I14-Hard X-ray Nanoprobe
I22-Small angle scattering & Diffraction
|
Diamond Proposal Number(s):
[19466, 26220, 22709]
Open Access
Abstract: The deposition of calcium phosphate mineral within a collagen matrix plays a pivotal role in the formation of bone and teeth, yet understanding its precise mechanism and time-dependence remains a significant challenge. Conventional approaches are often constrained to ex situ studies and as a result the intrinsic dynamics governing collagen mineralization remains unclear. To address this knowledge gap, we developed a custom thermal flow cell to enable in situ characterization using Raman spectroscopy and small/wide-angle X-ray scattering for comparable sample settings. This approach allowed us to monitor the intricate process of collagen matrix mineralization from the initial infiltration of precursor phases to the formation of intermediate phosphate phases, and ultimately to the predominant growth of hydroxyapatite. Our findings reveal a striking expansion of the collagen matrix during initial infiltration, followed by compression in the early stages of mineralization, likely driven by water expulsion, which suggests the development of pre-stress similar to that observed in bone. As mineralization progressed, the matrix expanded once again, correlated with crystal growth. Post-mortem analyses confirmed the presence of intrafibrillar mineralization, with remarkable agreement to bone formation at up to 9 h of mineralization before over-mineralization occurred. Our study further identified a tessellated mineralization pattern within the collagen matrix, a feature also seen in bone, pointing to a highly regulated physico-chemical control of the mineralization dynamics. These insights deepen our understanding of the fundamental processes governing bone mineralization with broad implications for designing advanced biomaterials.
|
Oct 2025
|
|
I13-2-Diamond Manchester Imaging
|
Diamond Proposal Number(s):
[29784]
Open Access
Abstract: Biological tissues are exposed to X-rays in medical applications (such as diagnosis and radiotherapy) and in research studies (for example microcomputed X-ray tomography: microCT). Radiotherapy may deliver doses up to 50Gy to both tumour and healthy tissues, resulting in undesirable clinical side effects which can compromise quality of life. Whilst cellular responses to X-rays are relatively well-characterised, X-ray-induced structural damage to the extracellular matrix (ECM) is poorly understood. This study tests the hypotheses that ECM proteins and ECM-rich tissues (purified collagen I and rat tail tendons respectively) are structurally compromised by exposure to X-ray doses used in breast radiotherapy. Protein gel electrophoresis demonstrated that breast radiotherapy equivalent doses can induce fragmentation of the constituent α chains in solubilised purified collagen I. However, assembly into fibrils, either in vitro or in vivo, prevented X-ray-induced fragmentation but not structural changes (as characterised by LC-MS/MS and peptide location fingerprinting: PLF). In subsequent experiments exposure to higher (synchrotron) X-ray doses induced substantial fragmentation of solubilised and fibrillar (chicken tendon) collagen I. LC-MS/MS and PLF analysis of synchrotron-irradiated tendon identified structure-associated changes in collagens I, VI, XII, proteoglycans including aggrecan, decorin, and fibromodulin, and the elastic fibre component fibulin-1. Thus, exposure to radiotherapy X-rays can affect the structure of key tissue ECM components, although additional studies will be required to understand dose dependent effects.
|
Mar 2025
|
|
I13-2-Diamond Manchester Imaging
|
Begum
Okutan
,
Uwe Y.
Schwarze
,
Hansjörg
Habisch
,
Kamila
Iskhakova
,
Hanna
Cwieka
,
Cláudia
Ribeiro-Machado
,
Julian
Moosmann
,
Clement
Blanchet
,
Iva
Brcic
,
Susana G.
Santos
,
Tobias
Madl
,
Berit
Zeller-Plumhoff
,
Annelie M.
Weinberg
,
D. C. Florian
Wieland
,
Nicole G.
Sommer
Diamond Proposal Number(s):
[25485]
Open Access
Abstract: Magnesium (Mg)-based implants have become an attractive alternative to conventional permanent implants in the orthopedic field. While biocompatibility, degradation kinetics, and osseointegration of Mg-based implants have been mostly investigated, the impact of degradation products on bone remodeling and potential systemic effects remains unclear. The aim of this study was to evaluate the early and mid-term local and systemic tissue responses of degrading ultrahigh-purity ZX00 (Mg–Zn–Ca alloy) and ultrahigh-purity Mg (XHP-Mg) pins in a juvenile healthy rat model. The potential differences between implant types (degradable vs. permanent), implantation, and age-related changes were investigated using titanium (Ti), sham-operated, and control groups (non-intervention), respectively. Degradation products of ZX00 and XHP-Mg pins promote osteogenesis in the medullary cavity by upregulating the expression levels of Bmp2 and Opg within 14 days post-surgery. The higher degradation rate of XHP-Mg resulted in the accumulation of degradation products starting from day 3 and upregulation of different genes, particularly Ccl2 and Cepbp. Besides good osseointegration and new bone tissue formation, we found a more parallel hydroxyapatite/collagen orientation along Mg-based pins in the perimeter region compared to Ti pins. In the liver, reduced glycogen levels in Mg-based pins indicated that degradation products promote glycogenolysis, while only the ZX00 group showed a higher serum glucagon level on day 14. Results suggest that degrading ZX00 and XHP-Mg pins stimulate osteogenesis mainly via Bmp2 and Opg and promote glycogenolysis in the liver, while the higher degradation rate of XHP-Mg pins resulted in upregulation of different genes and metabolites.
|
Feb 2025
|
|
I18-Microfocus Spectroscopy
|
Diamond Proposal Number(s):
[35162]
Open Access
Abstract: The quantitative characterization of the structure of biomineral surfaces is needed for guiding regenerative strategies. Current techniques are compromised by a requirement for extensive sample preparation, limited length-scales, or the inability to repeatedly measure the same surface over time and monitor structural changes. We aim to address these deficiencies by developing Calcium (Ca) K-edge Polarisation Induced Contrast X-ray Fluorescence (PIC-XRF) to quantify hydroxyapatite (HAp) crystallite structural arrangements in high and low textured surfaces. Minimally prepared human dental enamel was used as an exemplar to quantify initial surface structures, and the disruption caused by short dietary acid exposures. By measuring surfaces at different rotational angles relative to a polarised focused (2x2µm) monochromatic X-ray source (at either 4049.2 and 4051.1 eV) it was possible to discriminate the principal and secondary orientations of surface crystallites, along with their texture. It was also possible to quantify the organisation of crystallites in both low (enamel cross-sections) and highly textured (facial enamel) surfaces including the identification of crystallites aligned perpendicular to the surface—a challenge for other synchrotron techniques. Surface modifications following short term acid erosion (affecting <20µm of the enamel surface depth) were detected as significant shifts in principal crystallite orientation (p<0.001) and as a marked reduction in surface texture (p<0.001). Findings suggest preferential dissolution of HAp based on crystallite angular orientation. We demonstrate that PIC-XRF is a powerful tool to quantify biomineral surfaces, with minimal sample preparation that enables monitoring of surface structural changes through repeated measurements.
|
Jan 2025
|
|
E01-JEM ARM 200CF
|
Diamond Proposal Number(s):
[32822]
Open Access
Abstract: Growth plate cartilage (GP) serves as a dynamic site of active mineralization and offers a unique opportunity to investigate the cell-regulated matrix mineralization process. Transmission electron microscopy (TEM) provides a means for the direct observation of these mechanisms, offering the necessary resolution and chemical analysis capabilities. However, as mineral crystallinity is prone to artifacts using aqueous fixation protocols, sample preparation techniques are critical to preserve the mineralized tissue in its native form. We optimized cryofixation by high-pressure freezing followed by freeze substitution in anhydrous acetone containing 0.5% uranyl acetate to prepare murine GP for TEM analysis. This sample preparation workflow maintains cellular and extracellular protein structural integrity with sufficient contrast for observation and without compromising mineral crystallinity. By employing appropriate sample preparation techniques, we were able to observe two parallel mineralization processes driven by chondrocytes: 1) intracellular- and 2) extracellular-originating mineralized vesicles. Both mechanisms are based on sequestering calcium phosphate (CaP) within a membrane-limited structure, albeit originating from different compartments of the chondrocytes. In the intracellular originating pathway, CaP accumulates within mitochondria as globular CaP granules, which are incorporated into intracellular vesicles (500-1000 nm) and transported as granules to the extracellular matrix (ECM). In contrast, membrane budding vesicles with a size of approximately 100-200nm, filled with needle-shaped minerals were observed only in the ECM. Both processes transport CaP to the collagenous matrix via vesicles, they can be differentiated based on the vesicle size and mineral morphologies. Their individual importance to the cartilage mineralization process is yet to be determined.
|
Nov 2024
|
|
I22-Small angle scattering & Diffraction
|
Diamond Proposal Number(s):
[29651]
Open Access
Abstract: Crystal structure and morphology dictate the mechanical, thermal, and degradation properties of poly l-lactide (PLLA), the structural polymer of the first clinically approved bioresorbable vascular scaffolds (BVS). New experimental methods are developed to reveal the underlying mechanisms governing structure formation during the crimping step of the BVS manufacturing process. Our research specifically examines the “U-bends” – the region where the curvature is highest and stress is maximised during crimping, which can potentially lead to failure of the device with dramatic consequences on patient life. A custom-made crimping rig operated at a synchrotron beamline enabled collection of wide- and small-angle X-ray scattering (WAXS/SAXS) to probe local variations of the polymer morphology as a function of position in the crest of multiple U-bends with 5 μm resolution in situ after crimping and expansion. Additionally, polarised light microscopy (PLM) images of these deformed U-bends revealed areas with varying stress distribution developed during crimping and expansion. These variations were dependant on the initial biaxial stretching processing step. The integrated X-ray scattering-microscopy approach offered a comprehensive work-flow for uncovering the intricate relationship between processing conditions and the corresponding spatially-resolved semicrystalline morphology of a BVS.
|
Nov 2024
|
|
I22-Small angle scattering & Diffraction
|
Diamond Proposal Number(s):
[25197]
Open Access
Abstract: Chemically crosslinked acellular bovine pericardium (ABP) has been widely used in clinical practice as bioprostheses. To ensure its consistency and durability, crosslinkers are used in excess, with stability guided by indicators including the hydrothermal denaturation temperature, the enzymatic resistance and the degree of crosslinking. Yet, understanding of the intermolecular structure in collagen fibrils which imparts the intrinsic stability of the ABPs is lacking, and the discrepancies in the stability criteria in varied conditions are poorly explained. In this study, synchrotron small-angle X-ray scattering (SAXS) in combination with thermal and colorimetric methods are employed to investigate the changes in the structure and the stability of ABPs during crosslinking using glutaraldehyde (GA) or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) at different concentrations. Based on the findings, a mechanism is proposed to explicate the crosslinking effects on collagen structure and the relationship between the structure and each stability indicator. At low crosslinker concentrations, the telopeptidyl-helical linkages are preferred which cause rearrangements in the intermolecular structure of collagen, and efficiently contribute to its stabilities. Excess crosslinking is revealed by a revert trend in structural changes and the plateauing of the stabilities, assigning to the helical-helical linkages and monovalent bindings. The former would improve thermal stability but not collagenase resistance, whereas the latter have negligible effects. Overall, this study provides mechanistic understanding of the chemical crosslinking of ABPs which will contribute to the future development of more efficient and economically viable strategies to produce bioprostheses.
|
Aug 2022
|
|
I13-2-Diamond Manchester Imaging
|
Nicole
Sommer
,
Daniela
Hirzberger
,
Lisa
Paar
,
Leopold
Berger
,
Hanna
Cwieka
,
Uwe Y.
Schwarze
,
Valentin
Herber
,
Begum
Okutan
,
Andrew J.
Bodey
,
Regine
Willumeit-Römer
,
Berit
Zeller-Plumhoff
,
Jörg F.
Löffler
,
Annelie M.
Weinberg
Diamond Proposal Number(s):
[25485]
Open Access
Abstract: Implant removal is unnecessary for biodegradable magnesium (Mg)-based implants and, therefore, the related risk for implant-induced fractures is limited. Aging, on the other hand, is associated with low bone-turnover and decreased bone mass and density, and thus increased fracture risk. Osteoporosis is accompanied by Mg deficiency, therefore, we hypothesized that Mg-based implants may support bone formation by Mg2+ ion release in an ovariectomy-induced osteoporotic rat model. Hence, we investigated osseointegration and implant degradation of a low-alloyed, degrading Mg–Zn–Ca implant (ZX00) in ovariectomy-induced osteoporotic (Osteo), old healthy (OH), and juvenile healthy (JH) groups of female Sprague Dawley rats via in vivo micro-computed tomography (µCT). For the Osteo rats, we demonstrate diminished trabecular bone already after 8 weeks upon ovariectomy and significantly enhanced implant volume loss, with correspondingly pronounced gas formation, compared to the OH and JH groups. Sclerotic rim development was observed in about half of the osteoporotic rats, suggesting a prevention from foreign-body and osteonecrosis development. Synchrotron radiation-based µCT confirmed lower bone volume fractions in the Osteo group compared to the OH and JH groups. Qualitative histological analysis additionally visualized the enhanced implant degradation in the Osteo group. To date, ZX00 provides an interesting implant material for young and older healthy patients, but it may not be of advantage in pharmacologically untreated osteoporotic conditions.
|
May 2022
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I22-Small angle scattering & Diffraction
|
Diamond Proposal Number(s):
[11316, 8458]
Open Access
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.
|
Jan 2022
|
|
I13-2-Diamond Manchester Imaging
|
Diamond Proposal Number(s):
[19322]
Open Access
Abstract: Many soft tissues, such as the intervertebral disc (IVD), have a hierarchical fibrous composite structure which suffers from regional damage. We hypothesise that these tissue regions have distinct, inherent fibre structure and structural response upon loading. Here we used synchrotron computed tomography (sCT) to resolve collagen fibre bundles (∼5μm width) in 3D throughout an intact native rat lumbar IVD under increasing compressive load. Using intact samples meant that tissue boundaries (such as endplate-disc or nucleus-annulus) and residual strain were preserved; this is vital for characterising both the inherent structure and structural changes upon loading in tissue regions functioning in a near-native environment. Nano-scale displacement measurements along >10,000 individual fibres were tracked, and fibre orientation, curvature and strain changes were compared between the posterior-lateral region and the anterior region. These methods can be widely applied to other soft tissues, to identify fibre structures which cause tissue regions to be more susceptible to injury and degeneration. Our results demonstrate for the first time that highly-localised changes in fibre orientation, curvature and strain indicate differences in regional strain transfer and mechanical function (e.g. tissue compliance). This included decreased fibre reorientation at higher loads, specific tissue morphology which reduced capacity for flexibility and high strain at the disc-endplate boundary.
|
Oct 2021
|
|
