Blaine A. Christiansen

Guidelines for assessment of bone microstructure in rodents using micro-computed tomography

Use of high‐resolution micro–computed tomography (µCT) imaging to assess trabecular and cortical bone morphology has grown immensely. There are several commercially available µCT systems, each with different approaches to image acquisition, evaluation, and reporting of outcomes. This lack of consistency makes it difficult to interpret reported results and to compare findings across different studies. This article addresses this critical need for standardized terminology and consistent reporting of parameters related to image acquisition and analysis, and key outcome assessments, particularly with respect to ex vivo analysis of rodent specimens. Thus the guidelines herein provide recommendations regarding (1) standardized terminology and units, (2) information to be included in describing the methods for a given experiment, and (3) a minimal set of outcome variables that should be reported. Whereas the specific research objective will determine the experimental design, these guidelines are intended to ensure accurate and consistent reporting of µCT‐derived bone morphometry and density measurements. In particular, the methods section for papers that present µCT‐based outcomes must include details of the following scan aspects: (1) image acquisition, including the scanning medium, X‐ray tube potential, and voxel size, as well as clear descriptions of the size and location of the volume of interest and the method used to delineate trabecular and cortical bone regions, and (2) image processing, including the algorithms used for image filtration and the approach used for image segmentation. Morphometric analyses should be based on 3D algorithms that do not rely on assumptions about the underlying structure whenever possible. When reporting µCT results, the minimal set of variables that should be used to describe trabecular bone morphometry includes bone volume fraction and trabecular number, thickness, and separation. The minimal set of variables that should be used to describe cortical bone morphometry includes total cross‐sectional area, cortical bone area, cortical bone area fraction, and cortical thickness. Other variables also may be appropriate depending on the research question and technical quality of the scan. Standard nomenclature, outlined in this article, should be followed for reporting of results.

Musculoskeletal changes following non-invasive knee injury using a novel mouse model of post-traumatic osteoarthritis

OBJECTIVE Post-traumatic osteoarthritis (PTOA) is a common consequence of traumatic joint injury, with 50% of anterior cruciate ligament (ACL) rupture patients developing PTOA within 10-20 years. Currently accepted mouse models of PTOA initiate symptoms using various methods, none of which faithfully mimic clinically-relevant injury conditions. In this study we characterize a novel non-invasive mouse model of PTOA that injures the ACL with a single load of tibial compression overload. We utilize this model to determine the time course of articular cartilage and subchondral bone changes following knee injury. DESIGN Mice were euthanized 1, 3, 7, 14, 28, or 56 days after non-invasive knee injury. Knees were scanned using micro-computed tomography (μCT) in order to quantify subchondral trabecular bone, subchondral bone plate, and non-native bone formation (heterotopic ossification). Development of osteoarthritis (OA) was graded using the osteoarthritis research society international (OARSI) scale on histological sections of injured and uninjured knees. RESULTS Following injury we observed a rapid loss of trabecular bone in injured knees compared to uninjured knees by 7 days post-injury, followed by a partial recovery of trabecular bone to a new steady state by 28 days post-injury. We also observed considerable non-native bone formation by 56 days post-injury. Grading of histological sections revealed deterioration of articular cartilage by 56 days post-injury, consistent with development of mild OA. CONCLUSIONS This study establishes a novel mouse model of PTOA, and describes the time course of musculoskeletal changes following knee injury, helping to establish the window of opportunity for preventative treatment.

Mechanical contributions of the cortical and trabecular compartments contribute to differences in age‐related changes in vertebral body strength in men and women assessed by QCT‐based finite element analysis

The biomechanical mechanisms underlying sex‐specific differences in age‐related vertebral fracture rates are ill defined. To gain insight into this issue, we used finite element analysis of clinical computed tomography (CT) scans of the vertebral bodies of L3 and T10 of young and old men and women to assess age‐ and sex‐related differences in the strength of the whole vertebra, the trabecular compartment, and the peripheral compartment (the outer 2 mm of vertebral bone, including the thin cortical shell). We sought to determine whether structural and geometric changes with age differ in men and women, making women more susceptible to vertebral fractures. As expected, we found that vertebral strength decreased with age 2‐fold more in women than in men. The strength of the trabecular compartment declined significantly with age for both sexes, whereas the strength of the peripheral compartment decreased with age in women but was largely maintained in men. The proportion of mechanical strength attributable to the peripheral compartment increased with age in both sexes and at both vertebral levels. Taken together, these results indicate that men and women lose vertebral bone differently with age, particularly in the peripheral (cortical) compartment. This differential bone loss explains, in part, a greater decline in bone strength in women and may contribute to the higher incidence of vertebral fractures among women than men.

Biomechanics of Vertebral Fractures and the Vertebral Fracture Cascade

Vertebral fractures (VFxs) are the most common osteoporotic fracture, and are a strong risk factor for future fracture. The presence of a VFx greatly increases the risk of sustaining subsequent VFxs—a phenomenon often referred to as the “vertebral fracture cascade.” VFxs do not occur uniformly along the spine, but occur more often at the mid-thoracic and thoracolumbar regions than elsewhere. It is likely that both the vertebral fracture cascade and the bimodal distribution of VFx along the spine are attributable to biomechanical factors. VFxs occur when the forces applied to the vertebral body exceed its strength. Loading on the spine is primarily determined by a person’s height, weight, muscle forces, and the task or movement performed, but can also be affected by other factors, such as spinal curvature and invertebral disk deterioration. Vertebral strength is determined mainly by bone size, shape, and bone mineral density, and secondarily by bone microarchitecture, collagen characteristics, and microdamage. Better understanding of VFx etiology is hampered by the fact that most VFxs do not come to clinical attention; therefore, the factors and activities that cause VFxs remain ill defined, including possible differences in the etiology of acute fractures versus those of slow onset. Additional research is needed to elucidate the precise mechanical, morphologic, and biological mechanisms that underlie VFx to improve strategies for assessing VFx risk and preventing the vertebral fracture cascade.

Partial reductions in mechanical loading yield proportional changes in bone density, bone architecture, and muscle mass

Although the musculoskeletal system is known to be sensitive to changes in its mechanical environment, the relationship between functional adaptation and below‐normal mechanical stimuli is not well defined. We investigated bone and muscle adaptation to a range of reduced loading using the partial weight suspension (PWS) system, in which a two‐point harness is used to offload a tunable amount of body weight while maintaining quadrupedal locomotion. Skeletally mature female C57Bl/6 mice were exposed to partial weight bearing at 20%, 40%, 70%, or 100% of body weight for 21 days. A hindlimb unloaded (HLU) group was included for comparison in addition to age‐matched controls in normal housing. Gait kinematics was measured across the full range of weight bearing, and some minor alterations in gait from PWS were identified. With PWS, bone and muscle changes were generally proportional to the degree of unloading. Specifically, total body and hindlimb bone mineral density, calf muscle mass, trabecular bone volume of the distal femur, and cortical area of the femur midshaft were all linearly related to the degree of unloading. Even a load reduction to 70% of normal weight bearing was associated with significant bone deterioration and muscle atrophy. Weight bearing at 20% did not lead to better bone outcomes than HLU despite less muscle atrophy and presumably greater mechanical stimulus, requiring further investigation. These data confirm that the PWS model is highly effective in applying controllable, reduced, long‐term loading that produces predictable, discrete adaptive changes in muscle and bone of the hindlimb.

Long-term administration of AMD3100, an antagonist of SDF-1/CXCR4 signaling, alters fracture repair.

Fracture healing involves rapid stem and progenitor cell migration, homing, and differentiation. SDF‐1 (CXCL12) is considered a master regulator of CXCR4‐positive stem and progenitor cell trafficking to sites of ischemic (hypoxic) injury and regulates their subsequent differentiation into mature reparative cells. In this study, we investigated the role of SDF‐1/CXCR4 signaling in fracture healing where vascular disruption results in hypoxia and SDF‐1 expression. Mice were injected with AMD3100, a CXCR4 antagonist, or vehicle twice daily until euthanasia with the intent to impair stem cell homing to the fracture site and/or their differentiation. Fracture healing was evaluated using micro‐computed tomography, histology, quantitative PCR, and mechanical testing. AMD3100 administration resulted in a significantly reduced hyaline cartilage volume (day 14), callus volume (day 42) and mineralized bone volume (day 42) and reduced expression of genes associated with endochondral ossification including collagen Type 1 alpha 1, collagen Type 2 alpha 1, vascular endothelial growth factor, Annexin A5, nitric oxide synthase 2, and mechanistic target of rapamycin. Our data suggest that the SDF‐1/CXCR4 signaling plays a central role in bone healing possibly by regulating the recruitment and/or differentiation of stem and progenitor cells.

A Biomechanical Model for Estimating Loads on Thoracic and Lumbar Vertebrae

BACKGROUND Biomechanical models are commonly used to estimate loads on the spine. Current models have focused on understanding the etiology of low back pain and have not included thoracic vertebral levels. Using experimental data on the stiffness of the thoracic spine, ribcage, and sternum, we developed a new quasi-static stiffness-based biomechanical model to calculate loads on the thoracic and lumbar spine during bending or lifting tasks. METHODS To assess the sensitivity of the model to our key assumptions, we determined the effect of varying ribcage and sternal stiffness, maximum muscle stress, and objective function on predicted spinal loads. We compared estimates of spinal loading obtained with our model to previously reported in vivo intradiscal pressures and muscle activation patterns. FINDINGS Inclusion of the ribs and sternum caused an average decrease in vertebral compressive force of 33% for forward flexion and 18% in a lateral moment task. The impact of maximum muscle stress on vertebral force was limited to a narrow range of values. Compressive forces predicted by our model were strongly correlated to in vivo intradiscal pressure measurements in the thoracic (r=0.95) and lumbar (r=1) spine. Predicted trunk muscle activity was also strongly correlated (r=0.95) with previously published EMG data from the lumbar spine. INTERPRETATION The consistency and accuracy of the model predictions appear to be sufficient to justify the use of this model for investigating the relationships between applied loads and injury to the thoracic spine during quasi-static loading activities.

Non-invasive mouse models of post-traumatic osteoarthritis

Animal models of osteoarthritis (OA) are essential tools for investigating the development of the disease on a more rapid timeline than human OA. Mice are particularly useful due to the plethora of genetically modified or inbred mouse strains available. The majority of available mouse models of OA use a joint injury or other acute insult to initiate joint degeneration, representing post-traumatic osteoarthritis (PTOA). However, no consensus exists on which injury methods are most translatable to human OA. Currently, surgical injury methods are most commonly used for studies of OA in mice; however, these methods may have confounding effects due to the surgical/invasive injury procedure itself, rather than the targeted joint injury. Non-invasive injury methods avoid this complication by mechanically inducing a joint injury externally, without breaking the skin or disrupting the joint. In this regard, non-invasive injury models may be crucial for investigating early adaptive processes initiated at the time of injury, and may be more representative of human OA in which injury is induced mechanically. A small number of non-invasive mouse models of PTOA have been described within the last few years, including intra-articular fracture of tibial subchondral bone, cyclic tibial compression loading of articular cartilage, and anterior cruciate ligament (ACL) rupture via tibial compression overload. This review describes the methods used to induce joint injury in each of these non-invasive models, and presents the findings of studies utilizing these models. Altogether, these non-invasive mouse models represent a unique and important spectrum of animal models for studying different aspects of PTOA.

Comparison of loading rate-dependent injury modes in a murine model of post-traumatic osteoarthritis.

Post‐traumatic osteoarthritis (PTOA) is a common long‐term consequence of joint injuries such as anterior cruciate ligament (ACL) rupture. In this study we used a tibial compression overload mouse model to compare knee injury induced at low speed (1 mm/s), which creates an avulsion fracture, to injury induced at high speed (500 mm/s), which induces midsubstance tear of the ACL. Mice were sacrificed at 0 days, 10 days, 12 weeks, or 16 weeks post‐injury, and joints were analyzed with micro‐computed tomography, whole joint histology, and biomechanical laxity testing. Knee injury with both injury modes caused considerable trabecular bone loss by 10 days post‐injury, with the Low Speed Injury group (avulsion) exhibiting a greater amount of bone loss than the High Speed Injury group (midsubstance tear). Immediately after injury, both injury modes resulted in greater than twofold increases in total AP joint laxity relative to control knees. By 12 and 16 weeks post‐injury, total AP laxity was restored to uninjured control values, possibly due to knee stabilization via osteophyte formation. This model presents an opportunity to explore fundamental questions regarding the role of bone turnover in PTOA, and the findings of this study support a biomechanical mechanism of osteophyte formation following injury.

QCT Measures of Bone Strength at the Thoracic and Lumbar Spine: The Framingham Study

We used volumetric quantitative computed tomography (QCT) scans to evaluate volumetric bone density (vBMD), geometry, and strength in the thoracic (T8 to T10) and lumbar (L3 to L5) spine and determined how these parameters varied with age, sex, and spinal region. Participants included 690 participants of the Framingham Study, 40 to 87 years old (mean, 61 years). In both women and men, trabecular vBMD declined with age similarly for lumbar and thoracic regions, whereas cortical vBMD and integral vBMD, vertebral strength, and compressive force declined more at the lumbar spine than thoracic spine (interaction, p < 0.01). Notably, in men, cortical vBMD increased (β = 0.0004, p = 0.01), and vertebral strength did not change (β = 1.9305, p = 0.66) at the thoracic spine with age. In both women and men, vertebral cross‐sectional area increased less and the factor‐of‐risk increased more with age at the lumbar than at the thoracic region (interaction, p < 0.01). For example, in women, the factor‐of‐risk for forward flexion increased (worsened) with age 6.8‐fold more in the lumbar spine (β = 0.0157), compared with the thoracic spine (β = 0.0023). vBMD and vertebral strength declined more and the factor‐of‐risk increased more with age in women than men (interaction, p < 0.01). For instance, integral vBMD for the lumbar spine declined 36% from 40 to 75 years of age in women compared with 18% in men. There was little or no age‐related change in the forces applied to the thoracic vertebrae in either women or men. Age‐related changes were greater in the lumbar spine than in the thoracic region and greater in women than men. Whereas women lost bone density and strength at both the thoracic and lumbar spine, in men, vertebral strength declined only at the lumbar spine. Our study confirms the importance of evaluating determinants of vertebral strength in both the thoracic and lumbar spine and in both women and men to understand mechanisms underlying the structural failure of vertebral bodies with aging.