Detection and imaging of gadolinium accumulation in human bone tissue by micro- and submicro-XRF

Gadolinium-based contrast agents (GBCAs) are frequently used in patients undergoing magnetic resonance imaging. In GBCAs gadolinium (Gd) is present in a bound chelated form. Gadolinium is a rare-earth element, which is normally not present in human body. Though the blood elimination half-life of contrast agents is about 90 minutes, recent studies demonstrated that some tissues retain gadolinium, which might further pose a health threat due to toxic effects of free gadolinium. It is known that the bone tissue can serve as a gadolinium depot, but so far only bulk measurements were performed. Here we present a summary of experiments in which for the first time we mapped gadolinium in bone biopsy from a male patient with idiopathic osteoporosis (without indication of renal impairment), who received MRI 8 months prior to biopsy. In our studies performed by means of synchrotron radiation induced micro- and submicro-X-ray fluorescence spectroscopy (SR-XRF), gadolinium was detected in human cortical bone tissue. The distribution of gadolinium displays a specific accumulation pattern. Correlation of elemental maps obtained at ANKA synchrotron with qBEI images (quantitative backscattered electron imaging) allowed assignment of Gd structures to the histological bone structures. Follow-up beamtimes at ESRF and Diamond Light Source using submicro-SR-XRF allowed resolving thin Gd structures in cortical bone, as well as correlating them with calcium and zinc

Heterogeneity of the osteocyte lacuno-canalicular network architecture and material characteristics across different tissue types in healing bone

Various tissue types, including fibrous connective tissue, bone marrow, cartilage, woven and lamellar bone, coexist in healing bone. Similar to most bone tissue type, healing bone contains a lacuno-canalicular network (LCN) housing osteocytes. These cells are known to orchestrate bone remodeling in healthy bone by sensing mechanical strains and translating them into biochemical signals. The structure of the LCN is hypothesized to influence mineralization processes. Hence, the aim of the present study was to visualize and match spatial variations in the LCN topology with mineral characteristics, within and at the interfaces of the different tissue types that comprise healing bone. We applied a correlative multi-method approach to visualize the LCN architecture and quantify mineral particle size and orientation within healing femoral bone in a mouse osteotomy model (26 weeks old C57BL/6 mice). This approach revealed structural differences across several length scales during endochondral ossification within the following regions: calcified cartilage, bony callus, cortical bone and a transition zone between the cortical and callus region analyzed 21 days after the osteotomy. In this transition zone, we observed a continuous convergence of mineral characteristics and osteocyte lacunae shape as well as discontinuities in the lacunae volume and LCN connectivity. The bony callus exhibits a 34% higher lacunae number density and 40% larger lacunar volume compared to cortical bone. The presented correlations between LCN architecture and mineral characteristics improves our understanding of how bone develops during healing and may indicate a contribution of osteocytes to bone (re)modeling

Electrode configurations study for alkaline direct ethanol fuel cells

The direct electrochemical conversion of ethanol, a sustainable fuel, is an alternative sustainable technology of the future. In this study, membrane electrode assemblies with different electrode configurations for an alkaline direct ethanol fuel cell were fabricated and tested in a fuel cell device. The configurations include a catalyst-coated substrate (CCS), a catalyst-coated membrane (CCM), and a mixture of these two fabrication options. Two different anion exchange membranes were used to perform a comprehensive analysis. The fabricated CCSs and CCMs were characterized with single cell measurements, electro­chemical impedance spectroscopy and scanning electron microscopy. In addition, the swelling behavior of the membranes in alkaline solution was investigated in order to obtain information for CCM production. The results of the experimental electrochemical tests show that the CCS approach provides higher power densities (42.4 mW cm-2) than the others, regardless of the membrane type

Curvature in Biological Systems: Its quantification, Emergence and Implications Across the Scales

Surface curvature both emerges from, and influences the behavior of, living objects at length scales ranging from cell membranes to single cells to tissues and organs. The relevance of surface curvature in biology has been supported by numerous recent experimental and theoretical investigations in recent years. In this review, we first give a brief introduction to the key ideas of surface curvature in the context of biological systems and discuss the challenges that arise when measuring surface curvature. Giving an overview of the emergence of curvature in biological systems, its significance at different length scales becomes apparent. On the other hand, summarizing current findings also shows that both single cells and entire cell sheets, tissues or organisms respond to curvature by modulating their shape and their migration behavior. Finally, we address the interplay between the distribution of morphogens or micro-organisms and the emergence of curvature across length scales with examples demonstrating these key mechanistic principles of morphogenesis. Overall, this review highlights that curved interfaces are not merely a passive by-product of the chemical, biological and mechanical processes but that curvature acts also as a signal that co-determines these processes

Supplementary data for the article: Petrović, M. M.; Roschger, C.; Chaudary, S.; Zierer, A.; Mladenović, M.; Marković, V.; Trifunović, S.; Joksović, M. D. Low Cytotoxic Quinoline-4-Carboxylic Acids Derived from Vanillin Precursors as Potential Human Dihydroorotate Dehydrogenase Inhibitors. Bioorganic & Medicinal Chemistry Letters 2021, 46, 128194. https://doi.org/10.1016/j.bmcl.2021.128194.

Supplementary material for: [https://doi.org/10.1016/j.bmcl.2021.128194]Related to published version: [https://cherry.chem.bg.ac.rs/handle/123456789/4550

Increased zinc accumulation in mineralized osteosarcoma tissue measured by confocal synchrotron radiation micro X-ray fluorescence analysis

Abnormal tissue levels of certain trace elements such as zinc (Zn) were reported in various types of cancer. Little is known about the role of Zn in osteosarcoma. Using confocal synchrotron radiation micro X-ray fluorescence analysis, we characterized the spatial distribution of Zn in high-grade sclerosing osteosarcoma of nine patients (four women/five men; seven knee/one humerus/one femur) following chemotherapy and wide surgical resection. Levels were compared with adjacent normal tissue. Quantitative backscattered electron imaging as well as histological examinations was also performed. On average, the ratio of medians of Zn count rates (normalized to calcium) in mineralized tumor tissue was about six times higher than in normal tissue. There was no difference in Zn levels between tumor fraction areas with a low fraction and a high fraction of mineralized tissue, which were clearly depicted using quantitative backscattered electron imaging. Moreover, we found no correlation between the Zn values and the type of tumor regression according to the Salzer-Kuntschik grading. The underlying mechanism of Zn accumulation remains unclear. Given the emerging data on the role of trace elements in other types of cancer, our novel results warrant further studies on the role of trace elements in bone cancer

Quantitative analysis of local mineral content and composition during bone growth and remodeling

Das Ziel der Studien, die im Rahmen dieser Arbeit vorgestellt werden, war es neue Informationen über die elementare Zusammensetzung des mineralisierten Knochens zu gewinnen. In einer ersten Studie wurden zwei Parameter verglichen, die beide eng mit der Knochenmineralisierung verknüpft sind. So zeigte die Gegenüberstellung des mineral/matrix Raman-Wertes und der Kalziumkonzentration gute Übereinstimmung mit theoretischen Überlegungen. Diese Methoden wurden auch verwendet um Knochengewebe von Mäusen zu charakterisieren bei denen ein genetischer Defekt zu einem Mangel von Sclerostin führte. So war es möglich nachzuweisen, dass eine hierdurch verstärkte Knochenneubildung zu einer veränderten Mineralisationskinetik des Knochens führen kann. Nachdem zukünftig Sclerostinantikörper für die Behandlung von Knochenkrankheiten eingesetzt werden sollen, haben diese Erkenntnisse große medizinische Bedeutung. Es wurde auch die Mineraldichteverteilung eines Mausmodells mit fragilem Knochen (Osteogenesis Imperfecta, OI) untersucht. Die Mäuse wurden mit Sclerostinantikörpern behandelt. Es zeige sich ein signifikanter Knochenzuwachs doch die Mineraldichteverteilung veränderte sich gleichermaßen für gesunde und für OI Mäuse. In einer Studie am humanen Knochen konnten der Zusammenhang zwischen Osteozytennetzwerk und Knochenzusammensetzung untersucht werden. Elemente wie Na, Mg und S wiesen typische Konzentrationsverteilungen auf. Die Routinen wurden auch verwendet um Mineralisationsfronten zu charakterisieren. Es zeigte sich, dass die Konzentrationen von K, Mg, Na und Cl abhängig von dem analysierten anatomischen Ort, stark voneinander abweichen. Abschließend kann gesagt werden, dass durch die Entwicklung neuer Routinen zusätzliche Erkenntnisse über die Knochenmineralisierung und Zusammensetzung gewonnen werden konnten. Die Resultate sind von medizinischer und biologischer Bedeutung und tragen zu aktuellen Debatten über die Knochenentwicklung bei.The purpose of the presented work was to gain new insight into the elemental composition of mineralized bone matrix at different sites of human bone tissue, and in mouse models linked to human genetic diseases. Using novel tools and routines, human (femur cross sections from healthy adults and children) and murine samples (femur long-and cross sections of two mouse models) were analyzed with focus on the elemental composition. In a methodological study the consistency of matrix mineralization measured by Raman microspectroscopy (e.g. the mineral/matrix ratio) and the Calcium content (wt%Ca) as measured by qBEI was proved. Both methods were applied to a mouse model exhibiting induced bone overgrowth due to a genetic defect causing a lack of Sclerostin, which is a negative regulator for bone formation. We found changes in the mineralization kinetics depending on the anatomical site. This result is of clinical importance since Sclerostin antibodies are suggested for future treatment of diseases characterized by fragile bone. Hence, also a mouse model of a brittle bone disease (Osteogenesis Imperfecta) was analyzed with and without Sclerostin antibody treatment. A significant increase in bone mass was documented while the mineralization pattern revealed no interaction between genotype and treatment. The correlation between OLCN and the composition of the mineralized matrix was examined in the same regions of human compact bone. Characteristic distributions of the minor elements (Mg, Na, S) were found. The developed tools were also used to investigate mineralization fronts, reflecting a critical stage of bone development. Differences in the Ca/P ratio and in the concentrations of K, Mg, Na and Cl depending on the anatomical site were revealed. In conclusion, using newly developed measurement routines, it was possible to gain novel information of bone mineralization and composition. The results contribute to actively debated issues of biological and medical importance

Localization of trace elements in bone tissue using synchrotron radiation induced micro-XRF in combination with quantitative backscattered electron imaging

Zsfassung in dt. SpracheDie Verwendung von Synchrotronstrahlung induzierter mikro-Röntgenfuoreszenanalyse (SR-micro-XRF) stellte sich als eine Methode heraus, die hervorragend zur ortsaufgelösten, zerstörungsfreien Multielement-Untersuchung von Knochen und Gelenksknorpel geeignet ist.Ergänzend kann quantitative Rückstreuelektronenmikroskopie (qBEI) genaue Informationen über den Grad der Mineralisierung liefern und ermöglicht es zwischen verschiedenen histologischen Regionen zu unterscheiden.In Rahmen dieser Arbeit wurden mikro-XRF und qBEI kombiniert, um die Verteilung der Spurenelemente Blei (Pb), Zink (Zn) und Strontium (Sr) in Knochen und Gelenkknorpel zu charakterisieren. Diese Kombination erlaubt es die verschiedenen histologischen Regionen (ausgewählt anhand des qBEI Bildes) den Fluoreszenzmaps zuzuordnen. So können zum ersten Mal die 2-dimensionale Verteilung von Spurenelementen sowie die relativen Konzentrationen in Knochen und Knorpel untersucht werden. In 6 Hüftköpfen und 3 Kniescheiben wurden die Tidemarks untersucht (die TM ist die Grenzfläche zwischen mineralisiertem und nicht mineralisiertem Gelenksknorpel). Eine Verdoppelung der TM steht vermutlich in Zusammenhang mit äußeren mechanischen Reizen. Sowohl doppelte als auch einzelne TMs wurden auf die Spurenelementverteilung hin untersucht. Um weiters die Verteilung im Knochen zu erforschen wurden 5 frakturierte Oberschenkelhälse von osteoporotischen postmenopausalen Frauen, sowie 5 unfrakturierte Oberschenkelhälse von einer Kontrollgruppe gemessen. Zusätzlich wurde die Spurenelementverteilung in einem mit Cisplatin behandelten, sklerosierenden Osteosarcom (Knochentumor) ermittelt. In Regionen mit doppelten TMs erwiesen sich die Zn-Konzentrationen beiden TMs annähernd gleich. Die Analyse der relativen Pb-Konzentrationen in doppelten TMs zeigte durchschnittlich eine 2.6-fache Erhöhung des Pb Gehaltes in der inneren TM verglichen mit der äußeren. Das Pb Verhältnis zwischen den TMs schwankte stark.Relativ zu den Elementkonzentrationen im darunterliegenden Knochengewebe zeigten die TMs drastisch erhöhte Zn und der Pb Anreicherungen (bis zu 58-fach für Pb und bis zu 7-fach für Zn). Einfache TMs wiesen Konzentrationen vergleichbar mit der inneren TM von doppel-TM Regionen auf.In den Oberschenkelhalsproben konnte kein Unterschied in der Elementverteilung zwischen osteoporotischem Knochen und den nichtfrakturierten Proben der Kontrollgruppe festgestellt werden. In nahezu allen Proben zeigen die Zementlinien (Grenzflächen zwischen 2 Knochenpaketen) signifikant erhöhte Zn und Pb Werte, verglichen mit den Knochenpaketen. Es ist auffällig, dass Knochenränder und Ränder von Osteonen oft hohe Zn Konzentrationen aufwiesen. Weiters konnte bei der Untersuchung der Spurenelementkonzentration bezüglich des Ca-Gehaltes ein überproportional starker Anstieg von Pb und Sr in den Knochenpaketen beobachtet werden. Das sklerosierende Osteosarcom zeigte eine 13-fach erhöhte Zn Konzentration bezogen auf das umliegende gesunde Knochengewebe. Es wurde kein Platin in dieser Probe gefunden.Synchrotron radiation induced micro X-ray fluorescence analysis (SR-micro-XRF) is a suitable method for spatial resolved, non-destructive, multi-element examination of bone and articular cartilage. Quantitative backscatter electron imaging (qBEI) accounts for information of the degree of mineralization and allows distinguishing between histological regions. In this thesis micro-XRF and qBEI were used for a combined data evaluation of the trace elements lead (Pb), zinc (Zn) and strontium (Sr) in bone and articular cartilage. This combination allows allocating the histological regions (labelled in the qBEI image) to elemental fluorescence maps. Hence, for the first time, the spatial distribution and the relative concentrations of trace elements in bone and cartilage were evaluated.In 3 patellae and 6 hip heads the tidemarks (the TM is the interface between mineralized and non-mineralized articular cartilage) were analyzed. Double TMs (TM duplication is assumed to be a response to micro damage) as well as single TMs were measured. From 5 femoral necks of osteoporotic postmenopausal women with hip fractures, as well as from 5 age-matched controls without a history of bone disease, information of the elemental distribution was gained. Furthermore, the evaluation of the elemental distribution of a cisplatin treated, sclerosing osteosarcoma was performed.The analysis of the double TMs showed that the Zn concentration is nearly equal in both TMs. Contrarily, the evaluation of Pb intensities in double TMs revealed in average a 2.6 fold higher level in the inner TM compared to the outer TM, whereby the Pb in the outer TM showed a high variation with respect to the inner TM. Compared to the bone level, the Pb and Zn contents are drastically increased (up to the 58 fold for Pb and 7 fold for Zn). Single TMs show an elemental distribution comparable to the inner tidemark of double tidemark samples.In the femoral neck samples no alterations in the distributions of Sr, Pb and Zn were found for osteoporotic bone compared to non-fractured controls. The cementlines (interfaces between two bone packets) show a significantly raised content of Zn and Pb in nearly all samples. The bone borders and osteons borders are also often enhanced in Zn.A disproportionate increase of Pb and Sr with respect to the bone packet concentration is observed.The sclerosing osteosarcoma showed a 13 fold raised Zn concentration compared to the adjacent healthy bone tissue. No Platinum was found in this sample.11

Effect of Sclerostin Antibody Treatment in a Mouse Model of Severe Osteogenesis Imperfecta

Children with Osteogenesis imperfecta (OI) still suffer from frequent fractures, despite bisphosphonate treatment. Thus new therapeutic approaches are needed. Sclerostin is a protein that is thought to inhibit bone formation. Treatment with sclerostin antibodies (SclAB) increases bone mass in animal models and in clinical trials and may be a rational therapy for OI as well. Transgenic (TgOI) Col1a1Jrt/+ mice were generated being a model of OI type IV. 8 weeks old TgOI (n=8) and wild type (Wt) (n=8) were analyzed after 4 weeks of SclAB treatment and compared to untreated animals (n=7, n=8). The bone mineralization density distribution (BMDD) of the cortical (Ct), metaphyseal spongiosa (MS) and epiphyseal spongiosa (ES) in the distal femur was measured by quantitative backscattered electron imaging. Additionally microCT parameters of the femurs were obtained. TgOI mice exhibit increased bone matrix mineralization compared to Wt (most frequently occurring Ca concentration: Ct:+6.8% MS:+5.2% ES:+8.2%, p<0.001). The mineralization was also more homogenous in Ct. This fits previous findings in OI models. The percentage of lowly mineralized areas was increased in MS and ES most likely due to the decrease of bone volume (-86.3%, p<0.001). Treatment with SclAB of Wt and TgOI mice overall led to shifts in the BMDD of the spongiosa towards higher and more homogenously mineralized bone. This is consistent with the observed decrease in percentage of lowly mineralized regions reflecting the increase in bone volume due to SclAB treatment. 2-Way ANOVA tests revealed no interaction between genotype and treatment. After treatment, BV/TV of the Wt (+77.7%, p<0.001) and the TgOI (+65.9%, p=0.02) animals were elevated. Wt/SclAB and the TgOI/SclAB mice exhibit more bone volume and a similar increase in matrix mineralization compared to the corresponding untreated animals. Therefore we conclude, that SclAB treatment has the similar effect on mineralization in TgOI and Wt mice