The Effects of Titanium Dioxide Nanoparticles on Osteoblasts Mineralization: A Comparison between 2D and 3D Cell Culture Models.

Although several studies assess the biological effects of micro and titanium dioxide nanoparticles (TiO2 NPs), the literature shows controversial results regarding their effect on bone cell behavior. Studies on the effects of nanoparticles on mammalian cells on two-dimensional (2D) cell cultures display several disadvantages, such as changes in cell morphology, function, and metabolism and fewer cell-cell contacts. This highlights the need to explore the effects of TiO2 NPs in more complex 3D environments, to better mimic the bone microenvironment. This study aims to compare the differentiation and mineralized matrix production of human osteoblasts SAOS-2 in a monolayer or 3D models after exposure to different concentrations of TiO2 NPs. Nanoparticles were characterized, and their internalization and effects on the SAOS-2 monolayer and 3D spheroid cells were evaluated with morphological analysis. The mineralization of human osteoblasts upon exposure to TiO2 NPs was evaluated by alizarin red staining, demonstrating a dose-dependent increase in mineralized matrix in human primary osteoblasts and SAOS-2 both in the monolayer and 3D models. Furthermore, our results reveal that, after high exposure to TiO2 NPs, the dose-dependent increase in the bone mineralized matrix in the 3D cells model is higher than in the 2D culture, showing a promising model to test the effect on bone osteointegration.

New Phenotype and Mineralization of Biogenic Iron Oxide in Magnetotactic Bacteria.

Many magnetotactic bacteria (MTB) biomineralize magnetite crystals that nucleate and grow inside intracellular membranous vesicles originating from invaginations of the cytoplasmic membrane. The crystals together with their surrounding membranes are referred to as magnetosomes. Magnetosome magnetite crystals nucleate and grow using iron transported inside the vesicle by specific proteins. Here, we tackle the question of the organization of magnetosomes, which are always described as constituted by linear chains of nanocrystals. In addition, it is commonly accepted that the iron oxide nanocrystals are in the magnetite-based phase. We show, in the case of a wild species of coccus-type bacterium, that there is a double organization of the magnetosomes, relatively perpendicular to each other, and that the nanocrystals are in fact maghemite. These findings were obtained, respectively, by using electron tomography of whole mounts of cells directly from the environment and high-resolution transmission electron microscopy and diffraction. Structure simulations were performed with the MacTempas software. This study opens new perspectives on the diversity of phenotypes within MTBs and allows to envisage other mechanisms of nucleation and formation of biogenic iron oxide crystals.

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.

Insight on thermal stability of magnetite magnetosomes: implications for the fossil record and biotechnology.

Magnetosomes are intracellular magnetic nanocrystals composed of magnetite (Fe3O4) or greigite (Fe3S4), enveloped by a lipid bilayer membrane, produced by magnetotactic bacteria. Because of the stability of these structures in certain environments after cell death and lysis, magnetosome magnetite crystals contribute to the magnetization of sediments as well as providing a fossil record of ancient microbial ecosystems. The persistence or changes of the chemical and magnetic features of magnetosomes under certain conditions in different environments are important factors in biotechnology and paleomagnetism. Here we evaluated the thermal stability of magnetosomes in a temperature range between 150 and 500 °C subjected to oxidizing conditions by using in situ scanning transmission electron microscopy. Results showed that magnetosomes are stable and structurally and chemically unaffected at temperatures up to 300 °C. Interestingly, the membrane of magnetosomes was still observable after heating the samples to 300 °C. When heated between 300 °C and 500 °C cavity formation in the crystals was observed most probably associated to the partial transformation of magnetite into maghemite due to the Kirkendall effect at the nanoscale. This study provides some insight into the stability of magnetosomes in specific environments over geological periods and offers novel tools to investigate biogenic nanomaterials.

Uptake and persistence of bacterial magnetite magnetosomes in a mammalian cell line: Implications for medical and biotechnological applications.

Magnetotactic bacteria biomineralize intracellular magnetic nanocrystals surrounded by a lipid bilayer called magnetosomes. Due to their unique characteristics, magnetite magnetosomes are promising tools in Biomedicine. However, the uptake, persistence, and accumulation of magnetosomes within mammalian cells have not been well studied. Here, the endocytic pathway of magnetite magnetosomes and their effects on human cervix epithelial (HeLa) cells were studied by electron microscopy and high spatial resolution nano-analysis techniques. Transmission electron microscopy of HeLa cells after incubation with purified magnetosomes showed the presence of magnetic nanoparticles inside or outside endosomes within the cell, which suggests different modes of internalization, and that these structures persisted beyond 120 h after internalization. High-resolution transmission electron microscopy and electron energy loss spectra of internalized magnetosome crystals showed no structural or chemical changes in these structures. Although crystal morphology was preserved, iron oxide crystalline particles of approximately 5 nm near internalized magnetosomes suggests that minor degradation of the original mineral structures might occur. Cytotoxicity and microscopy analysis showed that magnetosomes did not result in any apparent effect on HeLa cells viability or morphology. Based on our results, magnetosomes have significant biocompatibility with mammalian cells and thus have great potential in medical, biotechnological applications.

Localized iron accumulation precedes nucleation and growth of magnetite crystals in magnetotactic bacteria.

Many magnetotactic bacteria (MTB) biomineralize magnetite crystals that nucleate and grow inside intracellular membranous vesicles that originate from invaginations of the cytoplasmic membrane. The crystals together with their surrounding membranes are referred to magnetosomes. Magnetosome magnetite crystals nucleate and grow using iron transported inside the vesicle by specific proteins. Here we address the question: can iron transported inside MTB for the production of magnetite crystals be spatially mapped using electron microscopy? Cultured and uncultured MTB from brackish and freshwater lagoons were studied using analytical transmission electron microscopy in an attempt to answer this question. Scanning transmission electron microscopy was used at sub-nanometric resolution to determine the distribution of elements by implementing high sensitivity energy dispersive X-ray (EDS) mapping and electron energy loss spectroscopy (EELS). EDS mapping showed that magnetosomes are enmeshed in a magnetosomal matrix in which iron accumulates close to the magnetosome forming a continuous layer visually appearing as a corona. EELS, obtained at high spatial resolution, confirmed that iron was present close to and inside the lipid bilayer magnetosome membrane. This study provides important clues to magnetite formation in MTB through the discovery of a mechanism where iron ions accumulate prior to magnetite biomineralization.

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.

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.

Long-range crystalline order in spicules from the calcareous sponge Paraleucilla magna (Porifera, Calcarea).

We investigated the ultrastructure and crystallographic orientation of spicules from the calcareous sponge Paraleucilla magna (subclass Calcaronea) by transmission and scanning electron microscopy using two different methods of sample preparation: ultramicrotomy and focused ion beam (FIB). It was found that the unpaired actine from the spicules was oriented in the [211] zone axis. The plane that contains the unpaired actine and divides symmetrically the paired actines is the (-120). This plane is a mirror plane of the hexagonal lattice system. All the spicule types analyzed presented the same crystallographic orientation. Electron nanodiffraction maps from 4µm×4µm regions prepared by FIB showed disorientation of <2° between diffraction patterns obtained from neighbor regions, indicating the presence of a unique, highly aligned calcite crystalline phase. Among the eight FIB sections obtained, four presented high pore density. In one section perpendicular to the actine axis pores were observed only in the center of the spicule aligned in a circular pattern and surrounded by a faint circular contour with a larger radius. The presence of amorphous carbon representative of organic molecules detected by electron energy loss spectroscopy was correlated neither with porosity nor with specific lattice planes.

Effect of strontium ranelate on bone mineral: Analysis of nanoscale compositional changes.

Strontium ranelate has been used to prevent bone loss and stimulate bone regeneration. Although strontium may integrate into the bone crystal lattice, the chemical and structural modifications of the bone when strontium interacts with the mineral phase are not completely understood. The objective of this study was to evaluate apatite from the mandibles of rats treated with strontium ranelate in the drinking water and compare its characteristics with those from untreated rats and synthetic apatites with and without strontium. Electron energy loss near edge structures from phosphorus, carbon, calcium and strontium were obtained by electron energy loss spectroscopy in a transmission electron microscope. The strontium signal was detected in the biological and synthetic samples containing strontium. The relative quantification of carbon by analyzing the CK edge at an energy loss of ΔE = 284 eV showed an increase in the number of carbonate groups in the bone mineral of treated rats. A synthetic strontium-containing sample used as control did not exhibit a carbon signal. This study showed physicochemical modifications in the bone mineral at the nanoscale caused by the systemic administration of strontium ranelate.

Three-dimensional chemistry of multiphase nanomaterials by energy-filtered transmission electron microscopy tomography.

A three-dimensional (3D) study of multiphase nanostructures by chemically selective electron tomography combining tomographic approach and energy-filtered imaging is reported. The implementation of this technique at the nanometer scale requires careful procedures for data acquisition, computing, and analysis. Based on the performances of modern transmission electron microscopy equipment and on developments in data processing, electron tomography in the energy-filtered imaging mode is shown to be a very appropriate analysis tool to provide 3D chemical maps at the nanoscale. Two examples highlight the usefulness of analytical electron tomography to investigate inhomogeneous 3D nanostructures, such as multiphase specimens or core-shell nanoparticles. The capability of discerning in a silica-alumina porous particle the two different components is illustrated. A quantitative analysis in the whole specimen and toward the pore surface is reported. This tool is shown to open new perspectives in catalysis by providing a way to characterize precisely 3D nanostructures from a chemical point of view.

Ultrastructure of regenerated bone mineral surrounding hydroxyapatite-alginate composite and sintered hydroxyapatite.

We report the ultrastructure of regenerated bone surrounding two types of biomaterials: hydroxyapatite-alginate composite and sintered hydroxyapatite. Critical defects in the calvaria of Wistar rats were filled with micrometer-sized spherical biomaterials and analyzed after 90 and 120 days of implantation by high-resolution transmission electron microscopy and Fourier transform infrared attenuated total reflectance microscopy, respectively. Infrared spectroscopy showed that hydroxyapatite of both biomaterials became more disordered after implantation in the rat calvaria, indicating that the biological environment induced modifications in biomaterials structure. We observed that the regenerated bone surrounding both biomaterials had a lamellar structure with type I collagen fibers alternating in adjacent lamella with angles of approximately 90°. In each lamella, plate-like apatite crystals were aligned in the c-axis direction, although a rotation around the c-axis could be present. Bone plate-like crystal dimensions were similar in regenerated bone around biomaterials and pre-existing bone in the rat calvaria. No epitaxial growth was observed around any of the biomaterials. A distinct mineralized layer was observed between new bone and hydroxyapatite-alginate biomaterial. This region presented a particular ultrastructure with crystallites smaller than those of the bulk of the biomaterial, and was possibly formed during the synthesis of alginate-containing composite or in the biological environment after implantation. Round nanoparticles were observed in regions of newly formed bone. The findings of this work contribute to a better understanding of the role of hydroxyapatite based biomaterials in bone regeneration processes at the nanoscale.

Physical-chemical and biological behavior of an amorphous calcium phosphate thin film produced by RF-magnetron sputtering.

This work evaluates the thermal reactivity and the biological reactivity of an amorphous calcium phosphate thin film produced by radio frequency (RF) magnetron sputtering onto titanium substrates. The analyses showed that the sputtering conditions used in this work led to the deposition of an amorphous calcium phosphate. The thermal treatment of this amorphous coating in the presence of H2O and CO2 promoted the formation of a carbonated HA crystalline coating with the entrance of CO32- ions into the hydroxyl HA lattice. When immersed in culture medium, the amorphous and carbonated coatings exhibited a remarkable instability. The presence of proteins increased the dissolution process, which was confirmed by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) analyses. Moreover, the carbonated HA coating induced precipitation independently of the presence of proteins under dynamic conditions. Despite this surface instability, this reactive calcium phosphate significantly improved the cellular behavior. The cell proliferation was higher on the Ticp than on the calcium phosphate coatings, but the two coatings increased cellular spreading and stress fiber formation. In this sense, the presence of reactive calcium phosphate coatings can stimulate cellular behavior.

Direct observation of stacking faults and pore connections in ordered cage-type mesoporous silica FDU-12 by electron tomography.

The porous structure and the periodic array of cavities in ordered mesoporous materials with large, three-dimensionally arranged and interconnected pores is thoroughly described by combining electron tomography, small-angle X-ray diffraction, and nitrogen sorption techniques. We used the ability of the electron tomography to provide local three-dimensional information of a nano-object and compared the results to those of the other characterization techniques which furnish global information. We showed thus that the face-centered cubic (fcc) structure usually assigned to the FDU-12 materials is in fact an intergrowth of cubic and hexagonal close-packing structures. This agrees with small-angle X-ray scattering (SAXS) modeling, but for the first time a direct visualization of these stacking faults was achieved. Three-dimensional transmission electron microscopy (3D-TEM) provides also a direct and unique evidence of peculiar stacking defects ("z-shifted [111] areas"), as well as an estimate of their density, which have never been reported elsewhere. In addition, interstitial cavities were also observed, revealing the complex defective structure of this material. A direct observation of the nature of the connecting pores was also achieved for the first time, with a resolution limit of 2 nm. Finally, the characteristics of the porous network evidenced by 3D-TEM are used to explain and validate the results obtained by nitrogen sorption experiments.

3D electron microscopy study of metal particles inside multiwalled carbon nanotubes.

The location of palladium nanoparticles on and inside the multiwalled carbon nanotubes channel is presented for the first time using electron tomography (3D TEM). The palladium salt precursor was rapidly sucked inside the nanotube channel by means of capillarity that is favored by the hydrophilic character of the tube wall after acidic treatment at low temperature. Statistical analysis indicates that the palladium particles were well dispersed and the palladium particle size was relatively homogeneous, ranging from 3 to 4 nm regardless of their location within the nanotube, within the resolution limit of the technique for our experimental conditions, i.e., about 2 nm. Three-dimensional TEM analysis also revealed that introduction of foreign elements inside the tube channel is strongly influenced by the diameter of the tube inner channel, i.e., easy filling seems to occur with a tube channel >or=30 nm , whereas with tubes having a smaller channel (<15 nm), almost no filling by capillarity occurred leading to the deposition of the metal particles only on the outer wall of the tube.