Age-related changes in porosity and mineralization and in-service damage accumulation

It has been proposed that bone damageability (i.e. bone's susceptibility to formation of damage) increases with the elevation or suppression of bone turnover. Suppression of turnover via bisphosphonates increases local bone mineralization, which theoretically should increase the susceptibility of bone to microcrack formation. Elevation of bone turnover has also been proposed to increase bone microdamage through an increase in bone intracortical porosity and local stresses and strains. The goal of this paper was to investigate the above proposals, i.e., whether or not increases to mineral content and porosity increase bone in-service damageability. To do this, we measured in vivo diffuse damage area (Df.Dm.Ar, %) and microcrack density (Cr.Dn) (cracks/mm(2)) in the same specimen from human cortical bone of the midshaft of the proximal femur obtained from cadavers with an age range of eight decades and examined their relationships with porosity, mineralization and age. Results of this study showed that Cr.Dn and Df.Dm.Ar increased with a decrease in bulk mineralization. This finding does not appear to support the proposal that damage accumulation increases with low bone turnover that results in increases mineralization. It was proposed however that the negative correlation between damage accumulation and mineralization may be attributed to highly mineralized regions of bone existing with under-mineralized regions resulting in an overall decrease in average bone mineralization. It was also found that microdamage accumulates with increasing porosity which does appear to support the proposal that elevated bone turnover that results in increased porosity can accelerate microdamage accumulation. Finally, it was shown that linear microcracks and Df.Dm.Ar accumulate with age differently, but because they correlate with each other, one may be the precursor for the other.     

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.     

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.     

Detection of trabecular bone microdamage by micro-computed tomography

Microdamage is an important component of bone quality and affects bone remodeling. Improved techniques to assess microdamage without the need for histological sectioning would provide insight into the role of microdamage in trabecular bone strength by allowing the spatial distribution of damage within the trabecular microstructure to be measured. Nineteen cylindrical trabecular bone specimens were prepared and assigned to two groups. The specimens in group I were damaged to 3% compressive strain and labeled with BaSO(4). Group II was not loaded, but was labeled with BaSO(4). Micro-computed tomography (Micro-CT) images of the specimens were obtained at 10 microm resolution. The median intensity of the treated bone tissue was compared between groups. Thresholding was also used to measure the damaged area fraction in the micro-CT scans. The histologically measured damaged area fraction, the median CT intensity, and the micro-CT measured damaged area fraction were all higher in the loaded group than in the unloaded group, indicating that the micro-CT images could differentiate the damaged specimen group from the unloaded specimens. The histologically measured damaged area fraction was positively correlated with the micro-CT measured damaged area fraction and with the median CT intensity of the bone, indicating that the micro-CT images can detect microdamage in trabecular bone with sufficient accuracy to differentiate damage levels between samples. This technique provides a means to non-invasively assess the three-dimensional distribution of microdamage within trabecular bone test specimens and could be used to gain insight into the role of trabecular architecture in microdamage formation.     

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.     

Microdamage in porcine alveolar bone due to functional and orthodontic loading

Bone remodelling has been associated with microdamage. The aim of this study was to investigate the presence of microdamage in the alveolar bone and its potential role in the initiation of bone remodelling following the application of an orthodontic load. The three-dimensional morphology of the alveolar bone was investigated by means of high resolution micro-CT scanning. In 25, 3-month-old, male Danish land-race pigs, the alveolar bone around the lower right and left first molars was analysed. The right first molar was moved buccally with a force of 130 cN by means of a custom-made cantilever made of a TMA 0.017 x 0.025 inch wire. The left molar was left untreated. After 1, 2, 4, 7 and 15 days of treatment the regions containing the right and left molars were excised and en bloc stained in basic fuchsin and the presence of microdamage detected. Diffuse damage was present in the alveolar bone of both the treated and the untreated teeth on both sides. On the lingual sides, diffuse damage showed the same orientation as the periodontal fibres. Bone microcracks were also detected on both the treated and untreated teeth. On the buccal surfaces they where often observed in close proximity to scalloped resorption surfaces. After 1 day of treatment, the presence of microcracks on the buccal-treated side was particularly marked. To conclude, bone microdamage is present in porcine alveolar bone in form of both microcracks and diffuse damage, suggesting that microdamage-driven remodelling also occurs in the alveolar bone. The presence of bone microcracks in the direction of the orthodontic force at day 1 suggests that they could represent the first damage induced by the orthodontic load that has to be repaired.     

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.     

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.     

Structural role of osteocalcin and osteopontin in energy dissipation in bone

Non-collagenous proteins are a vital component of bone matrix. Amongst them, osteocalcin (OC) and osteopontin (OPN) hold special significance due to their intimate interaction with the mineral and collagenous matrix in bone. Both proteins have been associated with microdamage and fracture, but their structural role in energy dissipation is unclear. This study used bone tissue from genetic deficient mice lacking OC and/or OPN and subjected them to a series of creep-fatigue-creep tests. To this end, whole tibiae were loaded in four-point bending to 70% stiffness loss which captured the three characteristic phases of fatigue associated with initiation, propagation, and coalescence of microdamage. Fatigue loading preceded and followed creep tests to determine creep and dampening parameters. Microdamage in the form of linear microcracks and diffuse damage were analyzed by histology. It was shown that OC and OPN were ‘activated’ following stiffness loss associated with fatigue damage where they facilitated creep and dampening parameters (i.e. increased energy dissipation). More specifically, post-fatigue creep rate and dampening were significantly greater in wild-types (WTs) than genetic deficient mice (p < 0.05). These results were supported by microdamage analysis which showed significant increase in creep-associated diffuse damage formation in WTs compared to genetic deficient groups (p < 0.05). Based on these findings, we propose that during local yield events, OC and OPN rely on ionic interactions of their charged side chains and on hydrogen bonding to dissipate energy in bone.     

Spatial correlations of trabecular bone microdamage with local stresses and strains using rigid image registration

Although microdamage is known to accumulate in trabecular bone with overloading and aging, the tissue-level stresses and strains associated with local bone failure are not well known. Local correlation of microdamage with microstructural stresses and strains requires methods to accurately register histological sections with micro-computed tomography (micro-CT) based finite element models. In addition, the resolution of correlation (i.e., grid size) selected for analysis may affect the observed results. Therefore, an automated, repeatable, and accurate image registration algorithm was developed to determine the range of local stresses and strains associated with microdamage initiation. Using a two-dimensional rigid registration algorithm, bone structures from histology and micro-CT imaging were aligned. Once aligned, microdamaged regions were spatially correlated with local stresses and strains obtained from micro-CT based finite element analysis. Using this more sophisticated registration technique, we were able to analyze the effects of varying spatial grid resolution on local stresses and strains initiating microdamage. The results indicated that grid refinement to the individual pixel level (pixel-by-pixel method) more precisely defined the range of microdamage initiation compared to manually selected individual damaged and undamaged trabeculae. Using the pixel-by-pixel method, we confirmed that trabecular bone from younger cows sustained higher local strains prior to microdamage initiation compared to older bone.     

Trabecular bone microdamage and microstructural stresses under uniaxial compression

The balance between local remodeling and accumulation of trabecular bone microdamage is believed to play an important role in the maintenance of skeletal integrity. However, the local mechanical parameters associated with microdamage initiation are not well understood. Using histological damage labeling, micro-CT imaging, and image-based finite element analysis, regions of trabecular bone microdamage were detected and registered to estimated microstructural von Mises effective stresses and strains, maximum principal stresses and strains, and strain energy density (SED). Bovine tibial trabecular bone cores underwent a stepwise uniaxial compression routine in which specimens were micro-CT imaged following each compression step. The results indicate that the mode of trabecular failure observed by micro-CT imaging agreed well with the polarity and distribution of stresses within an individual trabecula. Analysis of on-axis subsections within specimens provided significant positive relationships between microdamage and each estimated tissue stress, strain and SED parameter. In a more localized analysis, individual microdamaged and undamaged trabeculae were extracted from specimens loaded within the elastic region and to the apparent yield point. As expected, damaged trabeculae in both groups possessed significantly higher local stresses and strains than undamaged trabeculae. The results also indicated that microdamage initiation occurred prior to apparent yield at local principal stresses in the range of 88-121 MPa for compression and 35-43 MPa for tension and local principal strains of 0.46-0.63% in compression and 0.18-0.24% in tension. These data provide an important step towards understanding factors contributing to microdamage initiation and establishing local failure criteria for normal and diseased trabecular bone.     

Heterogeneous glycation of cancellous bone and its association with bone quality and fragility

Non-enzymatic glycation (NEG) and enzymatic biochemical processes create crosslinks that modify the extracellular matrix (ECM) and affect the turnover of bone tissue. Because NEG affects turnover and turnover at the local level affects microarchitecture and formation and removal of microdamage, we hypothesized that NEG in cancellous bone is heterogeneous and accounts partly for the contribution of microarchitecture and microdamage on bone fragility. Human trabecular bone cores from 23 donors were subjected to compression tests. Mechanically tested cores as well as an additional 19 cores were stained with lead-uranyl acetate and imaged to determine microarchitecture and measure microdamage. Post-yield mechanical properties were measured and damaged trabeculae were extracted from a subset of specimens and characterized for the morphology of induced microdamage. Tested specimens and extracted trabeculae were quantified for enzymatic and non-enzymatic crosslink content using a colorimetric assay and Ultra-high Performance Liquid Chromatography (UPLC). Results show that an increase in enzymatic crosslinks was beneficial for bone where they were associated with increased toughness and decreased microdamage. Conversely, bone with increased NEG required less strain to reach failure and were less tough. NEG heterogeneously modified trabecular microarchitecture where high amounts of NEG crosslinks were found in trabecular rods and with the mechanically deleterious form of microdamage (linear microcracks). The extent of NEG in tibial cancellous bone was the dominant predictor of bone fragility and was associated with changes in microarchitecture and microdamage.     

Modeling microdamage behavior of cortical bone

Bone is a complex material which exhibits several hierarchical levels of structural organization. At the submicron-scale, the local tissue porosity gives rise to discontinuities in the bone matrix which have been shown to influence damage behavior. Computational tools to model the damage behavior of bone at different length scales are mostly based on finite element (FE) analysis, with a range of algorithms developed for this purpose. Although the local mechanical behavior of bone tissue is influenced by microstructural features such as bone canals and osteocyte lacunae, they are often not considered in FE damage models due to the high computational cost required to simulate across several length scales, i.e., from the loads applied at the organ level down to the stresses and strains around bone canals and osteocyte lacunae. Hence, the aim of the current study was twofold: First, a multilevel FE framework was developed to compute, starting from the loads applied at the whole bone scale, the local mechanical forces acting at the micrometer and submicrometer level. Second, three simple microdamage simulation procedures based on element removal were developed and applied to bone samples at the submicrometer-scale, where cortical microporosity is included. The present microdamage algorithm produced a qualitatively analogous behavior to previous experimental tests based on stepwise mechanical compression combined with in situ synchrotron radiation computed tomography. Our results demonstrate the feasibility of simulating microdamage at a physiologically relevant scale using an image-based meshing technique and multilevel FE analysis; this allows relating microdamage behavior to intracortical bone microstructure.     

Age-dependent fatigue behaviour of human cortical bone

Despite a general understanding that bone quality contributes to skeletal fragility, very little information exits on the age-dependent fatigue behavior of human bone. In this study four-point bending fatigue tests were conducted on aging bone in conjunction with the analysis of stiffness loss and preliminary investigation of nanoindentation based measurements of local tissue stiffness and histological evaluation of resultant tensile and compressive damage to identify the damage mechanism responsible for the increase in age-related bone fragility. The results obtained show that there is an exponential decrease in fatigue life with age, and old bone exhibits different modulus degradation profiles than young bone. In addition, this study provides preliminary evidence indicating that during fatigue loading, younger bone formed diffuse damage, lost local tissue stiffness on the tensile side. Older bone, in contrast, formed linear microcracks lost local tissue stiffness on the compressive side. Thus, the propensity of aging human bone to form more linear microcracks than diffuse damage may be a significant contributor to bone quality, and age related fragility in bone.     

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.     

Determinants of Microdamage in Elderly Human Vertebral Trabecular Bone

Previous studies have shown that microdamage accumulates in bone as a result of physiological loading and occurs naturally in human trabecular bone. The purpose of this study was to determine the factors associated with pre-existing microdamage in human vertebral trabecular bone, namely age, architecture, hardness, mineral and organic matrix. Trabecular bone cores were collected from human L2 vertebrae (n = 53) from donors 54–95 years of age (22 men and 30 women, 1 unknown) and previous cited parameters were evaluated. Collagen cross-link content (PYD, DPD, PEN and % of collagen) was measured on surrounding trabecular bone. We found that determinants of microdamage were mostly the age of donors, architecture, mineral characteristics and mature enzymatic cross-links. Moreover, linear microcracks were mostly associated with the bone matrix characteristics whereas diffuse damage was associated with architecture. We conclude that linear and diffuse types of microdamage seemed to have different determinants, with age being critical for both types.     

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.     

Bone remodelling algorithms incorporating both strain and microdamage stimuli

Biomechanical theories to predict bone remodelling have used either mechanical strain or microdamage as the stimulus driving cellular responses. Even though experimental data have implicated both stimuli in bone cell regulation, a mechano-regulatory system incorporating both stimuli has not yet been proposed. In this paper, we test the hypothesis that bone remodelling may be regulated by signals due to both strain and microdamage. Four mechano-regulation algorithms are studied where the stimulus is: strain, damage, combined strain/damage, and either strain or damage with damage-adaptive remodelling prioritised when damage is above a critical level. Each algorithm is implemented with both bone lining cell (surface) sensors and osteocyte cell (internal) sensors. Each algorithm is applied to prediction of a bone multicellular unit (BMU) remodelling on the surface of a bone trabecula. It is predicted that a regulatory system capable of responding to changes in either strain or microdamage but which prioritises removal of damaged bone when damage is above a critical level, is the only one that provides a plausible prediction of BMU behaviour. A mechanism for this may be that, below a certain damage threshold, osteocyte processes can sense changes in strain and fluid flow but above the threshold damage interferes with the signalling mechanism, or causes osteocyte apoptosis so that a remodelling response occurs to remove the dead osteocytes.     

Development of a fluorescent light technique for evaluating microdamage in bone subjected to fatigue loading.

A new method using fluorescent light microscopy has been developed to visualize and evaluate bone microdamage. We report the findings of two different experiments with a common aim of comparing the fluorescent light technique to the brightfield method for quantifying microdamage in bone. In Experiment 1, 36 canine femurs were tested in four-point cyclic bending until they had lost between 5 and 43% of their stiffness. The loaded portion of the bone was stained en bloc with basic fuchsin for the presence of damage. Standard point counting techniques were used to calculate fractional damaged area (Dm.Ar = Cr.Ar/B.Ar, mm2/mm2) under brightfield and fluorescent microscopy. In Experiment 2, bone microdamage adjacent to endosseous implants, subjected to fatigue loading (150,000 cycles, 2 Hz and 37 degrees C) ex vivo was examined. The bone around the implant was either allowed to heal (adapted specimen) for 12 weeks after placement in dog mid-femoral diaphyses prior to testing or was loaded immediately to simulate non-healed bone surrounding endosseous implants (non-adapted). Crack numerical density (Cr.Dn = Cr.N/B.Ar, #/mm2), crack surface density (Cr.S.Dn = Tt.Cr.Le/B.Ar, mm/mm2) and fractional damaged area were calculated separately by both techniques in the adapted and non-adapted specimens. In both Experiments 1 and 2, significantly more microdamage was detected by the fluorescent technique than by the brightfield method. Also, there was a trend towards higher intraobserver repeatability when using the fluorescent method. These results suggest that the brightfield technique underestimates microdamage accumulation and that the fluorescent technique better represents the actual amounts of microdamage present. The results demonstrate that the fluorescent method provides an accurate and precise approach for bone microdamage evaluation, and that it improves the prediction of stiffness loss from damage accumulation.     

The effects of tensile-compressive loading mode and microarchitecture on microdamage in human vertebral cancellous bone

The amount of microdamage in bone tissue impairs mechanical performance and may act as a stimulus for bone remodeling. Here we determine how loading mode (tension vs. compression) and microstructure (trabecular microarchitecture, local trabecular thickness, and presence of resorption cavities) influence the number and volume of microdamage sites generated in cancellous bone following a single overload. Twenty paired cylindrical specimens of human vertebral cancellous bone from 10 donors (47–78 years) were mechanically loaded to apparent yield in either compression or tension, and imaged in three dimensions for microarchitecture and microdamage (voxel size 0.7×0.7×5.0 μm³). We found that the overall proportion of damaged tissue was greater (p=0.01) for apparent tension loading (3.9±2.4%, mean±SD) than for apparent compression loading (1.9±1.3%). Individual microdamage sites generated in tension were larger in volume (p<0.001) but not more numerous (p=0.64) than sites in compression. For both loading modes, the proportion of damaged tissue varied more across donors than with bone volume fraction, traditional measures of microarchitecture (trabecular thickness, trabecular separation, etc.), apparent Young?s modulus, or strength. Microdamage tended to occur in regions of greater trabecular thickness but not near observable resorption cavities. Taken together, these findings indicate that, regardless of loading mode, accumulation of microdamage in cancellous bone after monotonic loading to yield is influenced by donor characteristics other than traditional measures of microarchitecture, suggesting a possible role for tissue material properties.