Interplay between mineral crystallinity and mineral accumulation in health and postmenopausal osteoporosis.

Osteoporosis is characterized by an imbalance between bone formation and resorption rates, resulting in bone loss. For ethical reasons, effects of antiosteoporosis drugs are compared against patients receiving vitamin D and calcium supplementation which is a mild antiresorptive regimen. Bone formation may be resolved into two phases: the initial formation of mineral crystals (primary nucleation) and the subsequent mineral accumulation (secondary nucleation and mineral growth) on them. In this study, we used Raman microspectroscopic analysis of iliac crest biopsies from healthy females (N = 108), postmenopausal osteoporosis patients receiving vitamin D and calcium supplementation (PMOP-S; N = 66), and treatment-naïve postmenopausal osteoporosis patients (PMOP-TN; N = 12) to test the hypothesis that at forming trabecular surfaces, mineral maturity / crystallinity of the youngest crystallites associates with the amount of subsequent mineral accumulation. The surfaces of analysis were chosen based on the presence of fluorescent double labels, defining three distinct tissue ages. The results indicated that when adjusted for age and tissue age, there were no differences in amount of mineral formed between healthy females and either PMOP-S or PMOP-TN, while both PMOP-S and PMOP-TN had larger crystallites compared to healthy females. Moreover, significant differences existed between PMOP-S and PMOP-TN in size of initial crystals formed as well as rate of mineral accumulation and maturation. These findings suggest an additional mechanism that may contribute to the decreased mineral content evident in PMOP, and provide a potential target for the development of new interventions. STATEMENT OF SIGNIFICANCE: We used Raman microspectroscopic analysis of iliac crest biopsies from healthy females and postmenopausal osteoporosis patients (PMOP) receiving placebo to test the hypothesis that at forming trabecular surfaces, mineral maturity / crystallinity (MMC) of the youngest crystallites associates with the amount of subsequent mineral accumulation. This can affect bone mechanical properties as larger crystallites have been shown to result in compromised mechanical attributes; and larger crystallites grow slower compared to smaller ones. The results of the present analysis indicate that increased MMC of the youngest formed mineral may contribute to the bone mineral loss evident in PMOP and the accompanying increased fracture risk independently of bone turnover rate.

Human and mouse bones physiologically integrate in a humanized mouse model while maintaining species-specific ultrastructure.

Humanized mouse models are increasingly studied to recapitulate human-like bone physiology. While human and mouse bone architectures differ in multiple scales, the extent to which chimeric human-mouse bone physiologically interacts and structurally integrates remains unknown. Here, we identify that humanized bone is formed by a mosaic of human and mouse collagen, structurally integrated within the same bone organ, as shown by immunohistochemistry. Combining this with materials science techniques, we investigate the extracellular matrix of specific human and mouse collagen regions. We show that human-like osteocyte lacunar-canalicular network is retained within human collagen regions and is distinct to that of mouse tissue. This multiscale analysis shows that human and mouse tissues physiologically integrate into a single, functional bone tissue while maintaining their species-specific ultrastructural differences. These results offer an original method to validate and advance tissue-engineered human-like bone in chimeric animal models, which grow to be eloquent tools in biomedical research.

Network architecture strongly influences the fluid flow pattern through the lacunocanalicular network in human osteons.

A popular hypothesis explains the mechanosensitivity of bone due to osteocytes sensing the load-induced flow of interstitial fluid squeezed through the lacunocanalicular network (LCN). However, the way in which the intricate structure of the LCN influences fluid flow through the network is largely unexplored. We therefore aimed to quantify fluid flow through real LCNs from human osteons using a combination of experimental and computational techniques. Bone samples were stained with rhodamine to image the LCN with 3D confocal microscopy. Image analysis was then performed to convert image stacks into mathematical network structures, in order to estimate the intrinsic permeability of the osteons as well as the load-induced fluid flow using hydraulic circuit theory. Fluid flow was studied in both ordinary osteons with a rather homogeneous LCN as well as a frequent subtype of osteons-so-called osteon-in-osteons-which are characterized by a ring-like zone of low network connectivity between the inner and the outer parts of these osteons. We analyzed 8 ordinary osteons and 9 osteon-in-osteons from the femur midshaft of a 57-year-old woman without any known disease. While the intrinsic permeability was 2.7 times smaller in osteon-in-osteons compared to ordinary osteons, the load-induced fluid velocity was 2.3 times higher. This increased fluid velocity in osteon-in-osteons can be explained by the longer path length, needed to cross the osteon from the cement line to the Haversian canal, including more fluid-filled lacunae and canaliculi. This explanation was corroborated by the observation that a purely structural parameter-the mean path length to the Haversian canal-is an excellent predictor for the average fluid flow velocity. We conclude that osteon-in-osteons may be particularly significant contributors to the mechanosensitivity of cortical bone, due to the higher fluid flow in this type of osteons.

Surface tension determines tissue shape and growth kinetics.

The collective self-organization of cells into three-dimensional structures can give rise to emergent physical properties such as fluid behavior. Here, we demonstrate that tissues growing on curved surfaces develop shapes with outer boundaries of constant mean curvature, similar to the energy minimizing forms of liquids wetting a surface. The amount of tissue formed depends on the shape of the substrate, with more tissue being deposited on highly concave surfaces, indicating a mechano-biological feedback mechanism. Inhibiting cell-contractility further revealed that active cellular forces are essential for generating sufficient surface stresses for the liquid-like behavior and growth of the tissue. This suggests that the mechanical signaling between cells and their physical environment, along with the continuous reorganization of cells and matrix is a key principle for the emergence of tissue shape.

Multiscale analyses reveal native-like lamellar bone repair and near perfect bone-contact with porous strontium-loaded bioactive glass.

The efficient healing of critical-sized bone defects using synthetic biomaterial-based strategies is promising but remains challenging as it requires the development of biomaterials that combine a 3D porous architecture and a robust biological activity. Bioactive glasses (BGs) are attractive candidates as they stimulate a biological response that favors osteogenesis and vascularization, but amorphous 3D porous BGs are difficult to produce because conventional compositions crystallize during processing. Here, we rationally designed a porous, strontium-releasing, bioactive glass-based scaffold (pSrBG) whose composition was tailored to deliver strontium and whose properties were optimized to retain an amorphous phase, induce tissue infiltration and encourage bone formation. The hypothesis was that it would allow the repair of a critical-sized defect in an ovine model with newly-formed bone exhibiting physiological matrix composition and structural architecture. Histological and histomorphometric analyses combined with indentation testing showed pSrBG encouraged near perfect bone-to-material contact and the formation of well-organized lamellar bone. Analysis of bone quality by a combination of Raman spectral imaging, small-angle X-ray scattering, X-ray fluorescence and focused ion beam-scanning electron microscopy demonstrated that the repaired tissue was akin to that of normal, healthy bone, and incorporated small amounts of strontium in the newly formed bone mineral. These data show the potential of pSrBG to induce an efficient repair of critical-sized bone defects and establish the importance of thorough multi-scale characterization in assessing biomaterial outcomes in large animal models.

The contribution of the pericanalicular matrix to mineral content in human osteonal bone.

The osteocyte lacunar-canalicular network (LCN) penetrates bone and houses the osteocytes and their processes. Despite its rather low volume fraction, the LCN represents an outstanding large surface that is possibly used by the osteocytes to interact with the surrounding mineralized bone matrix thereby contributing to mineral homeostasis. The aim of this study was to quantitatively describe such contributions by spatially correlating the local density of the LCN with the mineral content at the same location in micrometer-sized volume elements in human osteons. For this purpose, 65 osteons from the femur midshaft from healthy adults (n = 4) and children (n = 2) were structurally characterized with two different techniques. The 3D structure of the LCN in the osteons was imaged with confocal laser scanning microscopy after staining the bone samples with rhodamine. Subsequent image analysis provided the canalicular length density, i.e. the total length of the canaliculi per unit volume (µm/µm3). Quantitative information on the mineral content (wt%Ca) from the identical regions was obtained using quantitative backscattered electron imaging. As the LCN-porosity lowers the mineral content, a negative correlation between Ca content and network density was expected. Calculations predict a reduction of around -0.97 fmol Ca per µm of network. However, the experiment revealed for 62 out of 65 osteons a positive correlation resulting in an average additional Ca loading of +1.15 fmol per µm of canalicular network, i.e. an accumulation of mineral has occurred at dense network regions. We hypothesize that this accumulation happens in the close vicinity of canaliculi forming mineral reservoirs that can be utilized by osteocytes. Significant differences found between individuals indicate that the extent of mineral loading of the reservoir zone reflects an important parameter for mineral homeostasis.

Breaking the long-standing morphological paradigm: Individual prisms in the pearl oyster shell grow perpendicular to the c-axis of calcite.

Cross-sections of calcitic prismatic layers in mollusk shells, cut perpendicular to growth direction, reveal well-defined polygonal shapes of individual "grains" clearly visible by light and electron microscopy. For several kinds of shells, it was shown that the average number of edges in an individual prism approaches six during the growth process. Taking into account the rhombohedral symmetry of calcite, often presented in hexagonal axes, all this led to the long-standing opinion that calcitic prisms grow along the c-axis of calcite. In this paper, using X-ray diffraction and electron backscatter diffraction (EBSD), we unambiguously show that calcitic prisms in pearl oyster Pinctada margaritifera predominantly grow perpendicular to the c-axis. The obtained results imply that the hexagon-like habitus of growing crystallites may be not necessarily connected to calcite crystallography and, therefore, other factors should be taken into consideration. We analyze this phenomenon by comparing the organic contents in Pinctada margaritifera and Pinna nobilis shells, the later revealing regular growth of calcitic prisms along the c-axis.

Effect of collagen packing and moisture content on leather stiffness.

Applications for skin derived collagen materials, such as leather and acellular dermal matrices, usually require both strength and flexibility. In general, both the tensile modulus (which has an impact on flexibility) and strength are known to increase with fiber alignment, in the tensile direction, for practically all collagen-based tissues. The structural basis for flexibility in leather was investigated and the moisture content was varied. Small angle X-ray scattering was used to determine collagen fibril orientation, elongation and lateral intermolecular spacing in leather conditioned by different controlled humidity environments. Flexibility was measured by a three point bending test. Leather was prepared by tanning under biaxial loading to create leather with increased fibril alignment and thus strength, but this treatment also increased the stiffness. As collagen aligns, it not only strengthens the material but it also stiffens because tensile loading is then applied along the covalent chain of the collagen molecules, rather than at an angle to it. Here it has been shown that with higher moisture content greater flexibility of the material develops as water absorption inside collagen fibrils produces a larger lateral spacing between collagen molecules. It is suggested that water provides a lubricating effect in collagen fibrils, enabling greater freedom of movement and therefore greater flexibility. When collagen molecules align in the strain direction during tanning, leather stiffens not only by the fiber alignment itself but also because collagen molecules pack closer together, reducing the ability of the molecules to move relative to each other.

Cortical bone properties in the Brtl/+ mouse model of Osteogenesis imperfecta as evidenced by acoustic transmission microscopy.

Higher skeletal fragility has been established for the Brtl/+ mouse model of osteogenesis imperfecta at the whole bone level, but previous investigations of mechanical properties at the bone material level were inconclusive. Bone material was analyzed separately at endosteal (ER) and periosteal regions (PR) on transverse femoral midshaft sections for 2-month old mice (wild-type n = 6; Brtl/+ n = 6). Quantitative backscattered electron imaging revealed that the mass density computed from mineral density maps was higher in PR than in ER for both wild-type (+2.1%, p < 0.05) and Brtl/+ mice (+1.8%, p < 0.05). Electron induced X-ray fluorescence analysis indicated significantly lower atomic Ca/P ratios and higher Na/Ca, Mg/Ca and K/Ca ratios in PR bone compared to ER independently of genotype. Second harmonic generation microscopy indicated that the occurrence of periodically alternating collagen orientation in ER of Brtl/+ mice was strongly reduced compared to wild-type mice. Scanning acoustic microscopy in time of flight mode revealed that the sound velocity and Young's modulus (estimated based on sound velocity and mass density maps) were significantly greater in PR (respectively +6% and +15%) compared to ER in wild-type mice but not in Brtl/+ mice. ER sound velocity and Young's modulus were significantly increased in Brtl/+ mice (+9.4% and +22%, respectively) compared to wild-type mice. These data demonstrate that the Col1a1 G349C mutation in Brtl/+ mice affects the mechanical behavior of bone material predominantly in the endosteal region by altering the collagen orientation.

Data on collagen structures in leather with varying moisture contents from small angle X-ray scattering and three point bend testing.

The data presented in this article are related to the research article entitled "Effect of collagen packing and moisture content on leather stiffness" (Kelly et al., 2018). This article describes how moisture content affects collagen packing and leather stiffness. Structural changes were experimentally introduced into ovine leather through biaxial strain during tanning (׳stretch tanning׳). Leather samples produced normally without strain (׳non-stretch tanned׳) and those produced by stretch tanning, were conditioned in a range of relative humidity environments and then analysed by small angle X-ray scattering and three point bend testing. The collagen D-spacing, lateral intermolecular spacing and flexural properties were measured under these varying moisture contents.

The three-dimensional arrangement of the mineralized collagen fibers in elephant ivory and its relation to mechanical and optical properties.

Elephant tusks are composed of dentin or ivory, a hierarchical and composite biological material made of mineralized collagen fibers (MCF). The specific arrangement of the MCF is believed to be responsible for the optical and mechanical properties of the tusks. Especially the MCF organization likely contributes to the formation of the bright and dark checkerboard pattern observed on polished sections of tusks (Schreger pattern). Yet, the precise structural origin of this optical motif is still controversial. We hereby address this issue using complementary analytical methods (small and wide angle X-ray scattering, cross-polarized light microscopy and scanning electron microscopy) on elephant ivory samples and show that MCF orientation in ivory varies from the outer to the inner part of the tusk. An external cohesive layer of MCF with fiber direction perpendicular to the tusk axis wraps the mid-dentin region, where the MCF are oriented mainly along the tusk axis and arranged in a plywood-like structure with fiber orientations oscillating in a narrow angular range. This particular oscillating-plywood structure of the MCF and the birefringent properties of the collagen fibers, likely contribute to the emergence of the Schreger pattern, one of the most intriguing macroscopic optical patterns observed in mineralized tissues and of great importance for authentication issues in archeology and forensic sciences. STATEMENT OF SIGNIFICANCE: Elephant tusks are intriguing biological materials as they are composed of dentin (ivory) like teeth but have mineralized collagen fibers (MCF) similarly arranged to the ones of lamellar bones and function as bones or antlers. Here, we showed that ivory has a graded structure with varying MCF orientations and that MCF of the mid-dentin are arranged in plywood like layers with fiber orientations oscillating in a narrow angular range around the tusk axis. This organization of the MCF may contribute to ivory's mechanical properties and, together with the collagen fibers birefringence properties, strongly relates to its optical properties, i.e. the emergence of a macroscopic checkerboard pattern, well known as the Schreger pattern.

Crack driving force in twisted plywood structures.

Twisted plywood architectures can be observed in many biological materials with high fracture toughness, such as in arthropod cuticles or in lamellar bone. Main purpose of this paper is to analyze the influence of the progressive rotation of the fiber direction on the spatial variation of the crack driving force and, thus, on the fracture toughness of plywood-like structures. The theory of fiber composites is used to describe the stiffness matrix of a twisted plywood structure in a specimen-fixed coordinate system. The driving force acting on a crack propagating orthogonally to the fiber-rotation plane is studied by methods of computational mechanics, coupled with the concept of configurational forces. The analysis unfolds a spatial variation of the crack driving force with minima that are beneficial for the fracture toughness of the material. It is shown that the estimation of the crack driving force can be simplified by replacing the complicated anisotropic twisted plywood structure by an isotropic material with appropriate periodic variations of Young's modulus, which can be constructed based either on the local stiffness or local strain energy density variations. As practical example, the concepts are discussed for a specimen with a stiffness anisotropy similar to lamellar bone. STATEMENT OF SIGNIFICANCE: Twisted plywood-like structures exist in many natural fiber composites, such as bone or insect carapaces, and are known to be very fracture resistant. The crack driving force in such materials is analyzed quantitatively for the first time, using the concept of configurational forces. This tool, well established in the mechanics of materials, is introduced to the modeling of biological material systems with inhomogeneous and anisotropic material behavior. Based on this analysis, it is shown that the system can be approximated by an appropriately chosen inhomogeneous but isotropic material for the calculation of the crack driving force. The spatial variation of the crack driving force and, especially, its local minima are essential to describe the fracture properties of twisted plywood structures.

Mechanical behavior of idealized, stingray-skeleton-inspired tiled composites as a function of geometry and material properties.

Tilings are constructs of repeated shapes covering a surface, common in both manmade and natural structures, but in particular are a defining characteristic of shark and ray skeletons. In these fishes, cartilaginous skeletal elements are wrapped in a surface tessellation, comprised of polygonal mineralized tiles linked by flexible joints, an arrangement believed to provide both stiffness and flexibility. The aim of this research is to use two-dimensional analytical models to evaluate the mechanical performance of stingray skeleton-inspired tessellations, as a function of their material and structural parameters. To calculate the effective modulus of modeled composites, we subdivided tiles and their surrounding joint material into simple shapes, for which mechanical properties (i.e. effective modulus) could be estimated using a modification of traditional Rule of Mixtures equations, that either assume uniform strain (Voigt) or uniform stress (Reuss) across a loaded composite material. The properties of joints (thickness, Young's modulus) and tiles (shape, area and Young's modulus) were then altered, and the effects of these tessellation parameters on the effective modulus of whole tessellations were observed. We show that for all examined tile shapes (triangle, square and hexagon) composite stiffness increased as the width of the joints was decreased and/or the stiffness of the tiles was increased; this supports hypotheses that the narrow joints and high tile to joint stiffness ratio in shark and ray cartilage optimize composite tissue stiffness. Our models also indicate that, for simple, uniaxial loading, square tessellations are least sensitive and hexagon tessellations most sensitive to changes in model parameters, indicating that hexagon tessellations are the most "tunable" to specific mechanical properties. Our models provide useful estimates for the tensile and compressive properties of 2d tiled composites under uniaxial loading. These results lay groundwork for future studies into more complex (e.g. biological) loading scenarios and three dimensional structural parameters of biological tilings, while also providing insight into the mechanical roles of tessellations in general and improving the design of bioinspired materials.

BMP delivery complements the guiding effect of scaffold architecture without altering bone microstructure in critical-sized long bone defects: A multiscale analysis.

Scaffold architecture guides bone formation. However, in critical-sized long bone defects additional BMP-mediated osteogenic stimulation is needed to form clinically relevant volumes of new bone. The hierarchical structure of bone determines its mechanical properties. Yet, the micro- and nanostructure of BMP-mediated fast-forming bone has not been compared with slower regenerating bone without BMP. We investigated the combined effects of scaffold architecture (physical cue) and BMP stimulation (biological cue) on bone regeneration. It was hypothesized that a structured scaffold directs tissue organization through structural guidance and load transfer, while BMP stimulation accelerates bone formation without altering the microstructure at different length scales. BMP-loaded medical grade polycaprolactone-tricalcium phosphate scaffolds were implanted in 30mm tibial defects in sheep. BMP-mediated bone formation after 3 and 12 months was compared with slower bone formation with a scaffold alone after 12 months. A multiscale analysis based on microcomputed tomography, histology, polarized light microscopy, backscattered electron microscopy, small angle X-ray scattering and nanoindentation was used to characterize bone volume, collagen fiber orientation, mineral particle thickness and orientation, and local mechanical properties. Despite different observed kinetics in bone formation, similar structural properties on a microscopic and sub-micron level seem to emerge in both BMP-treated and scaffold only groups. The guiding effect of the scaffold architecture is illustrated through structural differences in bone across different regions. In the vicinity of the scaffold increased tissue organization is observed at 3 months. Loading along the long bone axis transferred through the scaffold defines bone micro- and nanostructure after 12 months.

Pediatric reference Raman data for material characteristics of iliac trabecular bone.

Bone material characteristics are important contributors in the determination of bone strength. Raman spectroscopic analysis provides information on mineral/matrix ratio, mineral maturity/crystallinity, relative pyridinoline (Pyd) collagen cross-link content, relative proteoglycan content and relative lipid content. However, published reference data are available only for adults. The purpose of the present study was to establish reference data of Raman outcomes pertaining to bone quality in trabecular bone for children and young adults. To this end, tissue age defined Raman microspectroscopic analysis was performed on bone samples from 54 individuals between 1.5 and 23 years with no metabolic bone disease, which have been previously used to establish histomorphometric and bone mineralization density distribution reference values. Four distinct tissue ages, three well defined by the fluorescent double labels representing early stages of bone formation and tissue maturation (days 3, 12, 20 of tissue mineralization) and a fourth representing old mature tissue at the geometrical center of the trabeculae, were analyzed. In general, significant dependencies of the measured parameters on tissue age were found, while at any given tissue age, sex and subject age were not confounders. Specifically, mineral/matrix ratio, mineral maturity/crystallinity index and relative pyridinoline collagen cross-link content index increased by 485%, 20% and 14%, respectively between days 3 and 20. The relative proteoglycan content index was unchanged between days 3 and 20 but was elevated in the old tissue compared to young tissue by 121%. The relative lipid content decreased within days 3 to 20 by -22%. Thus, the method allows not only the monitoring of material characteristics at a specific tissue age but also the kinetics of tissue maturation as well. The established reference Raman database will serve as sensitive tool to diagnose disturbances in material characteristics of pediatric bone biopsy samples.

Relationship of bone mineralization density distribution (BMDD) in cortical and cancellous bone within the iliac crest of healthy premenopausal women.

Bone mineralization density distribution (BMDD) is an important determinant of bone mechanical properties. The most available skeletal site for access to the BMDD is the iliac crest. Compared to cancellous bone much less information on BMDD is available for cortical bone. Hence, we analyzed complete transiliac crest bone biopsy samples from premenopausal women (n = 73) aged 25-48 years, clinically classified as healthy, by quantitative backscattered electron imaging for cortical (Ct.) and cancellous (Cn.) BMDD. The Ct.BMDD was characterized by the arithmetic mean of the BMDD of the cortical plates. We found correlations between Ct. and Cn. BMDD variables with correlation coefficients r between 0.42 and 0.73 (all p < 0.001). Additionally to this synchronous behavior of cortical and cancellous compartments, we found that the heterogeneity of mineralization densities (Ct.Ca(Width)), as well as the cortical porosity (Ct.Po) was larger for a lower average degree of mineralization (Ct.Ca(Mean)). Moreover, Ct.Po correlated negatively with the percentage of highly mineralized bone areas (Ct.Ca(High)) and positively with the percentage of lowly mineralized bone areas (Ct.Ca(Low)). In conclusion, the correlation of cortical with cancellous BMDD in the iliac crest of the study cohort suggests coordinated regulation of bone turnover between both bone compartments. Only in a few cases, there was a difference in the degree of mineralization of >1wt % between both cortices suggesting a possible modeling situation. This normative dataset of healthy premenopausal women will provide a reference standard by which disease- and treatment-specific effects can be assessed at the level of cortical bone BMDD.

Simultaneous Raman microspectroscopy and fluorescence imaging of bone mineralization in living zebrafish larvae.

Confocal Raman microspectroscopy and fluorescence imaging are two well-established methods providing functional insight into the extracellular matrix and into living cells and tissues, respectively, down to single molecule detection. In living tissues, however, cells and extracellular matrix coexist and interact. To acquire information on this cell-matrix interaction, we developed a technique for colocalized, correlative multispectral tissue analysis by implementing high-sensitivity, wide-field fluorescence imaging on a confocal Raman microscope. As a proof of principle, we study early stages of bone formation in the zebrafish (Danio rerio) larvae because the zebrafish has emerged as a model organism to study vertebrate development. The newly formed bones were stained using a calcium fluorescent marker and the maturation process was imaged and chemically characterized in vivo. Results obtained from early stages of mineral deposition in the zebrafish fin bone unequivocally show the presence of hydrogen phosphate containing mineral phases in addition to the carbonated apatite mineral. The approach developed here opens significant opportunities in molecular imaging of metabolic activities, intracellular sensing, and trafficking as well as in vivo exploration of cell-tissue interfaces under (patho-)physiological conditions.

The role of angular reflection in assessing elastic properties of bone by scanning acoustic microscopy.

For an assessment of the mechanical performance of bone, a quantitative description of its mechanical heterogeneity is necessary. Previously, scanning acoustic microscopy (SAM) was used as a non-destructive method to estimate bone stiffness on the micrometer scale. While up to now only the normal incidence of acoustic waves is taken into account, we extend in our study the evaluation procedure by considering the full opening of the acoustic lens. The importance of this technical aspect is demonstrated by determining the contrast in Young's modulus between newly formed osteons and the surrounding higher mineralized interstitial bone. Several regions of human cortical bone of a femur in cross-section were imaged. For all the regions quantitative backscattered-electron imaging (qBEI) to estimate the local mass density was combined with SAM measurements. These measurements reveal a non-monotonic dependence between acoustic reflectivity and Young's modulus, which shows that it is actually necessary to consider the lens opening in a quantitative way. This problem was experimentally and theoretically approached by using lenses with two different opening angles operated at different frequencies (52° at 400MHz and 80° at 820MHz) to image the same specimen. The mass density of bone in osteons was found to be 1930kg/m(3) on average, while the higher mineral content in interstitial bone results in a 9% increase of the density. The contrast in the effective Young's modulus E, as determined through SAM, is more pronounced, with an average value of 14GPa in osteons and a more than 60% increase in interstitial bone. Additionally, SAM maps show oscillations in E with a periodicity of the typical bone lamella thickness of approximately 7µm in both osteons and interstitial bone. This mechanical heterogeneity can be explained by the varying orientation of the mineralized collagen fibers.

Osteoclasts on bone and dentin in vitro: mechanism of trail formation and comparison of resorption behavior.

The main function of osteoclasts in vivo is the resorption of bone matrix, leaving behind typical resorption traces consisting of pits and trails. The mechanism of pit formation is well described, but less is known about trail formation. Pit-forming osteoclasts possess round actin rings. In this study we show that trail-forming osteoclasts have crescent-shaped actin rings and provide a model that describes the detailed mechanism. To generate a trail, the actin ring of the resorption organelle attaches with one side outside the existing trail margin. The other side of the ring attaches to the wall inside the trail, thus sealing that narrow part to be resorbed next (3­21 lm). This 3D configuration allows vertical resorption layer-by-layer from the surface to a depth in combination with horizontal cell movement. Thus, trails are not just traces of a horizontal translation of osteoclasts during resorption. Additionally, we compared osteoclastic resorption on bone and dentin since the latter is the most frequently used in vitro model and data are extrapolated to bone. Histomorphometric analyses revealed a material-dependent effect reflected by an 11-fold higher resorption area and a sevenfold higher number of pits per square centimeter on dentin compared to bone. An important material-independent aspect was reflected by comparable mean pit area (µm²) and podosome patterns. Hence, dentin promotes the generation of resorbing osteoclasts, but once resorption has started, it proceeds independently of material properties. Thus, dentin is a suitable model substrate for data acquisition as long as osteoclast generation is not part of the analyses.

Quantifying degradation of collagen in ancient manuscripts: the case of the Dead Sea Temple Scroll.

Since their discovery in the late 1940s, the Dead Sea Scrolls, some 900 ancient Jewish texts, have never stopped attracting the attention of scholars and the broad public alike, because they were created towards the end of the Second Temple period and the "time of Christ". Most of the work on them has been dedicated to the information contained in the scrolls' text, leaving physical aspects of the writing materials unexamined. They are, however, crucial for both historical insight and preservation of the scrolls. Although scientific analysis requires handling, it is essential to establish the state of degradation of these valued documents. Polarized Raman Spectroscopy (PRS) is a powerful tool for obtaining information on both the composition and the level of disorder of molecular units. In this study, we developed a non-invasive and non-destructive methodology that allows a quantification of the disorder (that can be related to the degradation) of protein molecular units in collagen fibers. Not restricted to collagen, this method can be applied also to other protein-based fibrous materials such as ancient silk, wool or hair. We used PRS to quantify the degradation of the collagen fibers in a number of fragments of the Temple Scroll (11Q19a). We found that collagen fibers degrade heterogeneously, with the ones on the surface more degraded than those in the core.