Significant deterioration in nanomechanical quality occurs through incomplete extrafibrillar mineralization in rachitic bone: Evidence from in-situ synchrotron X-ray scattering and backscattered electron imaging

Bone diseases such as rickets and osteoporosis cause significant reduction in bone quantity and quality, which leads to mechanical abnormalities. However, the precise ultrastructural mechanism by which altered bone quality affects mechanical properties is not clearly understood. Here we demonstrate the functional link between altered bone quality (reduced mineralization) and abnormal fibrillar-level mechanics using a novel, real-time synchrotron X-ray nanomechanical imaging method to study a mouse model with rickets due to reduced extrafibrillar mineralization. A previously unreported N-ethyl-N-nitrosourea (ENU) mouse model for hypophosphatemic rickets (Hpr), as a result of missense Trp314Arg mutation of the phosphate regulating gene with homologies to endopeptidase on the X chromosome (Phex) and with features consistent with X-linked hypophosphatemic rickets (XLHR) in man, was investigated using in situ synchrotron small angle X-ray scattering to measure real-time changes in axial periodicity of the nanoscale mineralized fibrils in bone during tensile loading. These determine nanomechanical parameters including fibril elastic modulus and maximum fibril strain. Mineral content was estimated using backscattered electron imaging. A significant reduction of effective fibril modulus and enhancement of maximum fibril strain was found in Hpr mice. Effective fibril modulus and maximum fibril strain in the elastic region increased consistently with age in Hpr and wild-type mice. However, the mean mineral content was ∼21% lower in Hpr mice and was more heterogeneous in its distribution. Our results are consistent with a nanostructural mechanism in which incompletely mineralized fibrils show greater extensibility and lower stiffness, leading to macroscopic outcomes such as greater bone flexibility. Our study demonstrates the value of in situ X-ray nanomechanical imaging in linking the alterations in bone nanostructure to nanoscale mechanical deterioration in a metabolic bone disease. Copyright

Complex crystallization dynamics in amorphous germanium observed with dynamic transmission electron microscopy

Crystallization of amorphous germanium (a-Ge) by laser or electron beam heating is a remarkably complex process that involves several distinct modes of crystal growth and the development of intricate microstructural patterns on the nanosecond to ten microsecond time scales. Here we use dynamic transmission electron microscopy (DTEM) to study the fast, complex crystallization dynamics with 10 nm spatial and 15 ns temporal resolution. We have obtained time-resolved real-space images of nanosecond laser-induced crystallization in a-Ge with unprecedentedly high spatial resolution. Direct visualization of the crystallization front allows for time-resolved snapshots of the initiation and roughening of the dendrites on submicrosecond time scales. This growth is followed by a rapid transition to a ledgelike growth mechanism that produces a layered microstructure on a time scale of several microseconds. This study provides insights into the mechanisms governing this complex crystallization process and is a dramatic demonstration of the power of DTEM for studying time-dependent material processes far from equilibrium.

In situ investigation of explosive crystallization in a-Ge: Experimental determination of the interface response function using dynamic transmission electron microscopy

The crystallization of amorphous semiconductors is a strongly exothermic process. Once initiated the release of latent heat can be sufficient to drive a self-sustaining crystallization front through the material in a manner that has been described as explosive. Here, we perform a quantitative in situ study of explosive crystallization in amorphous germanium using dynamic transmission electron microscopy. Direct observations of the speed of the explosive crystallization front as it evolves along a laser-imprinted temperature gradient are used to experimentally determine the complete interface response function (i.e., the temperature-dependent front propagation speed) for this process, which reaches a peak of 16 m/s. Fitting to the Frenkel-Wilson kinetic law demonstrates that the diffusivity of the material locally/immediately in advance of the explosive crystallization front is inconsistent with those of a liquid phase. This result suggests a modification to the liquid-mediated mechanism commonly used to describe this process that replaces the phase change at the leading amorphous-liquid interface with a change in bonding character (from covalent to metallic) occurring in the hot amorphous material.

Comparative leaf anatomy and micromorphology of the Chilean Myrtaceae: Taxonomic and ecological implications

The family Myrtaceae in Chile comprises 26 species in 10 genera. The species occur in a diverse rangeof environments including humid temperate forests, swamps, riparian habitats and coastal xeromorphicshrublands. Most of these species are either endemic to Chile or endemic to the humid temperate forestsof Chile and Argentina. Although many taxa have very restricted distributions and are of conservationconcern, little is known about their biology and vegetative anatomy. In this investigation, we describe andcompare the leaf anatomy and micromorphology of all Chilean Myrtaceae using standard protocols forlight and scanning electron microscopy. Leaf characters described here are related to epidermis, cuticle,papillae, stomata, hairs, mesophyll, crystals, secretory cavities and vascular system. Nearly all the specieshave a typical mesophytic leaf anatomy, but some species possess xerophytic characters such as doubleepidermis, hypodermis, pubescent leaves, thick adaxial epidermis and straight epidermal anticlinal walls,which correlate with the ecological distribution of the species. This is the first report on leaf anatomyand micromorphology in most of these species. We identified several leaf characters with potential tax-onomic and ecological significance. Some combinations of leaf characters can reliably delimitate genera,while others are unique to some species. An identification key using micromorphological and anatomicalcharacters is provided to distinguish genera and species.

Assessment of conjunctival goblet cell density using laser scanning confocal microscopy versus impression cytology

Purpose To determine the association between conjunctival goblet cell density (GCD) assessed using in vivo laser scanning confocal microscopy and conjunctival impression cytology in a healthy population. Methods Ninety (90) healthy participants undertook a validated 5-item dry eye questionnaire, non-invasive tear film break-up time measurement, ocular surface fluorescein staining and phenol red thread test. These tests where undertaken to diagnose and exclude participants with dry eye. The nasal bulbar conjunctiva was imaged using laser scanning confocal microscopy (LSCM). Conjunctival impression cytology (CIC) was performed in the same region a few minutes later. Conjunctival goblet cell density was calculated as cells/mm2. Results There was a strong positive correlation of conjunctival GCD between LSCM and CIC (ρ = 0.66). Conjunctival goblet cell density was 475 ± 41 cells/mm2 and 466 ± 51 cells/mm2 measured by LSCM and CIC, respectively. Conclusions The strong association between in vivo and in vitro cellular analysis for measuring conjunctival GCD suggests that the more invasive CIC can be replaced by the less invasive LSCM in research and clinical practice.

Study on microwave dielectric properties of epoxy resin mixtures used for rapid product development

This paper presents a preliminary study on the dielectric properties and curing of three different types of epoxy resins mixed at various stichiometric mixture of hardener, flydust and aluminium powder under microwave energy. In this work, the curing process of thin layers of epoxy resins using microwave radiation was investigated as an alternative technique that can be implemented to develop a new rapid product development technique. In this study it was observed that the curing time and temperature were a function of the percentage of hardener and fillers presence in the epoxy resins. Initially dielectric properties of epoxy resins with hardener were measured which was directly correlated to the curing process in order to understand the properties of cured specimen. Tensile tests were conducted on the three different types of epoxy resins with hardener and fillers. Modifying dielectric properties of the mixtures a significant decrease in curing time was observed. In order to study the microstructural changes of cured specimen the morphology of the fracture surface was carried out by using scanning electron microscopy.

Polyurethane (PU) scaffolds prepared by solvent casting/particulate leaching (SCPL) combined with centrifugation

This article reports an enhanced solvent casting/particulate (salt) leaching (SCPL) method developed for preparing three-dimensional porous polyurethane (PU) scaffolds for cardiac tissue engineering. The solvent for the preparation of the PU scaffolds was a mixture of dimethylformamide (DFM) and tetrahydrofuran (THF). The enhanced method involved the combination of a conventional SCPL method and a step of centrifugation, with the centrifugation being employed to improve the pore uniformity and the pore interconnectivity of scaffolds. Highly porous three-dimensional scaffolds with a well interconnected porous structure could be achieved at the polymer solution concentration of up to 20% by air or vacuum drying to remove the solvent. When the salt particle sizes of 212-295, 295-425, or 425-531 µm and a 15% w/v polymer solution concentration were used, the porosity of the scaffolds was between 83-92% and the compression moduli of the scaffolds were between 13 kPa and 28 kPa. Type I collagen acidic solution was introduced into the pores of a PU scaffold to coat the collagen onto the pore walls throughout the whole PU scaffold. The human aortic endothelial cells (HAECs) cultured in the collagen-coated PU scaffold for 2 weeks were observed by scanning electron microscopy (SEM). It was shown that the enhanced SCPL method and the collagen coating resulted in a spatially uniform distribution of cells throughout the collagen-coated PU scaffold.

Processing of polycaprolactone and polycaprolactone-based copolymers into 3D scaffolds and their cellular responses

Synthetic polymers have attracted much attention in tissue engineering due to their ability to modulate biomechanical properties. This study investigated the feasibility of processing poly(varepsilon-caprolactone) (PCL) homopolymer, PCL-poly(ethylene glycol) (PEG) diblock, and PCL-PEG-PCL triblock copolymers into three-dimensional porous scaffolds. Properties of the various polymers were investigated by dynamic thermal analysis. The scaffolds were manufactured using the desktop robot-based rapid prototyping technique. Gross morphology and internal three-dimensional structure of scaffolds were identified by scanning electron microscopy and micro-computed tomography, which showed excellent fusion at the filament junctions, high uniformity, and complete interconnectivity of pore networks. The influences of process parameters on scaffolds' morphological and mechanical characteristics were studied. Data confirmed that the process parameters directly influenced the pore size, porosity, and, consequently, the mechanical properties of the scaffolds. The in vitro cell culture study was performed to investigate the influence of polymer nature and scaffold architecture on the adhesion of the cells onto the scaffolds using rabbit smooth muscle cells. Light, scanning electron, and confocal laser microscopy showed cell adhesion, proliferation, and extracellular matrix formation on the surface as well as inside the structure of both scaffold groups. The completely interconnected and highly regular honeycomb-like pore morphology supported bridging of the pores via cell-to-cell contact as well as production of extracellular matrix at later time points. The results indicated that the incorporation of hydrophilic PEG into hydrophobic PCL enhanced the overall hydrophilicity and cell culture performance of PCL-PEG copolymer. However, the scaffold architecture did not significantly influence the cell culture performance in this study.

Cryoreservation of alginate-fibrin beads involving bone marrow derived mesenchymal stromal cells by vitrification

Application of cell-–biomaterial systems in regenerative medicine can be facilitated by their successful low temperature preservation. Vitrification, which avoids ice crystal formation by amorphous solidification, is an emerging approach to cryopreservation. Developing vitrification strategy, effective cryopreservation of alginate–fibrin beads with porcine mesenchymal stromal cells has been achieved in this study. The cell–biomaterial constructs were pre-cultured for 20 days before cryopreservation, allowing for cell proliferation and construct stabilization. Ethylene glycol (EG) was employed as the basic cryoprotectant for two equilibration solutions. Successful cryopreservation of the constructs was achieved using vitrification solution composed of penetrating (EG MW 62 Da) and non-penetrating (sucrose MW 342 Da) cryoprotectants. Stepwise procedure of introduction to and removal of cryoprotectants was brief; direct plunging into liquid nitrogen was applied. Cell viability, evaluated by combining live/death staining and confocal laser microscopy, was similar for both control and vitrified cells in the beads. No detectable damage of microstructure of cryopreserved beads was found as shown by scanning electron microscopy. Both osteogenically induced control and vitrified cells in the constructs were equally capable of mineral production and deposition. There was no statistically significant difference in metabolic activity and proliferation between both groups during the entire culture period. Our study leads to the conclusion that the developed cryopreservation protocol allowed to maintain the integrity of the beads while preserving the ability of the pig bone marrow derived mesenchymal stromal cells to proliferate and subsequently differentiate; demonstrating that vitrification is a promising approach for cryopreser-vation of “ready-to-use” cell–biomaterial constructs.

Synthesis, characterization of palygorskite supported zero-valent iron and its application for methylene blue adsorption

In this work, natural palygorskite impregnated with zero-valent iron (ZVI) was prepared and characterised. The combination of ZVI particles on surface of fibrous palygorskite can help to overcome the disadvantage of ultra-fine powders which may have strong tendency to agglomerate into larger particles, resulting in an adverse effect on both effective surface area and catalyst performance. There is a significant increase of methylene blue (MB) decolourized efficiency on acid treated palygorskite with ZVI grafted, within 5 mins, the concentration of MB in the solution was decreased from 94 mg/L to around 20 mg/L and the equilibration was reached at about 30 to 60 mins with only around 10 mg/L MB remained in solution. Changes in the surface and structure of prepared materials were characterized using X-ray diffraction (XRD), infrared (IR) spectroscopy, surface analysing and scanning electron microscopy (SEM) with element analysis and mapping. Comparing with zero-valent iron and palygorskite, the presence of zero-valent iron reactive species on the palygorskite surface strongly increases the decolourization capacity for methylene blue, and it is significant for providing novel modified clay catalyst materials for the removal of organic contaminants from waste water.

Earliest diagenesis in scleractinian coral skeletons : implications for palaeoclimate-sensitive geochemical archives

Live-collected samples of four common reef building coral genera (Acropora, Pocillopora, Goniastrea, Porites) from subtidal and intertidal settings of Heron Reef, Great Barrier Reef, show extensive early marine diagenesis where parts of the coralla less than 3 years old contain abundant macro- and microborings and aragonite, high-Mg calcite, low-Mg calcite, and brucite cements. Many types of cement are associated directly with microendoliths and endobionts that inhabit parts of the corallum recently abandoned by coral polyps. The occurrence of cements that generally do not precipitate in normal shallow seawater (e.g., brucite, low-Mg calcite) highlights the importance of microenvironments in coral diagenesis. Cements precipitated in microenvironments may not reXect ambient seawater chemistry. Hence, geochemical sampling of these cements will contaminate trace-element and stable-isotope inventories used for palaeoclimate and dating analysis. Thus, great care must be taken in vetting samples for both bulk and microanalysis of geochemistry. Visual inspection using scanning electron microscopy may be required for vetting in many cases.

Synthesis and characterization of boehmite nanofibers

Boehmite nanofibers of high quality were synthesized through a wet-gel conversion process without the use of a surfactant. The long nanofibers of boehmite with clear-cut edges were obtained by steaming the wet-gel precipitate at 170 ºC for 2 days under a pH 5. Hydrothermal treatment of the boehmite gels enabled self-assembly through directed crystal growth. Detailed characterization using X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), Infrared Emission Spectroscopy (IES) and Raman Spectroscopy is presented.

Synthesis and characterisation of cobalt hydroxide, cobalt oxyhydroxide and cobalt oxide nanodiscs

Cobalt hydroxide, cobalt oxyhydroxide and cobalt oxide nanomaterials were synthesized through simple soft chemistry. The cobalt hydroxide displays hexagonal morphology with clear edges 20 nm long. This morphology and nanosize is retained through to cobalt oxide Co3O4 through a topotactical relationship. Cobalt oxyhydroxide and cobalt oxide nanomaterials were synthesized through oxidation and low temperature calcination from the as-prepared cobalt hydroxide. Characterisation of these cobalt-based nanomaterials were fully developed, including X-ray diffraction, transmission electron microscopy combined with selected area electron diffraction, scanning electron microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy, and thermal gravimetric analysis. Bonding of the divalent cobalt hydroxide from the oxyhydroxide and oxides by studying their high resolution XPS spectra for Co 2p3/2 and O 1s. Raman spectroscopy of the as-prepared Co(OH)2, CoO(OH) and Co3O4 nanomaterials characterised each material. The thermal stability of the materials Co(OH)2 and CoO(OH) were established. This research has developed methodology for the synthesis of cobalt oxide and cobalt oxyhydroxide nanodiscs at low temperatures.

Taxonomy and diagnostics of fruit fly infesting Opiine Braconids in Australia and the South Pacific

Opiine wasps (Hymenoptera: Braconidae: Opiinae) are parasitoids of dacine fruit flies (Diptera: Tephritidae: Dacinae), the primary horticultural pests of Australia and the South Pacific. Effective use of opiines for biological control of fruit flies is limited by poor taxonomy and identification difficulties. To overcome these problems, this thesis had two aims: (i) to carry out traditional taxonomic research on the fruit fly infesting opine braconids of Australia and the South Pacific; and (ii) to transfer the results of the taxonomic research into user friendly diagnostic tools. Curated wasp material was borrowed from all major Australian museum collections holding specimens. This was supplemented by a large body of material gathered as part of a major fruit fly project in Papua New Guinea: nearly 4000 specimens were examined and identified. Each wasp species was illustrated using traditional scientific drawings, full colour photomicroscopy and scanning electron microscopy. An electronic identification key was developed using Lucid software and diagnostic images were loaded on the web-based Pest and Diseases Image Library (PaDIL). A taxonomic synopsis and distribution and host records for each of the 15 species of dacine-parasitising opiine braconids found in the South Pacific is presented. Biosteres illusorius Fischer (1971) was formally transferred to the genus Fopius and a new species, Fopius ferrari Carmichael and Wharton (2005), was described. Other species dealt with were Diachasmimorpha hageni (Fullaway, 1952), D. kraussii (Fullaway, 1951), D. longicaudata (Ashmead, 1905), D. tryoni (Cameron, 1911), Fopius arisanus (Sonan, 1932), F. deeralensis (Fullaway, 1950), F. schlingeri Wharton (1999), Opius froggatti Fullaway (195), Psyttalia fijiensis (Fullaway, 1936), P. muesebecki (Fischer, 1963), P. novaguineensis (Szépliget, 1900i) and Utetes perkinsi (Fullaway, 1950). This taxonomic component of the thesis has been formally published in the scientific literature. An interactive diagnostics package (“OpiineID”) was developed, the centre of which is a Lucid based multi-access key. Because the diagnostics package is computer based, without the space limitations of the journal publication, there is no pictorial limit in OpiineID and so it is comprehensively illustrated with SEM photographs, full colour photographs, line drawings and fully rendered illustrations. The identification key is only one small component of OpiineID and the key is supported by fact sheets with morphological descriptions, host associations, geographical information and images. Each species contained within the OpiineID package has also been uploaded onto the PaDIL website (www.padil.gov.au). Because the identification of fruit fly parasitoids is largely of concern to fruit fly workers, rather than braconid specialists, this thesis deals directly with an area of growing importance to many areas of pure and applied biology; the nexus between taxonomy and diagnostics. The Discussion chapter focuses on this area, particularly the opportunities offered by new communication and information tools as new ways delivering the outputs of taxonomic science.

Structure–property relationships of silk-modified mesoporous bioglass scaffolds

Porous mesopore-bioglass (MBG) scaffolds have been proposed as a new class of bone regeneration materials due to their apatite-formation and drug-delivery properties; however, the material’s inherent brittleness and high degradation and surface instability are major disadvantages, which compromise its mechanical strength and cytocompatibility as a biological scaffold. Silk, on the other hand, is a native biomaterial and is well characterized with respect to biocompatibility and tensile strength. In this study we set out to investigate what effects blending silk with MBG had on the physiochemical, drug-delivery and biological properties of MBG scaffolds with a view to bone tissue engineering applications. Transmission electron microscopy (TEM), scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) were the methods used to analyze the inner microstructure, pore size and morphology, and composition of MBG scaffolds, before and after addition of silk. The effect of silk modification on the mechanical property of MBG scaffolds was determined by testing the compressive strength of the scaffolds and also compressive strength after degradation over time. The drug-delivery potential was evaluated by the release of dexamethasone (DEX) from the scaffolds. Finally, the cytocompatibility of silk-modified scaffolds was investigated by the attachment, morphology, proliferation, differentiation and bone-relative gene expression of bone marrow stromal cells (BMSCs). The results showed that silk modification improved the uniformity and continuity of pore network of MBG scaffolds, and maintained high porosity (94%) and large-pore size (200–400 mm). There was a significant improvement in mechanical strength, mechanical stability, and control of burst release of DEX in silkmodified MBG scaffolds. Silk modification also appeared to provide a better environment for BMSC attachment, spreading, proliferation, and osteogenic differentiation on MBG scaffolds.