GENDER SPECIFIC RESPONSE TO EXERCISE IN C57BL6/129 MICE Wallace, J.M.1; Rajachar, R.M.1, Chen, X.2; Shi, S.2; Robey, P.G.2; Young, M.F.2; Kohn, D.H.1,3 1
2
Department of Biomedical Engineering, University of Michigan National Institute of Dental and Craniofacial Research/National Institutes of Health 3 Department of Biologic and Materials Sciences, University of Michigan E-mail:
[email protected]
INTRODUCTION Adult peak bone mass is an important predictor of fracture risk (Akhter 1998). The decline in bone mass and strength in men and women with age may contribute to increases in skeletal fragility with age. Therefore, maximizing peak bone mass may protect against bone loss and fracture (Iwamoto 1999). Mechanical loading is recognized as a valuable way to maintain peak bone mass and structural integrity (Yingling 2001, Frost 1987). Exercisedbased animal models have shown a link between mechanical loading and maintenance/increase of bone structural and mechanical properties (Yingling 2001, Sakakura 2001, Mosekilde 1999). However, most of these models apply an exercise regimen of 2-5 months, and few have documented gender-specific responses to exercise. The purpose of this study was to establish a physiologicallyrelevant murine exercise model and use this model to test the hypothesis that the geometric and mechanical properties of male bones are more responsive to exercise than female bones. METHODS Forty C57BL6/129 mice were bred inhouse at the NIH (NIDCR animal approval protocol # NIDCR 001-151). Animals were housed in standard caging and given unrestricted access to food and water, and cage activity was unrestricted. At 2
months of age, mice were assigned randomly to 1 of 2 groups (exercise and control; 10 males and 10 females in each group). Exercise consisted of running at 10 m/min on a treadmill at a 5° incline, 30 min/day for 21 days (Columbus Instruments, Columbus OH, model 1055M). Mice were sacrificed on day 24. Left femora and tibiae were harvested, stripped of soft tissue and stored in a buffered calcium solution (with 0.01% sodium azide as an antibiotic) at -80º C for later use. Bones were tested to failure in 4 point bending using a custom-designed, solenoid driven loading apparatus (Rajachar 2003) at a rate of 0.01 mm/sec. Femora were tested in the AP direction (posterior surface in compression) and tibiae were tested in the ML direction (lateral surface in compression). Load and deflection were recorded, from which strength, energy, stiffness and deformation properties were derived (Turner, 1993). After fracture, the bones were sectioned at the fracture site. Geometric parameters (cross sectional area, ML and AP width, cortical thickness, centroid and moment of inertia) were determined using an inverted light microscope and digital analysis software (Nikon Eclipse 300T, Image ProPlus v4.1, Matlab v5.3). Statistical analyses were performed using 2-way ANOVA (SigmaStat v2.0). RESULTS AND DISCUSSION Male tibiae responded to exercise via an
increase and redistribution of the amount of tissue, as indicated by the significant increases in cross sectional area (p=0.045) and ML width (p=0.036), and marginally significant increases in AP width and moment of inertia (Table 1). These geometric changes were accompanied by a significant increase in post-yield displacement (p=0.018), but a decrease in yield force (p=0.027) and ultimate strength (p=0.026). Exercise did not alter the geometric properties of female tibiae, but there was a trend toward increased strength. No exercise-induced effects were seen in male femora, but female femora exhibited a significant increase in section modulus (p=0.011) and marginally significant increases in stiffness (p=0.072) and ultimate strength (p=0.082). Although 3 weeks of exercise elicited a formation response, the tissue is still osteoid and does not have the mechanical integrity of fully organized and mineralized lamellar bone. It is possible that given more time between terminated exercise and sacrifice, bone would mature and increase in strength. Three weeks of exercise were able to elicit an osteogenic effect, compared to other exercise regimens, which prolonged exercise up to 8-18
weeks. This data establishes a simpler exercise protocol than those previously used. Gender-specific effects may generally be hypothesized to be hormonally related. A previous study demonstrated that the amount of load-stimulated bone formation in growing male rats is greater than in female rats (Mosley 2002). Our study represents the first report indicating that there is a gender-specific response to exercise in mice. REFERENCES Akhter, et al. (1998). Calif Tissue Int, 63, 442-449. Frost, (1987). Bone Miner, 2, 73-85. Iwamoto, et al. (1999). Bone, 24, 163-169. Mosekilde, et al. (1999) Bone, 24, 71-80. Mosley,Lanyon. (2002). Bone, 30, 314-319 Rajachar. (2003). Ph.D. Dissertation, University of Michigan Sakakura, et al. (2001) J Bone Min Metab 19, 159-167. Turner, Burr, (1993). Bone, 14, 595-607. Yingling, et al. (2001). Calcif Tissue Int, 68, 235-239. ACKNOWLEDGEMENTS NIH IPA Award
Table 1: Geometric and Mechanical Properties of Tibiae (mean ± SEM) Property Male Control Male Run Female Control Female Run 2 a C/S Area (mm ) 0.574 ±0.048 0.586 ± 0.045 0.519 ± 0.042 0.633 ± 0.033 b AP Width (mm) 1.102 ± 0.053 1.244 ± 0.047 1.143 ± 0.084 1.117 ± 0.028 ML Width (mm) 0.913 ± 0.078 0.953 ± 0.044 0.899 ± 0.054 1.043 ± 0.034a 4 b Section MOI (mm ) 0.037 ± 0.008 0.058 ± 0.006 0.046 ± 0.007 0.044 ± 0.005 Yield Force (N) 16.91 ± 3.25 23.03 ± 4.32 23.13 ± 2.81 15.96 ± 1.01a b 0.075 ± 0.014 0.046 ± 0.005 0.063 ± 0.024 0.065 ± 0.014 δ (elastic) (mm) a 0.038 ± 0.007 0.024 ± 0.008 0.059 ± 0.010 0.028 ± 0.010 δ (plastic) (mm) b Yield Energy (mJ) 1.176 ± 0.350 0.501 ± 0.072 0.814 ± 0.430 1.067 ± 0.353 Yield Stress (N/mm2) 150.54 ± 33.34 85.02 ± 8.63b 129.13 ± 48.52 194.14 ± 64.53 Ult. Stress (N/mm2) 197.37 ± 32.20 116.85 ± 11.28a 150.96 ± 41.16 194.43 ± 119.98 Strain (µε) 16887 ± 2576 28842 ± 5863b 13702 ± 1920 13697 ± 2682 a b Significant effect of exercise (Bold, p
