Development of an in vivo rabbit ulnar loading model.

Ulnar and tibial cyclic compression in rats and mice have become the preferred animal models for investigating the effects of mechanical loading on bone modeling/remodeling. Unlike rodents, rabbits provide a larger bone volume and normally exhibit intracortical Haversian remodeling, which may be advantageous for investigating mechanobiology and pharmaceutical interventions in cortical bone. Therefore, the objective of this study was to develop and validate an in vivo rabbit ulnar loading model. Ulnar tissue strains during loading of intact forelimbs were characterized and calibrated to applied loads using strain gauge measurements and specimen-specific finite element models. Periosteal bone formation in response to varying strain levels was measured by dynamic histomorphometry at the location of maximum strain in the ulnar diaphysis. Ulnae loaded at 3000 microstrain did not exhibit periosteal bone formation greater than the contralateral controls. Ulnae loaded at 3500, 4000, and 4500 microstrain exhibited a dose-dependent increase in periosteal mineralizing surface (MS/BS) compared with contralateral controls during the second week of loading. Ulnae loaded at 4500 microstrain exhibited the most robust response with significantly increased MS/BS at multiple time points extending at least 2weeks after loading was ceased. Ulnae loaded at 5250 microstrain exhibited significant woven bone formation. Rabbits required greater strain levels to produce lamellar and woven bone on periosteal surfaces compared with rats and mice, perhaps due to lower basal levels of MS/BS. In summary, bone adaptation during rabbit ulnar loading was tightly controlled and may provide a translatable model for human bone biology in preclinical investigations of metabolic bone disease and pharmacological treatments.

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.

A probabilistic damage model based on direct 3-D correlation of strain to damage formation following fatigue loading of rat femora.

Microdamage accumulates in bone due to repetitive or excessive mechanical loading, and accumulation of damage can lead to an increase in fracture susceptibility. Understanding the stress or strain criterion for damage formation would allow improved predictive modeling to better assess experimental results or evaluate training regimens. Finite element models coupled with three-dimensional measurements of damage were used to directly correlate damage formation to the local strain state in whole rat femora subjected to three-point bending fatigue. Images of accumulated damage from contrast-enhanced micro-CT were overlaid onto the calculated strain result to determine the strain associated with damage. Most microdamage accumulated in areas where the first principal strain exceeded 0.5%, but damage also occurred at lower strains when applied over sufficiently large volumes. As such, a single threshold strain was not a good predictor of damage. In order to capture the apparently stochastic nature of damage formation, a Weibull statistical model was applied. The model provided a good fit to the data, and a fit based on a subset of the data was able to predict the results in the remaining samples with an RMS error of 17%. These results demonstrate that damage formation is dependent on principal strain, but has a random component that is likely due to the presence of pores or flaws smaller than the resolution of the model that act as stress concentrations in bone.

Detection of fatigue microdamage in whole rat femora using contrast-enhanced micro-computed tomography.

Microdamage in bone tissue is typically studied using destructive, two-dimensional histological techniques. Contrast-enhanced micro-computed tomography (micro-CT) was recently demonstrated to enable non-destructive, three-dimensional (3-D) detection of microdamage in machined cortical and trabecular bone specimens in vitro. However, the accumulation of microdamage in whole bones is influenced by variations in the magnitude and mode of loading due to the complex whole bone morphology. Therefore, the objective of this study was to detect the presence, spatial location, and accumulation of fatigue microdamage in whole rat femora in vitro using micro-CT with a BaSO(4) contrast agent. Microdamage was detected and observed to accumulate at specific spatial locations within the cortex of femora loaded in cyclic three-point bending to a 5% or 10% reduction in secant modulus. The ratio of the segmented BaSO(4) stain volume (SV) to the total volume (TV) of cortical bone was adopted as a measure of damage. The amount of microdamage measured by micro-CT (SV/TV) was significantly greater for both loaded groups compared to the control group (p<0.05), but the difference between loaded groups was not statistically significant. At least one distinct region of microdamage, as indicated by the segmented SV, was observed in 85% of loaded specimens. A specimen-specific finite element model confirmed elevated tensile principal strains localized in regions of tissue corresponding to the accumulated microdamage. These regions were not always located where one might expect a priori based upon Euler-Bernoulli beam theory, demonstrating the utility of contrast-enhanced micro-CT for non-destructive, 3-D detection of fatigue microdamage in whole bones in vitro.

Contrast-enhanced micro-computed tomography of fatigue microdamage accumulation in human cortical bone.

Conventional methods used to image and quantify microdamage accumulation in bone are limited to histological sections, which are inherently invasive, destructive, two-dimensional, and tedious. These limitations inhibit investigation of microdamage accumulation with respect to volumetric spatial variation in mechanical loading, bone mineral density, and microarchitecture. Therefore, the objective of this study was to investigate non-destructive, three-dimensional (3-D) detection of microdamage accumulation in human cortical bone using contrast-enhanced micro-computed tomography (micro-CT), and to validate micro-CT measurements against conventional histological methods. Unloaded controls and specimens loaded in cyclic uniaxial tension to a 5% and 10% reduction in secant modulus were labeled with a precipitated BaSO4 stain for micro-CT and basic fuchsin for histomorphometry. Linear microcracks were similarly labeled by BaSO4 and basic fuchsin as shown by backscattered electron microscopy and light microscopy, respectively. The higher X-ray attenuation of BaSO4 relative to the bone extracellular matrix provided enhanced contrast for the detection of damage that was otherwise not able to be detected by micro-CT prior to staining. Therefore, contrast-enhanced micro-CT was able to nondestructively detect the presence, 3-D spatial location, and accumulation of fatigue microdamage in human cortical bone specimens in vitro. Microdamage accumulation was quantified on segmented micro-CT reconstructions as the ratio of BaSO4 stain volume (SV) to total bone volume (BV). The amount of microdamage measured by both micro-CT (SV/BV) and histomorphometry (Cr.N, Cr.Dn, Cr.S.Dn) progressively increased from unloaded controls to specimens loaded to a 5% and 10% reduction in secant modulus (p < 0.001). Group means for micro-CT measurements of damage accumulation were strongly correlated to those using histomorphometry (p < 0.05), validating the new methods. Limitations of the new methods in the present study included that the precipitated BaSO4 stain was non-specific and non-biocompatible, and that micro-CT measurements exhibited greater variability compared to conventional histology. Nonetheless, contrast-enhanced micro-CT enabled non-destructive imaging and 3-D spatial information, which are not possible using conventional histological methods.

Detection of dentinal cracks using contrast-enhanced micro-computed tomography.

A new technique using contrast enhanced micro-computed tomography (micro-CT) was developed to improve the ability to detect dentinal cracks in teeth and assess associated risks to oral health. Extracted, whole human molars that exhibited visual evidence of external cracks following extraction and machined, partially fractured elephant dentin specimens were labeled by BaSO(4) precipitation and imaged by micro-CT. Contrast-enhanced micro-CT was demonstrated in vitro to enable non-destructive, 3-D imaging of the presence, morphology and spatial location of dentinal cracks in whole human molars and machined specimens. BaSO(4) staining provided enhanced contrast for the detection of cracks that could not be detected prior to staining. Backscattered SEM micrographs showed that BaSO(4) was precipitated on the surfaces of dentinal cracks and within adjacent tubules. The new methods demonstrated in this study are expected to be useful for clinical and scientific studies investigating the etiology and treatment of dentinal cracks in teeth.