Selection for longer limbs in mice increases bone stiffness and brittleness, but does not alter bending strength.

The ability of a bone to withstand loads depends on its structural and material properties. These tend to differ among species with different modes of locomotion, reflecting their unique loading patterns. The evolution of derived limb morphologies, such as the long limbs associated with jumping, may compromise overall bone strength. We evaluated bone mechanical properties in the Longshanks mouse, which was selectively bred for increased tibia length relative to body mass. We combined analyses of 3D shape and cross-sectional geometry of the tibia, with mechanical testing and bone composition assays, to compare bone strength, elastic properties and mineral composition in Longshanks mice and randomly bred controls. Our data show that, despite being more slender, cortical geometry and predicted bending strength of the Longshanks tibia were similar to controls. In whole bone bending tests, measures of bone bending strength were similar across groups; however, Longshanks tibiae were significantly more rigid, more brittle, and required less than half the energy to fracture. Tissue-level elastic properties were also altered in Longshanks mice, but the bones did not differ from the control in water content, ash content or density. These results indicate that while Longshanks bones are as strong as control tibiae, selection for increased tibia length has altered its elastic properties, possibly through changes in organic bony matrix composition. We conclude that selection for certain limb morphologies, and/or selection for rapid skeletal growth, can lead to tissue-level changes that can increase the risk of skeletal fracture, which in turn may favor the correlated evolution of compensatory mechanisms to mitigate increased fracture risk, such as delayed skeletal maturity.

Cortical and trabecular morphology is altered in the limb bones of mice artificially selected for faster skeletal growth.

Bone strength is influenced by mineral density and macro- and microstructure. Research into factors that contribute to bone morphology and strength has focused on genetic, environmental and morphological factors (e.g., body mass index), but little is known regarding the impact of rates of skeletal elongation on adult skeletal morphology and strength. Using micro-CT, we examined the impact of rates of skeletal elongation on bone cortical and trabecular morphology, and on rates of estrogen-dependent bone loss in the tibia in CD-1 mice, and in mice with accelerated skeletal growth (Longshanks). Groups of adult mice (n = 7/group) were subjected to ovariectomy or sham surgeries, scanned for 6 weeks, and indices of bone morphology were collected. Results show that Longshanks mice had significantly less trabecular bone at skeletal maturity, characterized by fewer, thinner trabeculae, and furthermore lost trabecular bone more slowly in response to ovariectomy. Artificial selection for rapid skeletal growth relative to somatic growth thus had a significant impact on trabecular bone morphology in Longshanks. Our data do not unequivocally demonstrate a causal relationship between rapid bone growth and reduced trabecular bone quality, but suggest that rapid linear bone growth may influence the risk of cancellous bone fragility.

Stem cell-derived endochondral cartilage stimulates bone healing by tissue transformation.

Although bone has great capacity for repair, there are a number of clinical situations (fracture non-unions, spinal fusions, revision arthroplasty, segmental defects) in which auto- or allografts attempt to augment bone regeneration by promoting osteogenesis. Critical failures associated with current grafting therapies include osteonecrosis and limited integration between graft and host tissue. We speculated that the underlying problem with current bone grafting techniques is that they promote bone regeneration through direct osteogenesis. Here we hypothesized that using cartilage to promote endochondral bone regeneration would leverage normal developmental and repair sequences to produce a well-vascularized regenerate that integrates with the host tissue. In this study, we use a translational murine model of a segmental tibia defect to test the clinical utility of bone regeneration from a cartilage graft. We further test the mechanism by which cartilage promotes bone regeneration using in vivo lineage tracing and in vitro culture experiments. Our data show that cartilage grafts support regeneration of a vascularized and integrated bone tissue in vivo, and subsequently propose a translational tissue engineering platform using chondrogenesis of mesenchymal stem cells (MSCs). Interestingly, lineage tracing experiments show the regenerate was graft derived, suggesting transformation of the chondrocytes into bone. In vitro culture data show that cartilage explants mineralize with the addition of bone morphogenetic protein (BMP) or by exposure to human vascular endothelial cell (HUVEC)-conditioned medium, indicating that endothelial cells directly promote ossification. This study provides preclinical data for endochondral bone repair that has potential to significantly improve patient outcomes in a variety of musculoskeletal diseases and injuries. Further, in contrast to the dogmatic view that hypertrophic chondrocytes undergo apoptosis before bone formation, our data suggest cartilage can transform into bone by activating the pluripotent transcription factor Oct4A. Together these data represent a paradigm shift describing the mechanism of endochondral bone repair and open the door for novel regenerative strategies based on improved biology.

Prolonged unloading in growing rats reduces cortical osteocyte lacunar density and volume in the distal tibia.

Bone dynamically adapts its structure to the environmental demands placed upon it. Load-related stimuli play an important role in this adaptation. It has been postulated that osteocytes sense changes in these stimuli and initiate adaptive responses, across a number of scales, through a process known as mechanotransduction. While much research has focused on gross and tissue-level adaptation, relatively little is known regarding the relation between cellular-level features (e.g. osteocyte lacunar density, volume and shape) and loading. The increasing availability of high resolution 3D imaging modalities, including synchrotron-based techniques, has made studying 3D cellular-level features feasible on a scale not previously possible. The primary objective of this study was to test the hypothesis that unloading (sciatic neurectomy) during growth results in altered osteocyte lacunar density in the tibial diaphysis of the rat. Secondarily, we explored a potential effect of unloading on mean lacunar volume. Lacunar density was significantly (p<0.05) lower in immobilized bones (49,642 ± 11,955 lacunae per mm(3); n=6) than in control bones (63,138 ± 1956 lacunae per mm(3); n=6). Mean lacunar volume for immobilized bones (209 ± 72 µm(3); n=6) was significantly smaller (p<0.05) than that for the control bones (284 ± 28 µm(3); n=6). Our results demonstrate that extreme differences in loading conditions, such as those created by paralysis, do indeed result in changes in osteocyte lacunar density and volume. Further investigation is warranted to examine relations between these measures and more subtle variation in loading as well as pathological states, which have been linked to alterations in mechanotransduction.

The effects of immobilization on vascular canal orientation in rat cortical bone.

It is well established that bone is capable of adapting to changes in loading; however, little is known regarding how loading specifically affects the internal 3D microarchitecture of cortical bone. The aim of this study was to experimentally test the hypothesis that loading is a determinant of the 3D orientation of primary vascular canals in the rat tibial diaphysis. Left tibiae from 10 rats (30 weeks old) that had been immobilized (sciatic neurectomy) for 27 weeks, right SHAM-operated tibiae from these same rats (internal control) and right tibiae from 10 normal age-matched rats (external control) were scanned by micro-CT. Mean canal orientation (for the whole bone segment and by region), percent porosity, canal diameter and canal separation were quantitatively assessed in 3D. Canal orientation in the immobilized tibiae was significantly (P < 0.001) more radial (by 9.9°) compared to the external controls but did not differ from the internal controls (P = 0.310). Comparing the external and internal controls, orientation was significantly (P < 0.05) more radial in the internal control group (by 6.8°). No differences were found for percent porosity and canal separation. Canal diameter was significantly greater in the immobilized vs. internal (P < 0.001) and external control (P < 0.001) tibiae. The differences in orientation relative to the external controls indicated that the organization of cortical bone in the rat is affected by loading. Although the predicted difference in canal orientation was not detected between immobilized and internal control groups, the distributions of individual canal orientations, from which the mean values were derived, revealed distinctive patterns for all three groups. The internal controls exhibited an intermediate position between the immobilized and external controls, suggesting that paralysis on the contralateral side resulted in altered loading relative to the normal state represented by the external control. This was also evident in a regional analysis by quadrant. The loaded bones had the same cross-sectional shape; however, their internal structure differed. These results provide novel insights into the impact of loading on the 3D organization of primary cortical bone and have implications for understanding the relation between cortical bone adaptation, disease and mechanical properties.

The relation of femoral osteon geometry to age, sex, height and weight.

As computational modeling becomes an increasingly common tool for probing the regulation of bone remodeling, the need for experimental data to refine and validate such models also grows. For example, van Oers et al. (R.F. van Oers, R. Ruimerman, B. van Rietbergen, P.A. Hilbers, R. Huiskes, Relating osteon diameter to strain. Bone 2008;43: 476-482.) recently described a mechanism by which osteon size may be regulated (inversely) by strain. Empirical data supporting this relation, particularly in humans, are sparse. Therefore, we sought to determine if there is a link between body weight (the only measure related to loading available for a cadaveric population) and osteon geometry in human bone. We hypothesized that after controlling for age, sex and height, weight would be inversely related to femoral osteon size (area, On.Ar; diameter, On.Dm). Secondarily we sought to describe the relation between osteon circularity (On.Cr) and these parameters. Osteons (n=12,690) were mapped within microradiographs of femoral mid-diaphyseal specimens (n=88; 45 male, 43 female; 17-97 yrs). Univariate analysis of covariance was conducted (n=87; 1 outlier) with sex as a fixed factor and height, weight and log-transformed age as covariates. Weight was negatively related to On.Ar and On.Dm (p=0.006 and p=0.004, respectively). Age was significantly related to osteon and, it was also significantly related to circularity (all p<0.001). This relation was negative for On.Ar and On.Dm and positive for On.Cr (increasing circularity with age). On.Ar and On.Dm were found to be significantly different between the sexes (p=0.021 and p=0.019, respectively), with females having smaller osteons. No relation between sex and On.Cr was detected (p=0.449). Height was not significantly related to any of the geometric parameters. Partial eta-squared values revealed that age accounted for the largest proportion (On.Ar: 28%, On.Dm: 18%, On.Cr: 30%), weight accounted for the second largest (On.Ar: 9%, On.Dm: 10%) and sex accounted for the smallest proportion (On.Ar: 6%, On.Dm: 7%) of the variance in geometry. While previous studies have reported relations between osteon size and sex/age, we believe that our findings are the first to demonstrate a link with weight. We believe that this negative relation with weight is most probably mechanical in nature; however, alternative (endocrine) links between bone and adipose tissue cannot be ruled out by our design.