A bicarbonate-rich liquid condensed phase in non-saturated solutions in the absence of divalent cations.

Bicarbonate (HCO3 -) and sodium (Na+)-containing solutions contain droplets of a separate, bicarbonate-rich liquid condensed phase (LCP) that have higher concentrations of HCO3 - relative to the bulk solution in which they reside. The existence and composition of the LCP droplets has been investigated by nanoparticle tracking analysis, nuclear magnetic resonance spectroscopy, refractive index measurements and X-ray pair distribution function analysis. The bicarbonate-rich LCP species is a previously unaccounted-for, ionic phenomenon which occurs even in solutions with solely monovalent cations. Its existence requires re-evaluation of models used to describe and model aqueous solution physicochemistry, especially those used to describe and model carbonate mineral formation.

Monitoring silver diamine fluoride application with optical coherence tomography and thermal imaging: An in vitro proof of concept study.

OBJECTIVES: The purpose of this study was to show that optical coherence tomography (OCT) and thermal imaging can be used to monitor changes in the structure and activity of caries lesions over time after treatment with silver diamine fluoride (SDF). METHODS: Artificial caries lesions were formed on enamel and dentin bovine blocks. Each block was partitioned into five windows with the central three windows exposed to a demineralization solution to create lesions: one sound window served as a sound control (SC), one sound window was exposed to SDF to serve as a test control (SCT), one lesion window served as a lesion control (LC), one lesion window received one application of SDF (L1), while the other lesion window received two applications of SDF (L2). Each window was scanned using OCT before SDF application, and every week subsequently, for 12 weeks after initial SDF treatment. Changes in the mean intensity and the width of the peak of increased reflectivity due to the lesion and SDF along with the intensity at a depth of 180 µm from the surface representing optical penetration through the lesion were monitored. Changes in the heat lost, ΔQ (temperature integrated over time) of each window during drying with air were also monitored using a thermal imaging camera. Transverse microradiography (TMR), and high-resolution microscopy were also used for the analysis of selected samples. RESULTS: The reflectivity and optical penetration of sound and lesion areas of enamel and dentin manifested significant changes in OCT images after SDF application. Thermal imaging showed significant differences in ΔQ indicative of permeability changes in the sound and lesion areas of enamel and dentin after SDF application.

Enhanced Tooth Structure Via Silver Microwires Following Treatment with 38 Percent Silver Diamine Fluoride.

Purpose: American Academy of Pediatric Dentistry guidelines recommend treatment of primary teeth with 38 percent silver diamine fluoride (SDF) as a noninvasive option to arrest active dental caries lesions. A significant outcome of SDF treatment are lesions that clinically harden and become more resistant to further decay. Many practicing dentists believe that this increased hardening is due to the reaction of silver and fluoride with carious dentin. The purpose of this study was to focus on the structural and chemical effects of silver diamine fluoride treatment on the native tooth. Methods: In SDF-treated cavitated dentin lesions in teeth subsequently extracted for orthodontic reasons, the authors observed continuous, filamentous silver densities formed in situ from 50 to 2,100 µm in length and 0.25 to 7.0 µm in diameter using high-resolution synchrotron X-ray microcomputer tomography and field emission scanning electron microscopy. These "microwires" fill voids in the lesion caused by disease and permeate through surrounding dentinal tubules. Results: Spectroscopy confirmed that the chemical composition of the observed microwires is predominantly silver. Conclusions: These observations suggest mechanistic explanations for the structural reinforcement of carious dentin in addition to remineralization. It is hypothesized that silver diamine fluoride may achieve its antimicrobial functions by biochemical interactions and through its inherent ability to integrate into the native tooth structure.

On a Robust, Sensitive Cell-Free Method for Pseudomonas Sensing and Quantification in Microfluidic Templated Hydrogels.

Through the use of droplet microfluidics to integrate cell-free activity into inert hydrogel beads, we have developed a platform that can perform biologically relevant functions without the need for cells. Specifically, cell-free lysates serve a utility in performing cellular functions and providing biologically relevant metabolic products without requiring the optimal biological conditions for cell growth and proliferation. By teasing out specific biological components that enable transcription and translation to occur, these cell-like functions can be reconstituted in vitro without requiring the entire cell and milieu of cellular organelles. This enables the optimization of synthetic biological circuits, either by concentration or logic switches, simply through the addition or removal of genetic components (plasmids, inducers, or repressors) of regulatory elements. Here, we demonstrate an application of cell-free processes that is robust and portable, independent of a substrate, to apply for sensing and reporting functions of a quorum-sensing molecule N-3-oxododecanoyl homoserine lactone (3OC12HSL) found crucial for pathological Pseudomonas aeruginosa infection. We develop an agarose bead platform that is easily adaptable and simply programmable to fit a variety of biological and chemical sensing applications for the utility of ease of delivery and activation in remote environments-even in conditions with very little hydration.

Diamond Magnetic Microscopy of Malarial Hemozoin Nanocrystals.

Magnetic microscopy of malarial hemozoin nanocrystals is performed by optically detected magnetic resonance imaging of near-surface diamond nitrogen-vacancy centers. Hemozoin crystals are extracted from Plasmodium falciparum-infected human blood cells and studied alongside synthetic hemozoin crystals. The stray magnetic fields produced by individual crystals are imaged at room temperature as a function of the applied field up to 350 mT. More than 100 nanocrystals are analyzed, revealing the distribution of their magnetic properties. Most crystals (96%) exhibit a linear dependence of the stray-field magnitude on the applied field, confirming hemozoin's paramagnetic nature. A volume magnetic susceptibility of 3.4 × 10-4 is inferred with use of a magnetostatic model informed by correlated scanning-electron-microscopy measurements of crystal dimensions. A small fraction of nanoparticles (4/82 for Plasmodium falciparum-produced nanoparticles and 1/41 for synthetic nanoparticles) exhibit a saturation behavior consistent with superparamagnetism. Translation of this platform to the study of living Plasmodium-infected cells may shed new light on hemozoin formation dynamics and their interaction with antimalarial drugs.

Growth of second stage mineral in Lytechinus variegatus.

Purpose and Aims: Sea urchin teeth consist of calcite and form in two stages with different magnesium contents. The first stage structures of independently formed plates and needle-prisms define the shape of the tooth, and the columns of the second stage mineral cements the first stage structures together and control the fracture behavior of the mature tooth. This study investigates the nucleation and growth of the second stage mineral. MATERIALS AND METHODS: Scanning electron microscopy (SEM) and synchrotron microComputed Tomography characterized the structures of the second phase material found in developing of Lytechinus variegatus teeth. RESULTS: Although the column development is a continuous process, defining four phases of column formation captures the changes that occur in teeth of L. variegatus. The earliest phase consists of small 1-2 µm diameter hemispheres, and the second of 5-10 µm diameter, mound-like structures with a nodular surface, develops from the hemispheres. The mounds eventually bridge the syncytium between adjacent plates and form hyperboloid structures (phase three) that appear like mesas when plates separate during the fracture. The mesa diameter increases with time until the column diameter is significantly larger than its height, defining the fourth phase of column development. Energy dispersive x-ray spectroscopy confirms that the columns contain more magnesium than the underlying plates; the ratios of magnesium to calcium are consistent with compositions derived from x-ray diffraction. CONCLUSION: Columns grow from both bounding plates. The presence of first phase columns interspersed among third stage mesas indicates very localized control of mineralization.

Neurofibromin inactivation impairs osteocyte development in Nf1Prx1 and Nf1Col1 mouse models.

Neurofibromin has been identified as a critical regulator of osteoblast differentiation. Osteoblast specific inactivation of neurofibromin in mice results in a high bone mass phenotype and hyperosteoidosis. Here, we show that inactivation of the Nf1 gene also impairs osteocyte development. We analyzed cortical bone tissue in two conditional mouse models, Nf1Prx1 and Nf1Col1, for morphological and molecular effects. Backscattered electron microscopy revealed significantly enlarged osteocyte lacunae in Nf1Prx1 and Nf1Col1 mice (level E2: ctrl=1.90±0.52%, Nf1Prx1=3.40±0.95%; ctrl 1.60±0.47%, Nf1Col1 2.46±0.91%). Moreover, the osteocyte lacunae appeared misshaped in Nf1Prx1 and Nf1Col1 mice as indicated by increased Feret ratios. Strongest osteocyte and dendritic network disorganization was observed in proximity of muscle attachment sites in Nf1Prx1 humeri. In contrast to control cells, Nf1Prx1 osteocytes contained abundant cytosolic vacuoles and accumulated immature organic matrix within the perilacunar space, a phenotype reminiscent of the hyperosteoidosis shown Nf1 deficient mice. Cortical bone lysates further revealed approx. twofold upregulated MAPK signalling in osteocytes of Nf1Prx1 mice. This was associated with transcriptional downregulation of collagens and genes involved in mechanical sensing in Nf1Prx1 and Nf1Col1 bone tissue. In contrast, matrix gla protein (MGP), phosphate regulating endopeptidase homolog, X-linked (PHEX), and genes involved in lipid metabolism were upregulated. In line with previously described hyperactivation of Nf1 deficient osteoblasts, systemic plasma levels of the bone formation markers osteocalcin (OCN) and procollagen typ I N-propeptide (PINP) were approx. twofold increased in Nf1Prx1 mice. Histochemical and molecular analysis ascertained that osteocytes in Nf1Prx1 cortical bone were viable and did not undergo apoptosis or autophagy. We conclude that loss of neurofibromin is not only critical for osteoblasts but also hinders normal osteocyte development. These findings expand the effect of neurofibromin onto yet another cell type where it is likely involved in the regulation of mechanical sensing, bone matrix composition and mechanical resistance of bone tissue.

Investigating processes of nanocrystal formation and transformation via liquid cell TEM.

Recent ex situ observations of crystallization in both natural and synthetic systems indicate that the classical models of nucleation and growth are inaccurate. However, in situ observations that can provide direct evidence for alternative models have been lacking due to the limited temporal and spatial resolution of experimental techniques that can observe dynamic processes in a bulk solution. Here we report results from liquid cell transmission electron microscopy studies of nucleation and growth of Au, CaCO3, and iron oxide nanoparticles. We show how these in situ data can be used to obtain direct evidence for the mechanisms underlying nanoparticle crystallization as well as dynamic information that provide constraints on important energetic parameters not available through ex situ methods.

Multiscale, converging defects of macro-porosity, microstructure and matrix mineralization impact long bone fragility in NF1.

Bone fragility due to osteopenia, osteoporosis or debilitating focal skeletal dysplasias is a frequent observation in the Mendelian disease Neurofibromatosis type 1 (NF1). To determine the mechanisms underlying bone fragility in NF1 we analyzed two conditional mouse models, Nf1Prx1 (limb knock-out) and Nf1Col1 (osteoblast specific knock-out), as well as cortical bone samples from individuals with NF1. We examined mouse bone tissue with micro-computed tomography, qualitative and quantitative histology, mechanical tensile analysis, small-angle X-ray scattering (SAXS), energy dispersive X-ray spectroscopy (EDX), and scanning acoustic microscopy (SAM). In cortical bone of Nf1Prx1 mice we detected ectopic blood vessels that were associated with diaphyseal mineralization defects. Defective mineral binding in the proximity of blood vessels was most likely due to impaired bone collagen formation, as these areas were completely devoid of acidic matrix proteins and contained thin collagen fibers. Additionally, we found significantly reduced mechanical strength of the bone material, which was partially caused by increased osteocyte volume. Consistent with these observations, bone samples from individuals with NF1 and tibial dysplasia showed increased osteocyte lacuna volume. Reduced mechanical properties were associated with diminished matrix stiffness, as determined by SAM. In line with these observations, bone tissue from individuals with NF1 and tibial dysplasia showed heterogeneous mineralization and reduced collagen fiber thickness and packaging. Collectively, the data indicate that bone fragility in NF1 tibial dysplasia is partly due to an increased osteocyte-related micro-porosity, hypomineralization, a generalized defect of organic matrix formation, exacerbated in the regions of tensional and bending force integration, and finally persistence of ectopic blood vessels associated with localized macro-porotic bone lesions.

Roles of larval sea urchin spicule SM50 domains in organic matrix self-assembly and calcium carbonate mineralization.

The larval spicule matrix protein SM50 is the most abundant occluded matrix protein present in the mineralized larval sea urchin spicule. Recent evidence implicates SM50 in the stabilization of amorphous calcium carbonate (ACC). Here, we investigate the molecular interactions of SM50 and CaCO3 by investigating the function of three major domains of SM50 as small ubiquitin-like modifier (SUMO) fusion proteins - a C-type lectin domain (CTL), a glycine rich region (GRR) and a proline rich region (PRR). Under various mineralization conditions, we find that SUMO-CTL is monomeric and influences CaCO3 mineralization, SUMO-GRR aggregates into large protein superstructures and SUMO-PRR modifies the early CaCO3 mineralization stages as well as growth. The combination of these mineralization and self-assembly properties of the major domains synergistically enable the full-length SM50 to fulfill functions of constructing the organic spicule matrix as well as performing necessary mineralization activities such as Ca(2+) ion recruitment and organization to allow for proper growth and development of the mineralized larval sea urchin spicule.

Formation of aragonitic layered structures from kaolinite and amorphous calcium carbonate precursors.

Clay materials have been an ever-present accoutrement of modern civilization; improvements to process these materials have quickened their utilization for use in complex multiaxial load-bearing structures. Specifically, with better methods to organize the constituent metal oxide components in clay, the distribution of characteristic nematic and smectic phases can be controlled. In this work, we utilize the interactions of an amorphous calcium carbonate phase with kaolinite to form a complex composite that can be organized into distinct hierarchical structures. We demonstrate that these ACC-kaolinite composites can maintain characteristic long-range-ordered layer-by-layer structures across many length scales, from nano- to millimeter, through convenient and economical processing at room temperature.

Accelerated growth plate mineralization and foreshortened proximal limb bones in fetuin-A knockout mice.

The plasma protein fetuin-A/alpha2-HS-glycoprotein (genetic symbol Ahsg) is a systemic inhibitor of extraskeletal mineralization, which is best underscored by the excessive mineral deposition found in various tissues of fetuin-A deficient mice on the calcification-prone genetic background DBA/2. Fetuin-A is known to accumulate in the bone matrix thus an effect of fetuin-A on skeletal mineralization is expected. We examined the bones of fetuin-A deficient mice maintained on a C57BL/6 genetic background to avoid bone disease secondary to renal calcification. Here, we show that fetuin-A deficient mice display normal trabecular bone mass in the spine, but increased cortical thickness in the femur. Bone material properties, as well as mineral and collagen characteristics of cortical bone were unaffected by the absence of fetuin-A. In contrast, the long bones especially proximal limb bones were severely stunted in fetuin-A deficient mice compared to wildtype littermates, resulting in increased biomechanical stability of fetuin-A deficient femora in three-point-bending tests. Elevated backscattered electron signal intensities reflected an increased mineral content in the growth plates of fetuin-A deficient long bones, corroborating its physiological role as an inhibitor of excessive mineralization in the growth plate cartilage matrix--a site of vigorous physiological mineralization. We show that in the case of fetuin-A deficiency, active mineralization inhibition is a necessity for proper long bone growth.

Structure-property relationships of a biological mesocrystal in the adult sea urchin spine.

Structuring over many length scales is a design strategy widely used in Nature to create materials with unique functional properties. We here present a comprehensive analysis of an adult sea urchin spine, and in revealing a complex, hierarchical structure, show how Nature fabricates a material which diffracts as a single crystal of calcite and yet fractures as a glassy material. Each spine comprises a highly oriented array of Mg-calcite nanocrystals in which amorphous regions and macromolecules are embedded. It is postulated that this mesocrystalline structure forms via the crystallization of a dense array of amorphous calcium carbonate (ACC) precursor particles. A residual surface layer of ACC and/or macromolecules remains around the nanoparticle units which creates the mesocrystal structure and contributes to the conchoidal fracture behavior. Nature's demonstration of how crystallization of an amorphous precursor phase can create a crystalline material with remarkable properties therefore provides inspiration for a novel approach to the design and synthesis of synthetic composite materials.

Observations of multiscale, stress-induced changes of collagen orientation in tendon by polarized Raman spectroscopy.

Collagen is a versatile structural molecule in nature and is used as a building block in many highly organized tissues, such as bone, skin, and cornea. The functionality and performance of these tissues are controlled by their hierarchical organization ranging from the molecular up to macroscopic length scales. In the present study, polarized Raman microspectroscopic and imaging analyses were used to elucidate collagen fibril orientation at various levels of structure in native rat tail tendon under mechanical load. In situ humidity-controlled uniaxial tensile tests have been performed concurrently with Raman confocal microscopy to evaluate strain-induced chemical and structural changes of collagen in tendon. The methodology is based on the sensitivity of specific Raman scattering bands (associated with distinct molecular vibrations, such as the amide I) to the orientation and the polarization direction of the incident laser light. Our results, based on the changing intensity of Raman lines as a function of orientation and polarization, support a model where the crimp and gap regions of collagen hierarchical structure are straightened at the tissue and molecular level, respectively. However, the lack of measurable changes in Raman peak positions throughout the whole range of strains investigated indicates that no significant changes of the collagen backbone occurs with tensing and suggests that deformation is rather redistributed through other levels of the hierarchical structure.

The organization of the osteocyte network mirrors the extracellular matrix orientation in bone.

Bone is a dynamic tissue that is continually undergoing a process of remodeling - an effect due to the interplay between bone resorption by osteoclasts and bone formation by osteoblasts. When new bone is deposited, some of the osteoblasts are embedded in the mineralizing collagen matrix and differentiate to osteocytes, forming a dense network throughout the whole bone tissue. Here, we investigate the extent to which the organization of the osteocyte network controls the collagen matrix arrangement found in various bone tissues. Several tissue types from equine, ovine and murine bone have been examined using confocal laser scanning microscopy as well as polarized light microscopy and back-scattered electron imaging. From comparing the spatial arrangements of unorganized and organized bone, we propose that the formation of a highly oriented collagen matrix requires an alignment of osteoblasts whereby a substrate layer provides a surface such that osteoblasts can align and, collectively, build new matrix. Without such a substrate, osteoblasts act isolated and only form matrices without long range order. Hence, we conclude that osteoblasts synthesize and utilize scaffold-like primary tissue as a guide for the deposition of highly ordered and mechanically competent bone tissue by a collective action of many cells.

Inhomogeneous fibril stretching in antler starts after macroscopic yielding: indication for a nanoscale toughening mechanism.

Antler is a unique mineralized tissue, with extraordinary toughness as well as an ability to annually regenerate itself in its entirety. The high fracture resistance enables it to fulfill its biological function as a weapon and defensive guard during combats between deer stags in the rutting season. However, very little is quantitatively understood about the structural origin of the antler's high toughness. We used a unique combination of time-resolved synchrotron small angle X-ray diffraction together with tensile testing of antler cortical tissue under physiologically wet conditions. We measured the deformation at the nanoscale from changes in the meridional diffraction pattern during macroscopic stretch-to-failure tests. Our results show that on average fibrils are strained only half as much as the whole tissue and the fibril strain increases linearly with tissue strain, both during elastic and inelastic deformation. Most remarkably, following macroscopic yielding we observe a straining of some fibrils equal to the macroscopic tissue strain while others are hardly stretched at all, indicating an inhomogeneous fibrillar strain pattern at the nanoscale. This behavior is unlike what occurs in plexiform bovine bone and may explain the extreme toughness of antler compared to normal bone.

Cooperative deformation of mineral and collagen in bone at the nanoscale.

In biomineralized tissues such as bone, the recurring structural motif at the supramolecular level is an anisotropic stiff inorganic component reinforcing the soft organic matrix. The high toughness and defect tolerance of natural biomineralized composites is believed to arise from these nanometer scale structural motifs. Specifically, load transfer in bone has been proposed to occur by a transfer of tensile strains between the stiff inorganic (mineral apatite) particles via shearing in the intervening soft organic (collagen) layers. This raises the question as to how and to what extent do the mineral particles and fibrils deform concurrently in response to tissue deformation. Here we show that both mineral nanoparticles and the enclosing mineralized fibril deform initially elastically, but to different degrees. Using in situ tensile testing with combined high brilliance synchrotron X-ray diffraction and scattering on the same sample, we show that tissue, fibrils, and mineral particles take up successively lower levels of strain, in a ratio of 12:5:2. The maximum strain seen in mineral nanoparticles (approximately 0.15-0.20%) can reach up to twice the fracture strain calculated for bulk apatite. The results are consistent with a staggered model of load transfer in bone matrix, exemplifying the hierarchical nature of bone deformation. We believe this process results in a mechanism of fibril-matrix decoupling for protecting the brittle mineral phase in bone, while effectively redistributing the strain energy within the bone tissue.

The localization of occluded matrix proteins in calcareous spicules of sea urchin larvae.

The sea urchin embryo forms calcareous endoskeletal spicules composed of calcite and an occluded protein matrix. Though the latter is approximately 0.1% of of the mass, the composite has substantially altered material properties, e.g., conchoidal fracture planes and increased hardness. Experiments were conducted to examine the localization of matrix proteins occluded in the mineral by use of immunocytochemistry coupled with scanning electron microscopy (SEM). The isolated, unfixed spicules were etched under relatively gentle conditions and exposed to affinity purified antibodies made against two different matrix proteins, as well as an antibody to the entire constellation of matrix proteins. Immunogold tagged secondary antibody was used to observe antibody localization in the back scatter mode of SEM. All proteins examined were very widely distributed throughout the calcite, supporting a model of the structure in which a multiprotein assemblage is woven with fine texture around microcrystalline domains of calcite. Gentle etching revealed a laminar arrangement of calcite solubility, consistent with a stepwise deposition of matrix and mineral to increase girth of the spicule.