Multi-resolution Correlative Ultrastructural and Chemical Analysis of Carious Enamel by Scanning Microscopy and Tomographic Imaging.

Caries, a major global disease associated with dental enamel demineralization, remains insufficiently understood to devise effective prevention or minimally invasive treatment. Understanding the ultrastructural changes in enamel is hampered by a lack of nanoscale characterization of the chemical spatial distributions within the dental tissue. This leads to the requirement to develop techniques based on various characterization methods. The purpose of the present study is to demonstrate the strength of analytic methods using a correlative technique on a single sample of human dental enamel as a specific case study to test the accuracy of techniques to compare regions in enamel. The science of the different techniques is integrated to genuinely study the enamel. The hierarchical structures within carious tissue were mapped using the combination of focused ion beam scanning electron microscopy with synchrotron X-ray tomography. The chemical changes were studied using scanning X-ray fluorescence (XRF) and X-ray wide-angle and small-angle scattering using a beam size below 80 nm for ångström and nanometer length scales. The analysis of XRF intensity gradients revealed subtle variations of Ca intensity in carious samples in comparison with those of normal mature enamel. In addition, the pathways for enamel rod demineralization were studied using X-ray ptychography. The results show the chemical and structural modification in carious enamel with differing locations. These results reinforce the need for multi-modal approaches to nanoscale analysis in complex hierarchically structured materials to interpret the changes of materials. The approach establishes a meticulous correlative characterization platform for the analysis of biomineralized tissues at the nanoscale, which adds confidence in the interpretation of the results and time-saving imaging techniques. The protocol demonstrated here using the dental tissue sample can be applied to other samples for statistical study and the investigation of nanoscale structural changes. The information gathered from the combination of methods could not be obtained with traditional individual techniques.

Characteristics of fine and ultrafine aerosols in the London underground.

Underground railway systems are recognised spaces of increased personal pollution exposure. We studied the number-size distribution and physico-chemical characteristics of ultrafine (PM0.1), fine (PM0.1-2.5) and coarse (PM2.5-10) particles collected on a London underground platform. Particle number concentrations gradually increased throughout the day, with a maximum concentration between 18:00 h and 21:00 h (local time). There was a maximum decrease in mass for the PM2.5, PM2.5-10 and black carbon of 3.9, 4.5 and ~ 21-times, respectively, between operable (OpHrs) and non-operable (N-OpHrs) hours. Average PM10 (52 µg m-3) and PM2.5 (34 µg m-3) concentrations over the full data showed levels above the World Health Organization Air Quality Guidelines. Respiratory deposition doses of particle number and mass concentrations were calculated and found to be two- and four-times higher during OpHrs compared with N-OpHrs, reflecting events such as train arrival/departure during OpHrs. Organic compounds were composed of aromatic hydrocarbons and polycyclic aromatic hydrocarbons (PAHs) which are known to be harmful to health. Specific ratios of PAHs were identified for underground transport that may reflect an interaction between PAHs and fine particles. Scanning transmission electron microscopy (STEM) chemical maps of fine and ultrafine fractions show they are composed of Fe and O in the form of magnetite and nanosized mixtures of metals including Cr, Al, Ni and Mn. These findings, and the low air change rate (0.17 to 0.46 h-1), highlight the need to improve the ventilation conditions.

Nanostars Carrying Multifunctional Neurotrophic Dendrimers Protect Neurons in Preclinical In Vitro Models of Neurodegenerative Disorders.

A challenge in neurology is the lack of efficient brain-penetrable neuroprotectants targeting multiple disease mechanisms. Plasmonic gold nanostars are promising candidates to deliver standard-of-care drugs inside the brain but have not been trialed as carriers for neuroprotectants. Here, we conjugated custom-made peptide dendrimers (termed H3/H6), encompassing motifs of the neurotrophic S100A4-protein, onto star-shaped and spherical gold nanostructures (H3/H6-AuNS/AuNP) and evaluated their potential as neuroprotectants and interaction with neurons. The H3/H6 nanostructures crossed a model blood-brain barrier, bound to plasma membranes, and induced neuritogenesis with the AuNS, showing higher potency/efficacy than the AuNP. The H3-AuNS/NP protected neurons against oxidative stress, the H3-AuNS being more potent, and against Parkinson's or Alzheimer's disease (PD/AD)-related cytotoxicity. Unconjugated S100A4 motifs also decreased amyloid beta-induced neurodegeneration, introducing S100A4 as a player in AD. Using custom-made dendrimers coupled to star-shaped nanoparticles is a promising route to activate multiple neuroprotective pathways and increase drug potency to treat neurodegenerative disorders.

Reconstruction of ovine axonal cytoarchitecture enables more accurate models of brain biomechanics.

There is an increased need and focus to understand how local brain microstructure affects the transport of drug molecules directly administered to the brain tissue, for example in convection-enhanced delivery procedures. This study reports a systematic attempt to characterize the cytoarchitecture of commissural, long association and projection fibres, namely the corpus callosum, the fornix and the corona radiata, with the specific aim to map different regions of the tissue and provide essential information for the development of accurate models of brain biomechanics. Ovine samples are imaged using scanning electron microscopy combined with focused ion beam milling to generate 3D volume reconstructions of the tissue at subcellular spatial resolution. Focus is placed on the characteristic cytological feature of the white matter: the axons and their alignment in the tissue. For each tract, a 3D reconstruction of relatively large volumes, including a significant number of axons, is performed and outer axonal ellipticity, outer axonal cross-sectional area and their relative perimeter are measured. The study of well-resolved microstructural features provides useful insight into the fibrous organization of the tissue, whose micromechanical behaviour is that of a composite material presenting elliptical tortuous tubular axonal structures embedded in the extra-cellular matrix. Drug flow can be captured through microstructurally-based models using 3D volumes, either reconstructed directly from images or generated in silico using parameters extracted from the database of images, leading to a workflow to enable physically-accurate simulations of drug delivery to the targeted tissue.

An overview of methods of fine and ultrafine particle collection for physicochemical characterisation and toxicity assessments.

Particulate matter (PM) is a crucial health risk factor for respiratory and cardiovascular diseases. The smaller size fractions, ≤2.5 µm (PM2.5; fine particles) and ≤0.1 µm (PM0.1; ultrafine particles), show the highest bioactivity but acquiring sufficient mass for in vitro and in vivo toxicological studies is challenging. We review the suitability of available instrumentation to collect the PM mass required for these assessments. Five different microenvironments representing the diverse exposure conditions in urban environments are considered in order to establish the typical PM concentrations present. The highest concentrations of PM2.5 and PM0.1 were found near traffic (i.e. roadsides and traffic intersections), followed by indoor environments, parks and behind roadside vegetation. We identify key factors to consider when selecting sampling instrumentation. These include PM concentration on-site (low concentrations increase sampling time), nature of sampling sites (e.g. indoors; noise and space will be an issue), equipment handling and power supply. Physicochemical characterisation requires micro- to milli-gram quantities of PM and it may increase according to the processing methods (e.g. digestion or sonication). Toxicological assessments of PM involve numerous mechanisms (e.g. inflammatory processes and oxidative stress) requiring significant amounts of PM to obtain accurate results. Optimising air sampling techniques are therefore important for the appropriate collection medium/filter which have innate physical properties and the potential to interact with samples. An evaluation of methods and instrumentation used for airborne virus collection concludes that samplers operating cyclone sampling techniques (using centrifugal forces) are effective in collecting airborne viruses. We highlight that predictive modelling can help to identify pollution hotspots in an urban environment for the efficient collection of PM mass. This review provides guidance to prepare and plan efficient sampling campaigns to collect sufficient PM mass for various purposes in a reasonable timeframe.

Nanoscale Chemical Heterogeneity in Aromatic Polyamide Membranes for Reverse Osmosis Applications.

Reverse osmosis membranes are used within the oil and gas industry for seawater desalination on off-shore oilrigs. The membranes consist of three layers of material: a polyester backing layer, a polysulfone support and a polyamide (PA) thin film separating layer. It is generally thought that the PA layer controls ion selectivity within the membrane but little is understood about its structure or chemistry at the molecular scale. This active polyamide layer is synthesized by interfacial polymerization at an organic/aqueous interface between m-phenylenediamine and trimesoyl chloride, producing a highly cross-linked PA polymer. It has been speculated that the distribution of functional chemistry within this layer could play a role in solute filtration. The only technique potentially capable of probing the distribution of functional chemistry within the active PA layer with sufficient spatial and energy resolution is scanning transmission electron microscopy combined with electron energy-loss spectroscopy (STEM-EELS). Its use is a challenge because organic materials suffer beam-induced damage at relatively modest electron doses. Here we show that it is possible to use the N K-edge to map the active layer of a PA film using monochromated EELS spectrum imaging. The active PA layer is 12 nm thick, which supports previous neutron reflectivity data. Clear changes in the fine structure of the C K-edge across the PA films are measured and we use machine learning to assign fine structure at this edge. Using this method, we map highly heterogeneous intensity variations in functional chemistry attributed to N-C═C bonds within the PA. Similarities are found with previous molecular dynamics simulations of PA showing regions with a higher density of amide bonding as a result of the aggregation process at similar length scales. The chemical pathways that can be deduced may offer a clearer understanding of the transport mechanisms through the membrane.

Frizzled-7-targeted delivery of zinc oxide nanoparticles to drug-resistant breast cancer cells.

There is a need for novel strategies to treat aggressive breast cancer subtypes and overcome drug resistance. ZnO nanoparticles (NPs) have potential in cancer therapy due to their ability to potently and selectively induce cancer cell apoptosis. Here, we tested the in vitro chemotherapeutic efficacy of ZnONPs loaded via a mesoporous silica nanolayer (MSN) towards drug-sensitive breast cancer cells (MCF-7: estrogen receptor-positive, CAL51: triple-negative) and their drug-resistant counterparts (MCF-7TX, CALDOX). ZnO-MSNs were coated on to gold nanostars (AuNSs) for future imaging capabilities in the NIR-II range. Electron and confocal microscopy showed that MSN-ZnO-AuNSs accumulated close to the plasma membrane and were internalized by cells. High-resolution electron microscopy showed that MSN coating degraded outside the cells, releasing ZnONPs that interacted with cell membranes. MSN-ZnO-AuNSs efficiently reduced the viability of all cell lines, and CAL51/CALDOX cells were more susceptible than MCF7/MCF-7-TX cells. MSN-ZnO-AuNSs were then conjugated with the antibody to Frizzled-7 (FZD-7), the receptor upregulated by several breast cancer cells. We used the disulphide (S-S) linker that could be cleaved with a high concentration of glutathione normally observed within cancer cells, releasing Zn2+ into the cytoplasm. FZD-7 targeting resulted in approximately three-fold amplified toxicity of MSN-ZnO-AuNSs towards the MCF-7TX drug-resistant cell line with the highest FZD-7 expression. This study shows that ZnO-MSs are promising tools to treat triple-negative and drug-resistant breast cancers and highlights the potential clinical utility of FZD-7 for delivery of nanomedicines and imaging probes specifically to these cancer types.

Limb bone scaling in hopping macropods and quadrupedal artiodactyls.

Bone adaptation is modulated by the timing, direction, rate and magnitude of mechanical loads. To investigate whether frequent slow, or infrequent fast, gaits could dominate bone adaptation to load, we compared scaling of the limb bones from two mammalian herbivore clades that use radically different high-speed gaits, bipedal hopping (suborder Macropodiformes; kangaroos and kin) and quadrupedal galloping (order Artiodactyla; goats, deer and kin). Forelimb and hindlimb bones were collected from 20 artiodactyl and 15 macropod species (body mass M 1.05-1536 kg) and scanned in computed tomography or X-ray microtomography. Second moment of area (I max) and bone length (l) were measured. Scaling relations (y = axb ) were calculated for l versus M for each bone and for I max versus M and I max versus l for every 5% of length. I max versus M scaling relationships were broadly similar between clades despite the macropod forelimb being nearly unloaded, and the hindlimb highly loaded, during bipedal hopping. I max versus l and l versus M scaling were related to locomotor and behavioural specializations. Low-intensity loads may be sufficient to maintain bone mass across a wide range of species. Occasional high-intensity gaits might not break through the load sensitivity saturation engendered by frequent low-intensity gaits.

Nanoanalytical electron microscopy of events predisposing to mineralisation of turkey tendon.

The macro- and micro-structures of mineralised tissues hierarchy are well described and understood. However, investigation of their nanostructure is limited due to the intrinsic complexity of biological systems. Preceding transmission electron microscopy studies investigating mineralising tissues have not resolved fully the initial stages of mineral nucleation and growth within the collagen fibrils. In this study, analytical scanning transmission electron microscopy and electron energy-loss spectroscopy were employed to characterise the morphology, crystallinity and chemistry of the mineral at different stages of mineralization using a turkey tendon model. In the poorly mineralised regions, calcium ions associated with the collagen fibrils and ellipsoidal granules and larger clusters composed of amorphous calcium phosphate were detected. In the fully mineralised regions, the mineral had transformed into crystalline apatite with a plate-like morphology. A change in the nitrogen K-edge was observed and related to modifications of the functional groups associated with the mineralisation process. This transformation seen in the nitrogen K-edge might be an important step in maturation and mineralisation of collagen and lend fundamental insight into how tendon mineralises.

Electron Microscopy Reveals Structural and Chemical Changes at the Nanometer Scale in the Osteogenesis Imperfecta Murine Pathology.

Alternations of collagen and mineral at the molecular level may have a significant impact on the strength and toughness of bone. In this study, scanning transmission electron microscopy (STEM) and electron energy-loss spectroscopy (EELS) were employed to study structural and compositional changes in bone pathology at nanometer spatial resolution. Tail tendon and femoral bone of osteogenesis imperfecta murine (oim, brittle bone disease) and wild type (WT) mice were compared to reveal defects in the architecture and chemistry of the collagen and collagen-mineral composite in the oim tissue at the molecular level. There were marked differences in the substructure and organization of the collagen fibrils in the oim tail tendon; some regions have clear fibril banding and organization, while in other regions fibrils are disorganized. Malformed collagen fibrils were loosely packed, often bent and devoid of banding pattern. In bone, differences were detected in the chemical composition of mineral in oim and WT. While mineral present in WT and oim bone exhibited the major characteristics of apatite, examination in EELS of the fine structure of the carbon K ionization edge revealed a significant variation in the presence of carbonate in different regions of bone. Variations have been also observed in the fine structure and peak intensities of the nitrogen K-edge. These alterations are suggestive of differences in the maturation of collagen nucleation sites or cross-links. Future studies will aim to establish the scale and impact of the modifications observed in oim tissues. The compositional and structural alterations at the molecular level cause deficiencies at larger length scales. Understanding the effect of molecular alterations to pathologic bone is critical to the design of effective therapeutics.

Probing carbonate in bone forming minerals on the nanometre scale.

To devise new strategies to treat bone disease in an ageing society, a more detailed characterisation of the process by which bone mineralises is needed. In vitro studies have suggested that carbonated mineral might be a precursor for deposition of bone apatite. Increased carbonate content in bone may also have significant implications in altering the mechanical properties, for example in diseased bone. However, information about the chemistry and coordination environment of bone mineral, and their spatial distribution within healthy and diseased tissues, is lacking. Spatially resolved analytical transmission electron microscopy is the only method available to probe this information at the length scale of the collagen fibrils in bone. In this study, scanning transmission electron microscopy combined with electron energy-loss spectroscopy (STEM-EELS) was used to differentiate between calcium-containing biominerals (hydroxyapatite, carbonated hydroxyapatite, beta-tricalcium phosphate and calcite). A carbon K-edge peak at 290 eV is a direct marker of the presence of carbonate. We found that the oxygen K-edge structure changed most significantly between minerals allowing discrimination between calcium phosphates and calcium carbonates. The presence of carbonate in carbonated HA (CHA) was confirmed by the formation of peak at 533 eV in the oxygen K-edge. These observations were confirmed by simulations using density functional theory. Finally, we show that this method can be utilised to map carbonate from the crystallites in bone. We propose that our calibration library of EELS spectra could be extended to provide spatially resolved information about the coordination environment within bioceramic implants to stimulate the development of structural biomaterials.

Whole-bone scaling of the avian pelvic limb.

Birds form the largest extant group of bipedal animals and occupy a broad range of body masses, from grams to hundreds of kilograms. Additionally, birds occupy distinct niches of locomotor behaviour, from totally flightless strong runners such as the ratites (moa, kiwi, ostrich) to birds that may walk, dabble on water or fly. We apply a whole-bone approach to investigate allometric scaling trends in the pelvic limb bones (femur, tibiotarsus, tarsometatarsus) from extant and recently extinct birds of greatly different size, and compare scaling between birds in four locomotor groups; flightless, burst-flying, dabbling and flying. We also compare scaling of birds' femoral cross-sectional properties to data previously collected from cats. Scaling exponents were not significantly different between the different locomotor style groups, but elevations of the scaling relationships revealed that dabblers (ducks, geese, swans) have particularly short and slender femora compared with other birds of similar body mass. In common with cats, but less pronounced in birds, the proximal and distal extrema of the bones scaled more strongly than the diaphysis, and in larger birds the diaphysis occupied a smaller proportion of bone length than in smaller birds. Cats and birds have similar femoral cross-sectional area (CSA) for the same body mass, yet birds' bone material is located further from the bone's long axis, leading to higher second and polar moments of area and a greater inferred resistance to bending and twisting. The discrepancy in the relationship between outer diameter to CSA may underlie birds' reputation for having 'light' bones.

3D Morphometric and posture study of felid scapulae using statistical shape modelling.

We present a three dimensional (3D) morphometric modelling study of the scapulae of Felidae, with a focus on the correlations between forelimb postures and extracted scapular shape variations. Our shape modelling results indicate that the scapular infraspinous fossa becomes larger and relatively broader along the craniocaudal axis in larger felids. We infer that this enlargement of the scapular fossa may be a size-related specialization for postural support of the shoulder joint.

Trabecular bone scales allometrically in mammals and birds.

Many bones are supported internally by a latticework of trabeculae. Scaling of whole bone length and diameter has been extensively investigated, but scaling of the trabecular network is not well characterized. We analysed trabecular geometry in the femora of 90 terrestrial mammalian and avian species with body masses ranging from 3 g to 3400 kg. We found that bone volume fraction does not scale substantially with animal size, while trabeculae in larger animals' femora are thicker, further apart and fewer per unit volume than in smaller animals. Finite element modelling indicates that trabecular scaling does not alter the bulk stiffness of trabecular bone, but does alter strain within trabeculae under equal applied loads. Allometry of bone's trabecular tissue may contribute to the skeleton's ability to withstand load, without incurring the physiological or mechanical costs of increasing bone mass.

BoneJ: Free and extensible bone image analysis in ImageJ.

Bone geometry is commonly measured on computed tomographic (CT) and X-ray microtomographic (µCT) images. We obtained hundreds of CT, µCT and synchrotron µCT images of bones from diverse species that needed to be analysed remote from scanning hardware, but found that available software solutions were expensive, inflexible or methodologically opaque. We implemented standard bone measurements in a novel ImageJ plugin, BoneJ, with which we analysed trabecular bone, whole bones and osteocyte lacunae. BoneJ is open source and free for anyone to download, use, modify and distribute.