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Biallelic ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) deficiency induces vascular/soft tissue calcifications in generalized arterial calcification of infancy (GACI), and low bone mass with phosphate-wasting rickets in GACI survivors (autosomal hypophosphatemic rickets type-2). ENPP1 haploinsufficiency induces early-onset osteoporosis and mild phosphate wasting in adults. Both conditions demonstrate the unusual combination of reduced accrual of skeletal mineral, yet excess and progressive heterotopic mineralization. ENPP1 is the only enzyme that generates extracellular pyrophosphate (PPi), a potent inhibitor of both bone and heterotopic mineralization. Life-threatening vascular calcification in ENPP1 deficiency is due to decreased plasma PPi; however, the mechanism by which osteopenia results is not apparent from an understanding of the enzyme's catalytic activity. To probe for catalysis-independent ENPP1 pathways regulating bone, we developed a murine model uncoupling ENPP1 protein signaling from ENPP1 catalysis, Enpp1

Background The fracture obliquity of supination-external rotation injury of the fibula is often amenable to lag screw insertion. The purpose of the study was to determine whether biomechanical differences exist between lag screws inserted from an anterior to posterior direction and from a posterior to anterior direction and the thickness of the anterior and posterior fibular cortices were correlated with biomechanical testing. Methods Ten cadaver fibulae were harvested and submitted to material testing following 3.5-mm cortical screw insertion from either an anterior to posterior direction or a posterior to anterior direction. Screw torsional insertion strength and axial pullout strength were measured. Computed tomography images of 40 consecutive patients undergoing preoperative planning for fractures excluding the fibula were examined to define fibular cortical thickness and correlate anatomic findings with the biomechanical testing. Results The axial pullout strength of lag screws inserted from posterior to anterior was significantly greater than that of lag screws inserted from anterior to posterior (p < 0.05). Screw insertion torque measurements demonstrated a similar trend although the data did not reach statistical significance (p = 0.056). The anterior cortex of the distal fibula exhibited a radiographically greater thickness than that of the posterior cortex at the same level (p < 0.001). Conclusions For oblique fractures of the distal fibula, posterior to anterior lag screw insertion exhibited improved biomechanical properties when compared with a similar screw inserted from anterior to posterior. These results correlated with the thicker cortical bone present along the anterior fibula.

Genome-wide association studies (GWASs) have revolutionized our understanding of the genetics of complex diseases, such as osteoporosis; however, the challenge has been converting associations to causal genes. Studies have demonstrated the utility of transcriptomics data in linking disease-associated variants to genes; though for osteoporosis, few population transcriptomics datasets have been generated on bone or bone cells, and an even smaller number have profiled individual cell-types. To begin to evaluate approaches to address this challenge, we profiled the transcriptomes of bone marrow-derived stromal cells (BMSCs) cultured under osteogenic conditions, a popular model of osteoblast differentiation and activity, from five Diversity Outbred (DO) mice using single-cell RNA-seq (scRNA-seq). The goal of the study was to determine if BMSCs could serve as a model for the generation of cell-type specific transcriptomic profiles of mesenchymal lineage cells derived from large populations of mice to inform genetic studies. We demonstrate that dissociation of BMSCs from a heavily mineralized matrix had little effect on viability or their transcriptomic signatures. Furthermore, we show that BMSCs cultured under osteogenic conditions are diverse and consist of cells with characteristics of mesenchymal progenitors, marrow adipogenic lineage precursors (MALPs), osteoblasts, osteocyte-like cells, and immune cells. Importantly, all cells were nearly identical from a transcriptomic perspective to cells isolated directly from bone. We also demonstrated the ability to multiplex single cells and subsequently assign cells to their “mouse-of-origin” using demultiplexing approaches based on genotypes inferred from coding SNPs. We employed scRNA-seq analytical tools to confirm the biological identity of profiled cell-types. SCENIC was used to reconstruct gene regulatory networks (GRNs) and we showed that identified cell-types show GRNs expected of osteogenic and pre-adipogenic lineage cells. Further, CELLECT analysis showed that osteoblasts, osteocyte-like cells, and MALPs captured a significant component of BMD heritability. Together, these data suggest that BMSCs cultured under osteogenic conditions coupled with scRNA-seq can be used as a scalable and biologically informative model to generate cell-type specific transcriptomic profiles of mesenchymal lineage cells in large mouse, and potentially human, populations.

It is unclear howmetal augments,polyethylene (PE) liner thickness, and length of cemented stemcontribute to load transferwhen reconstructing uncontained tibial metaphyseal bone loss of Anderson Orthopedic Research Institute (AORI) Type II defects during revision total knee arthroplasty (rTKA).The aimof this study is to understand the impact of these three variableson load transfer through the tibial baseplate. For a fixed defect depth, we hypothesized that there is a particular combination of liner and augment thickness and stem length that minimizes bone stress, reducing the risk of aseptic loosening.We conducted a finite element analysis (FEA) to model stresses at the bone-cement interface with different iterations of metal augments, PE liner thicknesses andfully-cemented stems lengths.For a 20 mm tibial defect, constructs with thicker metal augments and thinner polyethylene liners were superior. Constructswith a fully cemented stem further reduced bone stress on the tibial plateau. Bone stress was lowest when a 100 mm fully-cemented stem was used, while stems between 30 mm - 80 mm produced similar results.When addressing a tibial bone defect of AORI Type II in rTKA, our FEA model demonstrates that surgeons should opt to use the thickest metal augments in combination with afully-cemented stem with an added length of at least 30 mm, which allows for surgical flexibility together with the most stable construct.Our study is notably limited by lack of modeling of knee joint moments, which are important when considering micromotion, bone-implant interface and stem effectiveness.

Context X-Linked hypophosphatemia (XLH) is a lifelong metabolic disease with musculoskeletal comorbidities that dominate the adult clinical presentation. Objective The adult XLH disorder has yet to be quantified on the basis of the physical and functional limitations that can affect activities of daily living. Our goal was to report the impact of the musculoskeletal manifestations on physical function. Design and setting Musculoskeletal function was evaluated by validated questionnaires and in an interdisciplinary clinical space where participants underwent full-body radiologic imaging, goniometric range of motion (ROM) measurements, general performance tests, and kinematic gait analysis. Patients Nine adults younger than 60 years with a diagnosis of XLH and self-reported musculoskeletal disability, but able to independently ambulate, were selected to participate. Passive ROM and gait analysis were also performed on age-approximated controls to account for differences between individual laboratory instrumentation. Results Enthesophytes, degenerative arthritis, and osteophytes were found to be consistently bilateral and diffusely present at the spine and synovial joints across participants, with predominance at weight-bearing joints. Passive ROM in adults with XLH was decreased at the cervical spine, hip, knee, and ankle compared to controls. Gait analysis relative to controls revealed increased step width, markedly increased lateral trunk sway, and physical restriction at the hip, knees, and ankle joints that translated into limitations through the gait cycle. Conclusions The functional impact of XLH musculoskeletal comorbidities supports the necessity for creating an interprofessional health-care team with the goal of establishing a longitudinal plan of care that considers the manifestations of XLH across the lifespan.

A major comorbidity of X-linked hypophosphatemia (XLH) is fibrocartilaginous tendinous insertion site mineralization resulting in painful enthesophytes that contribute to the adult clinical picture and significantly impact physical function. Enthesophytes in Hyp mice, a murine model of XLH are the result of a hyperplastic expansion of resident alkaline phosphatase, Sox9-positive mineralizing fibrochondrocytes. Here, we hypothesized hyperplasia as a compensatory physical adaptation to aberrant mechanical stresses at the level of the entheses interface inserting into pathologically soft bone. To test this hypothesis, we examined the Achilles insertion of the triceps surae developed under normal and impaired loading conditions in Hyp and WT mice. Tensile stiffness, ultimate strength, and maximum strain were measured and compared. Biomechanical testing revealed that under normal loading conditions, despite inserting into a soft bone matrix, both the enthesophyte development (9 weeks) and progression (6-8 months) of Hyp mice were equivalent to the mechanical properties of WT mice. Unloading the insertion during development significantly reduced alkaline phosphatase, Sox9-positive fibrochondrocytes. In WT mice, this correlated with a decrease in stiffness and ultimate strength relative to the control limb, confirming the critical role of mechanical loading in the development of the enthesis. Most significantly, in response to unloading, maximum strain was increased in tensile tests only in the setting of subchondral osteomalacia of Hyp mice. These data suggest that mineralizing fibrochondrocyte expansion in XLH occurs as a compensatory adaptation to the soft bone matrix.

Biallelic ENPP1 deficiency in humans induces generalized arterial calcification of infancy (GACI) and/or autosomal recessive hypophosphatemic rickets type 2 (ARHR2). The latter is characterized by markedly increased circulating FGF23 levels and renal phosphate wasting, but aberrant skeletal manifestations associated with heterozygous ENPP1 deficiency are unknown. Here, we report three adult men with early onset osteoporosis who presented with fractures in the thoracic spine and/or left radius, mildly elevated circulating FGF23, and hypophosphatemia. Total hip bone mineral density scans demonstrated osteoporosis (Z‐score

Postoperative bone loss and increased fracture risk associated with Roux-en-Y gastric bypass (RYGB) have been attributed to vitamin D/calcium malabsorption and resultant secondary hyperparathyroidism (HPT). Adequate vitamin D supplementation (VDS), particularly in an older female population, reduces incidence of secondary HPT but the effect on bone loss and fracture risk remains unclear. To investigate whether VDS corrects the RYGB bone phenotype, 41 obese adult female rats were randomized to RYGB with 1000 IU (R1000) or 5000 IU (R5000) vitamin D/kg food or a sham surgical procedure with either paired (PF) or ad libitum (AL) feeding. Bone turnover markers, urinary calcium/creatinine ratio (CCR), and serum calciotropic and gut hormones were assessed throughout a 14-week postoperative period. Femurs were analyzed by micro-computed tomography (μCT), three-point bending test, and histomorphometry. 1000 IU animals had low 25‑hydroxyvitamin D (25(OH)D), high serum parathyroid hormone (PTH), and very low urine CCR levels. 5000 IU corrected the 25(OH)D and secondary HPT but did not increase urine CCR or serum levels of 1,25‑dihydroxyvitamin D (1,25(OH)D) significantly between RYGB groups. Compared to sham animals at 14 weeks, RYGB animals had significantly higher serum osteocalcin (OCN) and C-terminal telopeptide (CTX) levels. The gut hormone peptide tyrosine tyrosine hormone (PYY) was higher in the RYGB groups, and leptin was lower. μCT and biomechanical testing revealed RYGB females had decreased cortical and trabecular bone volume and weaker, stiffer bone than controls. Histomorphometry showed decreased bone volume and increased osteoid volume with increased mineral apposition rate in RYGB compared to controls. No differences in bone phenotype were identified between 1000 IU and 5000 IU groups, and osteoclast numbers were comparable across all four groups. Thus, in our model, 5000 IU VDS corrected vitamin D deficiency and secondary HPT but did not rescue RYGB mineralization rate nor the osteomalacia phenotype. Longer studies in this model are required to evaluate durability of these detrimental effects. Our findings not only underscore the importance of lifelong repletion of both calcium and vitamin D but also suggest that additional factors affect skeletal health in this population.

Bone crystallite chemistry and structure change during bone maturation. However, these properties of bone can also be affected by limited uptake of the chemical constituents of the mineral by the animal. This makes probing the effect of bone-mineralization-related diseases a complicated task. Here it is shown that the combination of vibrational spectroscopy with two-dimensional X-ray diffraction can provide unparalleled information on the changes in bone chemistry and structure associated with different bone pathologies (phosphate deficiency) and/or health conditions (pregnancy, lactation). Using a synergistic analytical approach, it was possible to trace the effect that changes in the remodelling regime have on the bone mineral chemistry and structure in normal and mineral-deficient (hypophosphatemic) mice. The results indicate that hypophosphatemic mice have increased bone remodelling, increased carbonate content and decreased crystallinity of the bone mineral, as well as increased misalignment of crystallites within the bone tissue. Pregnant and lactating mice that are normal and hypophosphatemic showed changes in the chemistry and misalignment of the apatite crystals that can be related to changes in remodelling rates associated with different calcium demand during pregnancy and lactation., The following funding is acknowledged: European Commission Seventh Framework Programme (grant No. 2012-328731 to HEK); Universidad de Granada (grant Nos. CGL2015- 64683-P and UCE PP 2016.05 to ABRN).

Genome-wide association studies (GWASs) for osteoporotic traits have identified over 1000 associations; however, their impact has been limited by the difficulties of causal gene identification and a strict focus on bone mineral density (BMD). Here, we use Diversity Outbred (DO) mice to directly address these limitations by performing a systems genetics analysis of 55 complex skeletal phenotypes. We apply a network approach to cortical bone RNA-seq data to discover 66 genes likely to be causal for human BMD GWAS associations, including the genes SERTAD4 and GLT8D2. We also perform GWAS in the DO for a wide-range of bone traits and identify Qsox1 as a gene influencing cortical bone accrual and bone strength. In this work, we advance our understanding of the genetics of osteoporosis and highlight the ability of the mouse to inform human genetics., Osteoporosis GWAS faces two challenges, causal gene discovery and a lack of phenotypic diversity. Here, the authors use the Diversity Outbred mouse population to inform human GWAS using networks and map genetic loci for 55 bone traits, identifying new potential bone strength genes.

BACKGROUND: Restoration of fibular length is the main determinant in preventing mal-union and early ankle arthritis in lateral malleolus fractures. A 1/3 tubular plate fashioned into a mini-blade plate can be used to distract the distal fragment and achieve length in a controlled fashion over time. The purpose of this study was to describe the surgical technique and perform a biomechanical comparison of the blade plate to a locking plate. METHODS: A 1/3 tubular plate is fashioned into a 135° blade plate. Blades are seated into the lateral malleolus and a distally directed force is applied on the plate to obtain length. A lateral malleolus fracture was created in 20 cadaveric ankles. The distal fragment was fixed with either a blade plate (BP, n = 10) or a locking plate (LP, n = 10). A distally directed force was applied by an Instron machine and fracture distraction, maximal load and construct stiffness were measured and compared. RESULTS: The average maximal load was 262.06 N compared to 255.52 N for the BP and LP groups, respectively. The maximal distraction was 3.57 mm compared to 4.57 mm for the BP and LP groups, respectively. The loading pattern of the blade plate over time differed from that of a locking plate as the blades seat into bone. CONCLUSION: A 1/3 tubular mini-blade plate demonstrates biomechanical similarities in terms of load and distraction to the more expensive locking plate. We recommend using this technique for fractures with late presentation or with significant shortening. LEVEL OF EVIDENCE: Level V—Mechanism-based reasoning.

BACKGROUND: To be FDA approved, acetabular shells must undergo rigorous testing. To prevent implant failure, acetabular shells must be able to tolerate peak loads during impaction with minimal deformation. The implants must therefore be validated in order to ensure that their structural integrity can tolerate peak loads. The current ISO 7206–12 recommends manufacturing an expensive single-purpose testing device to measure the deformation of acetabular shells. In the article, we provide an open access methodology, that can be conducted with minimal expense, for testing acetabular shell deformation. METHODS: We designed our experimental setup to utilize a servohydraulic materials testing device (Instron) commonly found in biomechanics laboratories and then validated the measurements optically with optical data. Furthermore, we designed an inexpensive acetabular screw fastener which acts as an adaptor that can be used to mount a variety of acetabular shell types just as effectively as current methods and is in compliance with the standards outlined in ISO 7206–12:2016. RESULTS: A Bland-Altman plot comparing the Instron and optical displacement measurements found the standard deviation of bias to be 0.046 mm and an insignificant systemic bias. CONCLUSION: We have developed and validated a low-cost open-source system that can effectively test acetabular shell deformation that meets ISO standards.

The COVID-19 pandemic has challenged the status quo of engineering education, especially in highly interactive, hands-on design classes. Here, we present an example of how we effectively adjusted an intensive hands-on, group project-based engineering course, Medical Device Design & Innovation, to a remote learning curriculum. We first describe the modifications we made. Drawing from student pre and post feedback surveys and our observations, we conclude that our adaptations were overall successful. Our experience may guide educators who are transitioning their engineering design courses to remote learning. Supplementary information The online version of this article (10.1007/s13246-020-00967-z) contains supplementary material, which is available to authorized users.

Genome-wide association studies (GWASs) for osteoporotic traits have identified over 1000 associations; however, their impact has been limited by the difficulties of causal gene identification and a strict focus on bone mineral density (BMD). Here, we used Diversity Outbred (DO) mice to directly address these limitations by performing the first systems genetics analysis of 55 complex skeletal phenotypes. We applied a network approach to cortical bone RNA-seq data to discover 72 genes likely to be causal for human BMD GWAS associations, including the novel genes SERTAD4 and GLT8D2. We also performed GWAS in the DO for a wide-range of bone traits and identified Qsox1 as a novel gene influencing cortical bone accrual and bone strength. Our results provide a new perspective on the genetics of osteoporosis and highlight the ability of the mouse to inform human genetics.

Minimally invasive percutaneous polymethyl methacrylate cement augmentation procedures offer numerous clinical advantages for patients with periacetabular osteolytic metastatic bone defects in contrast to open reconstructive procedures that are associated with many complications. Several techniques, such as Ablation-Osteoplasty-Reinforcement-Internal Fixation (AORIF), cementoplasty alone, and screw fixation alone are currently used. There is no consensus on optimal skeletal reinforcement of diseased bones. The purpose of this study was to determine the most effective technique of percutaneous acetabular augmentation for joint preservation, with respect to resilience on cyclic loading and fracture pattern at maximal load to failure.Five cohorts of hemipelvis composite bones with uniform periacetabular defects and various types of reinforcement techniques were utilized to simulate osteolytic metastasis in the weight bearing dome of the acetabulum. Five groups of hemipelves underwent finite element analysis and biomechanical testing for load to failure, energy absorption to failure, stress relaxation on cyclic loading, and fracture locations.The combination of screws and bone cement augmentation demonstrated significant higher energy absorption than the cement or screw only groups (p 0.05), and better protection of acetabulum from displaced intraarticular fractures than the screws alone oror cement only groups (p 0.05). Resilience to cyclic loading was higheest in the screw with cement fixation group than the screw only repair (p 0.01), though not the cement fixation only group.These data support the hypothesis that cementoplasty combined with screw augmentation such as the AORIF technique provides the best protection of acetabulum from massive metastatic cancer-induced acetabular fractures compared to augmentation with screws or cement alone.

Foot stiffness underlies its mechanical function, and is central to the evolution of human bipedal locomotion. The stiff and propulsive human foot has two distinct arches, the longitudinal and transverse. By contrast, the feet of non-human primates are flat and softer. Current understanding of foot stiffness is based on studies that focus solely on the longitudinal arch, and little is known about the mechanical function of the transverse arch. However, common experience suggests that transverse curvature dominates the stiffness; a drooping dollar bill stiffens significantly upon curling it along the transverse direction, not the longitudinal. We derive a normalized curvature parameter that encapsulates the geometric principle underlying the transverse curvature-induced stiffness. We show that the transverse arch accounts for almost all the difference in stiffness between human and monkey feet (vervet monkeys and pig-tailed macaques) by comparing transverse curvature-based predictions against published data on foot stiffness. Using this functional interpretation of the transverse arch, we trace the evolution of hominin feet and show that a human-like stiff foot likely predates Homo by $\sim 1.5$ million years, and appears in the $\sim 3.4$ million year old fossil from Burtele. A distinctly human-like transverse arch is also present in early members of Homo, including Homo naledi, Homo habilis, and Homo erectus. However, the $\sim 3.2$ million year old Australopithecus afarensis is estimated to have possessed a transitional foot, softer than humans and stiffer than other extant primates. A foot with human-like stiffness probably evolved around the same time as other lower limb adaptations for regular bipedality, and well before the emergence of Homo, the longitudinal arch, and other adaptations for endurance running., Comment: 22 pages, 7 figures, 4 tables

PGC-1α is a transcriptional co-activator associated with PPARγ that regulates thermogenic gene expression in brown fat. In this issue, Yu et al. (2018) show that PGC-1α regulates marrow mesenchymal stromal cell lineage allocation in vivo, inhibiting marrow adipogenesis and associated bone loss in the aging skeleton and following ovariectomy-induced osteoporosis.

Parathyroid hormone-related protein (PTHrP) is widely expressed in the fibrous outer layer of the periosteum (PO), and the PTH/PTHrP type I receptor (PTHR1) is expressed in the inner PO cambial layer. The cambial layer gives rise to the PO osteoblasts (OBs) and osteoclasts (OCs) that model/remodel the cortical bone surface during development as well as during fracture healing. PTHrP has been implicated in the regulation of PO modeling during development, but nothing is known as regards a role of PTHrP in this location during fracture healing. We propose that PTHrP in the fibrous layer of the PO may be a key regulatory factor in remodeling bone formation during fracture repair. We first assessed whether PTHrP expression in the fibrous PO is associated with PO osteoblast induction in the subjacent cambial PO using a tibial fracture model in PTHrP-lacZ mice. Our results revealed that both PTHrP expression and osteoblast induction in PO were induced 3 days post-fracture. We then investigated a potential functional role of PO PTHrP during fracture repair by performing tibial fracture surgery in 10-week-old CD1 control and PTHrP conditional knockout (PTHrP cKO) mice that lack PO PTHrP. We found that callus size and formation as well as woven bone mineralization in PTHrP cKO mice were impaired compared to that in CD1 mice. Concordant with these findings, functional enzyme staining revealed impaired OB formation and OC activity in the cKO mice. We conclude that deleting PO PTHrP impairs cartilaginous callus formation, maturation and ossification as well as remodeling during fracture healing. These data are the initial genetic evidence suggesting that PO PTHrP may induce osteoblastic activity and regulate fracture healing on the cortical bone surface.

Rac1 and Rac2 are thought to have important roles in osteoclasts. Therefore, mice with deletion of both Rac1 and Rac2 in mature osteoclasts (DKO) were generated by crossing Rac1(flox/flox) mice with mice expressing Cre in the cathepsin K locus and then mating these animals with Rac2(-/-) mice. DKO mice had markedly impaired tooth eruption. Bone mineral density (BMD) was increased 21% to 33% in 4- to 6-week-old DKO mice at all sites when measured by dual-energy X-ray absorptiometry (DXA) and serum cross-linked C-telopeptide (CTx) was reduced by 52%. The amount of metaphyseal trabecular bone was markedly increased in DKO mice, but the cortices were very thin. Spinal trabecular bone mass was increased. Histomorphometry revealed significant reductions in both osteoclast and osteoblast number and function in 4- to 6-week-old DKO animals. In 14- to 16-week-old animals, osteoclast number was increased, although bone density was further increased. DKO osteoclasts had severely impaired actin ring formation, an impaired ability to generate acid, and reduced resorptive activity in vitro. In addition, their life span ex vivo was reduced. DKO osteoblasts expressed normal differentiation markers except for the expression of osterix, which was reduced. The DKO osteoblasts mineralized normally in vitro, indicating that the in vivo defect in osteoblast function was not cell autonomous. Confocal imaging demonstrated focal disruption of the osteocytic dendritic network in DKO cortical bone. Despite these changes, DKO animals had a normal response to treatment with once-daily parathyroid hormone (PTH). We conclude that Rac1 and Rac2 have critical roles in skeletal metabolism.

Background Failure of glenoid labrum and capsular healing after glenohumeral dislocation can lead to persistent shoulder instability. The purpose of this study was to determine the effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on the healing glenoid labrum and capsule after glenohumeral dislocation in a rat model. Methods Sixty-six rats had surgically induced anterior-inferior labral tears and anterior glenohumeral dislocation. Postoperatively, the animals were assigned to either normal (n = 32) or ibuprofen drinking water (n = 31). Animals were euthanized at 2 and 4 weeks postoperatively for biomechanical testing and histologic analysis. Results The maximum load increased from 2 to 4 weeks after injury in the NSAID groups but not in the control groups. At 2 weeks, the maximum load was lower in the NSAID group compared with the control group. In a matched comparison between injured and uninjured limbs, the maximum load was significantly decreased in the injured limb of the 2-week NSAID group. At 4 weeks, the NSAID group had decreased stiffness compared with the 4-week control group. Conclusions In a new rat model of glenohumeral instability, the postinjury administration of ibuprofen resulted in decreased capsulolabral healing. A matched pair analysis of injured to uninjured limbs supported the findings of impaired healing in the NSAID-treated animals. These findings demonstrate that the use of NSAIDs after glenohumeral dislocation may impair capsulolabral healing and should be limited or avoided to optimize glenohumeral stability.

Colony-stimulating factor 1 (CSF1) is known to promote osteoclast progenitor survival, but its roles in osteoclast differentiation and mature osteoclast function are less well understood. In a microarray screen, Jun dimerization protein 2 (JDP2) was identified as significantly induced by CSF1. Recent reports indicate that JDP2 is required for normal osteoclastogenesis and skeletal metabolism. Because there are no reports on the transcriptional regulation of this gene, the DNA sequence from -2612 to +682 bp (relative to the transcription start site) of the JDP2 gene was cloned, and promoter activity was analyzed. The T box-binding element (TBE) between -191 and -141 bp was identified as the cis-element responsible for CSF1-dependent JDP2 expression. Using degenerate PCR, Tbx3 was identified as the major isoform binding the TBE. Overexpression of Tbx3 induced JDP2 promoter activity, whereas suppressing Tbx3 expression substantially attenuated CSF1-induced transcription. Suppressing Tbx3 in osteoclast precursors reduced JDP2 expression and significantly impaired RANKL/CSF1-induced osteoclastogenesis. A MEK1/2-specific inhibitor was found to block CSF1-induced JDP2 expression. Consistent with these data, JDP2(-/-) mice were found to have increased bone mass. In summary, CSF1 up-regulates JDP2 expression by inducing Tbx3 binding to the JDP2 promoter. The downstream signaling cascade from activated c-Fms involves the MEK1/2-ERK1/2 pathway. Tbx3 plays an important role in osteoclastogenesis at least in part by regulating CSF1-dependent expression of JDP2.

Bone’s microporosities play important biologic and mechanical roles. Here, we quantified 3D changes in cortical osteocyte-lacunae and other small porosities induced by estrogen withdrawal and two different osteoporosis treatments. Unlike 2D measurements, these data collected via synchrotron radiation-based μCT describe the size and 3D spatial distribution of a large number of porous structures. Six-month old female Sprague-Dawley rats were separated into four groups of age-matched controls, untreated OVX, OVX treated with PTH, and OVX treated with Alendronate (ALN). Intracortical microporosity of the medial quadrant of the femoral diaphysis was quantified at endosteal, intracortical, and periosteal regions of the samples, allowing the quantification of osteocyte lacunae that were formed primarily before versus after the start of treatment. Across the overall thickness of the medial cortex, lacunar volume fraction (Lc.V/TV) was significantly lower in ALN treated rats compared to PTH. In the endosteal region, average osteocyte lacunar volume () of untreated OVX rats was significantly lower than in age-matched controls, indicating a decrease in osteocyte lacunar size in bone formed on the endosteal surface after estrogen withdrawal. The effect of treatment (OVX, ALN, PTH) on the number of lacunae per tissue volume (Lc.N/TV) was dependent on the specific location within the cortex (endosteal, intracortical, periosteal). In both the endosteal and intracortical regions, Lc.N/TV was significantly lower in ALN than in untreated OVX, suggesting a site-specific effect in osteocyte lacuna density with ALN treatment. There also were a significantly greater number of small pores (5–100 μm3 in volume) in the endosteal region for PTH compared to ALN. The mechanical impact of this altered microporosity structure is unknown, but might serve to enhance, rather than deteriorate bone strength with PTH treatment, as smaller osteocyte lacunae may be better able to absorb shear forces than larger lacunae. Together, these data demonstrate that current treatments of osteoporosis can alter the number, size, and distribution of microporosities in cortical rat lamellar bone.

Objectives: Rotator cuff tendon heals by fibrovascular scar that is weaker than native tissue leading to repairs that are prone to failure. Our objective was to investigate the ability of an amniotic membrane-derived human allograft to improve rotator cuff tendon-bone healing and skeletal muscle architecture in a chronic massive rotator cuff injury and repair model in rats. We hypothesized that application of human amniotic membrane to the tendon-bone interface at the time of repair will result in increased attachment strength and rotator cuff muscle fiber force secondary to improved bone formation and nthesis histomorphometry, less plasminogen activator inhibitor-1 (PAI-1) expression, and induced muscle repair gene expression. Methods: Eighty-five male Sprague-Dawley rats were allocated to one of four groups: (1) uninjured (U), (2) injury only (IO), (3) chronic injury and repair (CR), or (4) chronic injury and augmented repair (ER). The IO, CR, and ER groups underwent unilateral detachment of the supraspinatus and infraspinatus tendons with injection of the corresponding muscle with 3 u/kg Onabotulinum toxin A. Four weeks following injury, a surgical repair was performed in the CR and ER groups using transosseous suture fixation with human amniotic membrane applied to the tendon-bone repair site in the ER group. Animals were euthanized at four weeks following the repair. Biomechanical testing of the supraspinatus and infraspinatus tendon-bone complex and single fiber contractility testing of the supraspinatus muscle were performed. Microcomputed tomography was utilized to quantitate bone microstructure at the repair site. The healing tendon-bone interface was evaluated with histomorphometric quantification of fibrocartilage formation and collagen organization, and immunostaining with PAI-1. The rotator cuff muscle was evaluated with immunohistochemical localization for lipid deposition and total cross-sectional area. Real-time polymerase chain reaction evaluated relative expression of genes relevant to muscle repair. Statistical analysis was performed using a nonparametric Kruskal-Wallis test with significance set at pResults: Augmentation with amniotic membrane did not lead to increased load-to-failure or stiffness compared to CR. The cross-sectional area, maximum isometric force, and specific force of individual supraspinatus muscle fibers were significantly greater with ER compared to CR (Figure 1). Microcomputed tomography revealed a larger volume of newly formed bone present at the bony footprint with ER compared to CR. Histomorphometric analysis demonstrated significantly greater fibrocartilage area and collagen organization at the healing tendon-bone insertion site with ER compared to CR at 4 week. Qualitatively, there was less PAI-1 immunostaining noted at the tendon-bone interface in the ER group. There was less lipid deposition and greater cross-sectional area of the rotator cuff muscle fibers in the ER group. Rotator cuff muscle gene expression with ER demonstrated a downregulation of apoptosis regulatory genes BCL2 and Caspase-3, adipogenesis regulatory genes FITM1 and FITM2, TNF-alpha and TGF-beta 3 compared to CR. Conclusion: Augmentation with human amniotic membrane in a chronic, massive rotator cuff injury and repair model led to greater bone formation, more organized tendon enthesis, induced muscle repair gene expression, and greater rotator cuff muscle fiber force secondary to less PAI-1 expression.

Compensatory interactions among adult skeletal traits are critical for establishing strength but complicate the search for fracture susceptibility genes by allowing many genetic variants to exist in a population without loss of function. A better understanding of how these interactions arise during growth will provide new insight into genotype-phenotype relationships and the biological controls that establish skeletal strength. We tested the hypothesis that genetic variants affecting growth in width relative to growth in length (slenderness) are coordinated with movement of the inner bone surface and matrix mineralization to match stiffness with weight-bearing loads during postnatal growth. Midshaft femoral morphology and tissue-mineral density were quantified at ages of 1 day and at 4, 8, and 16 weeks for a panel of 20 female AXB/BXA recombinant inbred mouse strains. Path Analyses revealed significant compensatory interactions among outer-surface expansion rate, inner-surface expansion rate, and tissue-mineral density during postnatal growth, indicating that genetic variants affecting bone slenderness were buffered mechanically by the precise regulation of bone surface movements and matrix mineralization. Importantly, the covariation between morphology and mineralization resulted from a heritable constraint limiting the amount of tissue that could be used to construct a functional femur. The functional interactions during growth explained 56-99% of the variability in adult traits and mechanical properties. These functional interactions provide quantitative expectations of how genetic or environmental variants affecting one trait should be compensated by changes in other traits. Variants that impair this process or that cannot be fully compensated are expected to alter skeletal growth leading to underdesigned (weak) or overdesigned (bulky) structures.

Androgens are anabolic hormones that affect many tissues, including bone. However, an anabolic effect of androgen treatment on bone in eugonadal subjects has not been observed and clinical trials have been disappointing. The androgen receptor (AR) mediates biological responses to androgens. In bone tissue, both AR and the estrogen receptor (ER) are expressed. Since androgens can be converted into estrogen, the specific role of the AR in maintenance of skeletal homoeostasis remains controversial. The goal of this study was to use skeletally targeted overexpression of AR in differentiated osteoblasts as a means of elucidating the specific role(s) for AR transactivation in the mature bone compartment. Transgenic mice overexpressing AR under the control of the 2.3-kb α1(I)-collagen promoter fragment showed no difference in body composition, testosterone, or 17s-estradiol levels. However, transgenic males have reduced serum osteocalcin, CTx and TRAPC5b levels, and a bone phenotype was observed. In cortical bone, high-resolution micro-computed tomography revealed no difference in periosteal perimeter but a significant reduction in cortical bone area due to an enlarged marrow cavity. Endocortical bone formation rate was also significantly inhibited. Biomechanical analyses showed decreased whole bone strength and quality, with significant reductions in all parameters tested. Trabecular morphology was altered, with increased bone volume comprised of more trabeculae that were closer together but not thicker. Expression of genes involved in bone formation and bone resorption was significantly reduced. The consequences of androgen action are compartment-specific; anabolic effects are exhibited exclusively at periosteal surfaces, but in mature osteoblasts androgens inhibited osteogenesis with detrimental effects on matrix quality, bone fragility and whole bone strength. Thus, the present data demonstrate that enhanced androgen signaling targeted to bone results in low bone turnover and inhibition of bone formation by differentiated osteoblasts. These results indicate that direct androgen action in mature osteoblasts is not anabolic, and raise concerns regarding anabolic steroid abuse in the developing skeleton or high-dose treatment in eugonadal adults.

Biomechanical properties were assessed from the tibias of 17 adult males 17-46 years of age. Tissue-level mechanical properties varied with bone size. Narrower tibias were comprised of tissue that was more brittle and more prone to accumulating damage compared with tissue from wider tibias. Introduction: A better understanding of the factors contributing to stress fractures is needed to identify new prevention strategies that will reduce fracture incidence. Having a narrow (i.e., more slender) tibia relative to body mass has been shown to be a major predictor of stress fracture risk and fragility in male military recruits and male athletes. The intriguing possibility that slender bones, like those shown in animal models, may be composed of more damageable material has not been considered in the human skeleton. Materials and Methods: Polar moment of inertia, section modulus, and antero-posterior (AP) and medial-lateral (ML) widths were determined for tibial diaphyses from 17 male donors 17-46 years of age. A slenderness index was defined as the inverse ratio of the section modulus to tibia length and body weight. Eight prismatic cortical bone samples were generated from each tibia, and tissue-level mechanical properties including modulus, strength, total energy, postyield strain, and tissue damageability were measured by four-point bending from monotonic (n = 4/tibia) and damage accumulation (n = 4/tibia) test methods. Partial correlation coefficients were determined between each geometrical parameter and each tissue-level mechanical property while taking age into consideration. Results: Significant correlations were observed between tibial morphology and the mechanical properties that characterized tissue brittleness and damageability. Positive correlations were observed between measures of bone size (AP width) and measures of tissue ductility (postyield strain, total energy), and negative correlations were observed between bone size (moment of inertia, section modulus) and tissue modulus. Conclusions: The correlation analysis suggested that bone morphology could be used as a predictor of tissue fragility and stress fracture risk. The average mechanical properties of cortical tissue varied as a function of the overall size of the bone. Therefore, under extreme loading conditions (e.g., military training), variation in bone quality parameters related to damageability may be a contributing factor to the increased risk of stress fracture for individuals with more slender bones.

Rac1 and Rac2 are thought to have important roles in osteoclasts. Therefore, mice with deletion of both Rac1 and Rac2 in mature osteoclasts (DKO) were generated by crossing Rac1(flox/flox) mice with mice expressing Cre in the cathepsin K locus and then mating these animals with Rac2(-/-) mice. DKO mice had markedly impaired tooth eruption. Bone mineral density (BMD) was increased 21% to 33% in 4- to 6-week-old DKO mice at all sites when measured by dual-energy X-ray absorptiometry (DXA) and serum cross-linked C-telopeptide (CTx) was reduced by 52%. The amount of metaphyseal trabecular bone was markedly increased in DKO mice, but the cortices were very thin. Spinal trabecular bone mass was increased. Histomorphometry revealed significant reductions in both osteoclast and osteoblast number and function in 4- to 6-week-old DKO animals. In 14- to 16-week-old animals, osteoclast number was increased, although bone density was further increased. DKO osteoclasts had severely impaired actin ring formation, an impaired ability to generate acid, and reduced resorptive activity in vitro. In addition, their life span ex vivo was reduced. DKO osteoblasts expressed normal differentiation markers except for the expression of osterix, which was reduced. The DKO osteoblasts mineralized normally in vitro, indicating that the in vivo defect in osteoblast function was not cell autonomous. Confocal imaging demonstrated focal disruption of the osteocytic dendritic network in DKO cortical bone. Despite these changes, DKO animals had a normal response to treatment with once-daily parathyroid hormone (PTH). We conclude that Rac1 and Rac2 have critical roles in skeletal metabolism.

The modeling of long bone surfaces during linear growth is a key developmental process, but its regulation is poorly understood. We report here that parathyroid hormone-related peptide (PTHrP) expressed in the fibrous layer of the periosteum (PO) drives the osteoclastic (OC) resorption that models the metaphyseal–diaphyseal junction (MDJ) in the proximal tibia and fibula during linear growth. PTHrP was conditionally deleted (cKO) in the PO via Scleraxis gene targeting (Scx-Cre). In the lateral tibia, cKO of PTHrP led to a failure of modeling, such that the normal concave MDJ was replaced by a mound-like deformity. This was accompanied by a failure to induce receptor activator of NF-kB ligand (RANKL) and a 75% reduction in OC number (P ≤ 0.001) on the cortical surface. The MDJ also displayed a curious threefold increase in endocortical osteoblast mineral apposition rate (P ≤ 0.001) and a thickened cortex, suggesting some form of coupling of endocortical bone formation to events on the PO surface. Because it fuses distally, the fibula is modeled only proximally and does so at an extraordinary rate, with an anteromedial cortex in CD-1 mice that was so moth-eaten that a clear PO surface could not be identified. The cKO fibula displayed a remarkable phenotype, with a misshapen club-like metaphysis and an enlargement in the 3D size of the entire bone, manifest as a 40–45% increase in the PO circumference at the MDJ (P ≤ 0.001) as well as the mid-diaphysis (P ≤ 0.001). These tibial and fibular phenotypes were reproduced in a Scx-Cre-driven RANKL cKO mouse. We conclude that PTHrP in the fibrous PO mediates the modeling of the MDJ of long bones during linear growth, and that in a highly susceptible system such as the fibula this surface modeling defines the size and shape of the entire bone.

The developing cortical surfaces of long bones are sculpted and modeled by periosteal osteoclasts and osteoblasts. These surfaces also receive the insertions of tendons and ligaments, and these insertion sites too are modeled to form the root systems that anchor them into the cortical bone. The regulatory molecules that control modeling are poorly understood, but recent evidence suggests that parathyroid hormone-related protein (PTHrP) participates in this process. PTHrP functions principally as a paracrine regulatory molecule, and is known to be induced by mechanical loading in a number of sites. The most curious example of developmental modeling of the cortex is the migration of insertion sites such as that of the medial collateral ligament (MCL) along the bone surface during long-bone growth. We report here the mechanisms that mediate MCL migration using a combination of genetic, imaging and histological techniques. We describe a MCL migratory complex that comprises two components. The first is the MCL insertion site itself, which is a prototypical fibrous insertion site with coupled osteoclast and osteoblast activities, and its key feature is that it is anchored early in development, well before initiation of the long-bone growth spurt. Above the insertion site the periosteum is excavated by osteoclasts to form a migratory tract; this is mediated by wholly uncoupled osteoclastic bone resorption and remains as an unmineralized canal on the cortical surface in the adult. Load-induction of PTHrP appears to regulate the osteoclastic activity in both the insertion site and migratory tract.

To gain insight into past human physi- cal activity, anthropologists often infer functional loading history from the morphology of limb bone remains. It is assumed that, during life, loading had a positive, dose- dependent effect on bone structure that can be identified despite other effects. Here, we investigate the effects of genetic background and functional loading on limb bones using mice from an artificial selection experiment for high levels of voluntary wheel running. Growing males from four replicate high runner (HR) lines and four rep- licate nonselected control (C) lines were either allowed or denied wheel access for 2 months. Using lCT, femoral morphology was assessed at two cortical sites (mid-dia- physis, distal metaphysis) and one trabecular site (distal metaphysis). We found that genetic differences between the linetypes (HR vs. C), between the replicate lines within linetype, and between individuals with and with- out the so-called ''mini-muscle'' phenotype (caused by a Mendelian recessive gene that halves limb muscle mass) gave rise to significant variation in nearly all morpholog- ical indices examined. Wheel access also influenced fem- oral morphology, although the functional response did not generally result in enhanced structure. Exercise caused moderate periosteal enlargement, but relatively greater endocortical expansion, resulting in significantly thinner cortices and reduced bone area in the metaphy- sis. The magnitude of the response was independent of distance run. Mid-diaphyseal bone area and area moments, and trabecular morphology, were unaffected by exercise. These results underscore the strong influ- ence of genetics on bone structure and the complexity by which mechanical stimuli may cause alterations in it. Am J Phys Anthropol 148:24-35, 2012. V C

Conventional approaches to identifying quantitative trait loci (QTLs) regulating bone mass and fragility are limited because they examine cortical and trabecular traits independently. Prior work examining long bones from young adult mice and humans indicated that skeletal traits are functionally related and that compensatory interactions among morphological and compositional traits are critical for establishing mechanical function. However, it is not known whether trait covariation (i.e., phenotypic integration) also is important for establishing mechanical function in more complex, corticocancellous structures. Covariation among trabecular, cortical, and compositional bone traits was examined in the context of mechanical functionality for L(4) vertebral bodies across a panel of 16-wk-old female AXB/BXA recombinant inbred (RI) mouse strains. The unique pattern of randomization of the A/J and C57BL/6J (B6) genome among the RI panel provides a powerful tool that can be used to measure the tendency for different traits to covary and to study the biology of complex traits. We tested the hypothesis that genetic variants affecting vertebral size and mass are buffered by changes in the relative amounts of cortical and trabecular bone and overall mineralization. Despite inheriting random sets of A/J and B6 genomes, the RI strains inherited nonrandom sets of cortical and trabecular bone traits. Path analysis, which is a multivariate analysis that shows how multiple traits covary simultaneously when confounding variables like body size are taken into consideration, showed that RI strains that tended to have smaller vertebrae relative to body size achieved mechanical functionality by increasing mineralization and the relative amounts of cortical and trabecular bone. The interdependence among corticocancellous traits in the vertebral body indicated that variation in trabecular bone traits among inbred mouse strains, which is often thought to arise from genetic factors, is also determined in part by the adaptive response to variation in traits describing the cortical shell. The covariation among corticocancellous traits has important implications for genetic analyses and for interpreting the response of bone to genetic and environmental perturbations.

Androgens are anabolic hormones that affect many tissues, including bone. However, an anabolic effect of androgen treatment on bone in eugonadal subjects has not been observed and clinical trials have been disappointing. The androgen receptor (AR) mediates biological responses to androgens. In bone tissue, both AR and the estrogen receptor (ER) are expressed. Since androgens can be converted into estrogen, the specific role of the AR in maintenance of skeletal homoeostasis remains controversial. The goal of this study was to use skeletally targeted overexpression of AR in differentiated osteoblasts as a means of elucidating the specific role(s) for AR transactivation in the mature bone compartment. Transgenic mice overexpressing AR under the control of the 2.3-kb alpha1(I)-collagen promoter fragment showed no difference in body composition, testosterone, or 17ss-estradiol levels. However, transgenic males have reduced serum osteocalcin, CTx and TRAPC5b levels, and a bone phenotype was observed. In cortical bone, high-resolution micro-computed tomography revealed no difference in periosteal perimeter but a significant reduction in cortical bone area due to an enlarged marrow cavity. Endocortical bone formation rate was also significantly inhibited. Biomechanical analyses showed decreased whole bone strength and quality, with significant reductions in all parameters tested. Trabecular morphology was altered, with increased bone volume comprised of more trabeculae that were closer together but not thicker. Expression of genes involved in bone formation and bone resorption was significantly reduced. The consequences of androgen action are compartment-specific; anabolic effects are exhibited exclusively at periosteal surfaces, but in mature osteoblasts androgens inhibited osteogenesis with detrimental effects on matrix quality, bone fragility and whole bone strength. Thus, the present data demonstrate that enhanced androgen signaling targeted to bone results in low bone turnover and inhibition of bone formation by differentiated osteoblasts. These results indicate that direct androgen action in mature osteoblasts is not anabolic, and raise concerns regarding anabolic steroid abuse in the developing skeleton or high-dose treatment in eugonadal adults.

Complex corticocancellous skeletal sites such as the vertebra or proximal femur are connected networks of bone capable of transferring mechanical loads. Characterizing these structures as networks may allow us to quantify the load transferring behavior of the emergent system as a function of the connected cortical and trabecular components. By defining the relationship between certain physical bone traits and mechanical load transfer pathways, a clearer picture of the genetic determinants of skeletal fragility can be developed. We tested the hypothesis that the measures provided by network percolation theory will reveal that different combinations of cortical, trabecular, and compositional traits lead to significantly different load transfer pathways within the vertebral bodies among inbred mouse strains. Gross morphologic, micro-architectural, and compositional traits of L5 vertebrae from 15 week old A/J (A), C57BL6/J (B6), and C3H/HeJ (C3H) inbred mice ( n = 10/strain) were determined using micro-computed tomography. Measures included total cross-sectional area, bone volume fraction, trabecular number, thickness, spacing, cortical area, and tissue mineral density. Two-dimensional coronal sections were converted to network graphs with the cortical shell considered as one highly connected node. Percolation parameters including correlation length (average number of connected nodes between superior and inferior surfaces), chemical length (minimum number of connected nodes between surfaces), and backbone mass (strut number) were measured. Analysis of the topology of the connected bone networks showed that A and B6 mice transfer load through trabecular pathways in the middle of the vertebral body in addition to the cortical shell. C3H mice transfer load primarily through the highly mineralized cortical shell. Thus, the measures provided by percolation theory provide a quantitative approach to study how different combinations of cortical and trabecular traits lead to mechanically functional structures. The data further emphasize the interdependent nature of these physical bone traits suggesting similar genetic variants may affect both trabecular and cortical bone. Therefore, developing a network approach to study corticocancellous architecture during growth should further our understanding of the biological basis of skeletal fragility and, thus, provide novel engineering approaches to studying the genetic basis of fracture risk.

A path analysis was conducted to determine whether functional interactions exist among morphological, compositional, and microstructural traits for young adult human tibias. Data provided evidence that bone traits are co-adapted during ontogeny so that the sets of traits together satisfy physiological loading demands. However, certain sets of traits are expected to perform poorly under extreme load conditions. Introduction: Previous data from inbred mouse strains suggested that biological processes within bone co-adapt morphological and compositional traits during ontogeny to satisfy physiological loading demands. Similar work in young adult humans showed that cortical tissue from slender tibias was stiffer, less ductile, and more susceptible to accumulating damage. Here we tested whether the relationships among morphology and tissue level mechanical properties were the result of biological processes that co-adapt physical traits, similar to those observed for the mouse skeleton. Materials and Methods: Cross-sectional morphology, bone slenderness (Tt.Ar/Le), and tissue level mechanical properties were measured from tibias from 14 female (22–46 yr old) and 17 male (17–46 yr old) donors. Physical bone traits measured included tissue density, ash content, water content, porosity, and the area fractions of osteonal, interstitial, and circumferential lamellar tissues. Bivariate relationships among traits were determined using linear regression analysis. A path analysis was conducted to test the hypothesis that Tt.Ar/Le is functionally related to mineralization (ash content) and the proportion of total area occupied by cortical bone. Results: Ash content correlated negatively with several traits including Tt.Ar/Le and marrow area, indicating that slender bones were constructed of tissue with higher mineralization. Path analysis revealed that slender tibias were compensated by higher mineralization and a greater area fraction of bone. Conclusions: The results suggest that bone adapts by varying the relative amount of cortical bone within the diaphysis and by varying matrix composition. This co-adaptation is expected to lead to a particular set of traits that is sufficiently stiff and strong to support daily loads. However, increases in mineralization result in a more brittle and damageable material that would be expected to perform poorly under extreme load conditions. Therefore, focusing attention on sets of traits and the relationship among traits may advance our understanding of how genetic and environmental factors influence bone fragility.

Understanding how growth influences adult bone morphology and tissue quality should provide important insight into why females show a greater incidence of stress fractures early in life and fragility fractures later in life compared to males. The objective of this study was to test whether females acquire similar tissue-level mechanical properties as males by the time peak bone properties are established. Standardized beams of bone were machined from the tibial diaphyses of 14 young, adult females ranging in age from 22 to 46 years. Data for males (n=17, age=17-46 years) were taken from a prior study. Measures of tissue-level mechanical properties, including stiffness, strength, ductility, toughness, and damageability, were compared between sexes using t-tests. The relationship between cross-sectional morphology and tissue-level mechanical properties was also examined. Males and females showed nearly identical tissue-level mechanical properties. Both sexes also showed similar age-related degradation of mechanical properties and a similar relationship between cross-sectional morphology and tissue quality. However, for all body sizes, female tibiae were smaller relative to body size (i.e., less robust) compared to males. The results indicated that sex-specific growth patterns affected transverse bone size, but did not affect tissue-level mechanical properties. This, combined with the observation that young, adult female long bones are undersized relative to body size, suggests that adult females would be expected to accumulate more damage under intense loading compared to males. This may be a contributing factor to the greater incidence of stress fractures observed for female military recruits.