Elsevier

Bone

Volume 37, Issue 6, December 2005, Pages 810-818
Bone

Non-invasive axial loading of mouse tibiae increases cortical bone formation and modifies trabecular organization: A new model to study cortical and cancellous compartments in a single loaded element

https://doi.org/10.1016/j.bone.2005.07.022Get rights and content

Abstract

Systematic study of bones' responses to loading requires simple non-invasive models in appropriate experimental animals where the applied load is controllable and the changes in bone quantifiable. Herein, we validate a model for applying axial loads, non-invasively to murine tibiae. This allows the effects of mechanical loading in both cancellous and cortical bone to be determined within a single bone in which genetic, neuronal and functional influences can also be readily manipulated. Using female C57Bl/J6 mice, peak strains at the tibial mid-shaft were measured during walking (<300 με tension) and jumping (<600 με compression) with single longitudinally oriented strain gauges attached to the bone's lateral and medial surfaces. Identically positioned gauges were also used to determine, for calibration, the strains engendered by external applied compressive tibial loading between the flexed knee and ankle ex vivo. Applied loads between 5 and 13 N produced strains of 1150–2000 με on the lateral surface, and in vivo repetitions of these loads on alternate days for 2 weeks produced significant load magnitude-related increases in cortical bone formation that were similar in mice at 8, 12 and 20 weeks of age. Micro-CT scans showed that loading significantly increases trabecular bone volume in 8 week old mice, but modifies trabecular organization with decreases in trabecular bone volume in 12 and 20 week old mice. This model for loading the tibia has several advantages over other approaches, including scope to study the effects of loading in cancellous as well as cortical bone, against a background of either disuse or of treatment with osteotropic agents within a single bone in normal, mutant and transgenic mice.

Introduction

The capacity of individual bones to withstand habitual loads without fracture is established and maintained, at least partly, by functional adaptation to the strains that these loads engender [1]. Bones respond to changes in load-induced mechanical strains by altering (re)modeling activities to ensure appropriate cortical and trabecular bone morphology and mass. Although this hypothesis is generally accepted, the mechanisms that underpin it remain to be established. To address this, animal models that enable specific loading have been developed. These are useful in defining how loading modulates (re)modeling and allow examination of the mechanisms that initiate and coordinate these events [2], [3].

The first attempt to examine the response of bone to defined, controlled artificial loads involved passing Kirschner wires through rabbit tibiae [4]. This and other studies showed that adaptive responses were induced by dynamic but not static loading, and did not require central nervous input, but were inherent to the bone itself [4], [5]. However, loading in these models had a major disadvantage of requiring surgery which causes trauma, increased risk of infection and direct effects on bone cell metabolism [6], [7], [8]. A model, that once calibrated, allows controlled non-invasive loading is therefore desirable.

The advantage of non-invasive loading is inherent in exercise, tail suspension and disuse models, unfortunately these rely upon the imposition of a relatively uncontrolled mechanical input as the stimulus [9], [10]. These have, however, allowed for the study of trabecular bone and also highlight the convenience of using laboratory animals [11]. Thus, non-invasive models for applying controlled mechanical loads to a bone in which both cortical and trabecular responses can be examined in an appropriate laboratory animal are advantageous.

Although convenient and controlled, four-point bending of the tibia relies upon direct pressure to the diaphyseal periosteum [3]. While cantilever-like bending of the mouse tibia and axial loading of rat ulna [12], [13] overcome this disadvantage, neither approach allows responses to loading to be examined in trabecular bone. Non-invasive controlled axial loading of the mouse tibia overcomes this deficiency allowing both trabecular and cortical responses to be measured. In addition, the tibia is probably the commonest bone to be studied in relation to the effects of disuse, ovariectomy and corticosteroids on bone (re)modeling.

This article describes a new non-invasive model for loading the murine tibia through its normal points of articulation; this model allows loading-induced changes in endosteal, periosteal and trabecular bone to be revealed by double fluorochrome labeling and computed tomography, respectively. Development of this model allows both cortical and trabecular adaptation to mechanical loading to be studied against the background of ‘disuse’, with or without modification by other bone-targeting agents, in a single bone in normal, mutant and transgenic mice.

Section snippets

Animals

Female C57BL/J6 mice (Charles River Company, UK) were housed in polypropylene cages in groups of 4, subjected to 12 h light/dark cycle with room temperature at 21 ± 2°C and fed ad libitum with maintenance diet (Special Diet Services, Witham UK). All procedures complied with the Animals (Scientific Procedures) Act 1986 and local ethics committee.

In vivo strain measurements

Trimmed single element gauges (each <1 mm × 2 mm, EA-06-015LA-120, Measurements Group Inc., Basingstoke, UK) were attached to medial and lateral

In-vivo strains at tibial diaphysis and ex-vivo calibration of loading of the mouse tibia

To establish mechanical strain levels engendered by normal loading in vivo, two gauges were attached to medial and lateral aspects of the tibial mid-shaft (Fig. 1). All mice with attached strain gauges showed normal locomotor activities in walking or running immediately after recovery from anesthesia. Although some favoring of the limb was observed in slow gaits, this was barely detectable at faster speeds. Results from strain gauge recordings in 12 week old mice show that the predominant

Discussion

This study validates a model for non-invasively applying axial compressive loads to the mouse tibia through natural sites of articulation, in which the resultant changes in cortical and cancellous bone can be measured. Validation involved measurement of normal locomotor strains on the diaphyseal tibial bone surface; reassurance from the absence of any evidence of either microcracks or intra-cortical remodeling that there was no significant microdamage; and the demonstration of dose-dependent

Acknowledgments

We are grateful to Mark Harrison and Gordon W Blunn of The Royal National Orthopaedic Hospital, Stanmore, UK, for their help. We are also grateful to CAPES, Ministry of Education, Brazil and Wellcome Trust, UK for their funding. Funding: CAPES PhD Studentship, Ministry of Education, Brazil and Wellcome Trust, UK.

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