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The use of magnetic nanoparticles in association with scaffolds is considered an important way to transform typical passive scaffolds into active scaffolds. Manganese can develop magnetic properties in hydroxyapatites, as can iron, copper, cobalt and samarium, but lacks the high toxicity of these latter compounds. Because the magnetic properties exhibited by transition metal-containing hydroxyapatite are entirely dependent on site occupation and on the formation of magnetic oxides under heating, it is extremely important to understand how manganese behaves when inserted into hydroxyapatite. In this paper, we demonstrated by using X-ray difference Fourier maps that the insertion of Mn into the hydroxyapatite structure induces several perturbations in its hexagonal channels, with a preference for occupying Ca(2) sites, particularly when the hydroxyapatite lattice is poorly ordered and has CO32− inserted at PO43− sites. When CO32− is released at high temperature and the structure is better ordered, Ca(1) sites become more susceptible to occupation by Mn atoms, while the occupation of PO43− sites by MnO43− is reduced. However, with increasing time and calcination temperature, Mn atoms tend to be released from the hydroxyapatite structure in the form of Mn3O4 nanoparticles through the hydroxyl channels, occupying Ca(2) sites in the final stage of the segregation pathway. These findings are fundamental to the development of new strategies to synthesize active hydroxyapatite-based scaffolds under remote control by a magnetic field.

Ferritin biomineralization is essential to regulate the toxic Fe2+ iron ions in the human body. Unravelling the mechanism of biomineralization in ferritin facilitates our understanding of the cause...

The ions found in biological environment during biomineralization as well as the counterions present in syntheses, i.e., the reagents employed, play an important role in the precipitation and stabilization of apatites. In this work, precipitation of strontium-containing hydroxyapatites at different concentrations (0, 20, 40, 60, 80 and 100% Sr2+) was performed in the presence of two counterions typically present in biological environments: Na+ and Cl−. It was demonstrated that the presence of Na+/Cl− in the synthesis environment led to the precipitation of a biphasic hydroxyapatite (HAp) system formed by two non-miscible phases: Ca-rich HAp and Sr-rich HAp. The biphasic system was observed for intermediate Sr2+ concentrations (20, 40 and 60%) and exhibited greater lattice microstrain compared with the single-phase systems of Ca HAp (0%) and Sr HAp (100%). Although Na+/Cl− were inserted into both Ca- and Sr-rich HAp phases, Cl− ions were preferentially accommodated into the enlarged Sr-rich HAp structure. The presence of Cl− ions in the Sr-rich HAp phase decreased microstrain compared to the Ca-rich phase. After calcination, the biphasic system was transformed into a completely miscible Ca/Sr HAp phase without the formation of other phosphates or oxides.

Commercial poly methyl methacrylate (PMMA)-based cement is currently used in the field of orthopedics. However, it suffers from lack of bioactivity, mechanical weakness, and monomer toxicity. In this study, a PMMA-based cement nanocomposite reinforced with hydroxyapatite (HA) nanofibers and two-dimensional (2D) magnesium phosphate MgP nanosheets was synthesized and optimized in terms of mechanical property and cytocompatibility. The HA nanofibers and the MgP nanosheets were synthesized using a hydrothermal homogeneous precipitation method and tuning the crystallization of the sodium-magnesium-phosphate ternary system, respectively. Compressive strength and MTT assay tests were conducted to evaluate the mechanical property and the cytocompatibility of the PMMA-HA-MgP nanocomposites prepared at different ratios of HA and MgP. To optimize the developed nanocomposites, the standard response surface methodology (RSM) design known as the central composite design (CCD) was employed. Two regression models generated by CCD were analyzed and compared with the experimental results, and good agreement was observed. Statistical analysis revealed the significance of both factors, namely, the HA nanofibers and the MgP nanosheets, in improving the compressive strength and cell viability of the PMMA-MgP-HA nanocomposite. Finally, it was demonstrated that the HA nanofibers of 7.5% wt and the MgP nanosheets of 6.12% wt result in the PMMA-HA-MgP nanocomposite with the optimum compressive strength and cell viability.

Water is ubiquitous in natural systems, ranging from the vast oceans to the nanocapillaries in the earth crust or cellular organelles. In bulk or in intimate contact with solid surfaces, water molecules arrange themselves according to their hydrogen (H) bonding, which critically affects their short- and long-range molecular structures. Formation of H-bonds among water molecules designates the energy levels of certain nonbonding molecular orbitals of water, which are quantifiable by spectroscopic techniques. While the molecular architecture of water in nanoenclosures is of particular interest to both science and industry, it requires fine spectroscopic probes with nanometer spatial resolution and sub-eV energy sensitivity. Graphene liquid cells (GLCs), which feature opposing closely spaced sheets of hydrophobic graphene, facilitate high-resolution transmission electron microscopy (TEM) and electron energy-loss spectroscopy (EELS) measurements of attoliter water volumes encapsulated tightly in the GLC nanovessels. We perform in situ TEM and EELS analysis of water encased in thin GLCs exposed to room and cryogenic temperatures to examine the nanoscale arrangement of the contained water molecules. Simultaneous quantification of GLC thickness leads to the conclusion that H-bonding strengthens under increased water confinement. The present results demonstrate the feasibility of nanoscale chemical characterization of aqueous fluids trapped in GLC nanovessels and offer insights on water molecule arrangement under high-confinement conditions.

Emre Firlar,1,2 Meagan Ouy,1 Leigha Covnot,1 Yuan Xing,3 Daniel Lee,1,4 Alessandro Chan,1,4 Yi He,3 Boao Song,2 Solomon Afelik,4 Yong Wang,3 Reza Shahbazian-Yassar,2 Jose Oberholzer,1,3 Tolou Shokuhfar1 1Department of Bioengineering, University of Illinois at Chicago, Chicago, IL, USA; 2University of Illinois at Chicago, Department of Mechanical and Industrial Engineering, Chicago, IL, USA; 3University of Virginia, Department of Surgery, Charlottesville, VA, USA; 4University of Illinois at Chicago, Department of Surgery, Chicago, IL, USA Background: Islet cell transplantation is one of the key treatments for type 1 diabetes. Understanding the mechanisms of insulin fusion and exocytosis are of utmost importance for the improvement of the current islet cell transplantation and treatment of diabetes. These phenomena have not been fully evaluated due either to the lack of proper dynamic imaging, or the lack of proper cell preservation during imaging at nanoscales. Methods: By maintaining the native environment of pancreatic β-cells between two graphene monolayer sheets, we were able to monitor the subcellular events using in situ graphene liquid cell (GLC)-transmission electron microscopy (TEM) with both high temporal and high spatial resolution. Results: For the first time, the nucleation and growth of insulin particles until the later stages of fusion were imaged at nanometer scales. The release of insulin from plasma membrane involves the degradation of plasma membrane and drastic reductions in the shorter axis of the insulin particles. Sequential exocytosis results indicated the nucleation, growth and attachment of the new insulin particles to the already anchored ones, which is thermodynamically favorable due to the reduction in total surface, further reducing the Gibbs free energy. The retraction of the already anchored insulin toward the cell is also monitored for the first time live at nanoscale resolution. Conclusion: Investigation of insulin granule dynamics in β-cells can be investigated via GLC-TEM. Our findings with this technology open new realms for the development of novel drugs on pathological pancreatic β-cells, because this approach facilitates observing the effects of the stimuli on the live cells and insulin granules. Keywords: transmission electron microscopy, graphene liquid cell, insulin secretion, exocytosis

One of the biggest bottlenecks for structural analysis of proteins remains the creation of high-yield and high-purity samples of the target protein. Cell-free protein synthesis technologies are powerful and customizable platforms for obtaining functional proteins of interest in short timeframes, while avoiding potential toxicity issues and permitting high-throughput screening. These methods have benefited many areas of genomic and proteomics research, therapeutics, vaccine development and protein chip constructions. In this work, we demonstrate a versatile and multiscale eukaryotic wheat germ cell-free protein expression pipeline to generate functional proteins of different sizes from multiple host organism and DNA source origins. We also report on a robust purification procedure, which can produce highly pure (> 98%) proteins with no specialized equipment required and minimal time invested. This pipeline successfully produced and analyzed proteins in all three major geometry formats used for structural biology including single particle analysis with electron microscopy, and both two-dimensional and three-dimensional protein crystallography. The flexibility of the wheat germ system in combination with the multiscale pipeline described here provides a new workflow for rapid production and purification of samples that may not be amenable to other recombinant approaches for structural characterization. Electronic supplementary material The online version of this article (10.1186/s40679-018-0062-9) contains supplementary material, which is available to authorized users.

The area of cellular interactions of nanomaterials is an important research interest. The sensitivity of cells toward their extracellular matrix allows researchers to create microenvironments for guided stem cell differentiation. Among nanomaterials, graphene, often called the “wonder material,” and its derivatives are at the forefront of such endeavors. Graphene’s carbon backbone, paired with its biocompatibility and ease of functionalization, has been used as an enhanced method of controlled cell proliferation. Graphene’s honeycomb nature allows for compatibility with polymers and biological material for the creation of nanocomposite scaffolds that help differentiation into cell types that have otherwise been proven difficult. Such materials and their role in guiding cell growth can aid the construction of tissue grafts where shortages and patient compatibility create a low success rate. This review will bring together novel studies and techniques used to understand and optimizes graphene’s role in cell growth mechanisms.

Ferritin is a protein that regulates the iron ions in humans by storing them in the form of iron oxides. Despite extensive efforts to understand the ferritin iron oxide structures, it is still not clear how ferritin proteins with a distinct light (L) and heavy (H) chain subunit ratio impact the biomineralization process. In situ graphene liquid cell-transmission electron microscopy (GLC-TEM) provides an indispensable platform to study the atomic structure of ferritin mineral cores in their native liquid environment. In this study, we report differences in the iron oxide formation in human spleen ferritins (HSFs) and human heart ferritins (HHFs) using in situ GLC-TEM. Scanning transmission electron microscopy (STEM) along with selected area electron diffraction (SAED) of the mineral core and electron energy loss spectroscopy (EELS) analyses enabled the visualization of morphologies, crystal structures and the chemistry of iron oxide cores in HSFs and HHFs. Our study revealed the presence of metastable ferrihydrite (5Fe2O3·9H2O) as a dominant phase in hydrated HSFs and HHFs, while a stable hematite (α-Fe2O3) phase predominated in non-hydrated HSFs and HHFs. In addition, a higher Fe3+/Fe2+ ratio was found in HHFs in comparison with HSFs. This study provides new understanding on iron-oxide phases that exist in hydrated ferritin proteins from different human organs. Such new insights are needed to map ferritin biomineralization pathways and possible correlations with various iron-related disorders in humans.

This review summarizes the breakthroughs in the field of soft material characterization by in situ liquid-cell transmission electron microscopy (TEM). The focus of this review is mostly on soft biological species such as cells, bacteria, viruses, proteins and polymers. The comparison between the two main liquid-cell systems (silicon nitride membranes liquid cell and graphene liquid cell) is also discussed in terms of their spatial resolution and imaging/analytical capabilities. We have showcased how liquid-cell TEM can reveal the structural details of whole cells, enable the chemical probing of proteins, detect the structural conformation of viruses, and monitor the dynamics of polymerization. In addition, the challenges faced by decoupling electron beam effect on beam-sensitive soft materials are discussed. At the end, future perspectives of in situ liquid-cell TEM studies of soft materials are outlined.

Transmission electron microscopy of whole cells is hindered by the inherently large thickness and low atomic contrast intrinsic of cellular material. Liquid cell transmission electron microscopy allows samples to remain in their native hydrated state and may permit visualizing cellular dynamics in-situ. However, imaging biological cells with this approach remains challenging and identifying an optimal imaging regime using empirical data would help foster new advancements in the field. Recent questions about the role of the electron beam inducing morphological changes or damaging cellular structure and function necessitates further investigation of electron beam-cell interactions, but such comparisons are complicated by variability in imaging techniques used across various studies currently present in literature. The necessity for using low electron fluxes while imaging biological samples requires finding an imaging strategy which produces the strongest contrast and signal to noise ratio for the electron flux used. Here, we experimentally measure and evaluate signal to noise ratios and damage mechanisms between liquid and cryogenic samples of intact cells using multiple electron imaging modalities all on the same instrument and with equivalent beam parameters to standardize the comparison. We also discuss considerations for optimal electron microscopy imaging conditions for future studies on whole cells within liquid environments.

Transmission electron microscopy of whole cells is hindered by the inherently large thickness and low atomic contrast intrinsic of cellular material. Liquid cell transmission electron microscopy allows samples to remain in their native hydrated state and may permit visualizing cellular dynamics in-situ. However, imaging biological cells with this approach remains challenging and identifying an optimal imaging regime using empirical data would help foster new advancements in the field. Recent questions about the role of the electron beam inducing morphological changes or damaging cellular structure and function necessitates further investigation of electron beam-cell interactions, but is complicated by variability in imaging techniques used across various studies currently present in literature. The necessity for using low electron fluxes for imaging biological samples requires finding an imaging strategy which produces the strongest contrast and signal to noise ratio for the electron flux used. Here, we experimentally measure and evaluate signal to noise ratios and damage mechanisms between liquid and cryogenic samples for cells using multiple electron imaging modalities all on the same instrument and with equivalent beam parameters to standardize the comparison. We also discuss considerations for optimal electron microscopy imaging conditions for future studies on whole cells within liquid environments.

Lack of bioactivity and monomer toxicity are limiting factors of polymethyl methacrylate (PMMA) bone cement in orthopedic applications. Herein, we address these shortcomings by proposing two-dimensional magnesium phosphate (MgP) nanosheets and hydroxyapatite (HA) nanofibers as novel fillers in PMMA bone cement nanocomposites. Two-dimensional MgP nanosheets and one-dimensional HA nanofibers were synthesized by tuning the crystallization of the sodium-magnesium-phosphate ternary system and hydrothermal homogeneous precipitation, respectively. We show that MgP nanosheets exhibit antibacterial properties against Escherichia coli (E. coli). In addition, HA nanofibers with high level of bioactivity are the proper choice to induce cell viability in the nanocomposite. Results indicate that the combination of both fillers can act as deformation locks enhancing the compressive strength of the nanocomposites. The synthesized nanocomposite possesses excellent bioactivity, mechanical properties, and cytocompatibility potentially opening new paradigm in the design of next generation bone cement composites.

While titanium is the metal of choice for most prosthetics and inner body devices due to its superior biocompatibility, the discovery of Ti-containing species in the adjacent tissue as a result of wear and corrosion has been associated with autoimmune diseases and premature implant failures. Here, we utilize the in situ liquid cell transmission electron microscopy (TEM) in a liquid flow holder and graphene liquid cells (GLCs) to investigate, for the first time, the in situ nano–bio interactions between titanium dioxide nanoparticles and biological medium. This imaging and spectroscopy methodology showed the process of formation of an ionic and proteic bio-camouflage surrounding Ti dioxide (anatase) nanoparticles that facilitates their internalization by bone cells. The in situ understanding of the mechanisms of the formation of the bio-camouflage of anatase nanoparticles may contribute to the definition of strategies aimed at the manipulation of these NPs for bone regenerative purposes.

A controlled electron-water radiolysis process is used to generate predictable concentrations of radical and ionic species in graphene liquid cells, allowing the concept of a nanoscale chemical reactor. A differential scanning technique is used to generate the desired time- and space-varying electron dose rate. Precise control of the local concentration of H2 , the dominant radiolysis species, is demonstrated experimentally at the nanometer scale.

A series of in situ transmission electron microscopy combined with scanning tunneling microscopy measurements were carried out to investigate the effect of mechanical deformation on the electrical transport properties of amorphous TiO2 nanotubes. Under no mechanical straining, it was found that the TiO2 nanotubes behave as electrical insulators. However, the nanotubes show semiconducting behavior under a highly deformed state. On the basis of a metal–semiconductor–metal model, it was suggested that in-shell defects, surface defect-driven conduction modes, are responsible for the appearance of the semiconducting behavior.

In this work carbon nanotubes (CNTs) were used to increase the strength and toughness of the hydroxyapatite (HA) and consequently to reduce its brittleness. The combination of CNT, HA and polymethyl methacrylate (PMMA) has led to a new composite material, which has mechanical properties superior to those of conventional HA/PMMA for biomedical scaffold in tissue engineering. PMMA is a well known bone cement which is highly compatible with HA and also it can act as a functionalizing/linking material with HA. The mechanical properties of the new nanocomposite were predicted with a self-consistent computational model taking into account the structure morphology and the orientation of the CNTs. CNT reinforced HA composite is shown to be a promising coating material for high-load-bearing metal implants. The development of this new nanocomposite based on HA/PMMA and CNTs, may significantly contribute to the bond strength of the HA/PMMA metal interface and the overall mechanical properties of the HA/PMMA coating.

The paper reports on a freeze-granulation technique to prepare a novel nanocomposite of poly(methyl methacrylate) (PMMA)-modified hydroxyapatite (HA) with multiwalled carbon nanotubes (MWCNTs) as reinforcement for a new generation biomedical bone cement and implant coatings. By using this technique it is possible to increase material homogeneity and also enhance the dispersion of MWCNTs in the composite matrix. The phase composition and the surface morphology of the nanocomposite material were studied using X-ray diffraction, field-emission scanning electron microscopy, and micro-Raman an spectroscopy. Additionally, nanomechanical properties of different concentrations of MWCNT-reinforced nanocomposite were performed by a nanoindentation technique, which indicates that a concentration of 0.1 wt % MWCNTs in the PMMA/HA nanocomposite material gives the best mechanical properties.

A novel method for fabricating thin films of oriented and densely packed metal functionalised carbon nanotubes (m-CNT) on silicon substrates is described. Positively charged metal ion from their salt solution was adsorbed on carboxylic acid funtionalised (f) CNT by electrostatic interaction. Subsequently, the aqueous dispersion of m-CNT was cast on silicon substrate forming m-CNT thin films after evaporation. Thin films of f-CNT and m-CNT were characterized by Scanning Electron Microscopy (SEM), Fourier Transform Infrared (FTIR) and Raman spectroscopy analysis. The mechanism of functionalisation and orientation of CNTs is discussed briefly.

Atomic and electronic structures of hydrated ferritin are characterized using electron microscopy and spectroscopy through encapsulation in single layer graphene in a biocompatible manner. Graphene's ability to reduce radiation damage levels to hydrogen bond breakage is demonstrated. A reduction of iron valence from 3+ to 2+ is measured at nanometer-resolution in ferritin, showing initial stages of iron release by ferritin.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

A combination of Hydroxyapatite (HA), Polymethylmethacrylate (PMMA) and Carbon Nanotubes (CNTs) was used to synthesize a new composite material, which is superior in mechanical properties to the conventional HA as a biomedical scaffold in tissue engineering. PMMA is well-known as a bone cement highly compatible with HA and can act as a functionalising/linking and/or coupling agent with the HA-CNTs mixtures, while the unique and excellent structure and properties of CNTs, after functionalisation, are able to reinforce and strengthen the porous HA matrix. The evolution of the secondary phases of HA may impair the mechanical properties; however, the evolving species (calcium oxide, tetra-calcium and tri-calcium phosphates or amorphous calcium phosphates) are trapped in the CNTs-PMMA network yielding a nanocomposite with improved mechanical and longer lasting lifetime performance, based on preliminary observations, shows good biocompatibility, and a detailed study to evaluate its biocompatibility is underway. The experimental study was characterised by means of X-Ray Diffraction (XRD), vibrational Raman spectroscopy and Scanning Electron Microscopy (SEM).