X. Neil Dong

IN SITU ACCUMULATION OF ADVANCED GLYCATION ENDPRODUCTS (AGES) IN BONE MATRIX AND ITS CORRELATION WITH OSTEOCLASTIC BONE RESORPTION

Advanced glycation end products (AGEs) have been observed to accumulate in bone with increasing age and may impose effects on bone resorption activities. However, the underlying mechanism of AGEs accumulation in bone is still poorly understood. In this study, human cortical bone specimens from young (31±6years old), middle-aged (51±3years old) and elderly (76±4years old) groups were examined to determine the spatial-temporal distribution of AGEs in bone matrix and its effect on bone resorption activities by directly culturing osteoclastic cells on bone slices. The results of this study indicated that the fluorescence intensity (excitation wave length 360nm and emission wave length 470±40nm) could be used to estimate the relative distribution of AGEs in bone (pentosidine as its marker) under an epifluorescence microscope. Using the fluorescence intensity as the relative measure of AGEs concentration, it was found that the concentration of AGEs varied with biological tissue ages, showing the greatest amount in the interstitial tissue, followed by the old osteons, and the least amount in newly formed osteons. In addition, AGEs accumulation was found to be dependent on donor ages, suggesting that the younger the donor the less AGEs were accumulated in the tissue. Most interestingly, AGEs accumulation appeared to initiate from the region of cement lines, and spread diffusively to the other parts as the tissue aged. Finally, it was observed that the bone resorption activities of osteoclasts were positively correlated with the in situ concentration of AGEs and such an effect was enhanced with increasing donor age. These findings may help elucidate the mechanism of AGEs accumulation in bone and its association with bone remodeling process.

DIFFERENCES IN THE MECHANICAL BEHAVIOR OF CORTICAL BONE BETWEEN COMPRESSION AND TENSION WHEN SUBJECTED TO PROGRESSIVE LOADING

The hierarchical arrangement of collagen and mineral into bone tissue presumably maximizes fracture resistance with respect to the predominant strain mode in bone. Thus, the ability of cortical bone to dissipate energy may differ between compression and tension for the same anatomical site. To test this notion, we subjected bone specimens from the anterior quadrant of human cadaveric tibiae to a progressive loading scheme in either uniaxial tension or uniaxial compression. One tension (dog-bone shape) and one compression specimen (cylindrical shape) were collected each from tibiae of nine middle aged male donors. At each cycle of loading-dwell-unloading-dwell-reloading, we calculated maximum stress, permanent strain, modulus, stress relaxation, time constant, and three pathways of energy dissipation for both loading modes. In doing so, we found that bone dissipated greater energy through the mechanisms of permanent and viscoelastic deformation in compression than in tension. On the other hand, however, bone dissipated greater energy through the release of surface energy in tension than in compression. Moreover, differences in the plastic and viscoelastic properties after yielding were not reflected in the evolution of modulus loss (an indicator of damage accumulation), which was similar for both loading modes. A possible explanation is that differences in damage morphology between the two loading modes may favor the plastic and viscoelastic energy dissipation in compression, but facilitate the surface energy release in tension. Such detailed information about failure mechanisms of bone at the tissue-level would help explain the underlying causes of bone fractures.

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.

Progressive post-yield behavior of human cortical bone in compression for middle-aged and elderly groups

In this study, a progressive loading regimen (load-dwell-unloading-dwell-reloading) was applied on bone samples to examine the compressive post-yield response of bone at increasing strain levels. Cortical bone specimens from human tibiae of two age groups (middle-aged group: 53+/-2 years, 4 females and 4 males, elderly group: 83+/-6 years, 4 females and 4 males) were loaded in compression using the progressive loading scheme. Modulus degradation, plastic deformation, viscous response, and energy dissipation of bone during post-yield deformation were assessed. Although initial modulus was not significantly different between the two age groups, the degradation of modulus with the applied strain in the elderly group was faster than in the middle-aged group. The modulus loss (or microdamage accumulation) of bone occurred prior to plastic deformation. Plastic strain had a similar linear relationship with the applied strain for both middle-aged and the elderly group although middle-aged bone yielded at a greater strain. The viscoelastic time constant changed similarly with increasing strain for the two groups, whereas a higher magnitude of stress relaxation was observed in the middle-aged group. Energy dissipation was investigated through three pathways: elastic release strain energy, hysteresis energy, and plastic strain energy. The middle-aged group had significantly greater capacity of energy dissipation than the elderly group in all three pathways. The information obtained may provide important insights in age-related effects on bone fragility.

Probabilistic failure analysis of bone using a finite element model of mineral-collagen composites

Microdamage accumulation is a major pathway for energy dissipation during the post-yield deformation of bone. In this study, a two-dimensional probabilistic finite element model of a mineral-collagen composite was developed to investigate the influence of the tissue and ultrastructural properties of bone on the evolution of microdamage from an initial defect in tension. The probabilistic failure analyses indicated that the microdamage progression would be along the plane of the initial defect when the debonding at mineral-collagen interfaces was either absent or limited in the vicinity of the defect. In this case, the formation of a linear microcrack would be facilitated. However, the microdamage progression would be scattered away from the initial defect plane if interfacial debonding takes place at a large scale. This would suggest the possible formation of diffuse damage. In addition to interfacial debonding, the sensitivity analyses indicated that the microdamage progression was also dependent on the other material and ultrastructural properties of bone. The intensity of stress concentration accompanied with microdamage progression was more sensitive to the elastic modulus of the mineral phase and the nonlinearity of the collagen phase, whereas the scattering of failure location was largely dependent on the mineral to collagen ratio and the nonlinearity of the collagen phase. The findings of this study may help understanding the post-yield behavior of bone at the ultrastructural level and shed light on the underlying mechanism of bone fractures.

In situ mechanical behavior of mineral crystals in human cortical bone under compressive load using synchrotron X-ray scattering techniques

It is of great interest to delineate the effect of orientation distribution of mineral crystals on the bulk mechanical behavior of bone. Using a unique synergistic approach combining a progressive loading scheme and synchrotron X-ray scattering techniques, human cortical bone specimens were tested in compression to examine the in situ mechanical behavior of mineral crystals aligned in different orientations. The orientation distribution was quantitatively estimated by measuring the X-ray diffraction intensity from the (002) plane in mineral crystals. In addition, the average longitudinal (c-axis), transverse (a-axis), and shear strains of the subset of mineral crystals aligned in each orientation were determined by measuring the lattice deformation normal to three distinct crystallographic planes (i.e. 002, 310, and 213) in the crystals. The experimental results indicated that the in situ strain and stress of mineral crystals varied with orientations. The normal strain and stress in the longitudinally aligned mineral crystals were markedly greater than those in the transversely oriented crystals, whereas the shear stress reached a maximum for the crystals aligned in ±30° with respect to the loading direction. The maximum principal strain and stress were observed in the mineral crystals oriented along the loading axis, with a similar trend observed in the maximum shear strain and stress. By examining the in situ behavior, the contribution of mineral crystals to load bearing and the bulk behavior of bone are discussed.

Random field assessment of nanoscopic inhomogeneity of bone

Bone quality is significantly correlated with the inhomogeneous distribution of material and ultrastructural properties (e.g., modulus and mineralization) of the tissue. Current techniques for quantifying inhomogeneity consist of descriptive statistics such as mean, standard deviation and coefficient of variation. However, these parameters do not describe the spatial variations of bone properties. The objective of this study was to develop a novel statistical method to characterize and quantitatively describe the spatial variation of bone properties at ultrastructural levels. To do so, a random field defined by an exponential covariance function was used to represent the spatial uncertainty of elastic modulus by delineating the correlation of the modulus at different locations in bone lamellae. The correlation length, a characteristic parameter of the covariance function, was employed to estimate the fluctuation of the elastic modulus in the random field. Using this approach, two distribution maps of the elastic modulus within bone lamellae were generated using simulation and compared with those obtained experimentally by a combination of atomic force microscopy and nanoindentation techniques. The simulation-generated maps of elastic modulus were in close agreement with the experimental ones, thus validating the random field approach in defining the inhomogeneity of elastic modulus in lamellae of bone. Indeed, generation of such random fields will facilitate multi-scale modeling of bone in more pragmatic details.

Collagen mutation causes changes of the microdamage morphology in bone of an OI mouse model

Previous studies have postulated that ultrastructural changes may alter the pattern and capacity of microdamage accumulation in bone. Using an osteogenesis imperfecta (OI) mouse model, this study was performed to investigate the correlation of collagen mutation with the microdamage morphology and the associated brittleness of bone. In this study, femurs from mild OI and wild type mice were fatigued under four-point bending to create microdamage in the specimens. Then, the microdamage morphology of these specimens was examined using the bulk-staining technique with basic fuchsin. Similar with the results of previous studies, it was observed that linear microcracks were formed more easily in compression, whereas diffuse damage was induced more readily in tension for both wild-type and mild-type mice. However, less diffuse damage was found in the tensile side of mild OI mouse femurs (collagen mutation) compared with those of wild type mice, showing that the microdamage morphology is correlated to the brittleness of bone. The results of this study provide direct evidence that supports the prediction made by the previous numerical simulation studies, suggesting that microdamage morphology in bone is significantly correlated with the integrity of the collagen phase.

Mechanistic modeling of a nanoscratch test for determination of in situ toughness of bone

The objective of this study was to develop a nanoscratch technique that can be used to measure the in situ toughness of bone at micro/nanostructural levels. Among the currently possible techniques, the surface scratch test may be conducted on very small regions, thus exhibiting a potential in determining the in situ failure behavior of materials. To adapt such a technique for assessing bone toughness at the micro/nanostructural levels and for limited stocks in small animal bone models (e.g. zebra finish and mice), a simple but reasonably accurate mechanistic model for the nanoscratch test was developed in this study. This model was based on the assumption that the removal energy of the tissue required during the nanoscratch test is the manifestation of the in situ toughness and the shear flow stress during the removal process is a measure of the in situ strength of bone. In addition, the experimental methodologies were developed to determine the elastic recovery force and frictional coefficients between the scratch tip and bone specimens that are required by the model. Finally, the efficacy of the nanoscratch technique was verified by testing bone samples from control (wild type), mild, and severe osteogenesis imperfecta (OI) mice, which have a distinct degree of brittleness. The experimental results indicated that the nanoscratch test could sensitively detect the in situ brittleness and strength of bone from the animal models.

Finding of microdamage morphology differences in mouse femoral bones with distinct mineralization levels

Microdamage progression in bone is dependent on the ultrastructure of the tissue. Thus, any pathological changes in bone ultrastructure may be reflected in the pattern and capacity of microdamage accumulation. A previous numerical study of microdamage progression in bone using a probabilistic failure analysis approach predicts that the microdamage morphology (either linear microcracks or diffuse damage) is very sensitive to the level of mineralization in bone, which is also implicated in some experimental observations. To examine the prediction, femurs from two strains of mice (C57BL/6J, N = 10 and C3H/HeJ, N = 11) that have distinct mineralization levels were fatigued under four-point bending to create damage in the bone tissues. After testing, the microdamage morphology of the bone samples was examined using bulk-staining technique with basic fuchsin. The results demonstrate that more linear microcracks are observed in femurs of C3H/HeJ (higher mineralization), whereas more diffuse-like damage is found in C57BL/6J femurs (less mineralized). Compared with linear microcracks, the formation of diffuse damage tends to dissipate more energy and help bone to avoid catastrophic failures. Therefore, results from this study may help explain why highly mineralized bone tends to be more brittle. Observations from this study are consistent with the numerical prediction from the previous study, suggesting that mineralization has a significant effect on the microdamage morphology of bone.