Effects of Treadmill Running on Bone Density and Bone Strength in Young Mice

Objective : To investigate the effects of treadmill running exercise on bone density and bone strength in young mice. Methods : Four-week-old male C57BL/6 mice (n=10) were assigned to either the control sedentary group (CON, n = 5) or the treadmill running exercise group (TRE, n = 5). Mice in the TRE group were forced to run on a treadmill at a rate of 6-18 m/min, 30 min/day, 5 days/week for 16 weeks. The mice in both groups were euthanized at 20 weeks of age. The right and left lower limbs, skeletal muscle, and white adipose tissue were removed for analysis. Bone density (mg/cm 3 ) was measured in the right limb using microcomputed tomography, and maximal breaking force (N) was determined in the left femur using a three-point bending test as an indicator of bone strength. Results : The TRE and CON groups did not significantly differ with respect to daily food intake during the study period or body weight at 20 weeks of age. Differences in white adipose tissue and skeletal muscle weight of 20-week-old mice in the TRE and CON groups were also not significant. Cortical bone density was significantly higher in the TRE group than in the CON group (p<0.05). However, there were no significant differences in cancellous bone density, total bone density or maximum breaking force between the TRE and CON groups. Conclusions : Treadmill running exercise for 16 weeks can increase cortical bone density of tibia in young mice, but does not improve bone strength in femur.


Introduction
In an aging society, people are at higher risk of bone fracture and osteoporosis and would thus benefit from efforts aimed at maintaining lifelong skeletal health. In humans, bone mass increases until adolescence but gradually decreases thereafter. Therefore, measures aimed at increasing peak bone mass during the growing period are likely to be beneficial later in life.
Mechanical stress is imperative for normal bone turnover, which consists of bone formation and resorption. Exercise or physical activity that includes mechanical stress increases bone mass and bone strength, and is therefore critical for normal bone development and bone health. In studies conducted in humans, past physical activity was shown to be beneficial for bone health 1) 2) . Strope et al. demonstrated that physical activity in adolescence (13-18 years) and young adulthood (19-29 years) had positive effects on adult (30-65 years) bone mass 1) . Exercise training was also shown to improve bone density in children and adults 3)-6) . However, despite the confirmed effects of exercise in children and young adults with respect to improved bone mass and density during adulthood, whether physical activity and exercise also improve these bone parameters in children is unknown. In animals, various exercise models, including treadmill running, jumping exercise, resistance training, and swimming, have been developed to clarify the effect of exercise on bone mass and bone density. Both treadmill running and jumping exercise lead to a gain in bone mass 7)-10) . Hamrick et al. found that treadmill exercise for 4 weeks increased cortical bone in the long bones of 16-week-old female mice 11) . Huang et al. reported that endurance exercise for 8 weeks benefitted the biomaterial quality of bone in growing rats at 15 weeks of age 8) .
Although these studies demonstrated the effects of exercise on bone mass, bone density, and bone turnover, they did not examine bone biomechanical properties. Therefore, in this study we investigated the effects of treadmill running exercise on bone strength and bone density in young mice.

Animals
Four-week-old male C57BL/6 mice (n = 10) were purchased from Japan SLC (Hamamatsu, Japan). Mice were maintained in a climate-controlled room at a temperature of 20 ± 1℃ on a 12:12-h lightdark cycle. Both standard rodent chow (Labdiet, EQ5L37, PMI nutrition international) and water were provided ad libitum for 16 weeks. After a 1-week acclimatization period, the mice were randomly assigned to either the control sedentary group (CON, n = 5) or the treadmill running exercise group (TRE, n = 5).

Exercise training
Exercise training was conducted in accordance with the procedures used in a previous study 11) . Briefly, mice in the TRE group were forced to run on a motorized treadmill for 30 min/day, 5 days/week for 16 weeks. Exercise intensity was controlled by adjusting the running speed and was increased progressively (6-18 m/min) over the 16-week training period.

Tissue sampling
All mice were euthanized at 20 weeks of age. Their right and left lower limbs, skeletal muscles (soleus, gastrocnemius, plantaris, anterior tibialis, extensor digitorum longus, and quadriceps), and white adipose tissue were removed and their weights immediately measured using an electronic scale. The right limb was then fixed with buffered 4% paraformaldehyde. Bone mineral density was measured using microcomputed tomography within the following 2 days. The left femur was harvested for testing of bone strength.

Microcomputed tomography
The bone density (mg/cm 3 ) of the right limb was measured using micro-CT scanning (Latheta LCT-200, HITACHI ALOKA Medical, Tokyo, Japan), performed at a spatial resolution of 48 μm × 48 μm and a slice thickness of 96 μm. All other parameters used followed those recommended in the manufacturerʼs protocols.

Bone strength testing
Maximum breaking force (N), as a measure of bone strength, was assessed in the left femur by a three-point bending test (TK-252C, Muromachi, Japan). Briefly, the connective tissue was removed and the bone was attached between two bottomsupporting stands separated by 1.6 mm. The crosshead speed was 2.0 mm/min.

Statistical analysis
All data are expressed as the mean ± standard deviation (SD). Comparisons among groups were performed using Studentʼs t-test. A p value < 0.05 was considered to indicate statistical significance. All analyses were performed using SPSS ver. 18.0 (IBM).

Daily food consumption and body weight
The mean (± SD) body weights of the mice in the CON and TRE groups at the start of the experiment were 16.7 ± 1.0 g and 16.4 ± 1.0 g, respectively. At the end of the experiment, they were CON: 24.3 ± 0.9 g and TRE: 24.2 ± 1.1 g. There was no significant difference between the CON and TRE groups either at the start or the end of the experiment.

White adipose tissue and skeletal muscle weight
White adipose tissue weight was higher in the TRE group than in the CON group (CON: 284.7 ± 22.9 mg and TRE: 331.5 ± 84.4 mg). There was no significant difference between the CON and TRE groups. Differences in skeletal muscle weight were also not significant: soleus (CON: 8.3 ± 0.6 mg and TRE: 8.3 ± 0.5 mg), gastrocnemius (CON: 124.4 ± 5.6 mg and TRE: 121.0 ± 8.1 mg), plantaris (CON: 17.7 ± 2.3 mg and TRE: 17.9 ± 1.3 mg), anterior tibialis (CON: 49.3 ± 1.5 mg and TRE: 47.1 ± 2.7 mg), extensor digitorum longus (CON: 10.6 ± 0.7 mg and TRE: 10.7 ± 0.4 mg), and quadriceps (CON: 193.2 ± 14.3 mg and TRE: 181.1 ± 8.4 mg). The sum of the muscle weights was therefore 403.5 ± 22.0 mg in the CON group and 386.1 ± 12.8 mg in the TRE group. The difference between groups was not significant. Figure-1 and 2 shows the bone density at 20 weeks of age in the CON and TRE groups. The difference in cancellous bone density of the tibia was not significantly different between the two groups (CON: 327.5 ± 18.2 mg/cm 3 and TRE: 331.6 ± 12.3 mg/cm 3 ). However, cortical bone density of the tibia was significantly higher in the TRE group than in the CON group (904.9 ± 4.9 vs. 884.1 ± 19.7 mg/cm 3 , respectively, p < 0.05). Thus, total bone density of the tibia was significantly higher in the TRE group than in the CON group (594.9 ± 8.0 vs. 576.3 ± 12.6 mg/cm 3 , respectively, p < 0.05). In the femur, cancellous and cortical bone density were not significantly different between the two groups (CON: 327.5 ± 18.2 mg/cm 3 and TRE: 331.6 ± 12.3 mg/cm 3 at the cancellous, CON: 327.5 ± 18.2 mg/cm 3 and TRE: 331.6 ± 12.3 mg/cm 3 at the cortical).

Bone strength
The maximal femoral breaking force is shown in Figure-3. The difference between the CON and TRE groups was not significant (CON: 11.1 ± 1.3 N and TRE: 11.0 ± 0.4 N).

Discussion
This study investigated the effects of treadmill  running exercise on bone density and bone strength in growing male mice. Whereas treadmill running increased the cortical bone density of the tibia in young mice, there was no significant change in cancellous bone density. The improvement in cortical bone density in response to treadmill running is consistent with the results of previous studies 12) 13) . In a study using female mice, Hamrick et al. demonstrated that cortical thickness increased by 50% at the metaphysis and by 10% at the midshaft in the treadmill running group compared to the non-exercise control group 11) . However, after 4 weeks of treadmill running, the increase in bone mass was not accompanied by an increase in bone density. By contrast, Joo et al. found that 4 weeks of treadmill running exercise increased bone strength in young male rats. The increased bone strength was attributed to alterations of cortical geometry, bone density, and bone microarchitecture 13) . In another study, endurance treadmill running for 8 weeks did not alter bone mass and bone density in growing male rats 8) . These inconsistent results may reflect differences in the osteogenic response to exercise, which varies according to loading intensity, frequency, sex, and age 7) .
Bone density, structure, and composition are important determinants of bone strength and, along with bone density and bone mass, should therefore be measured in studies on the effect of exercise on bone health. Indeed, Isaksson et al. showed that voluntary wheel running exercise improved both the collagen network of bone and bone strength 14) . Similarly, Holy and Zérath reported that, in growing rats, short-term voluntary wheel running exercise increased markers of bone formation and improved the histological properties of bone tissue 15) . In this study, only bone density and bone strength, assessed using microcomputed tomography and a three-point bending test, respectively, were evaluated. Other bone quality parameters were not examined. Nonetheless, our data suggest that, in young mice, 16 weeks of treadmill running exercise do not affect bone strength, but is at least beneficial to site-specific bone density.

Conclusion
Our study in young mice showed that treadmill running exercise for 16 weeks can increase cortical bone density of the tibia, but does not alter bone strength of the femur.