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

Influence of magnetic fields on magneto-aerotaxis

The response of cells to changes in their physico-chemical micro-environment is essential to their survival. For example, bacterial magnetotaxis uses the Earth's magnetic field together with chemical sensing to help microorganisms move towards favoured habitats. The studies of such complex responses are lacking a method that permits the simultaneous mapping of the chemical environment and the response of the organisms, and the ability to generate a controlled physiological magnetic field. We have thus developed a multi-modal microscopy platform that fulfils these requirements. Using simultaneous fluorescence and high-speed imaging in conjunction with diffusion and aerotactic models, we characterized the magneto-aerotaxis of Magnetospirillum gryphiswaldense. We assessed the influence of the magnetic field (orientation; strength) on the formation and the dynamic of a micro-aerotactic band (size, dynamic, position). As previously described by models of magnetotaxis, the application of a magnetic field pointing towards the anoxic zone of an oxygen gradient results in an enhanced aerotaxis even down to Earth's magnetic field strength. We found that neither a ten-fold increase of the field strength nor a tilt of 45° resulted in a significant change of the aerotactic efficiency. However, when the field strength is zeroed or when the field angle is tilted to 90°, the magneto-aerotaxis efficiency is drastically reduced. The classical model of magneto-aerotaxis assumes a response proportional to the cosine of the angle difference between the directions of the oxygen gradient and that of the magnetic field. Our experimental evidence however shows that this behaviour is more complex than assumed in this model, thus opening up new avenues for research

Charge-Induced Structural Changes in a Single Molecule Investigated by Atomic Force Microscopy

Intramolecular structural relaxations occurring upon electron transfer are crucial in determining the rate of redox reactions. Here, we demonstrate that subangstrom structural changes occurring upon single-electron charging can be quantified by means of atomically resolved atomic force microscopy (AFM) for the case of single copper(II)phthalocyanine (CuPc) molecules deposited on an ultrathin NaCl film. Imaging the molecule in distinct charge states (neutral and anionic) reveals characteristic differences in the AFM contrast. In comparison to density functional theory simulations these changes in contrast can be directly related to relaxations of the molecule's geometric structure upon charging. The dominant contribution arises from a nonhomogeneous vertical relaxation of the molecule, caused by a change in the electrostatic interaction with the surface

Computational analysis of dynamic bone structure and processes

Das menschliche Skelett besteht aus einem dynamischen Material welches in der Lage ist zu heilen, sowie sich durch strukturellen Umbau an mechanische Beanspruchung anzupassen. In dieser Arbeit ist die mechanische Regulierung dieser Prozesse untersucht worden. Hierfür ist ein Computermodell, sowie die dreidimensionale Abbildung des Knochens und die Auswertung dieser Bilder benutzt worden. An dem Heilungsprozesses von Knochen sind verschiedene Gewebetypen beteiligt. Dabei hängt die räumliche und zeitliche Anordnung dieser Gewebe von der mechanischen Belastung ab. Ein Computermodell, welches den vollständigen Verlauf der Heilung beschreibt, wurde mit der dokumentierten Gewebeentwicklung eines Tierexperimentes verglichen. Verschiedene Hypothesen, wie die mechanische Stimulation die Bildung verschiedene Gewebe beeinflusst, wurden getestet. Zwar ließen sich durch den Vergleich mit dem Experiment keine der Hypothesen verwerfen, jedoch konnten wir Vorschläge machen, worauf bei zukünftigen Experimenten verstärkt geachtet werden soll. Es wird angenommen dass der Umbauprozesses des Knochens vom dichten Netzwerk der Osteozyten mechanisch reguliert wird. Diese Zellen sind in den Knochen eingebettet und über ein dichtes Netzwerk aus engen Kanälen, den sogenannten Canaliculi, miteinander verbunden. Dieses Netzwerk mittels konfokaler Mikrokopie dreidimensional abgebildet. Spezielle Routinen zur Auswertung der Netzwerkorientierung sowie dessen Dichte wurden entwickelt. Die Hauptorientierung des Netzwerkes entspricht der Richtung in der Knochengewebe aufgebaut wird. Die Orientierung des zu dieser Richtung senkrechten Anteils des Netzwerkes rotiert abhängig von der Position entlang der Aufbaurichtung. Dies verdeutlicht den Zusammenhang zwischen der Netzwerkorientierung und der Vorzugsrichtung des Kollagens, dem faserigen Bestandteils des Knochens. Darüber hinaus zeigt die Auswertung der Daten weitere strukturelle Unterschiede im Netzwerk.Our skeleton is composed of a dynamic material that is capable of healing and of adapting to changing mechanical loads through structural remodeling. In this thesis the mechano-regulation of these dynamic processes are addressed using computer modeling and 3-dimensional imaging and image analysis. During bone healing an intricate pattern of different newly formed tissues around the fracture site evolves in time and is influenced by the mechanical loading. Using a computer model which is describing this temporal-spatial evolution of tissue types for the full time-course of healing, this evolution is compared to the documented evolution of an animal experiment. Different hypotheses were tested how the mechanical stimulation results in the formation of different tissues. While the comparison with the outcome of the animal experiments does not allow to falsify any of the hypotheses, it suggests a different design of future animal experiments. Bone remodeling is thought to be mechano-regulated by the dense network of osteocytes. These osteocytes are embedded in bone and are connected to each other via a network of narrow canaliculi. The 3-dimensional structure of the network was imaged using rhodamine staining and laser scanning confocal microscopy. Image analysis tools were developed to determine the network topology and to analyze its density and orientation. The analysis focused on osteons, the building blocks of cortical bone. Within an osteon we found a large variability of the network density with extensive regions without network. Most of the network is oriented radially towards the center of the osteon, i.e.\ parallel to the direction in which the bone material is deposited. The network perpendicular to this direction twists when moving along the direction of bone deposition. A correlation with the main orientation the fibrous constituent of bone, collagen, was detected. Furthermore indicates our data additional structural changes in the network alignment

Direct identification and determination of conformational response in adsorbed individual non-planar molecular species using non-contact atomic force microscopy

In recent years atomic force microscopy (AFM) at highest resolution was widely applied to mostly planar molecules, while its application toward exploring species with structural flexibility and a distinct 3D character remains a challenge. Herein, the scope of noncontact AFM is widened by investigating subtle conformational differences occurring in the well-studied reference systems 2H-TPP and Cu-TPP on Cu(111). Different saddle shape conformations of both species can be recognized in conventional constant-height AFM images. To unambiguously identify the behavior of specific molecular moieties, we extend data acquisition to distances that are inaccessible with constant height measurements by introducing vertical imaging, that is, AFM mapping in a plane perpendicular to the sample surface. Making use of this novel technique the vertical displacement of the central Cu atom upon tip-induced conformational switching of Cu-TPP is quantified. Further, for 2H-TPP two drastically different geometries are observed, which are systematically characterized. Our results underscore the importance of structural flexibility in adsorbed molecules with large conformational variability and, consequently, the objective to characterize their geometry at the single-molecule level in real space

Bonding Motifs in Metal-Organic Compounds on Surfaces

The bonds in metal organic networks on surfaces govern the resulting geometry as well as the electronic properties. Here, we study the nature of these bonds by forming phenazine-copper complexes on a copper surface by means of atomic manipulation. The structures are characterized by a combination of scanning probe microscopy and density functional theory calculations. We observed an increase of the molecule-substrate distance upon covalent bond formation and an out-of-plane geometry that is in direct contradiction with the common expectation that these networks are steered by coordination bonds. Instead, we find that a complex energy balance of hybridization with the substrate, inhomogeneous Pauli repulsion, and elastic deformation drives the phenazine-copper interaction. Most remarkably, this attractive interaction is not driven by electron acceptor properties of copper but is of completely different donation/back-donation mechanism between molecular pi-like orbitals and sp-like metal states. Our findings show that the nature of bonds between constituents adsorbed on surfaces does not have to follow the common categories

Sol gel hybrid coatings with strontium-doped 45S5 glass particles for enhancing the performance of stainless steel implants: Electrochemical, bioactive and in vivo response

The protection of stainless-steel implants by applying a hybrid organic–inorganic coating generates a barrier for ion migration and a potential holder for functional particles. Chemical composition of bioactive silicate-glasses (BG) can be varied to tailor their rate of dissolution in the biological environment. The substitution of calcium by strontium (Sr) generates a locally-controlled release of Sr-ions to the media. Strontium is known to reduce bone resorption and stimulate bone formation. This work presents coatings made by sol–gel method containing tetraethoxysilane, methyl-triethoxysilane and silica nanoparticles as precursors, and functionalized either with BG or Sr-substituted BG particles onto surgical grade stainless steel. The coated implants were tested in vitro for corrosion resistance and bioactivity, and in vivo to analyze bone formation. The applied coating system provided an excellent protection to aggressive fluids, even after 30 days of immersion. The presence of hydroxyapatite is shown as a first evidence of bioactivity. The evaluation of in vivo tests in Wistar–Hokkaido rat femur 4 or 8 weeks after the implantation showed slight differences in the thickness of newly formed bone measured by ESEM, and remarkable changes in bone quality characterized with Raman microscopy. The in vivo response of the coatings containing Sr-substituted bioglass is better at early times of implantation as regards the bone morphology and quality making this functionalized coatings a very promising option for implant protection and bone regeneration.Fil: Omar, Sheila Ayelén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; ArgentinaFil: Repp, Felix. Institut Max Planck fuer Bioanorganische Chemie; AlemaniaFil: Desimone, Paula Mariela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; ArgentinaFil: Weinkamer, Richard. Institut Max Planck fuer Bioanorganische Chemie; AlemaniaFil: Wagermaier, Wolfgang. Institut Max Planck fuer Bioanorganische Chemie; AlemaniaFil: Ceré, Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; ArgentinaFil: Ballarre, Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; Argentin

Manipulation of atoms and molecules with the low temperature scanning tunneling microscope

The controlled manipulation with a scanning tunneling microscope (STM) down to the scale of small molecules and single atoms allows the buildup of molecular and atomic nanostructures. In the case of the lateral manipulation of adsorbed species, in which only tip/particle forces are used, three different manipulation modes (pushing, pulling, sliding) can be discerned. Vertical manipulation of Xe and CO is demonstrated, leading to the formation of functionalized tips, which can be used for improved imaging and even to perform vibrational spectroscopy on single molecules. Furthermore, we describe how we have reproduced a full chemical reaction with single molecules, whereby all basic steps, namely, preparation of the reactants, diffusion and association, are induced with the STM tip

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

Mental and Physical Well-Being and Burden in Palliative Care Nursing: A Cross-Setting Mixed-Methods Study

The working routines in palliative care nursing are associated with a variety of causes of stress with regional and setting-specific differences in Germany. This mixed-methods study aimed to investigate the mental and physical well-being among nurses in German palliative and hospice care and to gain a deeper understanding of procedural and structural aspects that may influence the mental and physical burden in palliative nursing care. The mixed-methods approach combined qualitative interviews, (n = 16) an online survey (n = 101), and subsequent data validation in a focus group (n = 6). Interview and focus group data were analysed using structured qualitative content analysis. Survey data were analysed using descriptive statistics and an explorative quantitative analysis. Moderate to very high levels of stress were reported across all settings, but were highest for nurses in specialized outpatient palliative care settings. Underlying causes of stress related to working conditions in the nursing profession across all palliative care settings were poor working hours, perceived inadequate remuneration, and high demands for documentation. To ensure sustainable high-quality palliative care, adaptations to working conditions that target causes of stress and burden in palliative care nurses are required.Article 6240191