Investigating the Fibrillar Ultrastructure and Mechanics in Keloid Scars Using In Situ Synchrotron X-ray Nanomechanical Imaging.

Fibrotic scarring is prevalent in a range of collagenous tissue disorders. Understanding the role of matrix biophysics in contributing to fibrotic progression is important to develop therapies, as well as to elucidate biological mechanisms. Here, we demonstrate how microfocus small-angle X-ray scattering (SAXS), with in situ mechanics and correlative imaging, can provide quantitative and position-resolved information on the fibrotic matrix nanostructure and its mechanical properties. We use as an example the case of keloid scarring in skin. SAXS mapping reveals heterogeneous gradients in collagen fibrillar concentration, fibril pre-strain (variations in D-period) and a new interfibrillar component likely linked to proteoglycans, indicating evidence of a complex 3D structure at the nanoscale. Furthermore, we demonstrate a proof-of-principle for a diffraction-contrast correlative imaging technique, incorporating, for the first time, DIC and SAXS, and providing an initial estimate for measuring spatially resolved fibrillar-level strain and reorientation in such heterogeneous tissues. By application of the method, we quantify (at the microscale) fibrillar reorientations, increases in fibrillar D-period variance, and increases in mean D-period under macroscopic tissue strains of ~20%. Our results open the opportunity of using synchrotron X-ray nanomechanical imaging as a quantitative tool to probe structure-function relations in keloid and other fibrotic disorders in situ.

Tropocollagen springs allow collagen fibrils to stretch elastically.

The mechanical properties of connective tissues are tailored to their specific function, and changes can lead to dysfunction and pathology. In most mammalian tissues the mechanical environment is governed by the micro- and nano-scale structure of collagen and its interaction with other tissue components, however these hierarchical properties remain poorly understood. In this study we use the human cornea as a model system to characterise and quantify the dominant deformation mechanisms of connective tissue in response to cyclic loads of physiological magnitude. Synchronised biomechanical testing, x-ray scattering and 3D digital image correlation revealed the presence of two dominant mechanisms: collagen fibril elongation due to a largely elastic, spring-like straightening of tropocollagen supramolecular twist, and a more viscous straightening of fibril crimp that gradually increased over successive loading cycles. The distinct mechanical properties of the two mechanisms suggest they have separate roles in vivo. The elastic, spring-like mechanism is fast-acting and likely responds to stresses associated with the cardiac cycle, while the more viscous crimp mechanism will respond to slower processes, such as postural stresses. It is anticipated that these findings will have broad applicability to understanding the normal and pathological functioning of other connective tissues such as skin and blood vessels that exhibit both helical structures and crimp. STATEMENT OF SIGNIFICANCE: The tropocollagen spring mechanism allows collagen fibrils from some tissues to elongate significantly under small loads, and its recent discovery has the potential to change our fundamental understanding of how tissue deforms. This time-resolved study quantifies the contribution of the spring mechanism to the local strain in stretched tissue and compares it to the contribution associated with the straightening of fibril waviness, the widely accepted primary low-load strain mechanism. The spring mechanism contributed more to the local tissue strain than fibril straightening, and was found to be elastic while fibril straightening was more viscous. The results suggest that the viscoelastic behaviour of a biomaterial is controlled, at least in part, by the relative amount of fibril-scale crimp and tropocollagen supramolecular twist.

Beamline B21: high-throughput small-angle X-ray scattering at Diamond Light Source.

B21 is a small-angle X-ray scattering (SAXS) beamline with a bending magnet source in the 3 GeV storage ring at the Diamond Light Source Ltd synchrotron in the UK. The beamline utilizes a double multi-layer monochromator and a toroidal focusing optic to deliver 2 × 1012 photons per second to a 34 × 40 µm (FWHM) focal spot at the in-vacuum Eiger 4M (Dectris) detector. A high-performance liquid chromatography system and a liquid-handling robot make it possible to load solution samples into a temperature-controlled in-vacuum sample cell with a high level of automation. Alternatively, a range of viscous or solid materials may be loaded manually using a range of custom sample cells. A default scattering vector range from 0.0026 to 0.34 Å-1 and low instrument background make B21 convenient for measuring a wide range of biological macromolecules. The beamline has run a full user programme since 2013.

Body wall structure in the starfish Asterias rubens.

The body wall of starfish is composed of magnesium calcite ossicles connected by collagenous tissue and muscles and it exhibits remarkable variability in stiffness, which is attributed to the mechanical mutability of the collagenous component. Using the common European starfish Asterias rubens as an experimental animal, here we have employed a variety of techniques to gain new insights into the structure of the starfish body wall. The structure and organisation of muscular and collagenous components of the body wall were analysed using trichrome staining. The muscle system comprises interossicular muscles as well as muscle strands that connect ossicles with the circular muscle layer of the coelomic lining. The collagenous tissue surrounding the ossicle network contains collagen fibres that form loop-shaped straps that wrap around calcite struts near to the surface of ossicles. The 3D architecture of the calcareous endoskeleton was visualised for the first time using X-ray microtomography, revealing the shapes and interactions of different ossicle types. Furthermore, analysis of the anatomical organisation of the ossicles indicates how changes in body shape may be achieved by local contraction/relaxation of interossicular muscles. Scanning synchrotron small-angle X-ray diffraction (SAXD) scans of the starfish aboral body wall and ambulacrum were used to study the collagenous tissue component at the fibrillar level. Collagen fibrils in aboral body wall were found to exhibit variable degrees of alignment, with high levels of alignment probably corresponding to regions where collagenous tissue is under tension. Collagen fibrils in the ambulacrum had a uniformly low degree of orientation, attributed to macrocrimp of the fibrils and the presence of slanted as well as horizontal fibrils connecting antimeric ambulacral ossicles. Body wall collagen fibril D-period lengths were similar to previously reported mammalian D-periods, but were significantly different between the aboral and ambulacral samples. The overlap/D-period length ratio within fibrils was higher than reported for mammalian tissues. Collectively, the data reported here provide new insights into the anatomy of the body wall in A. rubens and a foundation for further studies investigating the structural basis of the mechanical properties of echinoderm body wall tissue composites.

Selective molecular annealing: in situ small angle X-ray scattering study of microwave-assisted annealing of block copolymers.

Microwave annealing has emerged as an alternative to traditional thermal annealing approaches for optimising block copolymer self-assembly. A novel sample environment enabling small angle X-ray scattering to be performed in situ during microwave annealing is demonstrated, which has enabled, for the first time, the direct study of the effects of microwave annealing upon the self-assembly behavior of a model, commercial triblock copolymer system [polystyrene-block-poly(ethylene-co-butylene)-block-polystyrene]. Results show that the block copolymer is a poor microwave absorber, resulting in no change in the block copolymer morphology upon application of microwave energy. The block copolymer species may only indirectly interact with the microwave energy when a small molecule microwave-interactive species [diethylene glycol dibenzoate (DEGDB)] is incorporated directly into the polymer matrix. Then significant morphological development is observed at DEGDB loadings ≥6 wt%. Through spatial localisation of the microwave-interactive species, we demonstrate targeted annealing of specific regions of a multi-component system, opening routes for the development of "smart" manufacturing methodologies.

Microfluidic processing of concentrated surfactant mixtures: online SAXS, microscopy and rheology.

We investigate the effect of microfluidic flow on the microstructure and dynamics of a model surfactant mixture, combining synchrotron Small Angle X-ray Scattering (SAXS), microscopy and rheology. A system comprising a single-chain cationic surfactant, hexadecyl trimethyl ammonium chloride (C16TAC), a short-chain alcohol (1-pentanol) and water was selected for the study due to its flow responsiveness and industrial relevance. Model flow fields, including sequential contraction-expansion (extensional) and rotational flows, were investigated and the fluid response in terms of the lamellar d-spacing, orientation and birefringence was monitored in situ, as well as the recovery processes after cessation of flow. Extensional flows are found to result in considerable d-spacing increase (from approx 59 Å to 65 Å). However, under continuous flow, swelling decreases with increasing flow velocity, eventually approaching the equilibrium values at velocities ≃2 cm s(-1). Through individual constrictions we observe the alignment of lamellae along the flow velocity, accompanied by increasing birefringence, followed by an orientation flip whereby lamellae exit perpendicularly to the flow direction. The resulting microstructures are mapped quantitatively onto the flow field in 2D with 200 µm spatial resolution. Rotational flows alone do not result in appreciable changes in lamellar spacing and flow type and magnitude evidently impact the fluid microstructure under flow, as well as upon relaxation. The findings are correlated with rheological properties measured ex situ to provide a mechanistic understanding of the effect of flow imposed by tubular processing units in the phase behavior and performance of a model surfactant system with ubiquitous applications in personal care and coating industries.

Hybrid glasses from strong and fragile metal-organic framework liquids.

Hybrid glasses connect the emerging field of metal-organic frameworks (MOFs) with the glass formation, amorphization and melting processes of these chemically versatile systems. Though inorganic zeolites collapse around the glass transition and melt at higher temperatures, the relationship between amorphization and melting has so far not been investigated. Here we show how heating MOFs of zeolitic topology first results in a low density 'perfect' glass, similar to those formed in ice, silicon and disaccharides. This order-order transition leads to a super-strong liquid of low fragility that dynamically controls collapse, before a subsequent order-disorder transition, which creates a more fragile high-density liquid. After crystallization to a dense phase, which can be remelted, subsequent quenching results in a bulk glass, virtually identical to the high-density phase. We provide evidence that the wide-ranging melting temperatures of zeolitic MOFs are related to their network topologies and opens up the possibility of 'melt-casting' MOF glasses.

Patterned Diblock Co-Polymer Thin Films as Templates for Advanced Anisotropic Metal Nanostructures.

We demonstrate glancing-angle deposition of gold on a nanostructured diblock copolymer, namely polystyrene-block-poly(methyl methacrylate) thin film. Exploiting the selective wetting of gold on the polystyrene block, we are able to fabricate directional hierarchical structures. We prove the asymmetric growth of the gold nanoparticles and are able to extract the different growth laws by in situ scattering methods. The optical anisotropy of these hierarchical hybrid materials is further probed by angular resolved spectroscopic methods. This approach enables us to tailor functional hierarchical layers in nanodevices, such as nanoantennae arrays, organic photovoltaics, and sensor electronics.

Predicting the orientation of lipid cubic phase films.

Lipid cubic phase films are of increasingly widespread importance, both in the analysis of the cubic phases themselves by techniques including microscopy and X-ray scattering, and in their applications, especially as electrode coatings for electrochemical sensors and for templates for the electrodeposition of nanostructured metal. In this work we demonstrate that the crystallographic orientation adopted by these films is governed by minimization of interfacial energy. This is shown by the agreement between experimental data obtained using grazing-incidence small-angle X-ray scattering (GI-SAXS), and the predicted lowest energy orientation determined using a theoretical approach we have recently developed. GI-SAXS data show a high degree of orientation for films of both the double diamond phase and the gyroid phase, with the [111] and [110] directions respectively perpendicular to the planar substrate. In each case, this matches the lowest energy facet calculated for that particular phase.

Hydrostatic pressure effects on the lamellar to gyroid cubic phase transition of monolinolein at limited hydration.

Monoacylglycerol based lipids are highly important model membrane components and attractive candidates for drug encapsulation and as delivery agents. However, optimizing the properties of these lipids for applications requires a detailed understanding of the thermodynamic factors governing the self-assembled structures that they form. Here, we report on the effects of hydrostatic pressure, temperature, and water composition on the structural behavior and stability of inverse lyotropic liquid crystalline phases adopted by monolinolein (an unsaturated monoacylglycerol having cis-double bonds at carbon positions 9 and 12) under limited hydration conditions. Six pressure-temperature phase diagrams have been determined using small-angle X-ray diffraction at water contents between 15 wt % and 27 wt % water, in the range 10-40 °C and 1-3000 bar. The gyroid bicontinuous cubic (Q(II)(G)) phase is formed at low pressure and high temperatures, transforming to a fluid lamellar (L(α)) phase at high pressures and low temperature via a region of Q(II)(G)/L(α) coexistence. Pressure stabilizes the lamellar phase over the Q(II)(G) phase; at fixed pressure, increasing the water content causes the coexistence region to move to lower temperature. These trends are consistent throughout the hydration range studied. Moreover, at fixed temperature, increasing the water composition increases the pressure at which the Q(II)(G) to L(α) transition takes place. We discuss the qualitative effect of pressure, temperature, and water content on the stability of the Q(II)(G) phase.

Effects of isopropanol on collagen fibrils in new parchment.

BACKGROUND: Isopropanol is widely used by conservators to relax the creases and folds of parchment artefacts. At present, little is known of the possible side effects of the chemical on parchments main structural component- collagen. This study uses X-ray Diffraction to investigate the effects of a range of isopropanol concentrations on the dimensions of the nanostructure of the collagen component of new parchment. RESULTS: It is found in this study that the packing features of the collagen molecules within the collagen fibril are altered by exposure to isopropanol. The results suggest that this chemical treatment can induce a loss of structural water from the collagen within parchment and thus a rearrangement of intermolecular bonding. This study also finds that the effects of isopropanol treatment are permanent to parchment artefacts and cannot be reversed with rehydration using deionised water. CONCLUSIONS: This study has shown that isopropanol induces permanent changes to the packing features of collagen within parchment artefacts and has provided scientific evidence that its use to remove creases and folds on parchment artefacts will cause structural change that may contribute to long-term deterioration of parchment artefacts. This work provides valuable information that informs conservation practitioners regarding the use of isopropanol on parchment artefacts.

Automated high pressure cell for pressure jump x-ray diffraction.

A high pressure cell for small and wide-angle x-ray diffraction measurements of soft condensed matter samples has been developed, incorporating a fully automated pressure generating network. The system allows both static and pressure jump measurements in the range of 0.1-500 MPa. Pressure jumps can be performed as quickly as 5 ms, both with increasing and decreasing pressures. Pressure is generated by a motorized high pressure pump, and the system is controlled remotely via a graphical user interface to allow operation by a broad user base, many of whom may have little previous experience of high pressure technology. Samples are loaded through a dedicated port allowing the x-ray windows to remain in place throughout an experiment; this facilitates accurate subtraction of background scattering. The system has been designed specifically for use at beamline I22 at the Diamond Light Source, United Kingdom, and has been fully integrated with the I22 beamline control systems.

Formation of green rust sulfate: a combined in situ time-resolved X-ray scattering and electrochemical study.

The mechanism of green rust sulfate (GR-SO(4)) formation was determined using a novel in situ approach combining time-resolved synchrotron-based wide-angle X-ray scattering (WAXS) with highly controlled chemical synthesis and electrochemical (i.e., Eh and pH) monitoring of the reaction. Using this approach,GR-SO(4) was synthesized under strictly anaerobic conditions by coprecipitation from solutions with known Fe(II)/Fe(III) ratios (i.e., 1.28 and 2) via the controlled increase of pH. The reaction in both systems proceeded via a three-stage precipitation and transformation reaction. During the first stage,schwertmannite (Fe(8)O(8)(OH)(4.5)(SO(4))(1.75)) precipitated directly from solution at pH 2.8-4.5. With increasing pH (>5), Fe(2+) ions adsorb to the surface of schwertmannite and catalyze its transformation to goethite (alpha-FeOOH) during the second stage of the reaction. In the third stage, the hydrolysis of the adsorbed Fe(2+) ions on goethite initiates its transformation to GR-SO(4) at pH >7. The GR-SO(4) then continues to crystallize up to pH approximately 8.5. These results suggest that with an Fe(II)/Fe(III) ratio of < or = 2 in the initial solution the structural Fe(II)/Fe(III) of the GR-SO(4) will be close to that of the starting composition.