Traits of the skeletal system are coordinately adjusted to establish mechanical homeostasis in response to genetic and environmental factors. Prior work demonstrated that this `complex adaptive' process is not perfect, revealing a two-fold difference in whole bone stiffness of the tibia across a population. Robustness (specifically, total cross-sectional area relative to length) varies widely across skeletal sites and between sexes. However, it is unknown whether the natural variation in whole bone stiffness and strength also varies across skeletal sites and between men and women. We tested the hypotheses that: 1) all major long bones of the appendicular skeleton demonstrate inherent, systemic constraints in the degree to which morphological and compositional traits can be adjusted for a given robustness; and 2) these traits covary in a predictable manner independent of body size and robustness. We assessed the functional relationships among robustness, cortical area (Ct.Ar), cortical tissue mineral density (Ct.TMD), and bone strength index (BSI) across the long bones of the upper and lower limbs of 115 adult men and women. All bones showed a significant (p < 0.001) positive regression between BSI and robustness after adjusting for body size, with slender bones being 1.7–2.3 times less stiff and strong in men and 1.3–2.8 times less stiff and strong in women compared to robust bones. Our findings are the first to document the natural inter-individual variation in whole bone stiffness and strength that exist within populations and that is predictable based on skeletal robustness for all major long bones. Documenting and further understanding this natural variation in strength may be critical for differentially diagnosing and treating skeletal fragility.

Background The twofold greater lifetime risk of fracturing a bone for white women compared with white men and black women has been attributed in part to differences in how the skeletal system accumulates bone mass during growth. On average, women build more slender long bones with less cortical area compared with men. Although slender bones are known to have a naturally lower cortical area compared with wider bones, it remains unclear whether the relatively lower cortical area of women is consistent with their increased slenderness or is reduced beyond that expected for the sex-specific differences in bone size and body size. Whether this sexual dimorphism is consistent with ethnic background and is recapitulated in the widely used mouse model also remains unclear. Questions/purposes We asked (1) do black women build bones with reduced cortical area compared with black men; (2) do white women build bones with reduced cortical area compared with white men; and (3) do female mice build bones with reduced cortical area compared with male mice? Methods Bone strength and cross-sectional morphology of adult human and mouse bone were calculated from quantitative CT images of the femoral midshaft. The data were tested for normality and regression analyses were used to test for differences in cortical area between men and women after adjusting for body size and bone size by general linear model (GLM). Results Linear regression analysis showed that the femurs of black women had 11% lower cortical area compared with those of black men after adjusting for body size and bone size (women: mean = 357.7 mm 2 ; 95% confidence interval [CI], 347.9-367.5 mm 2 ; men: mean = 400.1 mm 2 ; 95% CI, 391.5-408.7 mm 2 ; effect size = 1.2; p \ 0.001, GLM). Likewise, the femurs of white women had 12% less cortical area compared with those of white men after adjusting for body size and bone size (women: mean = 350.1 mm 2 ; 95% CI, 340.4-359.8 mm 2 ; men: mean = 394.3 mm 2 ; 95% CI, 386.5-402.1 mm 2 ; effect size = 1.3; p \ 0.001, GLM). In contrast, female and male femora from recombinant inbred mouse strains showed the opposite trend; femurs from female mice had a 4% larger cortical area compared with those of male mice after adjusting for body size and bone size (female: mean = 0.73 mm 2 ; 95% CI, 0.71-0.74 mm 2 ; male: mean = 0.70 mm 2 ; 95% CI, 0.68-0.71 mm 2 ; effect size = 0.74; p = 0.04, GLM).

Advances in computed tomography (CT) imaging are opening new avenues toward more precise characterization and quantification of connective tissue microarchitecture. In the last two decades, micro-computed tomography (microCT) has significantly augmented destructive methods for the 3D micro-analysis of tissue structure, primarily in the bone research field. Recently, microCT has been employed in combination with contrast agents to generate contrast-enhanced images of soft tissues that are otherwise difficult to visualize due to their native radiodensity. More recent advances in CT technology have enabled ultra-high resolution imaging by utilizing a more powerful nano-focused X-ray source, such as that found in nano-computed tomography (nanoCT) systems. NanoCT imaging has facilitated the expansion of musculoskeletal research by reducing acquisition time and significantly expanding the range of samples that can be imaged in terms of size, age and tissue-type (bone, muscle, tendon, cartilage, vessels and adipose tissue). We present the application and early results of nanoCT imaging in various tissue types and how this ultra-high resolution imaging modality is capable of characterizing microstructures at levels of details previously not possible. Contrast-enhanced imaging techniques to enable soft-tissue visualization and characterization are also outlined.

Accurately estimating whole bone strength is critical for identifying individuals that may benefit from prophylactic treatments aimed at reducing fracture risk. Strength is often estimated from stiffness, but it is not known whether the relationship between stiffness and strength varies with age and sex. Cadaveric proximal femurs (44 Male: 18-78 years; 40 Female: 24-95 years) and radial (36 Male: 18-89 years; 19 Female: 24-95 years) and femoral diaphyses (34 Male: 18-89 years; 19 Female: 24-95 years) were loaded to failure to evaluate how the stiffness-strength relationship varies with age and sex. Strength correlated significantly with stiffness at all sites and for both sexes, as expected. However, females exhibited significantly less strength for the proximal femur (58% difference, p<0.001). Multivariate regressions revealed that stiffness, age and PYD were significant negative independent predictors of strength for the proximal femur

Previously, we showed that cortical mineralization is coordinately adjusted to mechanically offset external bone size differences between A/J (narrow) and C57BL/6J (wide) mouse femora to achieve whole bone strength equivalence at adulthood. The identity of the genes and their interactions that are responsible for establishing this homeostatic state (i.e., canalization) remain unknown. We hypothesize that these inbred strains, whose inter-individual differences in bone structure and material properties mimic that observed among humans, achieve functional homeostasis by differentially adjusting key molecular pathways regulating external bone size and mineralization throughout growth. The cortices of A/J and C57BL/6J male mouse femora were phenotyped and gene expression levels were assessed across growth (i.e. 2, 4, 6, 8, 12, 16-weeks of age). A difference in total cross-sectional area (p < 0.01) and cortical tissue mineral density were apparent between mouse strains by 2-weeks of age and maintained at adulthood (p < 0.01). These phenotypic dissimilarities corresponded to gene expression level differences among key regulatory pathways throughout growth. A/J mice had a 1.55 – 7.65 fold greater expression among genes inhibitory to Wnt pathway induction while genes involved in cortical mineralization were largely upregulated 1.50 – 3.77 fold to compensate for their narrow diaphysis. Additionally, both mouse strains showed an upregulation among Wnt pathway antagonists corresponding to the onset of adult ambulation (i.e., increased physiological loads). This contrasts with other studies showing an increase in Wnt pathway activation following functionally isolated, experimental in vivo loading regimens. A/J and C57BL/6J long bones provide a model to develop a systems-based approach to identify individual genes and the gene-gene interactions that contribute to trait differences between the strains while being involved in the process by which these traits are coordinately adjusted to establish similar levels of mechanical function; thus providing insight into the process of canalization.