An improved labelling technique for monitoring microcrack growth in compact bone

Fatigue-induced damage plays an important role in bone remodelling and in the formation of stress and fragility fractures. Recently, a technique has been developed (Lee, T.C. et al., Sequential labelling of microdamage in bone using chelating agents. Journal of Orthopedic Research, 18 (2000) 322-325) which allows microcrack growth in trabecular bone to be monitored by the application of a series of chelating fluorochromes, however, some limitations were identified with the process. The aims of this study were to refine the method of detection using these agents in order to determine the optimal sequence of application and the optimal concentrations which allowed all the agents to fluoresce equally brightly using UV epifluorescence. A chemical analysis process, ion chromatography, followed by validation tests on bone samples showed that the optimal sequence of application and concentration of each agent was alizarin complexone (0.0005 M) followed by xylenol orange (0.0005 M), calcein (0.0005 M) and calcein blue (0.0001 M). A fifth agent, oxytetracycline was excluded from the study after recurring problems were found with its ability to chelate exposed calcium when applied in sequence with the other agents. This work has developed a sequential labelling technique, which allows for microcrack propagation during fatigue testing of bone specimens to be monitored without the problem of chelating agent substitution occurring.     

Cortical bone tissue resists fatigue fracture by deceleration and arrest of microcrack growth

Knowledge of kinetics of fatigue crack growth of microcracks is important so as to understand the dynamics of bone adaptation, remodeling, and the etiology of fatigue-based failures of cortical bone tissue. In this respect, theoretical models (Taylor, J. Biomech., 31 (1998) 587-592; Taylor and Prendergast, Proc. Instn. Mech. Engrs. Part H 211 (1997) 369-375) of microcrack growth in cortical bone have predicted a decreasing microcrack growth rate with increasing microcrack length. However, these predictions have not been observed directly. This study investigated microcrack growth and arrest through observations of surface microcracks during cyclic loading (R=0.1, 50-80MPa) of human femoral cortical bone (male, n=4, age range: 37-40yr) utilizing a video microscopy system. The change in crack length and orientation of eight surface microcracks were measured with the number of fatigue cycles from four specimens. At the applied cyclic stresses, the microcracks propagated and arrested in generally less than 10,000 cycles. The fatigue crack growth rate of all microcracks decreased with increasing crack length following initial identification, consistent with theoretical predictions. The growth rate of the microcracks was observed to be in the range of 5x10(-5) to 5x10(-7)mmcycle(-1). In addition, many of the microcracks were observed not to grow beyond 150 microm and a cyclic stress intensity factor of 0.5MNm(-3/2). The results of this study suggest that cortical bone tissue may resist fracture at the microscale by deceleration of fatigue crack growth and arrest of microcracks.     

A fatigue microcrack alters fluid velocities in a computational model of interstitial fluid flow in cortical bone

Targeted remodeling is activated by fatigue microcracks and plays an important role in maintaining bone integrity. It is widely believed that fluid flow-induced shear stress plays a major role in modulating the mechanotransduction process. Therefore, it is likely that fluid flow-induced shear stress plays a major role in the initiation of the repair of fatigue damage. Since no in vivo measurements of fluid flow within bone exist, computational and mathematical models must be employed to investigate the fluid flow field and the shear stress occurring within cortical bone. We developed a computational fluid dynamic model of cortical bone to examine the effect of a fatigue microcrack on the fluid flow field. Our results indicate that there are alterations in the fluid flow field as far as 150 microm away from the crack, and that at distances farther than this, the fluid flow field is similar to the fluid flow field of intact bone. Through the crack and immediately above and below it, the fluid velocity is higher, while at the lateral edges it is lower than that calculated for the intact model, with a maximum change of 29%. Our results suggest that the presence of a fatigue microcrack can alter the shear stress in regions near the crack. These alterations in shear stress have the potential to significantly alter mechanotransduction and may play a role in the initiation of the repair of fatigue microcracks.     

Microcrack accumulation at different intervals during fatigue testing of compact bone

Fatigue damage in bone occurs in the form of microcracks. This microdamage contributes to the formation of stress fractures and acts as a stimulus for bone remodelling. A technique has been developed, which allows microcrack growth to be monitored during the course of a fatigue test by the application of a series of fluorescent chelating agents. Specimens were taken from bovine tibiae and fatigue tested in cyclic compression at a stress range of 80MPa. The specimens were stained before testing with alizarin and up to three other chelating agents were applied during testing to label microcracks formed at different times. Microcracks initiated in interstitial bone in the early part of a specimen's life. Further accumulation of microcracks is then suppressed until the period late in the specimen's life. Microcracks were found to be longer in the longitudinal than in the transverse direction. Only a small proportion of cracks are actively propagating; these are longer than non-propagating cracks. These results support the concept of a microstructural barrier effect existing in bone, whereby cracks initiate easily but slow down or stop at barriers such as cement lines.     

The behaviour of microcracks in compact bone

This paper summarises four separate studies carried out by our group over the past number of years in the area of bone microdamage. The first study investigated the manner by which microcracks accumulate and interact with bone microstructure during fatigue testing of compact bone specimens. In a series of fatigue tests carried out at four different stress ranges between 50 and 80 MPA, crack density increased with loading cycles at a rate determined by the applied stress. Variations in the patterns of microdamage accumulation suggest that that at low stress levels, larger amounts of damage can build up without failure occurring. In a second study using a series of four-pont bending tests carried out on ovine bone samples, it was shown that bone microstructure influenced the ability of microcracks to propagate, with secondary osteons acting as barriers to crack growth. In a third study, the manner by which crack growth disrupts the canalicular processes connecting osteocytes was investigated. Analysis of individual cracks showed that disruption of the canalicular processes connecting osteocytes occurred due to shear displacement at the face of propagating microcracks, suggesting that this may play some role in the mechanism that signals bone remodelling. In a fourth in vivo study, it was shown that altering the mechanical load applied to the long bones of growing rats causes microcrack formation. In vivo microdamage was present in rats subjected to hindlimb suspension with a higher microcrack density found in the humeri than the femora. Microdamage was also found in control animals. This is the first study to demonstrate in vivo microcracks in normally loaded bones in a rat model.     

Microdamage accumulation in the cement layer of hip replacements under flexural loading.

Mechanical fatigue of bone cement leading to damage accumulation is implicated in the loosening of cemented hip components. Even though cracks have been identified in autopsy-retrieved mantles, damage accumulation by continuous growth and increase in number of microcracks has not yet been demonstrated experimentally. To determine just how damage accumulation occurs in the cement layer of a hip replacement, a physical model of the joint was used in an experimental study. The model regenerates the stress pattern found in the cement layers whilst at the same time allowing visualisation of microcrack initiation and growth. In this way the gradual process of damage accumulation can be determined. Six specimens were tested to 5 million cycles and a total of 1373 cracks were observed. It was found that, under the flexural loading allowed by the model, the majority of cracks come from pores in the bulk cement and not from the interfaces. Furthermore, the lateral and medial sides have statistically different damage accumulation behaviours, and pre-load cracks significantly accelerate the damage accumulation process. The experimental results confirm that damage accumulation commences early on in the loading history and that it is continuously increasing with load in the form of crack initiation and crack propagation. The results highlight the importance of replicating the loading and restraint conditions of clinical cement mantles when endeavouring to accurately model the damage accumulation process.     

Contribution, development and morphology of microcracking in cortical bone during crack propagation

A fracture mechanics study of cortical bone is presented to investigate the contribution, development morphology of microcracking in cortical bone during crack propagation. Post-hoc analyses of microcrack orientation, crack propagation velocity and fracture surface roughness were conducted on previously tested human and bovine bone compact tension specimens. It was found that, consistent with its higher toughness, bovine bone formed significantly more longitudinal, transverse and inclined microcracks than human bone. However, in human bone more of the microcracks that formed were longitudinal than transverse or inclined, a feature that would optimise bone's toughness. Crack propagation velocity in human and bovine bone displayed the same characteristic pattern with crack extension, where an increase in velocity is followed by a consequent decrease and vice versa. On the basis of this pattern, a model or crack propagation has been proposed. It provides a detailed account of mocrocrack formation and contribution towards the propagation of a fracture crack. Analyses of fracture surfaces indicated that, consistent with its higher toughness, bovine bone displays a rougher surface than human bone but they both have the same basic fractured element, i.e. a mineralised collagen fibril.     

Material heterogeneity,microstructure, and microcracks demonstrate differential influence on crack initiation and propagation in cortical bone

The recent studies have shown that long-term bisphosphonate use may result in a number of mechanical alterations in the bone tissue including a reduction in compositional heterogeneity and an increase in microcrack density. There are limited number of experimental and computational studies in the literature that evaluated how these modifications affect crack initiation and propagation in cortical bone. Therefore, in this study, the entire crack growth process including initiation and propagation was simulated at the microscale by using the cohesive extended finite element method. Models with homogeneous and heterogeneous material properties (represented at the microscale capturing the variability in material property values and their distribution) as well as different microcrack density and microstructure were compared. The results showed that initiation fracture resistance was higher in models with homogeneous material properties compared to heterogeneous ones, whereas an opposite trend was observed in propagation fracture resistance. The increase in material heterogeneity level up to 10 different material property sets increased the propagation fracture resistance beyond which a decrease was observed while still remaining higher than the homogeneous material distribution. The simulation results also showed that the total osteonal area influenced crack propagation and the local osteonal area near the initial crack affected the crack initiation behavior. In addition, the initiation fracture resistance was higher in models representing bisphosphonate treated bone (low material heterogeneity, high microcrack density) compared to untreated bone models (high material heterogeneity, low microcrack density), whereas an opposite trend was observed at later stages of crack growth. In summary, the results demonstrated that tissue material heterogeneity, microstructure, and microcrack density influenced crack initiation and propagation differently. The findings also elucidate how possible modifications in material heterogeneity and microcrack density due to bisphosphonate treatment may influence the initiation and propagation fracture resistance of cortical bone.     

Examination of mineralized nodule formation in living osteoblastic cultures using fluorescent dyes

Detecting the formation of mineralized nodules in osteogenic cell culture provides a means of assessing mature osteoblast cell function and the status of culture. In the present study, to continuously monitor the formation of mineralized nodules during the entire culture period, different concentrations of two fluorescent dyes (xylenol orange and calcein blue) were evaluated for their ability to specifically label calcified areas and their toxicity to cells in osteogenic cultures. Results showed that 20 microM xylenol orange and 30 microM calcein blue gave rise to distinct fluorescent staining for mineralized nodules, which were correlated exactly with von Kossa and alizarin red S staining at the same locations in cultures. In the assessment of toxicity, both dyes at the aforementioned concentrations did not alter cell viability or change the total DNA content in cultures. To demonstrate the advantage of using these fluorochromes to monitor mineralized nodules formation, consecutive fluorescent images of each staining were recorded at the same location of individual culture over the entire duration. The result indicates that both xylenol orange and calcein blue can provide contrasting fluorescent staining to continuously monitor mineralized nodules formation in living osteogenic cell cultures without deleterious effects.     

Microdamage propagation in trabecular bone due to changes in loading mode

Microdamage induced by falls or other abnormal loads that cause shear stress in trabecular bone could impair the mechanical properties of the proximal femur or spine. Existing microdamage may also increase the initiation and propagation of further microdamage during subsequent normal, on-axis, loading conditions, resulting in atraumatic or "spontaneous" fractures. Microdamage formation due to shear and compressive strains was studied in 14 on-axis cylindrical bovine tibial trabecular bone specimens. Microdamage was induced by a torsional overload followed by an on-axis compressive overload and quantified microscopically. Fluorescent agents were used to label microdamage and differentiate damage due to the two loading modes. Both the microcrack density and diffuse damage area caused by the torsional overload increased with increasing shear strain from the center to the edge of the specimen. However, the mean microcrack length was uniform across the specimen, suggesting that microcrack length is limited by microstructural features. The mean density of microcracks caused by compressive overloading was slightly higher near the center of the specimen, and the diffuse damage area was uniform across the specimen. Over 20% of the microcracks formed in the initial torsional overloading propagated during compression. Moreover the propagating microcracks were, on average, longer than microcracks formed by a single overload. As such, changes in loading mode can cause propagation of microcracks beyond the microstructural barriers that normally limit the length. Damage induced by in vivo off-axis loads such as falls may similarly propagate during subsequent normal loading, which could affect both remodeling activity and fracture susceptibility.     

Experimental validation of a microcracking-based toughening mechanism for cortical bone

It has been proposed that cortical bone derives its toughness by forming microcracks during the process of crack propagation (J. Biomech. 30 (1997) 763; J. Biomech. 33 (2000) 1169). The purpose of this study was to experimentally validate the previously proposed microcrack-based toughening mechanism in cortical bone. Crack initiation and propagation tests were conducted on cortical bone compact tension specimens obtained from the antlers of red deer. For these tests, the main fracture crack was either propagated to a predetermined crack length or was stopped immediately after initiating from the notch. The microcracks produced in both groups of specimens were counted in the same surface area of interest around and below the notch, and crack growth resistance and crack propagation velocity were analyzed. There were more microcracks in the surface area of interest in the propagation than in initiation specimens showing that the formation of microcracks continued after the initiation of a fracture crack. Crack growth resistance increased with crack extension, and crack propagation velocity vs. crack extension curves demonstrated the characteristic jump increase and decrease pattern associated with the formation of microcracks. The scanning electron micrographs of crack initiation and propagation displayed the formation of a frontal process zone and a wake, respectively. These results support the microcrack-based toughening mechanism in cortical bone. Bone toughness is, therefore, determined by its ability to form microcracks during fracture.     

Do microcracks decrease or increase fatigue resistance in cortical bone?

Fatigue of cortical bone produces microcracks; it has been hypothesized that these cracks are analogous to those occurring in engineered composite materials and constitute a similar mechanism for fatigue resistance. However, the numbers of these linear microcracks increase substantially with age, suggesting that they contribute to increased fracture incidence among the elderly. To test these opposing hypotheses, we fatigued 20 beams of femoral cortical bone from elderly men and women in load-controlled four point bending having initial strain ranges of 3000 or 5000 microstrain. Loading was stopped at fracture or 10(6) cycles, whichever occurred first, and microcrack density and length were measured in the loaded region and in a control region that was not loaded. We studied the dependence of fatigue life and induced microdamage on initial microdamage, cortical region, subject gender and age, and several other variables. When the effect of modulus variability was controlled, longer fatigue life was associated with higher rather than lower initial crack density, particularly in the medial cortex. The increase in crack density following fatigue loading was greater in specimens from older individuals and those initially having longer microcracks. Crack density increased as much in specimens fatigued short of the failure point as in those that fractured, and microcracks were, on average, shorter in specimens with greater numbers of resorption spaces, a measure of remodeling rate.     

Non Destructive Characterization of Cortical Bone Micro-Damage by Nonlinear Resonant Ultrasound Spectroscopy

The objective of the study was to evaluate the ability of a nonlinear ultrasound technique, the so-called nonlinear resonant ultrasound spectroscopy (NRUS) technique, for detecting early microdamage accumulation in cortical bone induced by four-point bending fatigue. Small parallelepiped beam-shaped human cortical bone specimens were subjected to cyclic four-point bending fatigue in several steps. The specimens were prepared to control damage localization during four-point bending fatigue cycling and to unambiguously identify resonant modes for NRUS measurements. NRUS measurements were achieved to follow the evolution of the nonlinear hysteretic elastic behavior during fatigue-induced damage. After each fatigue step, a small number of specimens was removed from the protocol and set apart to quantitatively assess the microcrack number density and length using synchrotron radiation micro-computed tomography (SR-µCT). The results showed a significant effect of damage steps on the nonlinear hysteretic elastic behavior. No significant change in the overall length of microcracks was observed in damaged regions compared to the load-free control regions. Only an increased number of shortest microcracks, those in the lowest quartile, was noticed. This was suggestive of newly formed microcracks during the early phases of damage accumulation. The variation of nonlinear hysteretic elastic behavior was significantly correlated to the variation of the density of short microcracks. Our results suggest that the nonlinear hysteretic elastic behavior is sensitive to early bone microdamage. Therefore NRUS technique can be used to monitor fatigue microdamage progression in in vitro experiments.     

The role of the lamellar interface during torsional yielding of human cortical bone

Fragility fractures are a result of alterations in bone quantity, tissue properties, applied loads, or a combination of these factors. The current study addresses the contribution of cortical bone tissue properties to skeletal fragility by characterizing the shear damage accumulation processes which occur during torsional yielding in normal bone. Samples of human femoral cortical bone were loaded in torsion and damaged at a post-yield twist level. The number of microcracks within osteons, interstitial tissue, and along cement lines were assessed using basic fuchsin staining. Damage density measures (number of cracks/mm2) were correlated with stiffness degradation and changes in relaxation. Damaged samples exhibited a wide variation in total microcrack density, ranging from 1.1 to 43.3 cracks/mm2 with a mean density of 19.7 +/- 9.8 cracks/mm2. Lamellar interface cracks comprised more than 75% of the total damage, indicating that the lamellar interface is weak in shear and is a principal site of shear damage accumulation. Damage density was positively correlated with secant stiffness degradation, but only explained 22% of the variability in degradation. In contrast, damage density was uncorrelated with the changes in relaxation, indicating that a simple crack counting measure such as microcrack density was not an appropriate measure of relaxation degradation. Finally, a nonuniform microcrack density distribution was observed, suggesting that internal shear stresses were redistributed within the torsion samples during post-yield loading. The results suggested that the lamellar interface in human cortical bone plays an important role in torsional yielding by keeping cracks physically isolated from each other and delaying microcrack coalescence in order to postpone the inevitable formation of the fatal crack.     

Synchrotron radiation micro-CT at the micrometer scale for the analysis of the three-dimensional morphology of microcracks in human trabecular bone

Bone quality is an important concept to explain bone fragility in addition to bone mass. Among bone quality factors, microdamage which appears in daily life is thought to have a marked impact on bone strength and plays a major role in the repair process. The starting point for all studies designed to further our understanding of how bone microdamage initiate or dissipate energy, or to investigate the impact of age, gender or disease, remains reliable observation and measurement of microdamage. In this study, 3D Synchrotron Radiation (SR) micro-CT at the micrometric scale was coupled to image analysis for the three-dimensional characterization of bone microdamage in human trabecular bone specimens taken from femoral heads. Specimens were imaged by 3D SR micro-CT with a voxel size of 1.4 μm. A new tailored 3D image analysis technique was developed to segment and quantify microcracks. Microcracks from human trabecular bone were observed in different tomographic sections as well as from 3D renderings. New 3D quantitative measurements on the microcrack density and morphology are reported on five specimens. The 3D microcrack density was found between 3.1 and 9.4/mm3 corresponding to a 2D density between 0.55 and 0.76 /mm2. The microcrack length and width measured in 3D on five selected microcrack ranged respectively from 164 μm to 209 μm and 100 μm to 120 μm. This is the first time that various microcracks in unloaded human trabecular bone--from the simplest linear crack to more complex cross-hatch cracks--have been examined and quantified by 3D imaging at this scale. The suspected complex morphology of microcracks is here considerably more evident than in the 2D observations. In conclusion, this technique opens new perspective for the 3D investigation of microcracks and the impact of age, disease or treatment.     

Postfailure modulus strongly affects microcracking and mechanical property change in human iliac cancellous bone: a study using a 2D nonlinear finite element method

A two-dimensional (2D) finite element (FE) method was used to estimate the ability of bone tissue to sustain damage as a function of postfailure modulus. Briefly, 2D nonlinear compact-tension FE models were created from quantitative back-scattered electron images taken of human iliac crest bone specimens. The effects of different postfailure moduli on predicted microcrack propagation were examined. The 2D FE models were used as surrogates for real bone tissues. The crack number was larger in models with higher postfailure modulus, while mean crack length and area were smaller in these models. The rate of stiffness reduction was greater in the models with lower postfailure modulus. Hence, the current results supported the hypothesis that hard tissue postfailure properties have strong effects on bone microdamage morphology and the rate of change in apparent mechanical properties.     

Computational determination of the critical microcrack size that causes a remodeling response in a trabecula: a feasibility study

Bone is a living tissue, which undergoes continuous renewal to repair local defects. Two separate processes, adaptation and remodeling, are involved when a defect appears. The defect produces stress concentrations that provoke regional adaptation, and is gradually repaired, first by resorption and then by deposition of new bone. Using a mathematical formulation of the adaptation mechanism in trabeculae of cancellous bone, we hypothesize that in some cases, where a microcrack is small enough relative to the dimensions of the trabecula, the adaptation response of the whole trabecula may be sufficient to regain homeostatic mechanical conditions (with no need for a remodeling process). The simulation results showed that for trabeculae with nominal length of 900 microm and nominal thickness of 80-800 microm, a microcrack with minimal length of 48 microm and minimal depth of 13% of the trabecula's thickness was required to initiate a remodeling process. A longer (100 microm) but shallower (depth of 7% of the trabecula's thickness) crack also triggered remodeling. These computational results support our hypothesis that when a microcrack small enough relative to the dimensions of the trabecula occurs, adaptation of the whole trabecula may be sufficient to regain homeostatic mechanical conditions with no need for a local remodeling process.     

The effect of cement creep and cement fatigue damage on the micromechanics of the cement–bone interface

The cement–bone interface provides fixation for the cement mantle within the bone. The cement–bone interface is affected by fatigue loading in terms of fatigue damage or microcracks and creep, both mostly in the cement. This study investigates how fatigue damage and cement creep separately affect the mechanical response of the cement–bone interface at various load levels in terms of plastic displacement and crack formation. Two FEA models were created, which were based on micro-computed tomography data of two physical cement–bone interface specimens. These models were subjected to tensile fatigue loads with four different magnitudes. Three deformation modes of the cement were considered: ‘only creep’, ‘only damage’ or ‘creep and damage’. The interfacial plastic deformation, the crack reduction as a result of creep and the interfacial stresses in the bone were monitored. The results demonstrate that, although some models failed early, the majority of plastic displacement was caused by fatigue damage, rather than cement creep. However, cement creep does decrease the crack formation in the cement up to 20%. Finally, while cement creep hardly influences the stress levels in the bone, fatigue damage of the cement considerably increases the stress levels in the bone. We conclude that at low load levels the plastic displacement is mainly caused by creep. At moderate to high load levels, however, the plastic displacement is dominated by fatigue damage and is hardly affected by creep, although creep reduced the number of cracks in moderate to high load region.     

Fatigue microcracks that initiate fracture are located near elevated intracortical porosity but not elevated mineralization

In vivo microcracks in cortical bone are typically observed within more highly mineralized interstitial tissue, but postmortem investigations are inherently limited to cracks that did not lead to fracture which may be misleading with respect to understanding fracture mechanisms. We hypothesized that the one fatigue microcrack which initiates fracture is located spatially adjacent to elevated intracortical porosity but not elevated mineralization. Therefore, the spatial correlation between intracortical porosity, elevated mineralization, and fatigue microdamage was investigated by combining, for the first time, sequential, nondestructive, three-dimensional micro-computed tomography (micro-CT) measurements of each in cortical bone specimens subjected to compressive fatigue loading followed by a tensile overload to fracture. Fatigue loading resulted in significant microdamage accumulation and compromised mechanical properties upon tensile overload compared to control specimens. The microdamage that initiated fracture upon tensile overload was able to be identified in all fatigue-loaded specimens using contrast-enhanced micro-CT and registered images. Two-point (or pair) correlation functions revealed a spatial correlation between microdamage at the fracture initiation site and intracortical porosity, but not highly mineralized tissue, confirming the hypothesis. This difference was unique to the fracture initiation site. Intracortical porosity and highly mineralized tissue exhibited a significantly lower and higher probability, respectively, of being located spatially adjacent to all sites of microdamage compared to the fracture initiation site. Therefore, the results of this study suggest that human cortical bone is tolerant of most microcracks, which are generally compartmentalized within the more highly mineralized interstitial tissue, but a single microcrack of sufficient size located in spatial proximity to intracortical porosity can compromise fracture resistance.     

Axial-shear interaction effects on microdamage in bovine tibial trabecular bone

Because many osteoporotic fractures occur during a fall, understanding the effect of off-axis loads on initiation and propagation of microdamage in trabecular bone should provide further insight into the biomechanics of age-related fractures. Fourteen on-axis cylindrical specimens were prepared from 12 bovine tibiae. Fluorescent stains were used to label the microdamage due to a sequence of compressive and torsional damaging loads. The mean decrease in Young's modulus was over four times greater than that in the shear modulus after the compressive overload, while there was no difference between the decrease in the axial and torsional stiffnesses after the torsional overload. The total microcrack density due to compression was uniform across the radius of the cylindrical specimens, while the mean density of microcracks due to torsional overloading increased from the axis of the cylindrical specimen to the circumference. The high density of microcracks near the axis of the specimen following torsional overloading was unexpected because of the low strains. Nearly 40% of the microcracks due to torsion propagated from pre-existing microcracks caused by axial compression, indicating that existing microcracks may extend at relatively low strain if the loading mode changes. The propagating microcracks were, on average, longer than the initiating microcracks due to either compressive or torsional loading. Damage due to axial compression appears to increase the susceptibility of trabecular bone to damage propagation during subsequent torsional loads, but it has little effect on the elastic properties in shear.