Full-Field Calcium K-Edge X-ray Absorption Near-Edge Structure Spectroscopy on Cortical Bone at the Micron-Scale: Polarization Effects Reveal Mineral Orientation.

Here, we show results on X-ray absorption near edge structure spectroscopy in both transmission and X-ray fluorescence full-field mode (FF-XANES) at the calcium K-edge on human bone tissue in healthy and diseased conditions and for different tissue maturation stages. We observe that the dominating spectral differences originating from different tissue regions, which are well pronounced in the white line and postedge structures are associated with polarization effects. These polarization effects dominate the spectral variance and must be well understood and modeled before analyzing the very subtle spectral variations related to the bone tissue variations itself. However, these modulations in the fine structure of the spectra can potentially be of high interest to quantify orientations of the apatite crystals in highly structured tissue matrices such as bone. Due to the extremely short wavelengths of X-rays, FF-XANES overcomes the limited spatial resolution of other optical and spectroscopic techniques exploiting visible light. Since the field of view in FF-XANES is rather large the acquisition times for analyzing the same region are short compared to, for example, X-ray diffraction techniques. Our results on the angular absorption dependence were verified by both site-matched polarized Raman spectroscopy, which has been shown to be sensitive to the orientation of bone building blocks and by mathematical simulations of the angular absorbance dependence. As an outlook we further demonstrate the polarization based assessment of calcium-containing crystal orientation and specification of calcium in a beta-tricalcium phosphate (ß-Ca3(PO4)2 scaffold implanted into ovine bone. Regarding the use of XANES to assess chemical properties of Ca in human bone tissue our data suggest that neither the anatomical site (tibia vs jaw) nor pathology (healthy vs necrotic jaw bone tissue) affected the averaged spectral shape of the XANES spectra.

3D Raman mapping of the collagen fibril orientation in human osteonal lamellae.

Chemical composition and fibrillar organization are the major determinants of osteonal bone mechanics. However, prominent methodologies commonly applied to investigate mechanical properties of bone on the micro scale are usually not able to concurrently describe both factors. In this study, we used polarized Raman spectroscopy (PRS) to simultaneously analyze structural and chemical information of collagen fibrils in human osteonal bone in a single experiment. Specifically, the three-dimensional arrangement of collagen fibrils in osteonal lamellae was assessed. By analyzing the anisotropic intensity of the amide I Raman band of collagen as a function of the orientation of the incident laser polarization, different parameters related to the orientation of the collagen fibrils and the degree of alignment of the fibrils were derived. Based on the analysis of several osteons, two major fibrillar organization patterns were identified, one with a monotonic and another with a periodically changing twist direction. These results confirm earlier reported twisted and oscillating plywood arrangements, respectively. Furthermore, indicators of the degree of alignment suggested the presence of disordered collagen within the lamellar organization of the osteon. The results show the versatility of the analytical PRS approach and demonstrate its capability in providing not only compositional, but also 3D structural information in a complex hierarchically structured biological material. The concurrent assessment of chemical and structural features may contribute to a comprehensive characterization of the microstructure of bone and other collagen-based tissues.

STED nanoscopy with mass-produced laser diodes.

We show that far-field fluorescence nanoscopy by stimulated emission depletion (STED) can be realized with compact off-the-shelf laser diodes, such as those used in laser pointers and DVDs. A spatial resolution of 40-50 nm is attained by pulsing a 660 nm DVD-diode. The efficacy of these low-cost STED microscopes in biological imaging is demonstrated by differentiating between clusters of the synaptic protein bassoon and transport vesicles in hippocampal neurons, based on the feature diameter. Our results facilitate the implementation of this all-molecular-transition based superresolution method in many applications ranging from nanoscale fluorescence imaging to nanoscale fluorescence sensing.

Multimodal correlative investigation of the interplaying micro-architecture, chemical composition and mechanical properties of human cortical bone tissue reveals predominant role of fibrillar organization in determining microelastic tissue properties.

UNLABELLED: The mechanical competence of bone is crucially determined by its material composition and structural design. To investigate the interaction of the complex hierarchical architecture, the chemical composition and the resulting elastic properties of healthy femoral bone at the level of single bone lamellae and entire structural units, we combined polarized Raman spectroscopy (PRS), scanning acoustic microscopy (SAM) and synchrotron X-ray phase contrast nano tomography (SR-nanoCT). In line with earlier studies, mutual correlation analysis strongly suggested that the characteristic elastic modulations of bone lamellae within single units are the result of the twisting fibrillar orientation, rather than compositional variations, modulations of the mineral particle maturity, or mass density deviations. Furthermore, we show that predominant fibril orientations in entire tissue units can be rapidly assessed from Raman parameter maps. Coexisting twisted and oscillating fibril patterns were observed in all investigated tissue domains. Ultimately, our findings demonstrate in particular the potential of combined PRS and SAM measurements in providing multi-scalar analysis of correlated fundamental tissue properties. In future studies, the presented approach can be applied for non-destructive investigation of small pathologic samples from bone biopsies and a broad range of biological materials and tissues. STATEMENT OF SIGNIFICANCE: Bone is a complex structured composite material consisting of collagen fibrils and mineral particles. Various studies have shown that not only composition, maturation, and packing of its components, but also their structural arrangement determine the mechanical performance of the tissue. However, prominent methodologies are usually not able to concurrently describe these factors on the micron scale and complementary tissue characterization remains challenging. In this study we combine X-ray nanoCT, polarized Raman imaging and scanning acoustic microscopy and propose a protocol for fast and easy assessment of predominant fibril orientations in bone. Based on our site-matched analysis of cortical bone, we conclude that the elastic modulations of bone lamellae are mainly determined by the fibril arrangement.

Synchrotron X-ray phase nano-tomography-based analysis of the lacunar-canalicular network morphology and its relation to the strains experienced by osteocytes in situ as predicted by case-specific finite element analysis.

Osteocytes are hypothesized to regulate bone remodeling guided by both biological and mechanical stimuli. Morphology of the lacunar-canalicular network of osteocytes has been hypothesized to be strongly related to the level of mechanical loading and to various bone diseases. Finite element modeling could help to better understand the mechanosensation process by predicting the physiological strain environment. The aims of this study were to (i) quantify the lacunar-canalicular morphology in the cortex of the human femur; (ii) predict the in situ local deformations around and in osteocytes by means of case-specific finite element models; and (iii) investigate the potential relationship between morphology and deformations. Human femoral cortical bone samples were imaged using synchrotron X-ray phase nano-tomography with 50 nm voxel size. Rectangular volumes of interest were selected to contain single osteocyte lacunae and the surrounding matrix. Lacunar-canalicular morphology was quantified and the cell geometry was artificially reconstructed based on a priori assumptions. Finite element models of the volumes of interest were generated, containing the extracellular matrix, osteocyte and peri-cellular matrix, and subjected to uniaxial compression. The morphological analysis revealed that canalicular number was dictated by lacunar size, that the spacing of canaliculi fell within a narrow range, suggesting that these pores are well distributed throughout the bone matrix and indicated the trend that lacunae at the outer osteon boundary were less elongated than others. No apparent relationship was found between the morphological parameters and the predicted strains. The globally applied strain was amplified locally by factors up to 10 and up to 70 in the extracellular matrix and the in cells, respectively. Cell deformations were localized mainly at the body-dendrite junctions, with magnitudes reaching the in vitro stimulatory threshold reported for osteocytes.

Canalicular network morphology is the major determinant of the spatial distribution of mass density in human bone tissue: evidence by means of synchrotron radiation phase-contrast nano-CT.

In bone remodeling, maturation of the newly formed osteonal tissue is associated with a rapid primary increase followed by a slower secondary increase of mineralization. This requires supply and precipitation of mineral into the bone matrix. Mineral delivery can occur only from the extracellular fluid via interfaces such as the Haversian system and the osteocyte pore network. We hypothesized that in mineralization, mineral exchange is achieved by the diffusion of mineral from the lacunar-canalicular network (LCN) to the bone matrix, resulting in a gradual change in tissue mineralization with respect to the distance from the pore-matrix interface. We expected to observe alterations in the mass density distribution with tissue age. We further hypothesized that mineral exchange occurs not only at the lacunar but also at the canalicular boundaries. The aim of this study was, therefore, to investigate the spatial distribution of mass density in the perilacunar and pericanalicular bone matrix and to explore how these densities are influenced by tissue aging. This is achieved by analyzing human jawbone specimens originating from four healthy donors and four treated with high-dosage bisphosphonate using synchrotron radiation phase-contrast nano-CT with a 50-nm voxel size. Our results provide the first experimental evidence that mass density in the direct vicinity of both lacunae (p < 0.001) and canaliculi (p < 0.001) is different from the mean matrix mass density, resulting in gradients with respect to the distance from both pore-matrix interfaces, which diminish with increasing tissue age. Though limited by the sample size, these findings support our hypotheses. Moreover, the density gradients are more pronounced around the lacunae than around the canaliculi, which are explained by geometrical considerations in the LCN morphology. In addition, we speculate that mineral exchange occurs at all interfaces of the LCN, not only in mineralization but also in mineral homeostasis.

On the elastic properties of mineralized turkey leg tendon tissue: multiscale model and experiment.

The key parameters influencing the elastic properties of the mineralized turkey leg tendon (MTLT) were investigated. Two structurally different tissue types appearing in the MTLT were considered: circumferential and interstitial tissue. These differ in their amount of micropores and their average diameter of the mineralized collagen fibril bundles. A multiscale model representing the apparent elastic stiffness tensor of MTLT tissue was developed using the Mori-Tanaka and the self-consistent homogenization schemes. The volume fraction of mineral (hydroxyapatite) in the fibril bundle, [Formula: see text], and the tissue microporosity are the variables of the model. The MTLT model was analyzed performing a global sensitivity analysis (Elementary Effects method) and a parametric study. The stiffnesses parallel (axial) and perpendicular (transverse) to the MTLT long axis were the only significantly sensitive components of the apparent stiffness tensor of MTLT tissue. The most important parameters influencing these apparent stiffnesses are [Formula: see text], tissue microporosity, as well as shape and distribution of the minerals in the fibril bundle (intra- vs. interfibrillar). The predicted apparent stiffness was converted to acoustic impedance for model validation. From measurements on embedded MTLT samples, including 50- and 200-MHz scanning acoustic microscopy as well as synchrotron radiation micro-computed tomography, we obtained site-matched acoustic impedance and [Formula: see text] data of circumferential and interstitial tissue. The experimental and the model data compare very well for both tissue types (relative error 6-8 %).

Alterations of mass density and 3D osteocyte lacunar properties in bisphosphonate-related osteonecrotic human jaw bone, a synchrotron µCT study.

Osteonecrosis of the jaw, in association with bisphosphonates (BRONJ) used for treating osteoporosis or cancer, is a severe and most often irreversible side effect whose underlying pathophysiological mechanisms remain largely unknown. Osteocytes are involved in bone remodeling and mineralization where they orchestrate the delicate equilibrium between osteoclast and osteoblast activity and through the active process called osteocytic osteolysis. Here, we hypothesized that (i) changes of the mineralized tissue matrix play a substantial role in the pathogenesis of BRONJ, and (ii) the osteocyte lacunar morphology is altered in BRONJ. Synchrotron µCT with phase contrast is an appropriate tool for assessing both the 3D morphology of the osteocyte lacunae and the bone matrix mass density. Here, we used this technique to investigate the mass density distribution and 3D osteocyte lacunar properties at the sub-micrometer scale in human bone samples from the jaw, femur and tibia. First, we compared healthy human jaw bone to human tibia and femur in order to assess the specific differences and address potential explanations of why the jaw bone is exclusively targeted by the necrosis as a side effect of BP treatment. Second, we investigated the differences between BRONJ and control jaw bone samples to detect potential differences which could aid an improved understanding of the course of BRONJ. We found that the apparent mass density of jaw bone was significantly smaller compared to that of tibia, consistent with a higher bone turnover in the jaw bone. The variance of the lacunar volume distribution was significantly different depending on the anatomical site. The comparison between BRONJ and control jaw specimens revealed no significant increase in mineralization after BP. We found a significant decrease in osteocyte-lacunar density in the BRONJ group compared to the control jaw. Interestingly, the osteocyte-lacunar volume distribution was not altered after BP treatment.

Multiscale, converging defects of macro-porosity, microstructure and matrix mineralization impact long bone fragility in NF1.

Bone fragility due to osteopenia, osteoporosis or debilitating focal skeletal dysplasias is a frequent observation in the Mendelian disease Neurofibromatosis type 1 (NF1). To determine the mechanisms underlying bone fragility in NF1 we analyzed two conditional mouse models, Nf1Prx1 (limb knock-out) and Nf1Col1 (osteoblast specific knock-out), as well as cortical bone samples from individuals with NF1. We examined mouse bone tissue with micro-computed tomography, qualitative and quantitative histology, mechanical tensile analysis, small-angle X-ray scattering (SAXS), energy dispersive X-ray spectroscopy (EDX), and scanning acoustic microscopy (SAM). In cortical bone of Nf1Prx1 mice we detected ectopic blood vessels that were associated with diaphyseal mineralization defects. Defective mineral binding in the proximity of blood vessels was most likely due to impaired bone collagen formation, as these areas were completely devoid of acidic matrix proteins and contained thin collagen fibers. Additionally, we found significantly reduced mechanical strength of the bone material, which was partially caused by increased osteocyte volume. Consistent with these observations, bone samples from individuals with NF1 and tibial dysplasia showed increased osteocyte lacuna volume. Reduced mechanical properties were associated with diminished matrix stiffness, as determined by SAM. In line with these observations, bone tissue from individuals with NF1 and tibial dysplasia showed heterogeneous mineralization and reduced collagen fiber thickness and packaging. Collectively, the data indicate that bone fragility in NF1 tibial dysplasia is partly due to an increased osteocyte-related micro-porosity, hypomineralization, a generalized defect of organic matrix formation, exacerbated in the regions of tensional and bending force integration, and finally persistence of ectopic blood vessels associated with localized macro-porotic bone lesions.