We demonstrated, using 3D specimen-specific geometry and a large sample, that the variability of microstructural properties within a bone is different between female and male vertebral cancellous bone, between white and black races and between T12 and L1 vertebrae in the same individual. We also found that the variability of microstructural properties within a bone changes differently with age between genders, races and vertebrae. Consistent with previous predictions [55
], we demonstrate that increases in microstructural variability contribute to increases in stress variability and decreases in bone stiffness independent from the contribution from intra-specimen average of the same microstructural parameter. Further, microstructural variability had a significant effect on bone stiffness and stress variability which was not accounted for by average microstructural measures including bone quantity, connectivity and anisotropy.
There are large differences in vertebral fracture risk between men and women. However, the exact sources of this difference are unclear. Very few studies noted gender-specific differences in vertebral cancellous bone microstructure [36
]. No difference was noted between men and women in vertebral cancellous bone strength, bone volume fraction or ash density, or in their respective relationship with age [12
] when bone samples of similar size and anatomic location were examined. Differences in the anisotropy, bone loss patterns and transverse strength have been observed between male and female vertebral cancellous bone in some age groups after age-stratification. However, the microstructural changes were not consistent from age group to age group so as to explain the strength changes. For example, a greater distance between horizontal trabeculae was noted in females from above 75 years of age compared to females in the 50–75 years of age group. This difference suggests a greater susceptibility to fracture in the former, especially when loaded in the horizontal direction. However, when the samples were tested by loading horizontally, their strength was not different (0.31±0.12 MPa to 0.30±0.18 MPa) (others found similar results [36
]), despite predictions to the contrary [21
]. Therefore, differences in uniaxial strength and average microstructure between male and female vertebral cancellous bone do not appear to explain the large differences in occurrence of vertebral fractures between them. The current results show that women have significantly greater cancellous bone variability than men in the anterior portion of T12 and L1 vertebrae. This previously unobserved difference in bone quality between males and females may be important in explaining the higher propensity of female vertebrae to fracture. There is some clinical evidence that the heterogeneity of cancellous bone properties can separate vertebral fracture patients from non-fracture patients [10
] suggesting that bone heterogeneity may be useful for predicting vertebral fracture risk.
Some of the differences in fracture risk between men and women may be attributable to the larger crossectional area of male vertebrae and their greater ability to expand with age than female vertebrae [4
]. It was predicted that female vertebrae will lose strength at a higher rate than male vertebrae with age, primarily due to a greater loss of BMD in females than in males rather than due to crossectional area changes [4
]. On the other hand, experimentally measured vertebral strength appears to decrease at a similar rate with age in men and women [37
]. Nonetheless, a greater loss of whole vertebral bone mass with age in females than in males, together with identical rate of age-related bone mass loss in the central anterior region in Mosekilde studies, suggests that the loss of bone mass in women is more regional than in men. Such a bone loss pattern would result in an increased heterogeneity of bone mass in women compared to men. Together, these results suggest that the differences in microstructural heterogeneity observed between men and women in the current study might exist at the whole bone level as well.
There are differences in vertebral fracture risk between black and white races as well; blacks have a lower incidence of fractures (eg. [49
]). Blacks are generally reported to have higher bone mass than whites, which is generally attributed to different rates of bone mass accumulation during growth [16
]. However, the differences in vertebral cancellous bone architecture between blacks and whites are less well understood. BV/TV, trabecular thickness and turnover rates were generally evaluated from iliac crest bone and the differences between blacks and whites are largely dependent upon gender and geographic origin. Higher Tb.Th has been reported for African blacks than for whites in the iliac crest bone [44
]. BV/TV has also been reported to be higher in African blacks than in whites but for men only [44
]. While turnover rate has been reported to be higher in African blacks than in whites [43
], it has been reported to be lower in American blacks than in whites [18
]. The relationship of microstructural parameters with age was also both gender- and race-dependent for African blacks: Trabecular thickness decreased in all groups except black males, trabecular number decreased in all groups except black females and, analogously with changes in trabecular number, trabecular separation increased in all groups except black females [44
]. It is difficult to know to what extent the previous findings from iliac crest can be extrapolated to vertebra due to substantial differences between iliac crest and vertebral cancellous bone properties within an individual [38
]. Nevertheless, given these previous results, it is not surprising that we have found high order interactions of race with age and gender for our parameters. To our knowledge, age-related changes in cancellous bone heterogeneity, moreover, differences by race and gender have not been documented previously. Further understanding of these differences may provide insight into the role of genetic and environmental factors in regulation of bone microstructure.
The significant differences observed in the variability of cancellous microstructure between T12 and L1 were less than 10% but were greater than were observed for the average microstructure () [59
]. The variability parameters were also less well-correlated between the T12 and L1 of the same individual than were the average parameters () [59
]. These results suggest that while variations in average bone microstructure can be explained by factors common to the T12-L1 segment of the spine, the organization of this microstructure as measured by the variability of trabecular morphology is more dependent on local factors. The differences in microstructural variability between vertebrae that are far apart is expected to be greater than between the T12 and L1 [58
]. As such, variability of trabecular microstructure within a vertebra may be a sufficiently sensitive parameter to detect small differences between vertebrae that are similar in their average microstructure.
The increase in VMCV and VMExp/σapp
with increasing variability of the microstructure is consistent with previous findings that cancellous bone strength decreases with increasing VMCV [14
] and vertebral strength decreases with increasing heterogeneity of bone mass [8
]. Together with the observation that increased microdamage content is associated with increased VMCV and VMExp/σapp
in vertebral bone, the current results suggest that microstructural heterogeneity is a measure of stress concentration in cancellous bone. The effect of microstructural variability on VMCV seems more prominent than on VMExp/σapp
, possibly due to the fact that VMExp/σapp
is a ratio of volume averages whereas the stress variability is directly incorporated in VMCV. The results of our multiple regression analyses indicate that microstructural heterogeneity contributes to trabecular stress variability independently from the average of the same parameter and independently from a set of conventionally measured microstructural parameters. These results suggest that vertebral strength prediction would be improved when microstructural heterogeneity information is available.
Consideration must be given to qualities of bone other than uniaxial strength, however. The existing data indicate that bone heterogeneity can reflect additional aspects of vertebral failure such as structural ductility [60
] and fatigue resistance [33
]. Further, cellular materials with irregularly shaped and distributed cells like cancellous bone have higher creep rates than those with regular cells even if they are stiffer [2
]. Similarly, analysis of the effect of random removal of struts within a foam on the creep rate indicates that removal of only a few percent of the struts can dramatically increase the creep rate by one to two orders of magnitude whereas the effect of uniform thinning is not as dramatic [23
]. The differences we found in microstructural variability between genders, race and anatomic sites may manifest differences in the fatigue and/or creep behavior of bone between these populations. Nonetheless, due to the complex nature of in vivo fractures, it is important that clinical data are obtained to ultimately test the utility of bone heterogeneity in predicting fracture.
Some limitations outlined in our previous study also apply to the current study [59
]. Briefly, voxel size used in the reconstruction of image volumes, homogeneous element properties used in the FE analyses, exclusion of vertebral regions other than the central anterior portion of the vertebral bodies and use of stereologic methods in calculation of microstructural parameters were identified as limitations. Slice-by-slice stereologic calculation of trabecular microstructure is subject to errors and will provide values that are different from those calculated by direct methods [9
]. However, dividing the sample volume in smaller regions of interest was necessary and the slice-by-slice approach provided a large number of data points from which distribution parameters could be calculated. It should be noted that BV/TV calculated from the slice-by-slice approach and that directly calculated by voxel counting had over 99% correlation (data not shown) in the current sample. This is consistent with a previous report that microstructural parameters calculated using direct methods are highly correlated to those calculated using a model-based method [9
]. While microstructural heterogeneity has information content distinct from the average as well as from connectivity and anisotropy, its relationship with other microstructural properties such plate- vs rod-likeness of trabeculae remains to be elucidated. Also, consideration of the spatial distribution in addition to the statistical distribution properties of microstructure could provide further information on the nature of the heterogeneity [61
]. This study was also limited to an investigation of the cancellous tissue from the anterior region. It is desirable to ultimately extend the results to the whole vertebrae. However, it is important to note that cancellous bone properties from the anterior region are strongly associated with whole vertebral strength [8
]. Thus, our study, while limited, is still very relevant to overall vertebral bone quality. The effect of modulus heterogeneity on finite element calculations of cancellous bone stiffness and stress distributions has been reported to be 6–20% [3
]. However, the determination of mineralization distributions and modulus assignment based on micro-CT gray levels can be problematic due to artifacts inherent in the micro-CT scans [25
] or due to lack of established relationships to accurately convert these values to modulus. Anatomic site, species and tissue processing differences could also result in a difference in the magnitude of this effect. It should also be pointed out that elastic moduli directly measured by nanoindentation had less variability than estimated by micro-CT gray levels and had little to no effect in FE calculation of apparent modulus of human vertebral bone in recent studies [7
]. Thus, these limitations are expected to have minor effects on our results but not affect our conclusions about cancellous tissue. In the end, the use of these conditions allows for direct comparisons between the current study and the previous ones that focused on average microstructural properties and trabecular stress variability [14
The current study did not focus on the biological factors underlying microstructural heterogeneity, although mechanical loading might be expected to play a role. For example, it has been reported that low magnitude vibrations caused changes in the microstructure of trabecular bone which were associated with a reduced skewness of trabecular stresses and strains [24
]. Previous work also suggested that status of the intervertebral disc and the end plate is associated with the regional microstructural and mechanical properties of cancellous bone [26
], stress distributions within a vertebra [13
] and the whole vertebral mechanical properties [39
The causal nature of these associations currently needs further research
A notable observation was the considerable degree of high order interaction in the data set. We consider the results that were significant only within a sub-group of interacting parameters to be of secondary nature since high order interactions result in a reduced sample size. These findings should be confirmed by larger scale studies.
In conclusion, microstructural variability contributes to trabecular stress distribution and cancellous bone stiffness independent from average microstructure in human vertebral bone. Microstructural variability is anatomic site-, age-, gender- and race-dependent. Consideration of microstructural variability may provide insight into the understanding of bone fragility and improve assessment of vertebral fracture risk.