Cancellous Bone Adaptation To Non-Invasive Mechanical Loading In The Murine Tibia

Introduction: Osteoporosis is a disease characterized by decreased bone mass and increased risk for fracture, especially at cancellous sites. Mechanical loading has been shown to induce bone adaptation through both increased formation and reduced resorption, and thus is a potential therapy for bone loss. The effects of loading and the mechanical signals that enhance bone formation are known primarily through studies of cortical bone, but cancellous bone adaptation is not wellcharacterized. We have established an osteogenic loading protocol using axial compression of the tibia in male mice. In growing mice, cancellous bone volume fraction and trabecular thickness increased after 6 weeks of loading [1]. In hormone-deficient mice administered similar loading, trabecular thickness was larger than in the sham-surgery control group and cancellous bone mineral content and bone volume fraction were fully recovered to sham levels [2]. The objectives of this study were to establish an osteogenic loading protocol in female mice and to determine the effects of sex on the response to loading. Materials and Methods: Thirty-eight C57Bl/6 mice (n=19/sex) underwent dynamic axial compression of the left tibia for 2 weeks. Loading was applied 5 days/week for 1200 cycles at 4 Hz. The average peak load magnitude was 11.5N, which was determined to engender strains of 1200 μe at the mid-shaft by in vivo strain gage measurement. The right tibia served as the unloaded, internal control. Normal cage activity was allowed between loading sessions. At end of the 2w loading period, mice were euthanized by CO2 inhalation. The tibiae of 24 mice (n=12/sex) were dissected and fixed in 4% paraformaldehyde. All procedures were approved by the IACUC at Cornell University. Each pair of tibiae was scanned using micro-computed tomography (MS-8 MicroCT, GE Healthcare Systems, London, Ontario, Canada). Scans were reconstructed at 11.6 μm isotropic resolution. A global threshold of 1100 HU was used to separate mineralized tissue from water and soft tissue. In each tibia, a single volume of interest (VOI) was defined in the proximal metaphysis excluding the cortical shell, beginning in the plane of the most proximal point beneath the growth plate and ending distally at 10% of the total tibia length. Measurable outcomes for the cancellous VOI included bone volume fraction (BV/TV), apparent mineral density (aBMD), tissue mineral density (tBMD), and trabecular thickness (Tb.Th). Statistical analyses were performed using image data from 10 females and 8 males ( JMP 6, SAS Institute Inc., Cary, NC). The effects of loading were determined using a paired t-test analysis. A two-way ANOVA was used to determine the effect of sex and loading. P-values of 0.05 or less signified statistical significance. Results: Over the 2 week period, the body mass of males and females increased an average of 2.30g and 3.36g, respectively. Significant effects of loading were present in both males and females. In female mice, BV/TV (40%), tMD (9%), aBMD (46%) and Tb.Th (40%) were significantly greater in the loaded tibiae relative to controls. Similarly in males, BV/TV (31%), tBMD (11%), aBMD (38%), and Tb.Th (44%) were significantly greater in the loaded tibiae relative to controls (Figure 1). The left and right tibiae lengths were not significantly different after 2 weeks of loading in either the males or the females (Table 1). BV/TV, tBMD, and aBMD demonstrated a significant effect of loading and sex, while Tb.Th. demonstrated only a significant effect of loading. The effect of loading was not different for male and female mice. Discussion: Noninvasive mechanical loading was osteogenic in the proximal metaphysis of male and female mice. The effect of loading was significant in all cancellous measures and similar between sexes. Bone volume fraction, tissue mineral density, and apparent mineral density were significantly lower in females than males. The male data presented here exhibit several differences compared to our previous results [1, 2], most notably in a greater effect on bone volume fraction (55% higher). These differences likely arise from two considerations. First, the 11.5N peak load magnitude used here was higher than previously, which corresponds to a similar study using tibial compression [3]. Second, a more comprehensive cancellous VOI was analyzed in the present study. Our VOIs are 27% larger on average and encompass the majority of the cancellous tissue in the proximal metaphysis. The improved definition of cancellous bone volume is certain to provide a clearer representation of the effects due to loading, particularly if local differences can be correlated with the mechanical stimulus experienced by the tissue. Future work will examine the effect of hormone-deficiency, aging, and genetic modification on the response to loading. References: [1] Fritton et al., Bone, 2005 [2] Fritton et al., ASME, 2005 [3] de Souza et al., Bone, 2005 Acknowledgements: NIH P30-AR46121 NIH R01-AG028664-01 Table 1: Summary table of outcome measurements for the proximal tibial metaphysis.