Nanostructure and elastic modulus of single trabecula in bovine cancellous bone

We aimed to investigate the elastic modulus of trabeculae using tensile tests and assess the effects of nanostructure at the hydroxyapatite (HAp) crystal scale on the elastic modulus. In the experiments, 18 trabeculae that were at least 3 mm in length in the proximal epiphysis of three adult bovine femurs were used. Tensile tests were conducted using a small tensile testing device coupled with microscopy under air-dried condition. The c-axis orientation of HAp crystals and the degree of orientation were measured by X-ray diffraction. To observe the deformation behavior of HAp crystals under tensile loading, the same tensile tests were conducted in X-ray diffraction measurements. The mineral content of specimens was evaluated using energy dispersive X-ray spectrometry. The elastic modulus of a single trabecula varied from 4.5 to 23.6 GPa, and the average was 11.5±5.0 GPa. The c-axis of HAp crystals was aligned with the trabecular axis and the crystals were lineally deformed under tensile loading. The ratio of the HAp crystal strain to the tissue strain (strain ratio) had a significant correlation with the elastic modulus (r=0.79; P<0.001). However, the mineral content and the degree of orientation did not vary widely and did not correlate with the elastic modulus in this study. It suggests that the strain ratio may represent the nanostructure of a single trabecula and would determine the elastic modulus as well as mineral content and orientation.     

Ductile sliding between mineral crystals followed by rupture of collagen crosslinks: Experimentally supported micromechanical explanation of bone strength

There is an ongoing discussion on how bone strength could be explained from its internal structure and composition. Reviewing recent experimental and molecular dynamics studies, we here propose a new vision on bone material failure: mutual ductile sliding of hydroxyapatite mineral crystals along layered water films is followed by rupture of collagen crosslinks. In order to cast this vision into a mathematical form, a multiscale continuum micromechanics theory for upscaling of elastoplastic properties is developed, based on the concept of concentration and influence tensors for eigenstressed microheterogeneous materials. The model reflects bone's hierarchical organization, in terms of representative volume elements for cortical bone, for extravascular and extracellular bone material, for mineralized fibrils and the extrafibrillar space, and for wet collagen. In order to get access to the stress states at the interfaces between crystals, the extrafibrillar mineral is resolved into an infinite amount of cylindrical material phases oriented in all directions in space. The multiscale micromechanics model is shown to be able to satisfactorily predict the strength characteristics of different bones from different species, on the basis of their mineral/collagen content, their intercrystalline, intermolecular, lacunar, and vascular porosities, and the elastic and strength properties of hydroxyapatite and (molecular) collagen.     

Flaw tolerant bulk and surface nanostructures of biological systems

Bone-like biological materials have achieved superior mechanical properties through hierarchical composite structures of mineral and protein. Gecko and many insects have evolved hierarchical surface structures to achieve extraordinary adhesion capabilities. We show that the nanometer scale plays a key role in allowing these biological systems to achieve their superior properties. We suggest that the principle of flaw tolerance may have had an overarching influence on the evolution of the bulk nanostructure of bone-like materials and the surface nanostructure of gecko-like animal species. We demonstrate that the nanoscale sizes allow the mineral nanoparticles in bone to achieve optimum fracture strength and the spatula nanoprotrusions in Gecko to achieve optimum adhesion strength. In both systems, strength optimization is achieved by restricting the characteristic dimension of the basic structure components to nanometer scale so that crack-like flaws do not propagate to break the desired structural link. Continuum modeling and atomistic simulations have been conducted to verify the concept of flaw tolerance at nanoscale.     

Structural development of the mineralized tissue in the human L4 vertebral body

Knowledge of the structural development of the human vertebrae from non-weight-bearing before birth to weight-bearing after birth is still poor. We studied the mineralized tissue of the developing lumbar L4 vertebral body at ages 15 weeks postconception to 97 years from the tissue level (trabecular architecture) to the material level (micro- and nanostructure). Trabecular architecture was investigated by 2D histomorphometry and the material level was examined by quantitative backscattered electron imaging (for typical calcium content, CaMaxFreq) and scanning small-angle X-ray scattering (for mean mineral particle thickness). During early development, the trabecular orientation changed from a radial to a vertical/horizontal pattern. For bone area per tissue area and trabecular width in postnatal cancellous bone, the maximum was reached at adolescence (20 years), while for trabecular number the maximum was reached at childhood (approximately 1 year). CaMaxFreq was lower in early bone (approximately 21 wt%) than in mineralized cartilage (approximately 29 wt%) and adolescent bone (approximately 23 wt%). In conclusion, the changes at the tissue level were observed to continue throughout life while the development of bone at the material level (CaMaxFreq, mineral particle thickness and orientation) is essentially complete after the first years of life. CaMaxFreq and mean particle thickness increase rapidly during the first years and reach saturation. Remarkably, when these parameters are plotted versus logarithm of age, they appear linear.     

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.     

Mineral phase in linguloid brachiopod shell: Lingula adamsi

The linguloid brachiopod shell family has been the focus of several studies because of the similarity in the composition of the mineral phase of these shells to that of human bone. However, ultrastructural features of Lingula shells have not yet been fully demonstrated at high magnification using Transmission Electron Microscopy (TEM) and Electron Diffraction. Ultrastructural characterization of the mineral phase in Lingula shells will improve our understanding of the biomineralization processes and mineral/organic interaction in more complex systems such as in bone or in other human mineralized tissues. In this study, the mineral phase of Lingula adamsi was characterized using a combination of ultrastructural and crystallographic techniques. The results showed that L. adamsi shells consist of apatite crystals of varying size, shape, and orientation in different areas of the shell. The c-axis of apatite was parallel to the shell surface and crystals were organized in different laminae. Compared to trabecular bovine bone, L. adamsi shells demonstrated a higher crystallinity and a lower amount of carbonate and organic compounds. This study therefore demonstrated how dissimilar organic matrix between L. adamsi shell and trabecular bone can modify the ultrastructural characteristics of apatite crystals in these two biomineralized tissues.     

Lateral packing of mineral crystals in bone collagen fibrils

Combined small-angle x-ray scattering and transmission electron microscopy studies of intramuscular fish bone (shad and herring) indicate that the lateral packing of nanoscale calcium-phosphate crystals in collagen fibrils can be represented by irregular stacks of platelet-shaped crystals, intercalated with organic layers of collagen molecules. The scattering intensity distribution in this system can be described by a modified Zernike-Prins model, taking preferred orientation effects into account. Using the model, the diffuse fan-shaped small-angle x-ray scattering intensity profile, dominating the equatorial region of the scattering pattern, could be quantitatively analyzed as a function of the degree of mineralization. The mineral platelets were found to be very thin (1.5 nm ∼ 2.0 nm), having a narrow thickness distribution. The thickness of the organic layers between adjacent mineral platelets within a stack is more broadly distributed with the average value varying from 6 nm to 10 nm, depending on the extent of mineralization. The two-dimensional analytical scheme also leads to quantitative information about the preferred orientation of mineral stacks and the average height of crystals along the crystallographic c axis.     

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.     

Coculturing denuded oocytes during the in vitro maturation of bovine cumulus oocyte complexes exerts a synergistic effect on embryo development

The present study examined the effect of coculturing cumulus oocyte complexes (COCs) and denuded oocytes (DOs) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation, zona pellucida (ZP) hardening, the pattern of fertilization and glutathione peroxidase 1 (GPX1) gene expression in the oocyte. Furthermore, the rate of embryonic development and the quality of blastocysts were examined for both COCs and DOs. Three IVM conditions were studied: 1) the coculture of 12 COCs and 60 DOs, 2) COC control with 12 COCs, and 3) DO control with 60 DOs. The IVM was performed in a 120-μl droplet of TCM199-based IVM medium. Following IVM, in vitro fertilization (IVF) and in vitro culture (IVC) were conducted separately for the COCs and DOs (DO coculture) from the IVM coculture group. Coculturing COCs and DOs increased the percentage of oocytes reaching the blastocyst stage and the total number of cells per blastocyst in both the COC coculture (44.4 ± 8.6 vs 26.7 ± 9.7%, P < 0.01, and 137.9 ± 24.9 vs 121.7 ± 21.1, P < 0.05) and the DO coculture (20.5 ± 5.0 vs 11.1 ± 2.5%, P < 0.01, and 121.9 ± 27.5 vs 112.3 ± 33.2, P < 0.05) compared to their respective control groups. The synergistic effects of coculturing were detected as increased nuclear and cytoplasmic maturation, the prevention of ZP hardening, increased monospermic fertilization and increased expression of GPX1 in the oocytes in response to endogenous oocyte-secreted factors. In conclusion, coculturing COCs and DOs may be an effective culture system for both intact COCs and immature DOs.     

Post-yield nanomechanics of human cortical bone in compression using synchrotron X-ray scattering techniques

The ultrastructural response to applied loads governs the post-yield deformation and failure behavior of bone, and is correlated with bone fragility fractures. Combining a novel progressive loading protocol and synchrotron X-ray scattering techniques, this study investigated the correlation of the local deformation (i.e., internal strains of the mineral and collagen phases) with the bulk mechanical behavior of bone. The results indicated that the internal strains of the longitudinally oriented collagen fibrils and mineral crystals increased almost linearly with respect to the macroscopic strain prior to yielding, but markedly decreased first and then gradually leveled off after yielding. Similar changes were also observed in the applied stress before and after yielding of bone. However, the collagen to mineral strain ratio remained nearly constant throughout the loading process. In addition, the internal strains of longitudinal mineral and collagen phases did not exhibit a linear relationship with either the modulus loss or the plastic deformation of bulk bone tissue. Finally, the time-dependent response of local deformation in the mineral phase was observed after yielding. Based on the results, we speculate that the mineral crystals and collagen fibrils aligned with the loading axis only partially explain the post-yield deformation, suggesting that shear deformation involving obliquely oriented crystals and fibrils (off axis) is dominant mechanism of yielding for human cortical bone in compression.     

Wet-spinning of amyloid protein nanofibers into multifunctional high-performance biofibers

Amyloid nanofibers derived from hen egg white lysozyme were processed into macroscopic fibers in a wet-spinning process based on interfacial polyion complexation using a polyanionic polysaccharide as cross-linker. As a result of their amyloid nanostructure, the hierarchically self-assembled protein fibers have a stiffness of up to 14 GPa and a tensile strength of up to 326 MPa. Fine-tuning of the polyelectrolytic interactions via pH allows to trigger the release of small molecules, as demonstrated with riboflavin-5'-phophate. The amyloid fibrils, highly oriented within the gellan gum matrix, were mineralized with calcium phosphate, mimicking the fibrolamellar structure of bone. The formed mineral crystals are highly oriented along the nanofibers, thus resulting in a 9-fold increase in fiber stiffness.     

Orientation of bone mineral and its role in the anisotropic mechanical properties of bone--transverse anisotropy

An orientation of hydroxyapatite (HAP) crystals in bovine femur mineral was investigated by means of X-ray pole figure analysis (XPFA). It was found that the c-axis of HAP generally orients parallel to the longitudinal axis of bone (bone axis) and a significant amount of c-axis was oriented in other directions, in particular, perpendicular to the bone axis. Comparing these results with those of the small angle X-ray scattering (SAXS) investigation by Matsushima et al. (Jap. J. appl. Phys. 21, 186-189, 1982) at least two types of morphology of bone mineral were found; rod like bone mineral having the c-axis of HAP crystal parallel to the longitudinal axis of the rod and that having the c-axis not parallel, in a particular case, perpendicular to its longitudinal axis. Transverse anisotropy in mechanical properties of bone was reproduced by the estimation of Young's moduli by using the structural results mainly from XPFA. It is concluded that the anisotropy in mechanical properties of bone is well explained by taking account of the non-longitudinal (off-bone) axial distribution of orientation of bone mineral.     

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.     

Estimating nanoscale deformation in bone by X-ray diffraction imaging method

Knowledge of internal stress-strain in bone tissue is important for clinical diagnosis and remedies. The inorganic mineral phase of apatite crystals in bone composite, because of its crystalline nature, provides a reliable way of measurement through X-ray diffraction system. Use of two-dimensional detector, imaging plate (IP), is considered to expedite the process with much more information, hence, is widely applied in the study of organization, stress, strain, etc. for crystalline substance. The distortion of Debye rings in the image obtained by IP can be directly related to the deformation in lattice plane of the crystals. Since X-ray diffraction method involves measurement at nano-level, proper focus on the extraction of data and corresponding analysis is needed. In the current work, we considered weighted average value of intensity to locate radius vectors along azimuthal direction in the diffracted rings from the primary array of digital data in steps of pixels. The widely applied approaches for profile shift measurement--peak shift and full width at half maximum (FWHM) of a peak, and shift of center of gravity of profile--were compared with a new concept of segmental shift (SS) proposed previously by the authors. We observed reliable and effective outcomes with higher precision in the consideration of SS while using IP as a detector. Our approach in this work for intensity integration and radius vector positioning especially add precision in such applications.     

THE PROBLEM OF DEMINERALISATION IN THIN SECTIONS OF FULLY CALCIFIED BONE

Thin sections of embryonic avian bone decalcify during preparation for electron microscopy, creating a false impression of mineral distribution. The results of the experiments reported herein show that viscous embedding materials do not penetrate compact formed bone, and so, in thin sections, the calcium apatite crystals may be leeched out by water, both in the collecting trough and in aqueous solutions of stains used to enhance tissue electron opacity. To prevent decalcification, a simple technique is described in which the aqueous fluids that come in contact with thin sections are saturated with respect to calcium and phosphate ions, thereby preventing solution of the bone mineral. The theoretical basis of this technique is briefly discussed.     

Time Related Changes of Mineral and Collagen and Their Roles in Cortical Bone Mechanics of Ovariectomized Rabbits

As cortical bone has a hierarchical structure, the macroscopic bone strength may be affected by the alterations of mineral crystal and collagen, which are main components of cortical bone. Limited studies focused on the time related alterations of these two components in osteoporosis, and their contributions to bone mechanics at tissue level and whole-bone level. Therefore, the purpose of this study was to elucidate the time related changes of mineral and collagen in cortical bone of ovariectomized (OVX) rabbits, and to relate these changes to cortical bone nanomechanics and macromechanics. 40 Rabbits (7-month-old) were randomly allocated into two groups (OVX and sham). OVX group received bilateral ovariectomy operation. Sham group received sham-OVX operation. Cortical bone quality of five rabbits in each group were assessed by DXA, μCT, nanoindentation, Fourier transform infrared (FTIR) spectroscopy and biomechanical tests (3-point bending of femoral midshaft) at pre-OVX, 4, 6, and 8 weeks after OVX. As time increased from pre-OVX to 8 weeks, the mineral to matrix ratio decreased with time, while both collagen crosslink ratio and crystallinity increased with time in OVX group. Elastic modulus and hardness measured by nanoindentation, whole-bone strength measured by biomechanical tests all decreased in OVX group with time. Bone material properties measured by FTIR correlated well with nano or whole-bone level mechanics. However, bone mineral density (BMD), structure, tissue-level and whole-bone mechanical properties did not change with age in sham group. Our study demonstrated that OVX could affect the tissue-level mechanics and bone strength of cortical bone. And this influence was attributed to the time related alterations of mineral and collagen properties, which may help us to design earlier interventions and more effective treatment strategies on osteoporosis.     

Factors affecting the in vitro action of cumulus cells on the maturing mouse oocytes

The removal of cumulus cells (CCs) from oocytes at the germinal vesicle (GV) stage still represents a major limitation in such embryo techniques as GV transfer, somatic cell haploidization, and oocyte cryopreservation. However, no efficient in vitro maturation (IVM) system for CC-denuded oocytes (DOs) has been established in mammalian species. Although follicular cells are considered to play an important role in oocyte maturation, the specific role and mechanisms of action of different cell types are poorly understood. Reports on whether junctional association between CCs and the oocyte is essential for the beneficial effect of CC co-culture on oocyte maturation are in conflict. Our objective was to try to address these issues using the mouse oocyte model. The results indicated that while co-culture with the CC monolayer could only partially restore the developmental potential of DOs without corona cells, it restored the competence of corona-enclosed DOs completely. Culture in medium conditioned with CC monolayer also promoted maturation of DOs. However, co-culture with the monolayer of mural granulosa cells had no effect. The efficiency of CC co-culture was affected by various factors such as density and age of the CCs, the presence of gonadotropin in the maturation medium and the duration for in vivo (IVO) gonadotropin priming. It is concluded that mouse CCs produce a diffusible factor(s) that support DO maturation in a CC-oocyte junctional communication dependent manner. The data will contribute to our understanding the mechanisms by which CCs promote oocyte maturation and to the establishment of an efficient DO IVM system.     

Presence of cumulus cells during in vitro fertilization protects the bovine oocyte against oxidative stress and improves first cleavage but does not affect further development

The present study was conducted to evaluate the function of cumulus cells during bovine IVF Oocytes within cumulus-oocyte complexes (COCs) or denuded oocytes (DOs) were inseminated in control medium, or DOs were inseminated in cumulus cell conditioned medium (CCCM). DOs exhibited reduced cleavage and blastocyst formation rates when compared with intact COCs. The reduced blastocyst formation rate of DOs resulted from reduced first cleavage but subsequent embryo development was not changed. Live-dead staining and staining for apoptotic cells revealed no differences in blastocysts from oocytes fertilized as COC or DO. Fertilization of DOs in CCCM partially restored the cleavage rate, suggesting that factors secreted by cumulus cells are important for fertilization but that physical contact between oocytes and cumulus cells is required for optimal fertilization and first cleavage. Exposure of COCs to hydrogen peroxide shortly before fertilization reduced the cleavage rate, but did not lead to enhanced death of cumulus cells or oocyte death. Exposure of DOs to hydrogen peroxide, however, resulted in oocyte death and a complete block of first cleavage, suggesting that cumulus cells protect the oocyte against oxidative stress during fertilization.     

Bone quality assessment in individuals of different age, gender and body constitution

The concept of bone quality describes the sets of the characteristics of the osseous tissue that influence bone strength. The aim was to explore the influence of anthropometric parameters and age on the parameters of the bone architecture and bone mineral properties in the lumbar vertebral bone of men and women. Vertebral bone samples underwent bone histomorphometry, bone densitometry and atomic absorption spectrometry. Men have greater values of the bone volume and thicker bone trabeculae in relation to women, which indicates that vertebral bone architecture is better preserved in men than in women. Age is the best predictor of changes that affect bone architecture and bone mineral properties. Bone mineral density value and calcium concentration are both negatively predicted by age, but positively predicted by body mass index. Such result supports the opinion that low body mass index is associated with conditions of bone deficit such are osteopenia and osteoporosis.     

Stress distribution in the scapula studied by neutron diffraction.

High intensity neutron beams provide a method of measuring the preferred orientation of apatite crystals in bulk samples of bone. Measurements at seven different sites on the scapula show that the c axes of the crystals lie preferentially along the directions of pull of the attached muscles. The highest orientation is found at positions under the influence of only a single group of muscles, such as M. teres major or M. infraspinatus. In intermediate regions a multiple distribution of crystals is found, able to withstand stresses in more than one direction. The technique provides a method of assessing the distribution of stress in bones.