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BACKGROUND: To perform a detailed morphological analysis of the inorganic portion of two different clinical presentations of calcium-based deposits retrieved from subjects with SSc and identify a chemical dissolution of these deposits suitable for clinical use. METHODS: Chemical analysis using Fourier Transform IR spectroscopy ('FTIR'), Raman microscopy, Powder X-Ray Diffraction ('PXRD'), and Transmission Electron Microscopy ('TEM') was undertaken of two distinct types of calcinosis deposits: paste and stone. Calcinosis sample titration with ethylenediaminetetraacetic acid ('EDTA') assessed the concentration at which the EDTA dissolved the calcinosis deposits in vitro. RESULTS: FTIR spectra of the samples displayed peaks characteristic of hydroxyapatite, where signals attributable to the phosphate and carbonate ions were all identified. Polymorph characterization using Raman spectra were identical to a hydroxyapatite reference while the PXRD and electron diffraction patterns conclusively identified the mineral present as hydroxyapatite. TEM analysis showed differences of morphology between the samples. Rounded particles from stone samples were up to a few micron in size, while needle-like crystals from paste samples reached up to 0.5 µm in length. Calcium phosphate deposits were effectively dissolved with 3% aqueous solutions of EDTA, in vitro. Complete dissolution of both types of deposit was achieved in approximately 30 min using a molar ratio of EDTA/HAp of ≈ 300. CONCLUSIONS: Stone and paste calcium-based deposits both comprise hydroxyapatite, but the constituent crystals vary in size and morphology. Hydroxyapatite is the only crystalline polymorph present in the SSc-related calcinosis deposits. Hydroxyapatite can be dissolved in vitro using a dosage of EDTA considered safe for clinical application. Further research is required to establish the optimal medium to develop the medical product, determine the protocol for clinical application, and to assess the effectiveness of EDTA for local treatment of dystrophic calcinosis.
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Calcinosis , Ácido Edético , Ácido Edético/química , Humanos , Calcinosis/tratamiento farmacológico , Calcinosis/patología , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Microscopía Electrónica de Transmisión/métodos , Difracción de Rayos X/métodos , Espectrometría Raman/métodos , Femenino , Durapatita/química , Persona de Mediana Edad , Masculino , Quelantes del Calcio/químicaRESUMEN
We present a comparison of common electron microscopy sample preparation methods for studying crystallisation processes from solution using both scanning and transmission electron microscopy (SEM and TEM). We focus on two widely studied inorganic systems: calcium sulphate, gypsum (CaSO4·2H2O) and calcium carbonate (CaCO3). We find significant differences in crystallisation kinetics and polymorph selection between the different sample preparation methods, which indicate that drying and chemical quenching can induce severe artefacts that are capable of masking the true native state of the crystallising solution. Overall, these results highlight the importance of cryogenic (cryo)-quenching crystallising solutions and the use of full cryo-TEM as the most reliable method for studying the early stages of crystallisation.
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The ability to control crystal nucleation through the simple addition of a nucleating agent (nucleant) is desirable for a huge range of applications. However, effective nucleating agents are known for only a small number of systems, and many questions remain about the mechanisms by which they operate. Here, we explore the features that make an effective nucleant and demonstrate that the biological material hair-which naturally possesses a chemically and topographically complex surface structure-has excellent potential as an effective nucleating agent. Crystallization of poorly soluble compounds in the presence of hairs from a range of mammals shows that nucleation preferentially occurs at the cuticle step edges, while a novel microdroplet-based methodology was used to quantify the nucleating activities of different hairs. This showed that the activities of the hairs can be tuned over a wide range using chemical treatments. Analysis of the hair structure and composition using atomic force microscopy, scanning ion conductance microscopy, and X-ray photoelectron spectroscopy demonstrates that surface chemistry, surface topography, and surface charge all act in combination to create effective nucleation sites. This work therefore contributes to our understanding of heterogeneous nucleating agents and shows that surface topography as well as surface chemistry can be used in the design or selection of universal nucleating agents.
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The cylindrical pores of track-etched membranes offer excellent environments for studying the effects of confinement on crystallization as the pore diameter is readily varied and the anisotropic morphologies can direct crystal orientation. However, the inability to image individual crystals in situ within the pores in this system has prevented many of the underlying mechanisms from being characterized. Here, we study the crystallization of calcium sulfate within track-etched membranes and reveal that oriented gypsum forms in 200 nm diameter pores, bassanite in 25-100 nm pores and anhydrite in 10 nm pores. The crystallization pathways are then studied by coating the membranes with an amorphous titania layer prior to mineralization to create electron transparent nanotubes that protect fragile precursor materials. By visualizing the evolutionary pathways of the crystals within the pores we show that the product single crystals derive from multiple nucleation events and that orientation is determined at early reaction times. Finally, the transformation of bassanite to gypsum within the membrane pores is studied using experiment and potential mean force calculations and is shown to proceed by localized dissolution/reprecipitation. This work provides insight into the effects of confinement on crystallization processes, which is relevant to mineral formation in many real-world environments.
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Calcium carbonate biomineralization is remarkable for the ability of organisms to produce calcite or aragonite with perfect fidelity, where this is commonly attributed to specific anionic biomacromolecules. However, it is proven difficult to mimic this behavior using synthetic or biogenic anionic organic molecules. Here, it is shown that cationic polyamines ranging from small molecules to large polyelectrolytes can exert exceptional control over calcium carbonate polymorph, promoting aragonite nucleation at extremely low concentrations but suppressing its growth at high concentrations, such that calcite or vaterite form. The aragonite crystals form via particle assembly, giving nanoparticulate structures analogous to biogenic aragonite, and subsequent growth yields stacked aragonite platelets comparable to structures seen in developing nacre. This mechanism of polymorph selectivity is captured in a theoretical model based on these competing nucleation and growth effects and is completely distinct from the activity of magnesium ions, which generate aragonite by inhibiting calcite. Profiting from these contrasting mechanisms, it is then demonstrated that polyamines and magnesium ions can be combined to give unprecedented control over aragonite formation. These results give insight into calcite/aragonite polymorphism and raise the possibility that organisms may exploit both amine-rich organic molecules and magnesium ions in controlling calcium carbonate polymorph.
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Recent advances in X-ray instrumentation and sample injection systems have enabled serial crystallography of protein nanocrystals and the rapid structural analysis of dynamic processes. However, this progress has been restricted to large-scale X-ray free-electron laser (XFEL) and synchrotron facilities, which are often oversubscribed and have long waiting times. Here, we explore the potential of state-of-the-art laboratory X-ray systems to perform comparable analyses when coupled to micro- and millifluidic sample environments. Our results demonstrate that commercial small- and wide-angle X-ray scattering (SAXS/WAXS) instruments and X-ray diffractometers are ready to access samples and timescales (â³5â ms) relevant to many processes in materials science including the preparation of pharmaceuticals, nanoparticles and functional crystalline materials. Tests of different X-ray instruments highlighted the importance of the optical configuration and revealed that serial WAXS/XRD analysis of the investigated samples was only possible with the higher flux of a microfocus setup. We expect that these results will also stimulate similar developments for structural biology.
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Calcite crystals grow by means of molecular steps that develop on {10.4} faces. These steps can arise stochastically via two-dimensional (2D) nucleation or emerge steadily from dislocations to form spiral hillocks. Here, we determine the kinetics of these two growth mechanisms as a function of supersaturation. We show that calcite crystals larger than â¼1 µm favor spiral growth over 2D nucleation, irrespective of the supersaturation. Spirals prevail beyond this length scale because slow boundary layer diffusion creates a low surface supersaturation that favors the spiral mechanism. Sub-micron crystals favor 2D nucleation at high supersaturations, although diffusion can still limit the growth of nanoscopic crystals. Additives can change the dominant mechanism by impeding spiral growth or by directly promoting 2D nucleation.
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Incorporation of guest additives within inorganic single crystals offers a unique strategy for creating nanocomposites with tailored properties. While anionic additives have been widely used to control the properties of crystals, their effective incorporation remains a key challenge. Here, we show that cationic additives are an excellent alternative for the synthesis of nanocomposites, where they are shown to deliver exceptional levels of incorporation of up to 70 wt % of positively charged amino acids, polymer particles, gold nanoparticles, and silver nanoclusters within inorganic single crystals. This high additive loading endows the nanocomposites with new functional properties, including plasmon coupling, bright fluorescence, and surface-enhanced Raman scattering (SERS). Cationic additives are also shown to outperform their acidic counterparts, where they are highly active in a wider range of crystal systems, owing to their outstanding colloidal stability in the crystallization media and strong affinity for the crystal surfaces. This work demonstrates that although often overlooked, cationic additives can make valuable crystallization additives to create composite materials with tailored composition-structure-property relationships. This versatile and straightforward approach advances the field of single-crystal composites and provides exciting prospects for the design and fabrication of new hybrid materials with tunable functional properties.
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Incorporating additives within host single crystals is an effective strategy for producing composite materials with tunable mechanical, magnetic and optical properties. The type of guest materials that can be occluded can be limited, however, as incorporation is a complex process depending on many factors including binding of the additive to the crystal surface, the rate of crystal growth and the stability of the additives in the crystallisation solution. In particular, the size of occluded guests has been restricted to a few angstroms - as for single molecules - to a few hundred nanometers - as for polymer vesicles and particles. Here, we present a synthetic approach for occluding micrometer-scale objects, including high-complexity unicellular organisms and synthetic hollow calcite spheres within calcite single crystals. Both of these objects can transport functional additives, including organic molecules and nanoparticles that would not otherwise occlude within calcite. Therefore, this method constitutes a generic approach using calcite as a delivery system for active compounds, while providing them with effective protection against environmental factors that could cause degradation.
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Minerales , Nanopartículas , Carbonato de Calcio/química , Cristalización , Polímeros/químicaRESUMEN
Biomineralization in a starfish displays morphologically complex features.
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Estrellas de Mar , AnimalesRESUMEN
X-ray scattering techniques provide a powerful means of characterizing the formation of nanoparticles in solution. Coupling these techniques to segmented-flow microfluidic devices that offer well-defined environments gives access to in situ time-resolved analysis, excellent reproducibility, and eliminates potential radiation damage. However, analysis of the resulting datasets can be extremely time-consuming, where these comprise frames corresponding to the droplets alone, the continuous phase alone, and to both at their interface. We here describe a robust, low-cost, and versatile droplet microfluidics device and use it to study the formation of magnetite nanoparticles with simultaneous synchrotron SAXS and WAXS. Lateral outlet capillaries facilitate the X-ray analysis and reaction times of between a few seconds and minutes can be accommodated. A two-step data processing method is then described that exploits the unique WAXS signatures of the droplets, continuous phase, and interfacial region to identify the frames corresponding to the droplets. These are then sorted, and the background scattering is subtracted using an automated frame-by-frame approach, allowing the signal from the nanoparticles to be isolated from the raw data. Modeling these data gives quantitative information about the evolution of the sizes and structures of the nanoparticles, in agreement with TEM observations. This versatile platform can be readily employed to study a wide range of dynamic processes in heterogeneous systems.
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Microfluídica , Nanopartículas , Reproducibilidad de los Resultados , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Rayos XRESUMEN
Nanocarriers have tremendous potential for the encapsulation, storage and delivery of active compounds. However, current formulations often employ open structures that achieve efficient loading of active agents, but that suffer undesired leakage and instability of the payloads over time. Here, a straightforward strategy that overcomes these issues is presented, in which protein nanogels are encapsulated within single crystals of calcite (CaCO3). Demonstrating our approach with bovine serum albumin (BSA) nanogels loaded with (bio)active compounds, including doxorubicin (a chemotherapeutic drug) and lysozyme (an antibacterial enzyme), we show that these nanogels can be occluded within calcite host crystals at levels of up to 45 vol%. Encapsulated within the dense mineral, the active compounds are stable against harsh conditions such as high temperature and pH, and controlled release can be triggered by a simple reduction of the pH. Comparisons with analogous systems - amorphous calcium carbonate, mesoporous vaterite (CaCO3) polycrystals, and calcite crystals containing polymer vesicles - demonstrate the superior encapsulation performance of the nanogel/calcite system. This opens the door to encapsulating a broad range of existing nanocarrier systems within single crystal hosts for the efficient storage, transport and controlled release of various active guest species.
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The nucleation of ice crystals in clouds is poorly understood, despite being of critical importance for our planet's climate. Nucleation occurs largely at rare "active sites" present on airborne particles such as mineral dust, but the nucleation pathway is distinct under different meteorological conditions. These give rise to two key nucleation pathways where a particle is either immersed in a supercooled liquid water droplet (immersion freezing mode) or suspended in a supersaturated vapor (deposition mode). However, it is unclear if the same active sites are responsible for nucleation in these two modes. Here, we directly compare the sites that are active in these two modes by performing immersion freezing and deposition experiments on the same thin sections of two atmospherically important minerals (feldspar and quartz). For both substrates, we confirm that nucleation is dominated by a limited number of sites and show that there is little correlation between the two sets of sites operating in each experimental method: across both materials, only six out of 73 sites active for immersion freezing nucleation were also active for deposition nucleation. Clearly, different properties determine the activity of nucleation sites for each mode, and we use the pore condensation and freezing concept to argue that effective deposition sites have size and/or geometry requirements not of relevance to effective immersion freezing sites. Hence, the ability to nucleate is pathway dependent, and the mode of nucleation has to be explicitly considered when applying experimental data in cloud models.
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Bone metastasis in breast cancer is associated with high mortality. Biomechanical cues presented by the extracellular matrix play a vital role in driving cancer metastasis. The lack of in vitro models that recapitulate the mechanical aspects of the in vivo microenvironment hinders the development of novel targeted therapies. Organ-on-a-chip (OOAC) platforms have recently emerged as a new generation of in vitro models that can mimic cell-cell interactions, enable control over fluid flow and allow the introduction of mechanical cues. Biomaterials used within OOAC platforms can determine the physical microenvironment that cells reside in and affect their behavior, adhesion, and localization. Refining the design of OOAC platforms to recreate microenvironmental regulation of metastasis and probe cell-matrix interactions will advance our understanding of breast cancer metastasis and support the development of next-generation metastasis-on-a-chip platforms. In this mini-review, we discuss the role of mechanobiology on the behavior of breast cancer and bone-residing cells, summarize the current capabilities of OOAC platforms for modeling breast cancer metastasis to bone, and highlight design opportunities offered by the incorporation of mechanobiological cues in these platforms.
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The mineralized collagen fibril is the basic building block of bone, and is commonly pictured as a parallel array of ultrathin carbonated hydroxyapatite (HAp) platelets distributed throughout the collagen. This orientation is often attributed to an epitaxial relationship between the HAp and collagen molecules inside 2D voids within the fibril. Although recent studies have questioned this model, the structural relationship between the collagen matrix and HAp, and the mechanisms by which collagen directs mineralization remain unclear. Here, we use XRD to reveal that the voids in the collagen are in fact cylindrical pores with diameters of ~2 nm, while electron microscopy shows that the HAp crystals in bone are only uniaxially oriented with respect to the collagen. From in vitro mineralization studies with HAp, CaCO3 and γ-FeOOH we conclude that confinement within these pores, together with the anisotropic growth of HAp, dictates the orientation of HAp crystals within the collagen fibril.