Nanometer scale insight on the analysis of limpets mineralized teeth: Special focus on the silica-containing regions.

We report the electron microscopy-based analysis of the major lateral tooth of the limpet Colisella subrugosa during early and intermediate stages of development. We aimed to analyze the structural relationship among the needle-like crystals of the iron oxide goethite, the amorphous silica phase that forms the tooth base and occupy inter-crystalline spaces in the cusp, and the chitin fibers of the matrix. Goethite crystals followed the three dimensional organization pattern of the chitin fibers in the cusp. In the tooth base, spherical individual silica granules were found in regions where the chitin fibers cross. The spherical granules near the interface between the tooth base and the cusp (junction zone) formed an almost continuous medium that could easily be ultrathin-sectioned for further analysis. By contrast, the nearby silica-rich region localized on the other side of the junction zone contained needle-like goethite crystals immersed in the matrix and presented a conchoidal fracture. The chitin fibers from the silica granules of the tooth base were dotted or undulating in projection with a periodicity of about 6 nm when observed by high magnification transmission electron microscopy. Very thin goethite crystals were present in the base of the cusp near the junction zone surrounded by silica. On several occasions, crystals presented internal thin straight white lines parallel to the major axis, indicating a possible growth around fibers. We propose that silica and iron oxide phases mineralization may occur simultaneously at least for some period and that silica moderates the dimensions of the iron oxide crystals.

Swimming behavior of the multicellular magnetotactic prokaryote 'Candidatus Magnetoglobus multicellularis' near solid boundaries and natural magnetic grains.

The magnetotactic yet uncultured species 'Candidatus Magnetoglobus multicellularis' is a spherical, multicellular ensemble of bacterial cells able to align along magnetic field lines while swimming propelled by flagella. Magnetotaxis is due to intracytoplasmic, membrane-bound magnetic crystals called magnetosomes. The net magnetic moment of magnetosomes interacts with local magnetic fields, imparting the whole microorganism a torque. Previous works investigated 'Ca. M. multicellularis' behavior when free swimming in water; however, they occur in sediments where bumping into solid particles must be routine. In this work, we investigate the swimming trajectories of 'Ca. M. multicellularis' close to solid boundaries using video microscopy. We applied magnetic fields 0.25-8.0 mT parallel to the optical axis of a light microscope, such that microorganisms were driven upwards towards a coverslip. Because their swimming trajectories approach cylindrical helixes, circular profiles would be expected. Nevertheless, at fields 0.25-1.1 mT, most trajectory projections were roughly sinusoidal, and net movements were approximately perpendicular to applied magnetic fields. Closed loops appeared in some trajectory projections at 1.1 mT, which could indicate a transition to the loopy profiles observed at magnetic fields ≥ 2.15 mT. The behavior of 'Ca. M. multicellularis' near natural magnetic grains showed that they were temporarily trapped by the particle's magnetic field but could reverse the direction of movement to flee away. Our results show that interactions of 'Ca. M. multicellularis with solid boundaries and magnetic grains are complex and possibly involve mechano-taxis.

Effects of cytoskeletal drugs on actin cortex elasticity.

Mechanical properties of cells are known to be influenced by the actin cytoskeleton. In this article, the action of drugs that interact with the actin cortex is investigated by tether extraction and rheology experiments using optical tweezers. The influences of Blebbistatin, Cytochalasin D and Jasplakinolide on the cell mechanical properties are evaluated. The results, in contradiction to current views for Jasplakinolide, show that all three drugs and treatments destabilize the actin cytoskeleton, decreasing the cell membrane tension. The cell membrane bending modulus increased when the actin cytoskeleton was disorganized by Cytochalasin D. This effect was not observed for Blebbistatin and Jasplakinolide. All drugs decreased by two-fold the cell viscoelastic moduli, but only Cytochalasin D was able to alter the actin network into a more fluid-like structure. The results can be interpreted as the interplay between the actin network and the distribution of myosins as actin cross-linkers in the cytoskeleton. This information may contribute to a better understanding of how the membrane and cytoskeleton are involved in cell mechanical properties, underlining the role that each one plays in these properties.

Biomineralization of calcium carbonate in the cell wall of Lithothamnion crispatum (Hapalidiales, Rhodophyta): correlation between the organic matrix and the mineral phase.

Over the past few decades, progress has been made toward understanding the mechanisms of coralline algae mineralization. However, the relationship between the mineral phase and the organic matrix in coralline algae has not yet been thoroughly examined. The aim of this study was to describe the cell wall ultrastructure of Lithothamnion crispatum, a cosmopolitan rhodolith-forming coralline algal species collected near Salvador (Brazil), and examine the relationship between the organic matrix and the nucleation and growth/shape modulation of calcium carbonate crystals. A nanostructured pattern was observed in L. crispatum along the cell walls. At the nanoscale, the crystals from L. crispatum consisted of several single crystallites assembled and associated with organic material. The crystallites in the bulk of the cell wall had a high level of spatial organization. However, the crystals displayed cleavages in the (104) faces after ultrathin sectioning with a microtome. This organism is an important model for biomineralization studies as the crystallographic data do not fit in any of the general biomineralization processes described for other organisms. Biomineralization in L. crispatum is dependent on both the soluble and the insoluble organic matrix, which are involved in the control of mineral formation and organizational patterns through an organic matrix-mediated process. This knowledge concerning the mineral composition and organizational patterns of crystals within the cell walls should be taken into account in future studies of changing ocean conditions as they represent important factors influencing the physico-chemical interactions between rhodoliths and the environment in coralline reefs.

Cell wall physicochemical properties determine the thallus biomineralization pattern of Padina gymnospora (Phaeophyceae).

Approximately half of the Padina (Dictyotales, Phaeophyceae) species mineralize aragonite needles over the adaxial thallus surface, where mineral bands are interspersed with nonmineralized regions along the thallus from the apical to basal end. However, this calcification pattern and the related algal properties are not well understood. Therefore, this work was performed to elucidate a potential role of cell walls in the inhibition/induction of mineralization in the brown alga Padina gymnospora. In a comparison of specific thallus regions, differences were identified in the cellulose distribution, microfibrils arrangement and thickness, distribution and abundance of phenolic substances, and physical differences among the surfaces of the thallus (deformation, adhesion, topography, and nano-rugosity). In vitro mineralization assays indicated that phenolic substances are strong modulators of calcium carbonate crystals growth. In addition, de novo mineralization assays over cell wall surfaces that were used as templates, even without cellular activity, indicated that the cell wall remains a key factor in the induction/inhibition of mineralization. Overall, the current findings indicate a strong correlation between the physico-chemical and structural properties of the cell wall and the alternation pattern of the mineralization bands over the thallus of P. gymnospora.

Crystallographic orientation and concentric layers in spicules of calcareous sponges.

In this work, the crystallography of calcareous sponges (Porifera) spicules and the organization pattern of the concentric layers present in their inner structure were investigated in 10 species of the subclass Calcaronea and three species of the subclass Calcinea. Polished spicules had specific concentric patterns that varied depending on the plane in which the spicules were sectioned. A 3D model of the concentric layers was created to interpret these patterns and the biomineralization process of the triactine spicules. The morphology of the spicules was compared with the crystallographic orientation of the calcite crystals by analyzing the Kikuchi diffraction patterns using a scanning electron microscope. Triactine spicules from the subclass Calcinea had actines (rays) elongated in the 〈210〉 direction, which is perpendicular to the c-axis. The scale spicules of the hypercalcified species Murrayona phanolepis presented the c-axis perpendicular to the plane of the scale, which is in accordance with the crystallography of all other Calcinea. The triactine spicules of the calcaronean species had approximately the same crystallographic orientation with the unpaired actine elongated in the ∼[211] direction. Only one Calcaronea species, whose triactine was regular, had a different orientation. Three different crystallographic orientations were found in diactines. Spicules with different morphologies, dimensions and positions in the sponge body had similar crystallographic directions suggesting that the crystallographic orientation of spicules in calcareous sponges is conserved through evolution.

Culture-independent characterization of novel psychrophilic magnetotactic cocci from Antarctic marine sediments.

Magnetotactic bacteria (MTB) are a heterogeneous group of ubiquitous aquatic microorganisms capable of biomineralizing nano-sized, membrane-bound, magnetic iron-rich mineral particles called magnetosomes. MTB are found in chemically-stratified aquatic sediments and/or water columns with a wide range of salinities, moderate to high temperatures, and pH varying from neutral to strongly alkaline. MTB from very cold environments have not been investigated to any great degree and here we characterize MTB from the low temperature Antarctic maritime region. Sediment samples were collected at nine sampling sites within Admiralty Bay, King George Island (62°23'S 58°27'W) from 2009 to 2013. Samples from five sites contained MTB and those from two of these sites contained large number of magnetotactic cocci that were studied using electron microscopy and molecular techniques. The magnetotactic cocci contained magnetosomes either arranged as two or four chains or as a disorganized cluster. The crystalline habit and composition of all magnetosomes analyzed with high-resolution transmission electron microscopy and energy dispersive X-ray microanalysis were consistent with elongated prismatic crystals of magnetite (Fe3 O4 ). The retrieved 16S rRNA gene sequences from magnetically-enriched magnetotactic cocci clustered into three distinct groups affiliated with the Alphaproteobacteria class of the Proteobacteria. Novel sequences of each phylogenetic cluster were confirmed using fluorescent in situ hybridization. Metagenomic data analysis of magnetically-enriched magnetotactic cocci revealed the presence of mam genes and MTB-specific hypothetical protein coding genes. Sequence homology and phylogenetic analysis indicated that predicted proteins are related to those of cultivated alphaproteobacterial MTB. The consistent and continuous low temperature of the sediment where the magnetotactic cocci are present (always below 1°C) suggests that these MTB from maritime Antarctica are psychrophiles. Moreover, similar morphotypes and 16S gene sequences were retrieved from samples collected from different sites from maritime Antarctica for several years suggesting that these new strains of MTB are indigenous members of Antarctic microbiota.

North-Seeking Magnetotactic Gammaproteobacteria in the Southern Hemisphere.

UNLABELLED: Magnetotactic bacteria (MTB) comprise a phylogenetically diverse group of prokaryotes capable of orienting and navigating along magnetic field lines. Under oxic conditions, MTB in natural environments in the Northern Hemisphere generally display north-seeking (NS) polarity, swimming parallel to the Earth's magnetic field lines, while those in the Southern Hemisphere generally swim antiparallel to magnetic field lines (south-seeking [SS] polarity). Here, we report a population of an uncultured, monotrichously flagellated, and vibrioid MTB collected from a brackish lagoon in Brazil in the Southern Hemisphere that consistently exhibits NS polarity. Cells of this organism were mainly located below the oxic-anoxic interface (OAI), suggesting it is capable of some type of anaerobic metabolism. Magnetosome crystalline habit and composition were consistent with elongated prismatic magnetite (Fe3O4) particles. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that this organism belongs to a distinct clade of the Gammaproteobacteria class. The presence of NS MTB in the Southern Hemisphere and the previously reported finding of SS MTB in the Northern Hemisphere reinforce the idea that magnetotaxis is more complex than we currently understand and may be modulated by factors other than O2 concentration and redox gradients in sediments and water columns. IMPORTANCE: Magnetotaxis is a navigational mechanism used by magnetotactic bacteria to move along geomagnetic field lines and find an optimal position in chemically stratified sediments. For that, magnetotactic bacteria swim parallel to the geomagnetic field lines under oxic conditions in the Northern Hemisphere, whereas those in the Southern Hemisphere swim antiparallel to magnetic field lines. A population of uncultured vibrioid magnetotactic bacteria was discovered in a brackish lagoon in the Southern Hemisphere that consistently swim northward, i.e., the opposite of the overwhelming majority of other Southern Hemisphere magnetotactic bacteria. This finding supports the idea that magnetotaxis is more complex than previously thought.

Strontium ranelate changes the composition and crystal structure of the biological bone-like apatite produced in osteoblast cell cultures.

We evaluate the effects of strontium ranelate on the composition and crystal structure of the biological bone-like apatite produced in osteoblast cell cultures, a system that gave us the advantage of obtaining mineral samples produced exclusively during treatment. Cells were treated with strontium ranelate at concentrations of 0.05 and 0.5 mM Sr(2+). Mineral substances were isolated and analyzed by using a combination of methods: Fourier transform infrared spectroscopy, solid-state (1)H nuclear magnetic resonance, X-ray diffraction, micro-Raman spectroscopy and energy dispersive X-ray spectroscopy. The minerals produced in all cell cultures were typical bone-like apatites. No changes occurred in the local structural order or crystal size of the minerals. However, we noticed several relevant changes in the mineral produced under 0.5 mM Sr(2+): (1) increase in type-B CO3 (2-) substitutions, which often lead to the creation of vacancies in Ca(2+) and OH(-) sites; (2) incorporation of Sr(2+) by substituting slightly less than 10 % of Ca(2+) in the apatite crystal lattice, resulting in an increase in both lattice parameters a and c; (3) change in the PO4 (3-) environments, possibly because of the expansion of the lattice; (4) the Ca/P ratio of this mineral was reduced, but its (Ca+Sr)/P ratio was the same as that of the control, indicating that its overall cation/P ratio was preserved. Thus, strontium ranelate changes the composition and crystal structure of the biological bone-like apatite produced in osteoblast cell cultures.

Ferromagnetic resonance of intact cells and isolated crystals from cultured and uncultured magnetite-producing magnetotactic bacteria.

Most magnetotactic bacteria (MB) produce stable, single-domain magnetite nanocrystals with species-specific size, shape and chain arrangement. In addition, most crystals are elongated along the [111] direction, which is the easy axis of magnetization in magnetite, chemically pure and structurally perfect. These special characteristics allow magnetite crystal chains from MB to be recognized in environmental samples including old sedimentary rocks. Ferromagnetic resonance (FMR) has been proposed as a powerful and practical tool for screening large numbers of samples possibly containing magnetofossils. Indeed, several studies were recently published on FMR of cultured MB, mainly Magnetospirillum gryphiswaldense. In this work, we examined both uncultured magnetotactic cocci and the cultured MB M. gryphiswaldense using transmission electron microscopy (TEM) and FMR from 10 K to room temperature (RT). The TEM data supported the FMR spectral characteristics of our samples. The FMR spectra of both bacteria showed the intrinsic characteristics of magnetite produced by MB, such as extended absorption at the low field region of the spectra and a Verwey transition around 100 K. As previously observed, the spectra of M. gryphiswaldense isolated crystals were more symmetrical than the spectra obtained from whole cells, reflecting the loss of chain arrangement due to the small size and symmetrical shape of the crystals. However, the FMR spectra of magnetic crystals isolated from magnetotactic cocci were very similar to the FMR spectra of whole cells, because the chain arrangement was maintained due to the large size and prismatic shape of the crystals. Our data support the use of FMR spectra to detect magnetotactic bacteria and magnetofossils in samples of present and past environments. Furthermore, the spectra suggest the use of the temperature transition of spectral peak-to-peak intensity to obtain the Verwey temperature for these systems.

Optimization of magnetosome production and growth by the magnetotactic vibrio Magnetovibrio blakemorei strain MV-1 through a statistics-based experimental design.

The growth and magnetosome production of the marine magnetotactic vibrio Magnetovibrio blakemorei strain MV-1 were optimized through a statistics-based experimental factorial design. In the optimized growth medium, maximum magnetite yields of 64.3 mg/liter in batch cultures and 26 mg/liter in a bioreactor were obtained.

Strontium ranelate increases the formation of bone-like mineralized nodules in osteoblast cell cultures and leads to Sr incorporation into the intact nodules.

We describe effects of strontium ranelate treatment on intact mineralized nodules produced in osteoblast cell cultures. We analyzed the matrix directly at the cell culture surfaces following treatment with 0.05 and 0.5 mM Sr(2+). This method allowed for data to be obtained from intact nodules, rather than from extracted samples. The bone-like nature of the matrix was evaluated by using attenuated total reflection Fourier transform infrared spectroscopy and the incorporation of Sr into the nodules was investigated by using both energy dispersive X-ray spectroscopy and synchrotron radiation micro X-ray fluorescence. We observed typical mineralized nodules in all of the cell cultures. However, the formation of these nodules was markedly increased in cultures treated with 0.5 mM Sr(2+). In all of the cultures, the nature of the intact matrix was similar to that described in native bone tissue, being comprised of a poorly crystalline CO3 (2-)-containing apatite and a collagenous matrix. This indicated that treatment had no deleterious effects on the matrix. Moreover, the nodules presented Ca and P as the main chemical components, confirming their bone-like mineralized nature. The incorporation of Sr into the nodules was clearly observed in the treated cultures, with their relative Sr content [Sr/(Ca+Sr) ratio] being markedly increased in a dose-dependent manner. Thus, strontium ranelate promoted an increase in the formation of mineralized nodules in osteoblast cell cultures while preserving the bone-like nature of the matrix at the tissue level. We further demonstrated that Sr was incorporated into the intact nodules formed during treatment.

Swimming behaviour of the multicellular magnetotactic prokaryote 'Candidatus Magnetoglobus multicellularis' under applied magnetic fields and ultraviolet light.

Magnetotactic bacteria move by rotating their flagella and concomitantly are aligned to magnetic fields because they present magnetosomes, which are intracellular organelles composed by membrane-bound magnetic crystals. This results in magnetotaxis, which is swimming along magnetic field lines. Magnetotactic bacteria are morphologically diverse, including cocci, rods, spirilla and multicellular forms known as magnetotactic multicellular prokaryotes (MMPs). 'Candidatus Magnetoglobus multicellularis' is presently the best known MMP. Here we describe the helical trajectories performed by these microorganisms as they swim forward, as well as their response to UV light. We measured the radius of the trajectory, time period and translational velocity (velocity along the helix axis), which enabled the calculation of other trajectory parameters such as pitch, tangential velocity (velocity along the helix path), angular frequency, and theta angle (the angle between the helix path and the helix axis). The data revealed that 'Ca. M. multicellularis' swims along elongated helical trajectories with diameters approaching the diameter of the microorganism. In addition, we observed that 'Ca. M. multicellularis' responds to UV laser pulses by swimming backwards, returning to forward swimming several seconds after the UV laser pulse. UV light from a fluorescence microscope showed a similar effect. Thus, phototaxis is used in addition to magnetotaxis in this microorganism.

Cell adhesion, multicellular morphology, and magnetosome distribution in the multicellular magnetotactic prokaryote Candidatus Magnetoglobus multicellularis.

Candidatus Magnetoglobus multicellularis is an uncultured magnetotactic multicellular prokaryote composed of 17-40 Gram-negative cells that are capable of synthesizing organelles known as magnetosomes. The magnetosomes of Ca. M. multicellularis are composed of greigite and are organized in chains that are responsible for the microorganism's orientation along magnetic field lines. The characteristics of the microorganism, including its multicellular life cycle, magnetic field orientation, and swimming behavior, and the lack of viability of individual cells detached from the whole assembly, are considered strong evidence for the existence of a unique multicellular life cycle among prokaryotes. It has been proposed that the position of each cell within the aggregate is fundamental for the maintenance of its distinctive morphology and magnetic field orientation. However, the cellular organization of the whole organism has never been studied in detail. Here, we investigated the magnetosome organization within a cell, its distribution within the microorganism, and the intercellular relationships that might be responsible for maintaining the cells in the proper position within the microorganism, which is essential for determining the magnetic properties of Ca. M. multicellularis during its life cycle. The results indicate that cellular interactions are essential for the determination of individual cell shape and the magnetic properties of the organism and are likely directly associated with the morphological changes that occur during the multicellular life cycle of this species.

Strontium is incorporated in different levels into bones and teeth of rats treated with strontium ranelate.

The aim of this study was to evaluate the strontium incorporation into specific bones and teeth of rats treated with strontium ranelate. The relative strontium levels [Sr/(Ca + Sr) ratio] were obtained by synchrotron radiation micro X-ray fluorescence. The incisor teeth were further examined by energy dispersive X-ray spectroscopy (EDS) in a scanning electron microscope. The isolated mineral phase was investigated by EDS in a transmission electron microscope and X-ray diffraction. The strontium content was markedly increased in animals treated with strontium ranelate, with different incorporation levels found among specific bones, regions within the same bone and teeth. The highest strontium levels were observed in the iliac crest, mandible and calvaria, while the lowest were observed in the femoral diaphysis, lumbar vertebrae, rib and alveolar bone. The strontium content was higher in the femoral neck than in the diaphysis. The strontium levels also varied within the alveolar bone. High levels of strontium were found in the incisor tooth, with values similar to those in the iliac crest. Strontium was observed in both enamel and dentin. The strontium content of the molar tooth was negligible. Strontium was incorporated into the mineral substance, with up to one strontium replacing one out of 10 calcium ions within the apatite crystal lattice. The mineral from treated animals presented increased lattice parameters, which might be associated to their bone strontium contents. In conclusion, the incorporation of strontium occurred in different levels into distinct bones, regions within the same bone and teeth of rats treated with strontium ranelate.

Effects of induced precocious puberty on cranial growth in female Wistar rats.

This investigation examined the effects of pharmacologically induced precocious puberty on cranial growth in Wistar rats. Forty-eight female newborn Wistar rats were divided into two groups: a control group (C) and an experimental group (E), with four subgroups of six animals each. The time interval from birth until sacrifice differed between the subgroups, and was set at 30, 60, 90, and 120 days. An intramuscular single dose (300 µg) of steroid hormone danazol was administered on day 5 after birth, as a means of inducing precocious puberty. Alizarin (2 mg/100 g) was administered to three animals in each subgroup three days prior to sacrifice. Body mass and dates corresponding to the beginning of the oestrous cycle were recorded. Craniometric measurements were undertaken. Histological analysis using light and fluorescence microscopy was then carried out to qualitatively and quantitatively evaluate the spheno-occipital synchondrosis and to visualize bone deposition patterns. The results were analysed with a Student's t-test and analysis of variance. Precocious puberty was effectively induced and differences between groups denoted an earlier maturation in the experimental rats. In qualitative analysis, a significant increase of total synchondrosis width was noted only in group E60, in comparison with C60, and an increase in the E90 subgroup cortical bone width compared with the C90 subgroup. Histomorphometrically, a statistical difference between total width values of subgroups E60 (434.3 µm) and C60 (323.5 µm) was detected. However, body mass and macroscopic measurements did not show statistically significant differences. An appropriate model for studying bone growth associated with precocious puberty in Wistar female rats was not achieved using steroid hormone danazol, when evaluated at 30 day intervals.

In Vitro and In Vivo Cell-Interactions with Electrospun Poly (Lactic-Co-Glycolic Acid) (PLGA): Morphological and Immune Response Analysis.

Random electrospun three-dimensional fiber membranes mimic the extracellular matrix and the interfibrillar spaces promotes the flow of nutrients for cells. Electrospun PLGA membranes were analyzed in vitro and in vivo after being sterilized with gamma radiation and bioactivated with fibronectin or collagen. Madin-Darby Canine Kidney (MDCK) epithelial cells and primary fibroblast-like cells from hamster's cheek paunch proliferated over time on these membranes, evidencing their good biocompatibility. Cell-free irradiated PLGA membranes implanted on the back of hamsters resulted in a chronic granulomatous inflammatory response, observed after 7, 15, 30 and 90 days. Morphological analysis of implanted PLGA using light microscopy revealed epithelioid cells, Langhans type of multinucleate giant cells (LCs) and multinucleated giant cells (MNGCs) with internalized biomaterial. Lymphocytes increased along time due to undegraded polymer fragments, inducing the accumulation of cells of the phagocytic lineage, and decreased after 90 days post implantation. Myeloperoxidase+ cells increased after 15 days and decreased after 90 days. LCs, MNGCs and capillaries decreased after 90 days. Analysis of implanted PLGA after 7, 15, 30 and 90 days using transmission electron microscope (TEM) showed cells exhibiting internalized PLGA fragments and filopodia surrounding PLGA fragments. Over time, TEM analysis showed less PLGA fragments surrounded by cells without fibrous tissue formation. Accordingly, MNGC constituted a granulomatous reaction around the polymer, which resolves with time, probably preventing a fibrous capsule formation. Finally, this study confirms the biocompatibility of electrospun PLGA membranes and their potential to accelerate the healing process of oral ulcerations in hamsters' model in association with autologous cells.

Magnetosome chain superstructure in uncultured magnetotactic bacteria.

Magnetotactic bacteria produce magnetosomes, which are magnetic particles enveloped by biological membranes, in a highly controlled mineralization process. Magnetosomes are used to navigate in magnetic fields by a phenomenon called magnetotaxis. Two levels of organization and control are recognized in magnetosomes. First, magnetotactic bacteria create a spatially distinct environment within vesicles defined by their membranes. In the vesicles, the bacteria control the size, composition and purity of the mineral content of the magnetic particles. Unique crystal morphologies are produced in magnetosomes as a consequence of this bacterial control. Second, magnetotactic bacteria organize the magnetosomes in chains within the cell body. It has been shown in a particular case that the chains are positioned within the cell body in specific locations defined by filamentous cytoskeleton elements. Here, we describe an additional level of organization of the magnetosome chains in uncultured magnetotactic cocci found in marine and freshwater sediments. Electron microscopy analysis of the magnetosome chains using a goniometer showed that the magnetic crystals in both types of bacteria are not oriented at random along the crystal chain. Instead, the magnetosomes have specific orientations relative to the other magnetosomes in the chain. Each crystal is rotated either 60°, 180° or 300° relative to their neighbors along the chain axis, causing the overlapping of the (1 1 1) and [Formula in text] capping faces of neighboring crystals. We suggest that genetic determinants that are not present or active in bacteria with magnetosomes randomly rotated within a chain must be present in bacteria that organize magnetosomes so precisely. This particular organization may also be used as an indicative biosignature of magnetosomes in the study of magnetofossils in the cases where this symmetry is observed.

Fine structure and molecular content of human chondrocytes encapsulated in alginate beads.

Preservation of the chondrocytic phenotype in vitro requires a 3D (three-dimensional) culture model. Diverse biomaterials have been tested as scaffolds for culture of animal chondrocytes; however, to date, none is considered a gold standard in regenerative medicine. Here, we studied the fine structure and the GAGs (glycosaminoglycans) content of human chondrocytes encapsulated in alginate beads by using electron microscopy and radioactive sulfate [35S] incorporation, respectively. Cells were obtained from human cartilage, encapsulated in alginate beads and cultured for 28 days. [35S]Na2SO4 was added to the culture media and later isolated for quantification of the sulfated GAGs found in three compartments: IC (intracellular), IB (intra-bead) and EB (extra-bead). Round cells were seen isolated or forming small groups throughout the alginate. Human chondrocytes presented the features of active cells such as euchromatic nuclei, abundant RER (rough endoplasmic reticulum) and many transport vesicles. We observed an extracellular matrix rich in collagen fibres and electrondense material adjacent to the cells. Most of the GAGs produced (74%) were found in the culture medium (EB), indicating that alginate has a limited capacity to retain the GAGs. CS (chondroitin sulfate), the major component of aggrecan, was the most prominent GAG produced by the encapsulated cells. Human chondrocytes cultured in alginate can sustain their phenotype, confirming the potential application of this biomaterial for cartilage engineering.

Trabecular architecture during the healing process of a tibial diaphysis defect.

The adaptation of trabecular bone microstructure to mechanical loads has been intensively investigated. However, loading-unrelated aspects of trabecular architecture remain unclear. We used synchrotron radiation-based X-ray microtomography to study the 3D microarchitecture of newly formed trabecular tissue in a defect produced in the cortical region of the rat tibia diaphysis, in the absence (7, 14, and 21 days) or the presence (21 days) of carbonated hydroxyapatite/alginate (cHA) microspheres. This work provides the first evidence that the woven bone trabecular network, formed during the healing process, displays a well-organized 3D microarchitecture consisting of nodes with 3 (3-N), 4 (4-N) and 5 (5-N) connecting trabeculae, with a mean relative abundance of (3-N)/(4-N)/(5-N) = 66/24/7, for the analyzed periods. The measured inter-trabecular angles (ITA) distribution presented a Gaussian profile, with mean value at 115° for 3-N nodes, and 105° for 4-N nodes, close to the angles of idealized 3D regular structures (120° and 109.5°, respectively). Changes in the dispersion of ITA distribution suggested that a highly symmetric trabecular fabric organized under tensegrity principles is formed early during the bone healing process. Post-implantation, cHA disaggregated into multiple fragments (~20-400 µm), stimulating osteoconduction and bone growth toward the interior of the medullary cavity. The presence of biomaterials in bone defects affected the trabecular dimensions; however, it did not interfere with the formation of geometrical motifs with topological parameters similar to those found in the sham-defects. STATEMENT OF SIGNIFICANCE: The trabecular bone microstructure enables the tissue to meet the necessary mechanical and functional demands. However, the process of trabecular microarchitecture formation during healing, in the absence or presence of a bone graft, is not yet well understood. This work demonstrated that, from the beginning of its formation in cortical bone defects, the woven-bone trabecular network is spatially organized according to the principle of tensegrity. This microarchitecture is comprised of highly symmetric geometric motifs and is an intrinsic characteristic of trabecular growth, regardless of hierarchical scale or mechanical stimulation. The addition of a biodegradable nanostructured calcium phosphate graft did not disrupt trabecular microarchitecture; however, graft biodegradation should be controlled to optimize the reproduction of intrinsic trabecular motifs throughout the defect.