Orientation of mineral in bovine bone and the anisotropic mechanical properties of plexiform bone.

Angular dependent Young's modulus E phi presented by Bonfield and Grynpas [Nature 270, 453-454 (1977)] was simulated by using the distribution function of the orientation of mineral in plexiform bone introduced on the basis of an X-ray pole figure analysis (XPFA) and a small angle X-ray scattering (SAXS) results. Calculations were performed with the aid of a simple model which expresses well the geometrical characteristic of plexiform bone. Estimated angular dependent Young's modulus in terms of the distribution of mineral orientation reproduced the experimental results. The suitable aspect ratio of bone mineral for the reproduction of the empirical data was a reasonable value compared with the morphological study of bone mineral. It is concluded that the angular dependence of mechanical properties of plexiform bone is explained by the distribution of bone mineral orientation and its morphology.     

Self-assembly and mineralization of genetically modifiable biological nanofibers driven by β-structure formation

Bioinspired mineralization is an innovative approach to the fabrication of bone biomaterials mimicking the natural bone. Bone mineral hydroxylapatite (HAP) is preferentially oriented with c-axis parallel to collagen fibers in natural bone. However, such orientation control is not easy to achieve in artificial bone biomaterials. To overcome the lack of such orientation control, we fabricated a phage-HAP composite by genetically engineering M13 phage, a nontoxic bionanofiber, with two HAP-nucleating peptides derived from one of the noncollagenous proteins, Dentin Matrix Protein-1 (DMP1). The phage is a biological nanofiber that can be mass produced by infecting bacteria and is nontoxic to human beings. The resultant HAP-nucleating phages are able to self-assemble into bundles by forming β-structure between the peptides displayed on their side walls. The β-structure further promotes the oriented nucleation and growth of HAP crystals within the nanofibrous phage bundles with their c-axis preferentially parallel to the bundles. We proposed that the preferred orientation resulted from the stereochemical matching between the negatively charged amino acid residues within the β-structure and the positively charged calcium ions on the (001) plane of HAP crystals. The self-assembly and mineralization driven by the β-structure formation represent a new route for fabricating mineralized fibers that can serve as building blocks in forming bone repair biomaterials and mimic the basic structure of natural bones.     

The effect of hydration on mechanical anisotropy,topography and fibril organization of the osteonal lamellae

The effect of hydration on the mechanical properties of osteonal bone, in directions parallel and perpendicular to the bone axis, was studied on three length scales: (i) the mineralized fibril level (~100 nm), (ii) the lamellar level (~6 µm); and (iii) the osteon level (up to ~30 µm).We used a number of techniques, namely atomic force microscopy (AFM), nanoindentation and microindentation. The mechanical properties (stiffness, modulus and/or hardness) have been studied under dry and wet conditions. On all three length scales the mechanical properties under dry conditions were found to be higher by 30–50% compared to wet conditions. Also the mechanical anisotropy, represented by the ratio between the properties in directions parallel and perpendicular to the osteon axis (anisotropy ratio, designated here by AnR), surprisingly decreased somewhat upon hydration. AFM imaging of osteonal lamellae revealed a disappearance of the distinctive lamellar structure under wet conditions. Altogether, these results suggest that a change in mineralized fibril orientation takes place upon hydration.     

Cellulose orientation determines mechanical anisotropy in onion epidermis cell walls

The role of cellulose microfibril orientation in determining cell wall mechanical anisotropy and in the control of the wall plastic versus elastic properties was studied in the adaxial epidermis of onion bulb scales using the constant-load (creep) test. The mean or net cellulose orientation in the outer periclinal wall of the epidermis was parallel to the long axis of the cells. In vitro cell wall extensibility was 30-90% higher in the direction perpendicular to the net microfibril orientation than parallel to it. This was the case for the size of the initial deformation occurring just after the load application and for the rate of time-dependent creep. Loading/unloading experiments confirmed the presence of a real irreversible component in cell wall extension. The plastic component of the time-dependent deformation was higher perpendicular to the net cellulose orientation than parallel to it. An acid buffer (pH 4.5) increased the creep rate by 25-30% but this response was not related to cellulose orientation. The present data provide direct evidence that the net orientation of cellulose microfibrils confers mechanical anisotropy to the walls of seed plants, a characteristic that may be relevant to understanding anisotropic cell growth.     

Microstructure and crystallographic-texture of giant barnacle (Austromegabalanus psittacus) shell

Barnacle shell is a very complex and strong composite bioceramic composed of different structural units which consist of calcite 15 microcrystals of very uniform size. In the study reported herein, the microstructural organization of these units has been examinated in detail with optical and scanning electron microscopy, and X-ray diffraction techniques. These analyses showed that the external part of the shell has a massive microstructure consisting of randomly oriented crystals. Toward the interior, the shell became organized in mineral layers separated by thin organic sheets. Each of these mineral layers has a massive microstructure constituted by highly oriented calcite microcrystals with their c-axes aligned [(001) fibre texture] perpendicular to the organic sheets and the shell surface. Interestingly, in another structural unit, the shell shield, the orientation of the c-axis calcite crystals shifts from being perpendicular to being parallel to the shell surface across its thickness. This study provides evidence that the organic matrix is responsible for the organization of the shell mineral and exterts strong a strict control on the polymorphic type, size and orientation of shell-forming crystals.     

Cortical bone failure mechanisms during screw pullout

An experimental and computational study of screw pullout from cortical bone has been conducted. A novel modification of standard pullout tests providing real time image capture of damage mechanisms during screw pullout was developed. Pullout forces, measured using the novel test rig, have been validated against standard pullout tests. Pullout tests were conducted, considering osteon alignment, to investigate the effect of osteons aligned parallel to the axis of the orthopaedic screw (longitudinal pullout) as well as the effect of osteons aligned perpendicular to the axis of the screw (transverse pullout). Distinctive alternate failure mechanisms, for longitudinally and transversely orientated cortical bone during screw pullout, were uncovered. Vertical crack propagation, parallel to the axis of the screw, was observed for a longitudinal pullout. Horizontal crack propagation, perpendicular to the axis of the screw, was observed for a transverse pullout. Finite element simulation of screw pullout, incorporating material damage and crack propagation, was also performed. Simulations revealed that a homogenous material model for cortical bone predicts vertical crack propagation patterns for both longitudinal and transverse screw pullout. A bi-layered composite model representing cortical bone microstructure was developed. A unique set of material and damage properties was used for both transverse and longitudinal pullout simulations, with only layer orientations being changed. Simulations predicted: (i) higher pullout forces for transverse pullout; (ii) horizontal crack paths perpendicular to screw axis for transverse pullout, whereas vertical crack paths were computed for longitudinal pullout. Computed results agreed closely with experimental observations in terms of pullout force and crack propagation.     

Characteristics of mineral particles in the human bone/cartilage interface

Bone and cartilage consist of different organic matrices, which can both be mineralized by the deposition of nano-sized calcium phosphate particles. We have studied these mineral particles in the mineralized cartilage layer between bone and different types of cartilage (bone/articular cartilage, bone/intervertebral disk, and bone/growth cartilage) of individuals aged 54 years, 12 years, and 6 months. Quantitative backscattered electron imaging and scanning small-angle X-ray scattering at a synchrotron radiation source were combined with light microscopy to determine calcium content, mineral particle size and alignment, and collagen orientation, respectively. Mineralized cartilage revealed a higher calcium content than the adjacent bone (p<0.05 for all samples), whereas the highest values were found in growth cartilage. Surprisingly, we found the mineral platelet width similar for bone and mineralized cartilage, with the exception of the growth cartilage sample. The most striking result, however, was the abrupt change of mineral particle orientation at the interface between the two tissues. While the particles were aligned perpendicular to the interface in cartilage, they were oriented parallel to it in bone, reflecting the morphology of the underlying organic matrices. The tight bonding of mineralized cartilage to bone suggests a mechanical role for the interface of the two elastically different tissues, bone and cartilage.     

A Raman-scattering study on the net orientation of biomacromolecules in the outer epidermal walls of mature wheat stems (Triticum aestivum)

BACKGROUND AND AIMS: Raman spectroscopy can be used to examine the orientation of biomacromolecules using relatively thick samples of material, whereas more traditional means of analysing molecular structure require prior isolation of the components, which often destroys morphological features. In this study, Raman spectroscopy was used to examine the outer epidermal cell walls of wheat stems. METHODS: Polarized Raman spectra from the epidermal cell walls of wheat stem were obtained using near-infrared-Fourier transform Raman scattering. By comparing spectra taken with Raman light polarized perpendicular or parallel to the longitudinal axis of the cell, the orientation of macromolecules in the cell wall was investigated. KEY RESULTS: The net orientation of macromolecules varies in the epidermal cell walls of the different components of wheat stem. The net orientation of cellulose is parallel to the longitudinal axis of the cells, whereas the xylan and the phenylpropane units of lignin tend to lie perpendicular to the longitudinal axis of the cells, i.e. perpendicular to the net orientation of cellulose in the epidermal cell walls. CONCLUSIONS: The results imply that cellulose, lignin and xylan form a relatively ordered network that defines the mechanical and structural properties of the cell wall. Such results are likely to have a significant impact on the formulation of definitive models for the static and growing cell wall.     

Oriented spermatozoa in the spermatophore of the palaemonid shrimp, Macrobrachium rosenbergii

Estimates of the degree of orientation of spermatozoa within spermatophores of the freshwater shrimp, Macrobrachium rosenbergii, were made using electron micrographs of ultrathin sections cut transverse and parallel to the longitudinal axis of the spermatophores. Counts of the total number of longitudinal and non-longitudinal profiles of sperm spikes in transverse sections of spermatophores indicated that 67% of the spermatozoa were oriented with their spikes approximately perpendicular to the long axis of the spermatophore. In longitudinal sections, ~30% of the sperm spike profiles were oriented parallel to the longitudinal axis of the spermatophore, leaving ~70% of the profiles oriented in planes not parallel to the longitudinal spermatophore axis. Stereologic analyses based on the number of intersections of longitudinally sectioned sperm spike profiles per unit length of test lines placed over photographic images of ultrathin sections cut in both transverse and longitudinal planes of spermatophores gave a Saltykov approximation of 0.607, favoring the view that the spermatozoa were oriented with their spikes perpendicular to the long axis of the spermatophore. This orientation brings many of the sperm bases approximately parallel to the surface of the spermatophore, an orientation which may facilitate normal sperm-egg interaction during fertilization in this species.     

Fetal and postnatal mouse bone tissue contains more calcium than is present in hydroxyapatite

It has been shown for developing enamel and zebrafish fin that hydroxyapatite (HA) is preceded by an amorphous precursor, motivating us to examine the mineral development in mammalian bone, particularly femur and tibia of fetal and young mice. Mineral particle thickness and arrangement were characterized by (synchrotron) small-angle X-ray scattering (SAXS) combined with wide-angle X-ray diffraction (WAXD) and X-ray fluorescence (XRF) analysis. Simultaneous measurements of the local calcium content and the HA content via XRF and WAXD, respectively, revealed the total calcium contained in HA crystals. Interestingly, bones of fetal as well as newborn mice contained a certain fraction of calcium which is not part of the HA crystals. Mineral deposition could be first detected in fetal tibia at day 16.5 by environmental scanning electron microscopy (ESEM). SAXS revealed a complete lack of orientation in the mineral particles at this stage, whereas 1 day after birth particles were predominantly aligned parallel to the longitudinal bone axis, with the highest degree of alignment in the midshaft. Moreover, we found that mineral particle length increased with age as well as the thickness, while fetal particles were thicker but much shorter. In summary, this study revealed strong differences in size and orientation of the mineral particles between fetal and postnatal bone, with bulkier, randomly oriented particles at the fetal stage, and highly aligned, much longer particles after birth. Moreover, a part of the calcium seems to be present in other form than HA at all stages of development.     

Structural differences between "dark" and "bright" isolated human osteonic lamellae

This investigation presents new insights into the structure of human secondary lamellae. Lamellar specimens that appear dark and bright on alternate osteon transverse sections under circularly polarizing light were isolated using a new technique, and examined by polarizing light microscopy, synchrotron X-ray diffraction, and confocal microscopy. A distribution of unidirectional collagen bundles and of two overlapping oblique bundles appears on circularly polarizing light microscopy images in relation to the angle between the specimen and the crossed Nicols' planes. The unidirectional collagen bundles observed at 45 degrees run parallel to the osteon axis in the dark lamellar specimens and perpendicular to it in the bright ones. Small and wide-angle micro-focus X-ray diffraction indicates that the dark lamellae are structurally quite homogeneous, with collagen fibers and apatite crystals preferentially oriented parallel to the osteon axis. Bright lamellar specimens exhibit different orientation patterns with the dominant ones bidirectional at +/-45 degrees with respect to the osteon axis. Accordingly, confocal microscopy evidences the presence of longitudinal bundles in dark lamellar specimens and oblique bundles in the bright ones. Radial bundles are evidenced in both lamellar types. The alternate osteon structure is described by a rather continuous multidirectional pattern, in which dark and bright lamellae display different mechanical and possibly biological functions.     

Orientation of paramecium swimming in a DC magnetic field

We found that a ciliated protozoan, Paramecium, swam perpendicular to a static (DC) magnetic field (0.68 T). The swimming orientation was similar even when the ionic current through the cell membrane disappeared after saponin treatment. To determine the diamagnetic anisotropy of intracellular organs, macronuclei, cilia, and secretory vesicles, trichocysts, were selectively isolated. Both cilia and trichocysts tended to align their long axis parallel to the magnetic field (0.78 T). Paramecium mutants that lack trichocysts also swam perpendicular to the magnetic field, although the proportion fraction was smaller than the normal population. Since large numbers of cilia and trichocysts are arranged at right angles to the long axis of the cell, the diamagnetic anisotropies of cilia and trichocysts cause the long axis of the cell to align perpendicular to the magnetic field. In contrast to the DC magnetic field, an alternative (AC) magnetic field (60 Hz, 0.65 T) had almost no effect on the swimming orientation of Paramecium.     

Anisotropic structure of calcium-induced alginate gels by optical and small-angle X-ray scattering measurements

It was more than 50 years ago that an appearance of birefringence in alginate gels prepared under cation flow was reported for the first time, however, the anisotropic structure of the alginate gel has not been studied in detail. In the present study, anisotropic Ca-alginate gels were prepared within dialysis tubing in a high Ca(2+)-concentration external bath, and optical and small-angle X-ray scattering (SAXS) measurements were performed to characterize the structure of the gel. The observations of the gel with crossed polarizers and with circular polarizers revealed the molecular orientation perpendicular to the direction of Ca(2+) flow. Analyses of the SAXS intensity profiles indicated the formation of rod-like fibrils consisting of a few tens of alginate molecules and that the anisotropy of the gel was caused by the circumferential orientation of the large fibrils. From the observed asymmetric SAXS pattern, it was found that the axis of rotational symmetry of the anisotropic structure was parallel to the direction of Ca(2+) flow. The alignment factor (A(f)) calculated from the SAXS intensity data confirmed that the orientation of the fibrils was perpendicular to the direction of Ca(2+) flow.     

Orientation behavior of retinal photoreceptors in alternating electric fields

In alternating electric (AC) fields, particles experience polarizing effects that induce dipoles that orient elongated specimens either parallel or perpendicular to the field lines. In this work we studied the behavior of photoreceptor cells' rod outer segments (ROS) in AC fields of different frequencies. We showed that at low frequencies, ROS orient parallel to the field, whereas at higher frequencies they orient perpendicular to the field lines (in the frequency range from 100 Hz to 10 MHz). We found this behavior to be dependent on the physiological state of cells (due to modifications in their electrical properties). To simulate cell damage, the membrane conductivity was changed by treating the cell with gramicidin A, which resulted in a decrease of cytosol conductivity and, consequently, in a change of the orientation behavior of the treated cells. The change of cell orientation with cytosol conductivity is rather sharp, suggesting the potential of the method for accurate evaluation of the cell physiological status. We modeled the interaction between ROS and AC fields approximating the rod cell by a prolate spheroid with a very long axis. The internal compartment of the ellipsoid was considered to be filled with an inhomogeneous medium consisting of alternating layers of membrane and cytoplasm as media modeling the disks. This theoretical model proved to be in good agreement with the experimental results and enabled the derivation (by fitting with the experimental results) of the membrane and cytosol parameters for normal and damaged cells.     

Assessment of composition and anisotropic elastic properties of secondary osteon lamellae

Measurement of the elastic properties of single osteon lamellae is still one of the most demanding tasks in bone mechanics to be solved. By means of site-matched Raman microspectroscopy, acoustic microscopy and nanoindentation the structure, chemical composition and anisotropic elasticity of individual lamellae in secondary osteons were investigated. Acoustic impedance images (911-MHz) and two-dimensional Raman spectra were acquired in sections of human femoral bone. The samples were prepared with orientations at various observation angles theta relative to the femoral long axis. Nanoindentations provided local estimations of the elastic modulus and landmarks necessary for spatial fusion of the acoustic and spectral Raman images. Phosphate nu(1) (961 cm(-1)) and amide I (1665 cm(-1)) band images representing spatial distributions of mineral and collagen were fused with the acoustic images. Acoustic impedance was correlated with the indentation elastic modulus E(IT) (R(2)=0.61). Both parameters are sensitive to elastic tissue anisotropy. The lowest values were obtained in the direction perpendicular to the femoral long axis. Acoustic images exhibit a characteristic bimodal lamellar pattern of alternating high and low impedance values. Since this undulation was not associated with a variation of the phosphate nu(1)-band intensity in the Raman images, it was attributed to variations of the lamellar orientation. After threshold segmentation and conversion to elastic modulus the orientation and transverse isotropic elastic constants were derived for individual ensembles of apparent thin and thick lamellae. Our results suggest that this model represents the effective anisotropic properties of an asymmetric twisted plywood structure made of transverse isotropic fibrils. This is the first report that proves experimentally the ability of acoustic microscopy to map tissue elasticity in two dimensions with micrometer resolution. The combination with Raman microspectroscopy provides a unique way to study bone and mineral metabolism and the relation with mechanical function at the ultrastructural tissue level.     

A bone remodelling model including the directional activity of BMUs

Bone is able to adapt itself to the mechanical and biological environment by changing its porosity and/or orientation of its internal microstructure in a process known as bone remodelling. As a consequence, a change of bone mechanical properties is produced leading to an optimum structure, able to bear the external loads with the minimum weight. This adaptation is carried out by a temporal association of cells known as BMUs (basic multicellular units) that resorb old bone and sometimes produce new organic extracellular matrix (osteoid) that is later mineralized. This involves changes in porosity, damage level (density of microcracks accumulated by cyclic loads) and mineral content. All of these features were taken into account in a previous model, but the whole process and therefore the resulting bone constitutive behaviour was considered isotropic. The model proposed herein, recognizing that bone is actually anisotropic, tries to explain how BMUs modify the anisotropy by changing their progressing direction. We check the potential of the model to predict the alignment of the bone microstructure with the external loads in different situations. Then, the model is also applied to obtain the anisotropy and mechanical properties of the human proximal femur under physiological loads with initial conditions corresponding to a heterogeneous, but otherwise isotropic bone.     

Incorporating tissue anisotropy and heterogeneity in finite element models of trabecular bone altered predicted local stress distributions

Trabecular bone is composed of organized mineralized collagen fibrils, which results in heterogeneous and anisotropic mechanical properties at the tissue level. Recently, biomechanical models computing stresses and strains in trabecular bone have indicated a significant effect of tissue heterogeneity on predicted stresses and strains. However, the effect of the tissue-level mechanical anisotropy on the trabecular bone biomechanical response is unknown. Here, a computational method was established to automatically impose physiologically relevant orientation inherent in trabecular bone tissue on a trabecular bone microscale finite element model. Spatially varying tissue-level anisotropic elastic properties were then applied according to the bone mineral density and the local tissue orientation. The model was used to test the hypothesis that anisotropy in both homogeneous and heterogeneous models alters the predicted distribution of stress invariants. Linear elastic finite element computations were performed on a 3 mm cube model isolated from a microcomputed tomography scan of human trabecular bone from the distal femur. Hydrostatic stress and von Mises equivalent stress were recorded at every element, and the distributions of these values were analyzed. Anisotropy reduced the range of hydrostatic stress in both tension and compression more strongly than the associated increase in von Mises equivalent stress. The effect of anisotropy was independent of the spatial redistribution high compressive stresses due to tissue elastic heterogeneity. Tissue anisotropy and heterogeneity are likely important mechanisms to protect bone from failure and should be included for stress analyses in trabecular bone.     

Inactivation of Oriented Bacteria with Polarized Ultraviolet Light

Aligned deoxyribonucleic acid (DNA) molecules exhibit a large absorption anisotropy in the ultraviolet (UV) region of the spectrum (11). Also, the UV action spectra of most bacteria resemble the absorption spectrum of DNA (23), implying that inactivation is directly proportional to the UV absorbed by the bacterial DNA. Hence, the UV sensitivity of aligned uniaxial bacteria might be anisotropic with respect to polarization of the incident UV (17, 19). Any inactivation anisotropy would depend upon the orientation of DNA within the bacteria, as well as upon the alignment of bacteria, and could provide a more sensitive indication of in vivo DNA orientation than is presently available using optical methods (5, 12-16). Using an electric field of 3.5 × 10⁶ cycles/second, samples of bacteria of strain LS-301 were aligned in a quartz cell and were irradiated with UV (λ = 2652 A) polarized perpendicular and parallel to the alignment direction. The resultant survival curves resolved no inactivation anisotropy. This result is interpreted to mean that there was insufficient bacterial DNA alignment to give a detectable anisotropy. The minimum average DNA alignment necessary to have resolved an anisotropy is calculated to be 15 per cent in an axial direction (bases perpendicular to the bacterial axis) or 30 per cent in a radial direction (bases parallel to the bacterial axis).     

Oriented crystallization of octacalcium phosphate into beta-chitin scaffold

Composites of beta-chitin with octacalcium phosphate (OCP) or hydroxylapatite (HAP) were prepared by precipitation of the mineral into a chitin scaffold by means of a double diffusion system. The beta-chitin was obtained from the pen of the Loligo sp. squid. Only oriented precipitation of OCP was observed. The OCP crystals with the usual form of (001) blades grow inside chitin layers preferentially oriented with the [100] faces parallel to the surface of the squid pen and were more stable to the hydrolysis to HAP with respect to that precipitated in solution. Reasons are given why mechanical factors are thought to be the predominant cause for the orientation of the OCP crystals with the a-axis almost normal to the chitin fibers. We conclude that in these in vitro experiments the compartmentalized space in the chitin governs the orientation of the crystals, even if epitaxial factors may play a role in the nucleation processes.     

Surface structure and frictional properties of the skin of the Amazon tree boa <Emphasis Type="Italic">Corallus hortulanus</Emphasis> (Squamata,Boidae)

The legless locomotion of snakes requires specific adaptations of their ventral scales to maintain friction force in different directions. The skin microornamentation of the snake Corallus hortulanus was studied by means of scanning electron microscopy and the friction properties of the skin were tested on substrates of different roughness. Skin samples from various parts of the body (dorsal, lateral, ventral) were compared. Dorsal and lateral scales showed similar, net-like microornamentation and similar friction coefficients. Average friction coefficients for dorsal and lateral scales on the epoxy resin surfaces were 0.331 and 0.323, respectively. In contrast, ventral scales possess ridges running parallel to the longitudinal body axis. They demonstrated a significantly lower friction coefficient compared to both dorsal and lateral scales (0.191 on average). In addition, ventral scales showed frictional anisotropy comparing longitudinal and perpendicular direction of the ridges. This study clearly demonstrates that different skin microstructure is responsible for different frictional properties in different body regions.