The Small World of Osteocytes: Connectomics of the Lacuno-Canalicular Network in Bone

Osteocytes and their cell processes reside in a large, interconnected network of voids pervading the mineralized bone matrix of most vertebrates. This osteocyte lacuno-canalicular network (OLCN) is believed to play important roles in mechanosensing, mineral homeostasis, and for the mechanical properties of bone. While the extracellular matrix structure of bone is extensively studied on ultrastructural and macroscopic scales, there is a lack of quantitative knowledge on how the cellular network is organized. Using a recently introduced imaging and quantification approach, we analyze the OLCN in different bone types from mouse and sheep that exhibit different degrees of structural organization not only of the cell network but also of the fibrous matrix deposited by the cells. We define a number of robust, quantitative measures that are derived from the theory of complex networks. These measures enable us to gain insights into how efficient the network is organized with regard to intercellular transport and communication. Our analysis shows that the cell network in regularly organized, slow-growing bone tissue from sheep is less connected, but more efficiently organized compared to irregular and fast-growing bone tissue from mice. On the level of statistical topological properties (edges per node, edge length and degree distribution), both network types are indistinguishable, highlighting that despite pronounced differences at the tissue level, the topological architecture of the osteocyte canalicular network at the subcellular level may be independent of species and bone type. Our results suggest a universal mechanism underlying the self-organization of individual cells into a large, interconnected network during bone formation and mineralization

Evidence for an elementary process in bone plasticity with an activation enthalpy of 1 eV

The molecular mechanisms for plastic deformation of bone tissue are not well understood. We analysed temperature and strain-rate dependence of the tensile deformation behaviour in fibrolamellar bone, using a technique originally developed for studying plastic deformation in metals. We show that, beyond the elastic regime, bone is highly strain-rate sensitive, with an activation volume of ca 0.6 nm3. We find an activation energy of 1.1 eV associated with the basic step involved in the plastic deformation of bone at the molecular level. This is much higher than the energy of hydrogen bonds, but it is lower than the energy required for breaking covalent bonds inside the collagen fibrils. Based on the magnitude of these quantities, we speculate that disruption of electrostatic bonds between polyelectrolyte molecules in the extrafibrillar matrix of bone, perhaps mediated by polyvalent ions such as calcium, may be the rate-limiting elementary step in bone plasticity

Accelerated Growth Plate Mineralization and Foreshortened Proximal Limb Bones in Fetuin-A Knockout Mice

PMCID: PMC3473050This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Image-Based Bronchial Anatomy Codification for Biopsy Guiding in Video Bronchoscopy

Bronchoscopy examinations allow biopsy of pulmonary nodules with minimum risk for the patient. Even for experienced bronchoscopists, it is difficult to guide the bronchoscope to most distal lesions and obtain an accurate diagnosis. This paper presents an image-based codification of the bronchial anatomy for bronchoscopy biopsy guiding. The 3D anatomy of each patient is codified as a binary tree with nodes representing bronchial levels and edges labeled using their position on images projecting the 3D anatomy from a set of branching points. The paths from the root to leaves provide a codification of navigation routes with spatially consistent labels according to the anatomy observes in video bronchoscopy explorations. We evaluate our labeling approach as a guiding system in terms of the number of bronchial levels correctly codified, also in the number of labels-based instructions correctly supplied, using generalized mixed models and computer-generated data. Results obtained for three independent observers prove the consistency and reproducibility of our guiding system. We trust that our codification based on viewer's projection might be used as a foundation for the navigation process in Virtual Bronchoscopy systems

Pharmacodynamic effect of bempedoic acid and statin combinations: predictions from a dose–response model

Aims Many patients are unable to achieve guideline-recommended LDL cholesterol (LDL-C) targets, despite taking maximally tolerated lipid-lowering therapy. Bempedoic acid, a competitive inhibitor of ATP citrate lyase, significantly lowers LDL-C with or without background statin therapy in diverse populations. Because pharmacodynamic interaction between statins and bempedoic acid is complex, a dose-response model was developed to predict LDL-C pharmacodynamics following administration of statins combined with bempedoic acid. Methods And Results Bempedoic acid and statin dosing and LDL-C data were pooled from 14 phase 1-3 clinical studies. Dose-response models were developed for bempedoic acid monotherapy and bempedoic acid-statin combinations using previously published statin parameters. Simulations were performed using these models to predict change in LDL-C levels following treatment with bempedoic acid combined with clinically relevant doses of atorvastatin, rosuvastatin, simvastatin, and pravastatin. Dose-response models predicted that combining bempedoic acid with the lowest statin dose of commonly used statins would achieve a similar degree of LDL-C lowering as quadrupling that statin dose" for example, the predicted LDL-C lowering was 54% with atorvastatin 80 mg compared with 54% with atorvastatin 20 mg + bempedoic acid 180 mg, and 42% with simvastatin 40 mg compared with 46% with simvastatin 10 mg + bempedoic acid 180 mg. Conclusion These findings suggest bempedoic acid combined with lower statin doses offers similar LDL-C lowering compared with statin monotherapy at higher doses, potentially sparing patients requiring additional lipid-lowering therapies from the adverse events associated with higher statin doses

Characteristics of patients initiated on edoxaban in Europe:baseline data from edoxaban treatment in routine clinical practice for patients with atrial fibrillation (AF) in Europe (ETNA-AF-Europe)

Background: Non-vitamin K antagonist (VKA) oral anticoagulants (NOACs) have substantially improved anticoagulation therapy for prevention of stroke and systemic embolism in patients with atrial fibrillation (AF). The available routine care data have demonstrated the safety of different NOACs; however, such data for edoxaban are scarce. Here, we report baseline characteristics of 13,638 edoxaban-treated patients with AF enrolled between November 2016 and February 2018. Methods: ETNA-AF-Europe is a multinational, multi-centre, post-authorisation, observational study conducted in 825 sites in 10 European countries. Patients will be followed up for four years. Results: Overall, 13,980 patients were enrolled of which 342 patients were excluded from the analysis. Mean patient age was 73.6 years with an average creatinine clearance of 69.4 mL/min. 56.6% were male. The calculated CHA2DS2-VASc and HAS-BLED mean scores were 3.1 and 2.6, respectively. Overall, 3.3, 14.6 and 82.0% of patients had low (CHA2DS2-VASc = 0), intermediate (CHA2DS2-VASc = 1) and high (CHA2DS2-VASc≥2) risks of stroke, respectively. High-risk patients (those with prior stroke, prior major bleeding, prior intracranial bleed or CHA2DS2-VASc ≥4) comprised 38.4% of the overall population. For 75.1% of patients edoxaban was their first anticoagulant prescription, whilst 16.9% switched from a VKA and 8.0% from another NOAC. A total of 23.4% of patients in ETNA-AF-Europe received the reduced dose of edoxaban 30 mg. Overall, 83.8% of patients received an edoxaban dose in line with the criteria outlined in the label. Conclusion: Edoxaban was predominantly initiated in older, often anticoagulation-naïve, unselected European patients with AF, with a good overall adherence to the approved label. Trial registration: NCT02944019; Date of registration: October 24, 2016

1,25(OH)2D3 Alters Growth Plate Maturation and Bone Architecture in Young Rats with Normal Renal Function

Whereas detrimental effects of vitamin D deficiency are known over century, the effects of vitamin D receptor activation by 1,25(OH)2D3, the principal hormonal form of vitamin D, on the growing bone and its growth plate are less clear. Currently, 1,25(OH)2D3 is used in pediatric patients with chronic kidney disease and mineral and bone disorder (CKD-MBD) and is strongly associated with growth retardation. Here, we investigate the effect of 1,25(OH)2D3 treatment on bone development in normal young rats, unrelated to renal insufficiency. Young rats received daily i.p. injections of 1 µg/kg 1,25(OH)2D3 for one week, or intermittent 3 µg/kg 1,25(OH)2D3 for one month. Histological analysis revealed narrower tibial growth plates, predominantly in the hypertrophic zone of 1,25(OH)2D3-treated animals in both experimental protocols. This phenotype was supported by narrower distribution of aggrecan, collagens II and X mRNA, shown by in situ hybridization. Concomitant with altered chondrocyte maturation, 1,25(OH)2D3 increased chondrocyte proliferation and apoptosis in terminal hypertrophic cells. In vitro treatment of the chondrocytic cell line ATDC5 with 1,25(OH)2D3 lowered differentiation and increased proliferation dose and time-dependently. Micro-CT analysis of femurs from 1-week 1,25(OH)2D3-treated group revealed reduced cortical thickness, elevated cortical porosity, and higher trabecular number and thickness. 1-month administration resulted in a similar cortical phenotype but without effect on trabecular bone. Evaluation of fluorochrome binding with confocal microscopy revealed inhibiting effects of 1,25(OH)2D3 on intracortical bone formation. This study shows negative effects of 1,25(OH)2D3 on growth plate and bone which may contribute to the exacerbation of MBD in the CKD pediatric patients

Bone material characteristics influenced by osteocytes

In dieser Doktorarbeit wird die Hypothese geprüft, ob Osteozyten einen direkten Einfluss auf die Knocheneigenschaften in ihrer unmittelbaren Umgebung haben. Der zentrale Experimentieransatz ist dabei die Korrelation der Organisation des Osteozytennetzwerks mit den Mineraleigenschaften des Knochens auf der Submikrometerebene. Es wird gezeigt, dass bereits die anfängliche Ausrichtung der Osteoblasten entscheidend für die Synthese von hoch ausgerichtetem Knochenmaterial ist. Die dabei entstehenden Osteozytennetzwerke sind so organisiert, dass die Osteozyten und ihre Zellfortsätze jeweils einen möglichst kleinen Abstand zum Knochenmineral haben. Deshalb wird vermutet, dass genau diese Netzwerkorganisation mitentscheidend ist, wie gut die Zellen das Mineral in ih-rer Umgebung beeinflussen können. Messungen der Knochenmineraleigenschaften auf Submikrometerebene mit Röntgenkleinwinkelstreuung bestätigen diese Vermutung. Dabei wird deutlich, dass Knochenmaterial in der Nähe der Osteozyten durch andere Mineraleigenschaften geprägt ist. Um zu klären, wie Osteozyten Mineral in ihrer direkten Umgebung verändern können, werden Mechanismen der passiven Mineralherauslösung aus der mineralisierten Oberfläche des Osteozytennetzwerks untersucht. Es wird gezeigt, dass kalziumarme ionische Lösungen unter physiologischen Bedingungen große Mengen von Kalzium-Ionen aus dem Knochen lösen und diese dann durch die Osteozytennetzwerkstrukturen diffundieren können. Zum Abschluss wurde medullärer Knochen von Hühnern als ein Modellsystem für rasanten Knochenumbau untersucht. Dieser spezielle Knochentyp dient den Hennen als labiles Kalziumreservoir und ermöglicht dadurch die tägliche Eierschalenproduktion. Experimente am medullären Knochen-material zeigen insbesondere die Bedeutung von weniger stabilen Mineralstrukturen die benötigt werden um den Knochen an den schnellen, sich wiederholenden Knochenauf- sowie Abbau optimal anzupassen.This thesis aims to test the hypothesis whether osteocytes have a direct influence on bone material properties in their vicinity. In this regard, the concomitant ana-lysis of osteocyte network organization and bone ultrastructural properties on the submicron level is the central approach to answer this question. In this work, it is shown that already initial cell-cell alignment during the process of bone formation is crucial for the synthesis of highly organized bone. Furthermore it is proposed that the occurrence of highly ordered osteocyte networks visualized with confocal laser scanning microscopy (CLSM) has a strong impact on the ability of osteocytes to directly influence bone material properties. These highly organized networks are another consequence of initial cell-cell alignment and are found to be arranged such as to feature short mineral cell distances. Examination of sub-micron mineral properties with scanning small angle x-ray scattering (sSAXS) shows that bone material in the direct vicinity of osteocytes and their cell proc-esses shows different mineral properties compared to bone further away in the depth of the tissue. Moreover, mechanisms of passive mineral extraction from the mineralized surface of the osteocyte network, due to the treatment with calcium poor ionic solutions, are investigated. It is shown that this chemical process occurring under physiological conditions leads not only to the dissolution of considerable amounts of calcium, but also to efficient diffusion of these ions through the osteocyte network structures. Finally, medullary bone which is intended as a labile calcium source for daily egg shell formation in hens is used as a model system for rapid bone turnover rates. This bone type in particular indicates the importance of uniquely adapted, less stable mineral structures to fit the requirements for rapid bone resorption as well as reformation

Mineral formation in the Larval Zebrafish tail bone occurs via an acidic disordered calcium phosphate phase

\u3cp\u3eBoth in vivo and ex vivo observations support the hypothesis that bone mineral formation proceeds via disordered precursor phases. The characteristics of the precursor phases are not well defined, but octacalcium phosphate-like, amorphous calcium phosphate-like, and HPO\u3csub\u3e4\u3c/sub\u3e \u3csup\u3e2-\u3c/sup\u3e-enriched phases were detected. Here we use in vivo Raman spectroscopy and high-resolution wide-angle X-ray diffraction (WAXD) to characterize and map at 2 μm resolution the mineral phases in the rapidly forming tail fin bones of living zebrafish larvae and zebrafish larvae immediately after sacrifice, respectively. Raman spectroscopy shows the presence of an acidic disordered calcium phosphate phase with additional characteristic features of HPO\u3csub\u3e4\u3c/sub\u3e \u3csup\u3e2-\u3c/sup\u3e at the bone-cell interface. The complexity in the position and shape of the ν\u3csub\u3e1\u3c/sub\u3e PO\u3csub\u3e4\u3c/sub\u3e peak viewed by in vivo Raman spectroscopy emphasizes the heterogeneity of the mineral during bone formation. WAXD detects an additional isolated peak, appearing alone or together with the characteristic diffraction pattern of carbonated hydroxyapatite. This unidentified phase is located at the interface between the mature bone and the surrounding tissue, similar to the location at which the disordered phase was observed by Raman spectroscopy. The variable peak positions and profiles support the notion that this is an unstable disordered precursor phase, which conceivably crystallized during the X-ray diffraction measurement. Interestingly, this precursor phase is co-aligned with the c-axes of the mature bone crystals and thus is in intimate relation with the surrounding collagen matrix. We conclude that a major disordered precursor mineral phase containing HPO\u3csub\u3e4\u3c/sub\u3e \u3csup\u3e2-\u3c/sup\u3e is part of the deposition pathway of the rapidly forming tail fin bones of the zebrafish.\u3c/p\u3

Data for arXiv:1702.04117: "The Small World of Osteocytes: Connectomics of the Lacuno-Canalicular Network in Bone"

<p>This dataset contains the raw confocal image stacks of the osteocyte lacuno-canalicular network in woven bone from mouse and fibrolamellar bone from sheep as described in  "The Small World of Osteocytes: Connectomics of the Lacuno-Canalicular Network in Bone" (https://arxiv.org/abs/1702.04117). See the methods section of the manuscript for further detail. MATLAB code to reproduce the data plots in the figures can be downloaded from https://github.com/phi-max/OCY_connectomics.</p