I22-Small angle scattering & Diffraction
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Abstract: Background: An improved understanding of intervertebral disc (IVD) structure and function is required for treatment development. Loading induces micro-fractures at the interface between the nucleus pulposus (NP) and the annulus fibrosus (AF), which is hypothesized to induce a cascade of cellular changes leading to degeneration. However, there is limited understanding of the structural relationship between the NP and AF at this interface and particularly response to load. Here, X-ray scattering is utilised to provide hierarchical morphometric information of collagen structure across the IVD, especially the interface region under load. Methodology: IVDs were imaged using the I22 SAXS/WAXS beamline at Diamond Light Source. Peaks associated with the D-banded structure of collagen fibrils were fitted to quantify their azimuthal distribution, as well the magnitude and direction of internal strains under static and applied strain (0–20%). Results: IVD tissue regions exhibited structural “AF-like” and “NP-like” fingerprints. Demonstrating high internal strains on collagen fibres particularly within the NP region of the disc. AF and NP regions showed distinct collagen orientation and internal strains with an apparent lack of bracing structure seen at the interface between the differential mechanical tissues. X-ray scattering under tensile strain provided structural information at high resolution, with clear differences observed between normal and degenerate discs under load. Conclusion: X ray scattering has been utilised to develop an improved understanding of collagen structure across the intervertebral disc which can be utilised to gain an increased understanding of load induced propagation of micro fissures and disc degeneration.
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Oct 2022
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I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[19081]
Open Access
Abstract: The protective carapace of Skogsbergia lerneri, a marine ostracod, is scratch-resistant and transparent. The compositional and structural organisation of the carapace that underlies these properties is unknown. In this study, we aimed to quantify and determine the distribution of chemical elements and chitin within the carapace of adult ostracods, as well as at different stages of ostracod development, to gain insight into its composition. Elemental analyses included X-ray absorption near-edge structure, X-ray fluorescence and X-ray diffraction. Nonlinear microscopy and spectral imaging were performed to determine chitin distribution within the carapace. High levels of calcium (20.3%) and substantial levels of magnesium (1.89%) were identified throughout development. Amorphous calcium carbonate (ACC) was detected in carapaces of all developmental stages, with the polymorph, aragonite, identified in A-1 and adult carapaces. Novel chitin-derived second harmonic generation signals (430/5 nm) were detected. Quantification of relative chitin content within the developing and adult carapaces identified negligible differences in chitin content between developmental stages and adult carapaces, except for the lower chitin contribution in A-2 (66.8 ± 7.6%) compared to A-5 (85.5 ± 10%) (p = 0.03). Skogsbergia lerneri carapace calcium carbonate composition was distinct to other myodocopid ostracods. These calcium polymorphs and ACC are described in other biological transparent materials, and with the consistent chitin distribution throughout S. lerneri development, may imply a biological adaptation to preserve carapace physical properties. Realisation of S. lerneri carapace synthesis and structural organisation will enable exploitation to manufacture biomaterials and biomimetics with huge potential in industrial and military applications.
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Jun 2022
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I22-Small angle scattering & Diffraction
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Abstract: Purpose : To elucidate the hierarchical deformation mechanisms of human corneal collagen under controlled inflation of physiological and pathological magnitudes.
Methods : 6 human donor corneas with scleral rim were mounted onto a bespoke sealed cell, which was connected via two pressure regulators and an expansion vessel to a source of compressed nitrogen. The cell included a window transparent to X-rays and was connected to translation and rotation stages inside beamline I22 at the Diamond Light Source synchrotron, UK. Images were acquired naso-temporally across the cornea from limbus to limbus at intraocular pressures of 5.5 mmHg, 17.5 mmHg and 40 mmHg. These increments were chosen for comparison with previous static results in the literature and to mimic the normal physiological state and ocular hypertension.
Results : The inflation apparatus was able to maintain pressure to an accuracy better than 0.1 mmHg. Features corresponding to collagen fibril diameter, spacing, D-period and orientation as well as tropocollagen spacing and orientation were measured simultaneously. Preliminary analysis revealed a radial distribution of strain (manifested as a change in D-period) across the cornea, which was accentuated in the outer periphery and limbus. In general, the interfibrillar spacing in the periphery and limbus (where collagen fibrils are arranged circumferentially) was found to increase with pressure.
Conclusions : The trends in D-period strain are indicative of bulging under increased pressure, which is pronounced at the limbus and the outer periphery. This agrees with the general consensus that the outer periphery and limbus act as a buffer zone, which takes up most of the strain under changes in intraocular pressure, and thus minimises changes in focussing power of the cornea. Further in-depth analysis of this data will allow us to calculate the supramolecular twist (which gives rise to a spring-like stretch in collagen fibrils) and examine in more detail the hierarchical response of corneal collagen to changes in intraocular pressures.
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Jun 2022
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[11316, 8458]
Abstract: 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.
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Jan 2022
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I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[17839]
Open Access
Abstract: Primary crustacean cell culture was introduced in the 1960s, but to date limited cell lines have been established. Skogsbergia lerneri is a myodocopid ostracod, which has a body enclosed within a thin, durable, transparent bivalved carapace, through which the eye can see. The epidermal layer lines the inner surface of the carapace and is responsible for carapace synthesis. The purpose of the present study was to develop an in vitro epidermal tissue and cell culture method for S. lerneri. First, an optimal environment for the viability of this epidermal tissue was ascertained, while maintaining its cell proliferative capacity. Next, a microdissection technique to remove the epidermal layer for explant culture was established and finally, a cell dissociation method for epidermal cell culture was determined. Maintenance of sterility, cell viability and proliferation were key throughout these processes. This novel approach for viable S. lerneri epidermal tissue and cell culture augments our understanding of crustacean cell biology and the complex biosynthesis of the ostracod carapace. In addition, these techniques have great potential in the fields of biomaterial manufacture, the military and fisheries, for example, in vitro toxicity testing.
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Oct 2020
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I22-Small angle scattering & Diffraction
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Abstract: X-ray scattering enables the structure of collagen-rich tissues, such as the cornea, to be examined at both the molecular and fibrillar level. The high-intensity X-rays available at synchrotron radiation sources, coupled with minimal sample preparation requirements, facilitates the rapid generation of high-quality X-ray scattering data from corneal tissue at a close-to-physiological state of hydration. Analysis of resulting X-ray scatter patterns allows one to quantify numerous structural parameters relating to the average diameter, lateral arrangement and alignment of collagen fibrils within the cornea, as well as the axial and lateral arrangements of collagen molecules within the fibrils. Here we describe the typical experimental setup and considerations involved in the collection of X-ray scattering data from corneal tissue.
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Jun 2020
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I02-Macromolecular Crystallography
I22-Small angle scattering & Diffraction
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S.
Hayes
,
N.
Aldahlawi
,
A. L.
Marcovich
,
J.
Brekelmans
,
A.
Goz
,
A.
Scherz
,
R. D.
Young
,
J. S.
Bell
,
D. P.
O’brart
,
R. M. M. A.
Nuijts
,
K. M.
Meek
Diamond Proposal Number(s):
[8458, 11316]
Open Access
Abstract: A cross-linking technique involving application of Bacteriochlorophyll Derivative WST-11 mixed with dextran (WST-D) to the epithelium-debrided cornea and illumination with Near Infrared (NIR), has been identified as a promising therapy for stiffening pathologically weakened corneas. To investigate its effect on corneal collagen architecture, x-ray scattering and electron microscopy data were collected from paired WST-D/NIR treated and untreated rabbit corneas. The treated eye received 2.5 mg/mL WST-D and was illuminated by a NIR diode laser (755 nm, 10 mW/cm2). An increase in corneal thickness (caused by corneal oedema) occurred at 1-day post-treatment but resolved in the majority of cases within 4 days. The epithelium was fully healed after 6–8 days. X-ray scattering revealed no difference in average collagen interfibrillar spacing, fibril diameter, D-periodicity or intermolecular spacing between treated and untreated specimens. Similarly, electron microscopy images of the anterior and posterior stroma in healed WST-D/NIR corneas and untreated controls revealed no obvious differences in collagen organisation or fibril diameter. As the size and organisation of stromal collagen is closely associated with the optical properties of the cornea, the absence of any large-scale changes following treatment confirms the potential of WST-D/NIR therapy as a means of safely stiffening the cornea.
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Jun 2020
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[17757]
Open Access
Abstract: Elastic fibres provide tissues with elasticity and flexibility. In the healthy human cornea, elastic fibres are limited to the posterior region of the peripheral stroma, but their specific functional role remains elusive.
Here, we examine the physical and structural characteristics of the cornea during development in the mgΔloxPneo dominant-negative mouse model for Marfan syndrome, in which the physiological extracellular matrix of its elastic-fibre rich tissues is disrupted by the presence of a dysfunctional fibrillin-1 glycoprotein. Optical coherence tomography demonstrated a reduced corneal thickness in the mutant compared to wild type mice from embryonic day 16.5 until adulthood. X-ray scattering and electron microscopy revealed a disruption to both the elastic fibre and collagen fibril ultrastructure in the knockout mice, as well as abnormally low levels of the proteoglycan decorin. It is suggested that these alterations are a result of increased transforming growth factor beta signalling. To conclude, this study has demonstrated corneal structure and ultrastructure to be altered when fibrillin-1 is disrupted and has provided insights into the role of fibrillin-1 in developing a functional cornea.
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Mar 2020
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[5745, 8028]
Abstract: Many myodocopid ostracods are unusual in that they have well-developed compound eyes yet must view their environment through a shell. The cypridinid Macrocypridina castanea is relatively large among ostracods (about 5–10 mm) and is a pelagic predator. This species possess highly pigmented shells with a transparent region lying just above the eye. Here we examine the ultrastructure and transparency of this window using electron microscopy, serial-block face scanning electron microscopy and X-ray diffraction analysis and optical modelling. An internal, laminar stack was identified within the window region of the shell that formed a more regular half-wave reflector than in non-window regions, and where the distance between molecules in the chitin–protein fibrils decreases as compared to the non-window area. This results in excellent transmission properties—at around 99% transmission—for wavelengths between 350 and 630 nm due to its half-wave reflector organization. Therefore, blue light, common in the mid and deep sea, where this species inhabits, would be near-optimally transmitted as a consequence of the sub-micrometre structuring of the shell, thus optimizing the ostracod's vision. Further, pore canals were identified in the shell that may secrete substances to prevent microbial growth, and subsequently maintain transparency, on the shell surface.
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Feb 2019
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[6407]
Open Access
Abstract: Microwave keratoplasty is a thermo-refractive surgical procedure that can correct myopia (short-sightedness) and pathologic corneal steepening by using microwave energy to cause localised shrinkage around an annulus of the cornea leading to its flattening and vision correction. The effects on the corneal extracellular matrix, however, have not yet been evaluated, thus the current study to assess post-procedure ultrastructural changes in an in-vivo rabbit model. To achieve this a series of small-angle x-ray scattering (SAXS) experiments were carried out across whole transects of treated and untreated rabbit corneas at 0.25 mm intervals, which indicated no significant change in collagen intra-fibrillar parameters (i.e. collagen fibril diameter or axial D-period), whereas inter-fibrillar measures (i.e. fibril spacing and the degree of spatial order) were markedly altered in microwave-treated regions of the cornea. These structural matrix alterations in microwave-treated corneas have predicted implications for corneal biomechanical strength and tissue transparency, and, we contend, potentially render microwave-treated corneas resistant to surgical stabilization using corneal cross-linking procedures currently employed to combat refractive error caused by corneal steepening.
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Sep 2018
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