitates. Scanning and transmission electron microscopy showed that complete CaCO3(s) crystal encrustation of Arthrobacter sp. cells was common, while encrustation of Rhodococcus sp. cells did not occur. Several euhedral and anhedral mineral formations including disphenoid-like epitaxial plates, rhomboid-like aggregates with epitaxial rhombs, and spherulite aggregates were observed. While phylotype could not be linked to specific mineral formations, isolates tended to precipitate either euhedral or anhedral minerals, but not both. Three anhydrous CaCO3(s) polymorphs (calcite, aragonite, and vaterite) were identified by µ-XRD, and calcite and aragonite were also identified based on TEM lattice-fringe d value measurements. The presence of certain polymorphs was not indicative of biogenic origin, although several mineralogical features such as crystal-encrusted bacterial cells, or casts of bacterial cells embedded in mesocrystals are an indication of biogenic origin. In addition, some features such as the formation of vaterite and bacterial entombment appear to be linked to certain phylotypes. Identifying phylotypes consistent with certain mineralogical features is the first step toward discovering a link between these crystal features and the precise underlying molecular biology of the organism precipitating them.

14-3-3 protects against stress-induced apoptosis.

Expression of human Bax, a cardinal regulator of mitochondrial membrane permeabilization, causes death in yeast. We screened a human cDNA library for suppressors of Bax-mediated yeast death and identified human 14-3-3ß/α, a protein whose paralogs have numerous chaperone-like functions. Here, we show that, yeast cells expressing human 14-3-3ß/α are able to complement deletion of the endogenous yeast 14-3-3 and confer resistance to a variety of different stresses including cadmium and cycloheximide. The expression of 14-3-3ß/α also conferred resistance to death induced by the target of rapamycin inhibitor rapamycin and by starvation for the amino acid leucine, conditions that induce autophagy. Cell death in response to these autophagic stimuli was also observed in the macroautophagic-deficient atg1Δ and atg7Δ mutants. Furthermore, 14-3-3ß/α retained its ability to protect against the autophagic stimuli in these autophagic-deficient mutants arguing against so called 'autophagic death'. In line, analysis of cell death markers including the accumulation of reactive oxygen species, membrane integrity and cell surface exposure of phosphatidylserine indicated that 14-3-3ß/α serves as a specific inhibitor of apoptosis. Finally, we demonstrate functional conservation of these phenotypes using the yeast homolog of 14-3-3: Bmh1. In sum, cell death in response to multiple stresses can be counteracted by 14-3-3 proteins.

Cyanobacterial diversity and activity in modern conical microbialites.

Modern conical microbialites are similar to some ancient conical stromatolites, but growth, behavior and diversity of cyanobacteria in modern conical microbialites remain poorly characterized. Here, we analyze the diversity of cyanobacterial 16S rRNA gene sequences in conical microbialites from 14 ponds fed by four thermal sources in Yellowstone National Park and compare cyanobacterial activity in the tips of cones and in the surrounding topographic lows (mats), respectively, by high-resolution mapping of labeled carbon. Cones and adjacent mats contain similar 16S rRNA gene sequences from genetically distinct clusters of filamentous, non-heterocystous cyanobacteria from Subsection III and unicellular cyanobacteria from Subsection I. These sequences vary among different ponds and between two sampling years, suggesting that coniform mats through time and space contain a number of cyanobacteria capable of vertical aggregation, filamentous cyanobacteria incapable of initiating cone formation and unicellular cyanobacteria. Unicellular cyanobacteria are more diverse in topographic lows, where some of these organisms respond to nutrient pulses more rapidly than thin filamentous cyanobacteria. The densest active cyanobacteria are found below the upper 50 µm of the cone tip, whereas cyanobacterial cells in mats are less dense, and are more commonly degraded or encrusted by silica. These spatial differences in cellular activity and density within macroscopic coniform mats imply a strong role for diffusion limitation in the development and the persistence of the conical shape. Similar mechanisms may have controlled the growth, morphology and persistence of small coniform stromatolites in shallow, quiet environments throughout geologic history.

Degeneration of biogenic superparamagnetic magnetite.

Magnetite crystals precipitated as a consequence of Fe(III) reduction by Shewanella algae BrY after 265 h incubation and 5-year anaerobic storage were investigated with transmission electron microscopy, Mössbauer spectroscopy and X-ray diffraction. The magnetite crystals were typically superparamagnetic with an approximate size of 13 nm. The lattice constants of the 265 h and 5-year crystals are 8.4164A and 8.3774A, respectively. The Mössbauer spectra indicated that the 265 h magnetite had excess Fe(II) in its crystal-chemistry (Fe(3+) (1.990)Fe(2+) (1.015)O(4)) but the 5-year magnetite was Fe(II)-deficient in stoichiometry (Fe(3+) (2.388)Fe(2+) (0.419)O(4)). Such crystal-chemical changes may be indicative of the degeneration of superparamagnetic magnetite through the aqueous oxidization of Fe(II) anaerobically, and the concomitant oxidation of the organic phases (fatty acid methyl esters) that were present during the initial formation of the magnetite. The observation of a corona structure on the aged magnetite corroborates the anaerobic oxidation of Fe(II) on the outer layers of magnetite crystals. These results suggest that there may be a possible link between the enzymatic activity of the bacteria and the stability of Fe(II)-excess magnetite, which may help explain why stable nano-magnetite grains are seldom preserved in natural environments.

Ultrastructural matrix-mineral relationships in avian eggshell, and effects of osteopontin on calcite growth in vitro.

We investigated matrix-mineral relationships in the avian eggshell at the ultrastructural level using scanning and transmission electron microscopy combined with surface-etching techniques to selectively increase topography at the matrix-mineral interface. Moreover, we investigated the distribution of osteopontin (OPN) in the eggshell by colloidal-gold immunolabeling for OPN, and assessed the effects of this protein on calcite crystal growth in vitro. An extensive organic matrix network was observed within the calcitic structure of the eggshell that showed variable, region-specific organization including lamellar sheets of matrix, interconnected fine filamentous threads, thin film-like surface coatings of proteins, granules, vesicles, and isolated proteins residing preferentially on internal {104} crystallographic faces of fractured eggshell calcite. With the exception of the vesicles and granules, these matrix structures all were immunolabeled for OPN, as were occluded proteins on the {104} calcite faces. OPN inhibited calcite growth in vitro at the {104} crystallographic faces producing altered crystal morphology and circular growth step topography at the crystal surface resembling spherical voids in mineral continuity prominent in the palisades region of the eggshell. In conclusion, calcite-occluded and interfacial proteins such as OPN likely regulate eggshell growth by inhibiting calcite growth at specific crystallographic faces and compartmental boundaries to create a biomineralized architecture whose structure provides for the properties and functions of the eggshell.

Extracellular synthesis of magnetite and metal-substituted magnetite nanoparticles.

We have developed a novel microbial process that exploits the ability of Fe(III)-reducing microorganisms to produce copious amounts of extracellular magentites and metal-substituted magnetite nanoparticles. The Fe(III)-reducing bacteria (Theroanaerobacter ethanolicus and Shewanella sp.) have the ability to reduce Fe(III) and various metals in aqueous media and form various sized magnetite and metal-substituted magnetite nano-crystals. The Fe(III)-reducing bacteria formed metalsubstituted magnetites using iron oxide plus metals (e.g., Co, Cr, Mn, Ni) under conditions of relatively low temperature (<70 degrees C), ambient pressure, and pH values near neutral to slightly basic (pH = 6.5 to 9). Precise biological control over activation and regulation of the biosolid-state processes can produce magnetite particles of well-defined size (typically tens of nanometers) and crystallographic morphology, containing selected dopant metals into the magnetite (Fe(3-y)XyO4) structure (where X = Co, Cr, Mn, Ni). Magnetite yields of up to 20 g/L per day have been observed in 20-L vessels. Water-based ferrofluids were formed with the nanometer sized, magnetite, and metal-substituted biomagnetite particles.

Correlation of cell necrosis and tissue calcification with ischemia/reperfusion injury after liver transplantation.

BACKGROUND: The cellular events following liver ischemia/reperfusion (I/R) during transplantation are largely unknown. The spectrum of I/R damage to the liver can be clinically revealed by the development of primary graft dysfunction or nonfunction. Because viral-induced liver necrosis has been associated with the development of calcifications in an animal model, we investigated the spectrum of I/R changes identified at an ultrastructural level among livers after liver transplant (LT). MATERIALS AND METHODS: Random liver biopsies from five recipients with different degrees of liver dysfunction (LD) were processed for light (LM) and electron (EM) microscopic examination. The degree of calcification was estimated as mild-moderate or severe. The degree of cell vacuolization, used as a surrogate marker of cell necrosis, was reported as mild-moderate or severe. RESULTS: Two patients with severe LD had obvious calcifications by LM and EM examinations. Both showed significant vacuole formation, suggesting a severe degree of cell necrosis, and both succumbed to the sequelae of their LD. One patient showed evidence of mild calcifications at EM (but not LM) examination, with mild vacuole formation. The remaining two patients displayed no microcalcifications. Both presented only mild vacuole formation. Both patients recovered from LD and are currently alive. CONCLUSION: In this preliminary report, we conclude that the clinically observed degree of LD after orthotopic liver transplant (OLT)correlates well with ultrastructural modifications. These include calcification and vacuole formation. We believe that both findings can be used as surrogate markers of a clinically significant hepatic I/R injury.

Lung mineral fibers of former miners and millers from Thetford-Mines and asbestos regions: a comparative study of fiber concentration and dimension.

Fiber dimension and concentration may vary substantially between two necropsy populations of former chrysotile miners and millers of Thetford-Mines and Asbestos regions. This possibility could explain, at least in part, the higher incidence of respiratory diseases among workers from Thetford-Mines than among workers from the Asbestos region. The authors used a transmission electron microscope, equipped with an x-ray energy-dispersive spectrometer, to analyze lung mineral fibers of 86 subjects from the two mining regions and to classify fiber sizes into three categories. The most consistent difference was the higher concentration of tremolite in lung tissues of workers from Thetford-Mines, compared with workers from the Asbestos region. Amosite and crocidolite were also detected in lung tissues of several workers from the Asbestos region. No consistent and biologically important difference was found for fiber dimension; therefore, fiber dimension does not seem to be a factor that accounts for the difference in incidence of respiratory diseases between the two groups. The greater incidence of respiratory diseases among workers of Thetford-Mines can be explained by the fact that they had greater exposure to fibers than did workers at the Asbestos region. Among the mineral fibers studied, retention of tremolite fibers was most apparent.

Truncated hexa-octahedral magnetite crystals in ALH84001: presumptive biosignatures.

McKay et al. [(1996) Science 273, 924-930] suggested that carbonate globules in the meteorite ALH84001 contained the fossil remains of Martian microbes. We have characterized a subpopulation of magnetite (Fe(3)O(4)) crystals present in abundance within the Fe-rich rims of these carbonate globules. We find these Martian magnetites to be both chemically and physically identical to terrestrial, biogenically precipitated, intracellular magnetites produced by magnetotactic bacteria strain MV-1. Specifically, both magnetite populations are single-domain and chemically pure, and exhibit a unique crystal habit we describe as truncated hexa-octahedral. There are no known reports of inorganic processes to explain the observation of truncated hexa-octahedral magnetites in a terrestrial sample. In bacteria strain MV-1 their presence is therefore likely a product of Natural Selection. Unless there is an unknown and unexplained inorganic process on Mars that is conspicuously absent on the Earth and forms truncated hexa-octahedral magnetites, we suggest that these magnetite crystals in the Martian meteorite ALH84001 were likely produced by a biogenic process. As such, these crystals are interpreted as Martian magnetofossils and constitute evidence of the oldest life yet found.

A low temperature transfer of ALH84001 from Mars to Earth.

The ejection of material from Mars is thought to be caused by large impacts that would heat much of the ejecta to high temperatures. Images of the magnetic field of martian meteorite ALH84001 reveal a spatially heterogeneous pattern of magnetization associated with fractures and rock fragments. Heating the meteorite to 40 degrees C reduces the intensity of some magnetic features, indicating that the interior of the rock has not been above this temperature since before its ejection from the surface of Mars. Because this temperature cannot sterilize most bacteria or eukarya, these data support the hypothesis that meteorites could transfer life between planets in the solar system.

Elongated prismatic magnetite crystals in ALH84001 carbonate globules: potential Martian magnetofossils.

Using transmission electron microscopy (TEM), we have analyzed magnetite (Fe3O4) crystals acid-extracted from carbonate globules in Martian meteorite ALH84001. We studied 594 magnetites from ALH84001 and grouped them into three populations on the basis of morphology: 389 were irregularly shaped, 164 were elongated prisms, and 41 were whisker-like. As a possible terrestrial analog for the ALH84001 elongated prisms, we compared these magnetites with those produced by the terrestrial magnetotactic bacteria strain MV-1. By TEM again, we examined 206 magnetites recovered from strain MV-1 cells. Natural (Darwinian) selection in terrestrial magnetotactic bacteria appears to have resulted in the formation of intracellular magnetite crystals having the physical and chemical properties that optimize their magnetic moment. In this study, we describe six properties of magnetite produced by biologically controlled mechanisms (e.g., magnetotactic bacteria), properties that, collectively, are not observed in any known population of inorganic magnetites. These criteria can be used to distinguish one of the modes of origin for magnetites from samples with complex or unknown histories. Of the ALH84001 magnetites that we have examined, the elongated prismatic magnetite particles (similar to 27% of the total) are indistinguishable from the MV-1 magnetites in five of these six characteristics observed for biogenically controlled mineralization of magnetite crystals.

Paleomagnetic evidence of a low-temperature origin of carbonate in the Martian meteorite ALH84001.

Indirect evidence for life on Mars has been reported from the study of meteorite ALH84001. The formation temperature of the carbonates is controversial; some estimates suggest 20 degrees to 80 degrees C, whereas others exceed 650 degrees C. Paleomagnetism can be used to distinguish between these possibilities because heating can remagnetize ferrimagnetic minerals. Study of two adjacent pyroxene grains from the crushed zone of ALH84001 shows that each possesses a stable natural remanent magnetization (NRM), implying that Mars had a substantial magnetic field when the grains cooled. However, NRM directions from these particles differ, implying that the meteorite has not been heated significantly since the formation of the internal crushed zone about 4 billion years ago. The carbonate globules postdate this brecciation, and thus formed at low temperatures.

Search for past life on Mars: possible relic biogenic activity in martian meteorite ALH84001.

Fresh fracture surfaces of the martian meteorite ALH84001 contain abundant polycyclic aromatic hydrocarbons (PAHs). These fresh fracture surfaces also display carbonate globules. Contamination studies suggest that the PAHs are indigenous to the meteorite. High-resolution scanning and transmission electron microscopy study of surface textures and internal structures of selected carbonate globules show that the globules contain fine-grained, secondary phases of single-domain magnetite and Fe-sulfides. The carbonate globules are similar in texture and size to some terrestrial bacterially induced carbonate precipitates. Although inorganic formation is possible, formation of the globules by biogenic processes could explain many of the observed features, including the PAHs. The PAHs, the carbonate globules, and their associated secondary mineral phases and textures could thus be fossil remains of a past martian biota.

Occurrence of magnetic bacteria in soil.

Enrichment of the ferrimagnetic minerals magnetite and maghemite is frequently observed in the top layer of soil horizons. Although both inorganic and organic processes are known to produce magnetite, magnetite in soils has been ascribed to an inorganic origin. We report here the discovery of living magnetic bacteria, similar to those found in salt- and fresh-water sediments, in the A horizon of a well developed soil profile in a typical meadow environment in southern Bavaria. The bacteria were detected in fresh samples using an optical microscope equipped with a rotating magnetic field and a volumetrically calibrated depression slide, permitting accurate counts of the volume density of the organisms. We suggest that magnetic bacteria and their magnetofossils can contribute to the magnetic properties of soils.