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

Fragility of bone material controlled by internal interfaces

Bone material is built in a complex multiscale arrangement of mineralized collagen fibrils containing water, proteoglycans and some noncollagenous proteins. This organization is not static as bone is constantly remodeled and thus able to repair damaged tissue and adapt to the loading situation. In preventing fractures, the most important mechanical property is toughness, which is the ability to absorb impact energy without reaching complete failure. There is no simple explanation for the origin of the toughness of bone material, and this property depends in a complex way on the internal architecture of the material on all scales from nanometers to millimeters. Hence, fragility may have different mechanical origins, depending on which toughening mechanism is not working properly. This article reviews the toughening mechanisms described for bone material and attempts to put them in a clinical context, with the hope that future analysis of bone fragility may be guided by this collection of possible mechanistic origins

Understanding hierarchy and functions of bone using scanning x-ray scattering methods

International audienceBiological materials are often hierarchically structured from the nanometer to the macroscopic scale. Specific characterization methods are needed to characterize the structures at these different length scales. This chapter reviews -based on the example of bone- the use of X-ray scattering methods to explore representative and quantitative structure information as well as structure-function relations in hierarchically structured biological materials. X-ray scattering techniques are particularly well suited for the characterization of the form and organization of organic and inorganic components in those materials. When nanometer-sized structures are exposed to X-rays, details of the internal material structure can be revealed by the analysis of the resulting interference patterns. Fundamental aspects of wide and small angle X-ray scattering (WAXS and SAXS) are discussed with specific focus on bone studies. An important field of research using X-ray scattering techniques, is the in situ combination with mechanical testing, which allows investigating changes in structure under specific loading conditions. Another common application is the structural study of heterogeneities or local structures within a sample using a narrow focused X-ray beam. Furthermore, in scanning mode, where the specimen is displaced step by step across a microbeam while collecting a SAXS/WAXS pattern at each step, complex structural maps of the sample can be derived. A natural extension of the method toward imaging is described in the context of X-ray imaging with scattering contrast

Springback effect and structural features during the drying of silica aerogels tracked by in-situ synchrotron X-ray scattering

The springback effect during ambient pressure drying of aerogels is an interesting structural phenomenon, consisting of a severe shrinkage followed by almost complete re-expansion. The drying of gels causes shrinkage, whereas re-expansion is believed to be linked to repelling forces on the nanoscale. A multi-scale structural characterization of this significant volume change is key in controlling aerogel processing and properties. In this work, hydrophobic, monolithic silica aerogels with high specific surface areas were synthesized by modification with trimethylchlorosilane and ambient pressure drying. Here, we report a multi-method approach focusing on in-situ X-ray scattering to observe alterations of the nanostructured material during the drying of surface-modified and unmodified silica gels. Both show a porous fractal nanostructure, which partially collapses during drying and only recovers in surface-modified samples during the springback effect. Distinct changes of the X-ray scattering data were reproducibly associated with the shrinkage, re-expansion and drying of the gel network. Our findings may contribute to tailor aerogels with specific functionality, as the springback effect has a direct influence on properties (e.g., porosity, pore size distribution), which is directly affected by the degree of re-expansion

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

Breast cancer–secreted factors perturb murine bone growth in regions prone to metastasis

Breast cancer frequently metastasizes to bone, causing osteolytic lesions. However, how factors secreted by primary tumors affect the bone microenvironment before the osteolytic phase of metastatic tumor growth remains unclear. Understanding these changes is critical as they may regulate metastatic dissemination and progression. To mimic premetastatic bone adaptation, immunocompromised mice were injected with MDA-MB-231–conditioned medium [tumor-conditioned media (TCM)]. Subsequently, the bones of these mice were subjected to multiscale, correlative analysis including RNA sequencing, histology, micro–computed tomography, x-ray scattering analysis, and Raman imaging. In contrast to overt metastasis causing osteolysis, TCM treatment induced new bone formation that was characterized by increased mineral apposition rate relative to control bones, altered bone quality with less matrix and more carbonate substitution, and the deposition of disoriented mineral near the growth plate. Our study suggests that breast cancer–secreted factors may promote perturbed bone growth before metastasis, which could affect initial seeding of tumor cells

A 3D network of nanochannels for possible ion and molecule transit in mineralizing bone and cartilage

During crucial growth stages of vertebrate long bones, calcified cartilage beneath the growth plate is anchored to bone by a third mineralized component, the cement line. Proper skeletal development is contingent on the interplay of these three constituents, yet their mineralization processes and structural interactions are incompletely understood, in part from limited knowledge of their meso- and nanoscale features. Herein, focused ion beam-scanning electron microscopy (FIB-SEM) with serial surface imaging is applied to examine the cartilage–bone interface of mouse femoral heads at an unprecedented scale: FIB-SEM provides 3D, nanometer resolution of structural details for volumes encompassing metaphyseal calcified cartilage, bone, and the intervening cement line. A novel and complex structural network is revealed, comprising densely packed nanochannels smaller than bone canaliculi (≈10–50 nm diameter) within the calcified cartilage and bone extracellular matrices, but absent in the cement line. A structural correlation is demonstrated between the nanochannels and ellipsoidal mineral domains, which appear to coalesce during mineralization in a process analogous to powder sintering in metallurgy. A mineralization process is proposed, supported by energy-dispersive X-Ray spectroscopy of nanochannel contents, in which these unreported structures offer ion and molecule conduits to access the extracellular matrices of calcified cartilage and bone

Structure-function relationships at the human spinal disc-vertebra interface.

Damage at the intervertebral disc-vertebra interface associates with back pain and disc herniation. However, the structural and biomechanical properties of the disc-vertebra interface remain underexplored. We sought to measure mechanical properties and failure mechanisms, quantify architectural features, and assess structure-function relationships at this vulnerable location. Vertebra-disc-vertebra specimens from human cadaver thoracic spines were scanned with micro-computed tomography (μCT), surface speckle-coated, and loaded to failure in uniaxial tension. Digital image correlation (DIC) was used to calculate local surface strains. Failure surfaces were scanned using scanning electron microscopy (SEM), and adjacent sagittal slices were analyzed with histology and SEM. Seventy-one percent of specimens failed initially at the cartilage endplate-bone interface of the inner annulus region. Histology and SEM both indicated a lack of structural integration between the cartilage endplate (CEP) and bone. The interface failure strength was increased in samples with higher trabecular bone volume fraction in the vertebral endplates. Furthermore, failure strength decreased with degeneration, and in discs with thicker CEPs. Our findings indicate that poor structural connectivity between the CEP and vertebra may explain the structural weakness at this region, and provide insight into structural features that may contribute to risk for disc-vertebra interface injury. The disc-vertebra interface is the site of failure in the majority of herniation injuries. Here we show new structure-function relationships at this interface that may motivate the development of diagnostics, prevention strategies, and treatments to improve the prognosis for many low back pain patients with disc-vertebra interface injuries. © 2017 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 36:192-201, 2018