Mechanical loading is necessary for proper development and maintenance of the musculoskeletal system. Yet, despite the profound effects of reduced mechanical loading on muscle atrophy and skeletal fragility, there has been little investigation into the physiological effects of clinically relevant partial weight-bearing environments, such as bed rest, immobilization, stroke, cerebral palsy, muscular dystrophy, spinal cord injury or age-related reductions in physical activity.
The major obstacle to such research has been the lack of a suitable animal model. We have developed a novel model of titrated weight-bearing that offers a unique capability for exploring the chronic effects of reduced quadrupedal loading in mice. The system allows studies with controlled exposure to 10-80% weight-bearing compared to normally loaded controls in an identical environment [link].

Our long-term goal is to take advantage of this unique model to gain insight into the mechanisms underlying the musculoskeletal response to reduced mechanical loading, thereby identifying new targets for preventing musculoskeletal deterioration in due to age-, disease- or injury-induced reductions in mechanical loading. Thus, we propose to extensively characterize the mechanical stimuli associated with our partial weight-bearing model, and to determine the timing and magnitude of the musculoskeletal response to partial weight-bearing, as compared to both normal weight-bearing and full hindlimb unloading via tail suspension. Establishment of a model where quadrupedal gait is maintained, yet loads can be reduced by prescribed amounts will provide the opportunity to test long-held views about the minimal loading stimulus necessary to maintain bone and muscle tissue under conditions of disuse. A major advantage to developing a partial weight-bearing murine model is that it will be ideally suited for future studies designed to delineate the genetic, cellular and molecular mechanisms associated with musculoskeletal adaptation to altered loading environments.

We will use our partial weight-bearing model to quantify the musculoskeletal effects of 10, 21 and 35 days exposure to 20, 40, 60 or 100% body weight loading in adult female mice, and compare the response to that of tail suspension (0% body weight). Outcome assessments will include in vivo bone mineral density, body composition, as well as ex vivo muscle weight, bone architecture by µCT, and femoral biomechanics. Serum markers of bone turnover, marrow fat assessment, histology and dynamic histomorphometry will be used to delineate mechanisms underlying the response to partial weight-bearing.

We are also testing whether new therapeutic interventions can inhibit bone loss during disuse. In particular, we are testing whether a new anti-resorptive therapy, antibody to RANKL (denosumab) and whether a new anabolic therapy, sclerostin antibody, inhibits skeletal deterioration across the spectrum of partial weight bearing environments. Denosumab, a fully human monoclonal antibody to the receptor activator of nuclear factor-B ligand (RANKL), inhibits development and activity of osteoclasts, and thereby markedly decreases bone resorption. Semi-annual subcutaneous injections of denosumab significantly reduce vertebral and non-vertebral fracture risk in postmenopausal women with osteoporosis. The secreted protein sclerostin is a key negative regulator of bone formation. Humans with genetic mutations leading to sclerostin deficiency have increased bone mass, and in rodents, inhibition of sclerostin via pharmacologic antibody treatment or genetic manipulation leads to anabolic skeletal effects. Moreover, mice deficient in sclerostin are resistant to disuse-induced bone loss. Our preliminary studies demonstrate that treatment with sclerostin antibody leads to bone formation even is a disuse model. Images at the right show 3D rendering of representative microCT images of the mouse distal femur, with the unloaded, vehicle treated animal on the top and the unloaded, sclerostin-treated animal on the bottom.

Altogether, this work will provide novel information about musculoskeletal adaptation across a continuum of reduced mechanical loading, and insights into the fundamental relationship between mechanical loading and musculoskeletal adaptation. Moreover, the studies will provide experimental data that can be used to test existing quantitative theories about skeletal adaptation to altered mechanical loading. Finally, development of this model will establish a basis for future studies designed to delineate the cellular and molecular mechanisms underlying skeletal response to reduced loading, and will enhance the development of interventions to prevent muscle and bone atrophy during a variety of clinical conditions of reduced musculoskeletal loading due to disease, injury or inactivity.

This work is funded by NIH-NIAMS R21 AR057522, NASA NNX10AE39G, and a research grant from Amgen.

Recent publications:

Ellman R, Spatz J, Cloutier A, Palme R, Christiansen BA, Bouxsein ML. Partial reductions in mechanical loading yield proportional changes in bone density, bone architecture, and muscle mass. J Bone Miner Res 2013; 28(4): 875-85.

Ellman R, Grasso DJ, van Vliet M, Brooks DJ, Spatz JM, Conlon C, Bouxsein ML. Combined Effects of Botulinum Toxin Injection and Hind Limb Unloading on Bone and Muscle. Calcif Tissue Int 2013; 94(3): 327-337.

Wagner EB, Granzella NP, Saito H, Newman DJ, Young LR, Bouxsein ML. Partial weight suspension: a novel murine model for investigating adaptation to reduced musculoskeletal loading. J Appl Physiol 2010; 109(2):350-7. PDF

For several decades we have been investigating the biomechanical mechanisms underlying skeletal fragility in osteoporosis and other bone disorders, and how various interventions improve bone strength and reduce fracture risk. Our work has included studies in animal models, human cadaveric specimens and clinical studies. Our overall goals are to better understand the origins and causes of skeletal fragility, to better identify those at risk for fracture and to enhance the monitoring of treatment efficacy.

Predicting Femoral Strength:

Measurement of BMD by DXA is the current gold standard for diagnosis of osteoporosis. However, several studies have identified limitations in BMD measurements with regard to assessing fracture risk and monitoring efficacy of osteoporosis therapies. New imaging methods, combined with state-of-the art biomechanical analyses, may improve prediction of hip fracture risk. This main goal of this study is to evaluate the ability of different imaging modalities to predict the strength of human proximal femur in a sideways fall configuration. Secondary goals are to determine the relative contribution of BMD, femoral geometry, and cortical and trabecular bone microarchitecture to femoral strength.

Human cadaveric femora will be selected to represent the target population of individuals likely to suffer a hip fracture (ie, age > 65, BMD T-score < -1.5). Non-invasive imaging modalities will include image analysis of radiographs, DXA, multi-angle DXA, hip structural analysis, QCT, and QCT-based finite element analysis. Femurs will be divided into two groups, providing a “training set” for establishment of statistical models for prediction of bone strength (n=60 femurs) and a “test set” used to validate the model predictions (n=20 femurs). Several aspects of this study will be novel, as it will be the first to evaluate a wide variety of imaging modalities in the same experiment, to enroll specimens from individuals with low BMD only, and to use the training/test set approach to evaluate the fidelity of strength predictions for femurs tested in a sideways fall configuration. Results will provide strong evidence for qualification of surrogate markers for hip fracture risk.

We are also collaborating with Professor Tony Keaveny at UC Berkeley to examine micro-finite element models of the proximal femur to better understand how the femur fails in a sideways fall configuration, and what are the contributions of trabecular and cortical morphology to this failure.

Determinants of vertebral strength:

This work was done in collaboration with Julien Wergyzn and Jean-Paul from Professor Pierre Delmas’ and Roland Chapurlat’s group in Lyon, France. We used human lumbar vertebrae to determine the contribution of trabecular bone heterogeneity and cortical shell thickness to whole vertebral strength. In addition, we investigated the factors the influence the mechanical behavior of lumbar vertebra after simulating a mild vertebral fracture (ie, 25% deformation). We are also interested in the relative contribution of bone volume, collagen cross-links, mineralization and microdamage to mechanical behavior of human vertebral trabecular bone.

Assessment of bone material properties by reference point indentation:

Structural mechanics dictates that whole-bone mechanical behavior depends on bone size (or mass), geometry, and the intrinsic material properties of bone tissue. The effect of geometry on whole-bone strength is well documented, but the role of tissue material properties is less well understood. Indentation measurements offer an opportunity to study the material properties of bone tissue independent of geometrical and bone mass contributions. A novel microindentation instrument, termed reference probe indentation (RPI), uses cyclic loading to assess the ability of bone to resist crack generation and propagation. The insights gained from RPI may be useful for identifying mechanisms underlying different forms of skeletal fragility. We are examining a number of mouse models — either exposing the excised bones to conditions that alter the bone matrix or using genetically modified mice with alterations to the bone matrix. Images of bone indentation in mouse bone are shown in SEM images.

Recent publications:

Roberts BJ, Thrall E, Muller J, Bouxsein ML. Comparison of hip fracture risk by femoral aBMD to Comparison of hip fracture risk prediction by femoral BMD and by the factor-of-risk for hip fracture derived from direct measurements of femoral strength. Bone. 2010; 46(3):742-6. PDF

Roux J, Wegrzyn J, Arlot M, Guyen O, Delmas P, Chapurlat R, Bouxsein M. Contribution of trabecular and cortical components to biomechanical behavior of human vertebrae: an ex-vivo study. J Bone Miner Res. 2010; 25(2): 356-61. PDF

Wegrzyn J, Roux JP, Arlot ME, Boutroy S, Vilayphiou N, Guyen O, Delmas PD, Chapurlat R, Bouxsein ML. Role of trabecular microarchitecture and its heterogeneity parameters in the mechanical behavior of ex-vivo human L3 vertebrae. J Bone Miner Res. 2010; 25(11): 2324-31. PDF

Wegrzyn J, Roux JP, Arlot ME, Boutroy S, Vilayphiou N, Guyen O, Delmas PD, Chapurlat R, Bouxsein ML. Determinants of the mechanical behavior of human lumbar vertebrae after simulated mild fracture. J Bone Miner Res. 2011; 26(4):739-46. PDF

Follet H, Viguet-Carrin S, Burt-Pichat B, Depalle B, Bala Y, Gineyts E, Munoz F, Arlot M, Boivin G, Chapurlat R, Delmas PD, Bouxsein ML. Effects of preexisting microdamage, collagen cross-links, degree of mineralization, age and architecture on compressive mechanical properties of elderly human vertebral trabecular bone. J Orthop Res. 2011; 29(4):481-8. PDF