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Mice are widely used in studies of skeletal biology, and assessment of their bones by mechanical testing is a critical step when evaluating the functional effects of an experimental perturbation. For example, a gene knockout may target a pathway important in bone formation and result in a “low bone mass” phenotype. But how well does the skeleton bear functional loads; eg, how much do bones deform during loading and how resistant are bones to fracture? By systematic evaluation of bone morphological, densitometric, and mechanical properties, investigators can establish the “biomechanical mechanisms” whereby an experimental perturbation alters whole-bone mechanical function. The goal of this review is to clarify these biomechanical mechanisms and to make recommendations for systematically evaluating phenotypic changes in mouse bones, with a focus on long-bone diaphyses and cortical bone. Further, minimum reportable standards for testing conditions and outcome variables are suggested that will improve the comparison of data across studies. Basic biomechanical principles are reviewed, followed by a description of the cross-sectional morphological properties that best inform the net cellular effects of a given experimental perturbation and are most relevant to biomechanical function. Although morphology is critical, whole-bone mechanical properties can only be determined accurately by a mechanical test. The functional importance of stiffness, maximum load, postyield displacement, and work-to-fracture are reviewed. Because bone and body size are often strongly related, strategies to adjust whole-bone properties for body mass are detailed. Finally, a comprehensive framework is presented using real data, and several examples from the literature are reviewed to illustrate how to synthesize morphological, tissue-level, and whole-bone mechanical properties of mouse long bones.

Toughening in hierarchically structured materials like bone arises from the arrangement of constituent material elements and their interactions. Unlike microcracking, which entails micrometer-level separation, there is no known evidence of fracture at the level of bone's nanostructure. Here, we show that the initiation of fracture occurs in bone at the nanometer scale by dilatational bands. Through fatigue and indentation tests and laser confocal, scanning electron, and atomic force microscopies on human and bovine bone specimens, we established that dilatational bands of the order of 100 nm form as ellipsoidal voids in between fused mineral aggregates and two adjacent proteins, osteocalcin (OC) and osteopontin (OPN). Laser microdissection and ELISA of bone microdamage support our claim that OC and OPN colocalize with dilatational bands. Fracture tests on bones from OC and/or OPN knockout mice (OC −/− , OPN −/− , OC-OPN −/−;−/− ) confirm that these two proteins regulate dilatational band formation and bone matrix toughness. On the basis of these observations, we propose molecular deformation and fracture mechanics models, illustrating the role of OC and OPN in dilatational band formation, and predict that the nanometer scale of tissue organization, associated with dilatational bands, affects fracture at higher scales and determines fracture toughness of bone.noncollagenous proteins | diffuse damage | energy dissipation I n hierarchically structured materials, the composition and spatial arrangement of nanoscale elements are the key determinants of toughness (1, 2). In comparison with many man-made materials, cortical bone is well known for its superior toughness (3, 4). Bone's ability to resist crack propagation originates from its highly complex hierarchical material structure ( Fig. 1). At the highest level of material structure in adult human bone lie the osteons (0.1-0.2 mm in diameter) that contribute to toughness by trapping microcracks (5, 6) and participate in the formation of "uncracked ligaments" (7). Osteons are made of multiple 3-to 7-μm-thick sheets (lamellae) of mineralized matrix. Individual lamellae have the ability to slide past each other (8, 9), forming 60-to 130-μm-long linear microcracks (9) that provide resistance to fracture through microcrack toughening (10). Individual mineralized collagen fibrils <1μm thick, which make up the lamellae, bridge the crack surfaces and toughen the bone (7). Bone's ability to crack, and not fracture by propagating that crack, is therefore a key fundamental aspect of the toughening mechanisms at the microstructural level (10).Recent evidence suggests that bone's nanostructure contributes to bone toughness (11). The nonfibrillar and ductile extrafibrillar matrix components in bone can serve as a "glue" between stiffened mineralized collagen fibrils (11) and between fibrils and mineral platelets (12). Fibril matrix shearing (13) has been proposed to enhance bone toughness through mineral interparticle friction (14) and "sacrificial bonds," a nanoscale mechanism...

Post-translational modifications of collagen, such as non-enzymatic glycation (NEG), occur through the presence of extracellular sugars and cause the formation of advanced glycation end-products (AGEs). While AGEs have been shown to accumulate in a variety of collagenous human tissues and alter the tissues' functional behavior, the role of AGEs in modifying the mechanical properties of cancellous bone is not well understood. In this study, an in vitro ribosylation model was used to examine the effect of NEG on the mechanical behavior of cancellous bone. Cancellous bone cores and individual trabeculae were harvested from the femoral heads of eight fresh human cadavers and paired for ribosylation and control treatments. The cores were subjected to either unconfined compression tests or were demineralized and subjected to stress relaxation tests. The trabeculae were loaded to fracture in four-point bending. In vitro NEG significantly reduced the energy dissipation characteristics of the organic matrix as well as the post-yield properties including the stiffness loss of the individual trabeculae (p<0.05) and the damage fraction of cancellous bone (p<0.001). AGEs in cancellous bone cores from both treatment groups correlated with damage fraction (r(2)=0.36, p<0.05) and post-yield strain energy (r(2)=0.21, p<0.05); and with energy dissipation characteristics of the organic matrix (r(2)=0.35, p<0.05). In the control group, AGEs content increased up to six-fold with age (r(2)=0.95, p<0.008). This study shows that cancellous bone is susceptible to NEG that increases its propensity to fracture. Moreover, despite tissue turnover, cancellous bone may be susceptible to an age-related accumulation of AGEs.

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