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The purpose of this Chapter is to present a detailed description of methods for performing bone Micro-Computed Tomography (microCT) scanning and analysis. MicroCT is an x-ray imaging method capable of visualizing bone at the micro-structural scale, that is, 1-100 µm resolution. MicroCT is the gold-standard method for assessment of 3D bone morphology in studies of small animals. As applied to the small bones of mice or rats, microCT can efficiently and accurately assess bone structure (e.g., cortical bone area [Ct.Ar]) and micro-structure (e.g., trabecular bone volume fraction [Tb.BV/TV]). The particular application described herein is for post mortem mouse femur specimens. The material presented should be generally applicable to many commercially available laboratory microCT systems, although some details are specific to the system used in our lab (Scanco mCT 40; SCANCO Medical AG, Bruttisellen, Switzerland).

Obesity is generally protective against osteoporosis and bone fracture. However, recent studies indicate that the influence of obesity on the skeleton is complex and can be detrimental. We evaluated the effects of a high-fat, obesogenic diet on the femur and radius of 1100 mice (males and females) from the Large-by-Small advanced intercross line (F 34 generation). At age 5 months, bone morphology was assessed by microCT and mechanical properties by three-point bending. Mice raised on a high-fat diet had modestly greater cortical area, bending stiffness, and strength. Size-independent material properties were unaffected by a high-fat diet, indicating that diet influenced bone quantity but not quality. Bone size and mechanical properties were strongly correlated with body mass. However, the increases in many bone traits per unit increase in body mass were less in high-fat diet mice than low-fat diet mice. Thus, although mice raised on a high-fat diet have, on average, bigger and stronger bones than low-fat-fed mice, a high-fat diet diminished the positive relationship between body mass and bone size and whole-bone strength. The findings support the concept that there are diminishing benefits to skeletal health with increasing obesity. © 2019 American Society for Bone and Mineral Research., (© 2019 American Society for Bone and Mineral Research.)

Unlabelled: To examine the link between bone material properties and skeletal fragility, we analyzed the mechanical, histological, biochemical, and spectroscopic properties of bones from a murine model of skeletal fragility (SAMP6). Intact bones from SAMP6 mice are weak and brittle compared with SAMR1 controls, a defect attributed to reduced strength of the bone matrix. The matrix weakness is attributed primarily to poorer organization of collagen fibers and reduced collagen content., Introduction: The contribution of age-related changes in tissue material properties to skeletal fragility is poorly understood. We previously reported that bones from SAMP6 mice are weak and brittle versus age-matched controls. Our present objectives were to use the SAMP6 mouse to assess bone material properties in a model of skeletal fragility and to relate defects in the mechanical properties of bone to the properties of demineralized bone and to the structure and organization of collagen and mineral., Materials and Methods: Femora from 4- and 12-month-old SAMR1 (control) and SAMP6 mice were analyzed using bending and torsional mechanical testing of intact bones, tensile testing of demineralized bone, quantitative histology (including collagen fiber orientation), collagen cross-links biochemistry, and Raman spectroscopic analysis of mineral and collagen., Results: Intact bones from SAMP6 mice have normal elastic properties but inferior failure properties, with 60% lower fracture energy versus SAMR1 controls. The strength defect in SAMP6 bones was associated with a 23% reduction in demineralized bone strength, which in turn was associated with poorer collagen fiber organization, lower collagen content, and higher hydroxylysine levels. However, SAMP6 have normal levels of collagen cross-links and normal apatite mineral structure., Conclusions: Bones from SAMP6 osteoporotic mice are weak and brittle because of a defect in the strength of the bone matrix. This defect is attributed primarily to poorer organization of collagen fibers and reduced collagen content. These findings highlight the role of the collagen component of the bone matrix in influencing skeletal fragility.

Peripheral quantitative computed tomography (pQCT) is increasingly used for measurement of cortical bone geometry and density in mice. We evaluated the accuracy of pQCT for area and density measurements of thin-walled aluminum phantoms and mouse femora. Aluminum tubes with varying wall thicknesses and femora from 1- to 6-month-old C3H/HeJ (C3H) and C57B1/6J (B6) mice (average cortical thickness 0.14-0.29 mm) were scanned at 70- or 90-microm resolution. pQCT values of area were compared to optical values determined after sectioning, while pQCT density (vBMD) was compared to solid aluminum density or correlated to bone ash content. For the aluminum phantoms, the error in pQCT area and density depended strongly on wall thickness, and density was consistently underestimated. For mouse femora, threshold values were found that produced zero error in bone area for each strain and age group, although the optimal threshold differed between groups. pQCT vBMD correlated strongly with ash content (r2=0.7), although the regression equations differed between strains and the magnitude of the inter-strain difference in vBMD was fourfold greater than the difference in ash content. This finding suggests that pQCT can overestimate the differences in volumetric mineral density between inbred mouse strains whose bones are of different thickness (e.g., C3H vs. B6). In conclusion, both area and density values obtained by pQCT depend strongly on specimen thickness, consistent with a partial volume averaging artifact. Investigators using pQCT to assess cortical bones in mice should be aware of the potential for cortical thickness-dependent errors.

In vivo murine models are becoming increasingly important in bone research. To establish baseline data for researchers using these models, we studied the long bones from C57BL/6 female mice, a strain that is widely used in bone research. We determined the femoral structural and material properties in both torsion and four-point bending for mice at ages 4-24 weeks. Measurements of femoral cross-sectional geometry and tibial densitometric properties were also obtained. Results indicated that all structural properties (except ultimate energy), changed significantly with age (p < 0.001). Ultimate torque, ultimate moment, torsional rigidity, and bending rigidity all increased to peak values at 20 weeks, whereas ultimate rotation and ultimate displacement decreased to minimum values at 20 weeks. Our data indicate that increases in the material properties contributed more than increases in cross-sectional geometry to the changes in structural rigidity and ultimate load. For example, from 4-20 weeks torsional rigidity increased 1030%, while shear modulus increased 610% and the polar moment of inertia (a measure of the geometric resistance to rotation) increased by only 85%. Changes in the cross-sectional geometry with age were due to increases in periosteal diameter and decreases in endosteal diameter. In general, both torsion and bending techniques revealed large changes in structural and material properties with age. We conclude that peak bone strength is not achieved before 20 weeks in C57BL/6 female mice, and that torsion and four-point bending tests are equally well suited for evaluating mechanical properties of murine long bones.