Effects of treadmill with different intensities on bone quality and muscle properties in adult rats
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Bone is a dynamically hierarchical material that can be divided into length scales of several orders of magnitude. Exercise can cause bone deformation, which in turn affects bone mass and structure. This study aimed to study the effects of treadmill running with different intensities on the long bone integrity and muscle biomechanical properties of adult male rats.
Forty-eight 5-month-old male SD rats were randomly divided into 4 groups: i.e., sedentary group (SED), exercise with speed of 12 m/min group (EX12), 16 m/min group (EX16), and 20 m/min group (EX20). The exercise was carried out for 30 min every day, 5 days a week for 4 weeks. The femurs were examined using three-point bending test, microcomputer tomography scanning and nanoindentation test; the soleus muscle was dissected for tensile test; ALP and TRACP concentrations were measured by serum analysis.
The failure load was significantly increased by the EX12 group, whereas the elastic modulus was not significantly changed. The microstructure and mineral densities of the trabecular and cortical bone were significantly improved by the EX12 group. The mechanical properties of the soleus muscle were significantly increased by treadmill exercise. Bone formation showed significant increase by the EX12 group. Statistically higher nanomechanical properties of cortical bone were detected in the EX12 group.
The speed of 12 m/min resulted in significant changes in the microstructure and biomechanical properties of bone; besides, it significantly increased the ultimate load of the soleus muscle. The different intensities of treadmill running in this study provide an experimental basis for the selection of exercise intensity for adult male rats.
KeywordsTreadmill exercise Exercise intensity Femurs Biomechanical properties Multiscale analysis
tartrate-resistant acid phosphatase
region of interest
bone mineral density
bone volume fraction
structure model index
cortical bone density
cortical bone thickness
cortical bone porosity
longitudinal cortical bone
transverse cortical bone
modulus of indentation
ratio of indentation modulus to indentation hardness
longitudinal modulus of indentation
transverse modulus of indentation
longitudinal indentation hardness
transverse indentation hardness
ratio of longitudinal indentation modulus to indentation hardness
ratio of transverse indentation modulus to indentation hardness
Osteoporosis is a type of bone disease characterized by decrease in bone quality and deterioration of bone microstructure, which leads to an increase in bone fragility and susceptibility to fractures [1, 2]. More than 200 million people suffer from osteoporosis worldwide . As a major public health problem all over the world, osteoporosis imposes severe health threats and financial burdens on the patients and their families.
Currently, a wide variety of drugs, such as alendronate sodium, raloxifene, and teriparatide, are used for the treatment of osteoporosis. However, these drugs are associated with serious adverse reactions, e.g., high blood pressure, hectic fever, and thrombus formation . Alternatively, non-drug therapy has become a popular method for treating osteoporosis due to its low price, applicability, and good treatment outcomes. According to the previous studies, exercises such as jumping, jogging, and swimming can improve bone quality, density, and strength [5, 6, 7]. Physical exercise, in particular treadmill running, has been widely used in animal models associated with osteoporosis, as its mechanism is basically similar to human running on a treadmill [8, 9].
The data obtained from animal experiments do not fully represent the condition of the human body. However, animal experiments can replicate the development of the disease and can be studied for specific situations, and can be combined with a variety of experimental methods to further study the mechanism of disease development. In addition, animal experiments can explore the effectiveness of different therapeutic options and provide a theoretical basis for their clinical application. Due to the similarities of bone morphology and structure between rat and human, rats have become an excellent animal model for studying bone diseases. Rats are also the most commonly used experimental animals for studying the effects of exercise on the musculoskeletal system. Compared to female rats, male rats can reduce estrogen intervention and are more suitable for treadmill running.
Bone strength is related to not only its structure and geometry, but also the external load and the bone material properties [10, 11]. Studies have demonstrated that there is a “feedback system” in bone tissue, which is able to sense strain changes inside the bone . Furthermore, In addition to age, gender, nutritional status and energy intake, the strain environment around the bone is also an important factor affecting bone mass, which decreased in the position where strain is reduced and added in the position where strain is increased, affecting the size, shape, and structure of the bone. Bone has a multi-scale hierarchical structure at macroscopic, microscopic, and nanoscopic levels with various characteristics at different scales . Thus, studies at different scales are required to better understand the relationship between bone strength and structure.
Since the mechanical stimulus generated by treadmill running is mostly generated by impact with the ground or muscle contraction, it is essential to investigate the muscle force. The soleus and the gastrocnemius muscles are both posterior muscles of the lower limbs, and belong to the antigravity muscles that maintain body posture or exercise against gravity. Moreover, the soleus and gastrocnemius muscles are dominated by slow twitch (ST) and fast twitch (FT) fibers, respectively . Contraction of the slow twitch fibers is dominant during endurance training; while the fast twitch fibers also participate in contraction during prolonged exercises. Therefore, treadmill running affects the soleus muscle first, and then the gastrocnemius muscle is affected. There are many reports about the effects of exercise on the soleus muscle under micro-gravity [15, 16, 17, 18], which revealed that treadmill running could alleviate muscle atrophy to a certain extent in astronauts caused by long-term exposure to micro-gravity. Moreover, high-intensity treadmill running led to 59% less muscle loss in astronauts compared to low-intensity exercise . As one of the main muscles providing calf tension and directly participating in the eccentric contraction, the soleus muscle has important significance in terms of its biomechanical properties.
A recent study of a 1-month-old female rat backpack with weight-bearing and moderate intensity treadmill found that when rats ran at moderate intensity with an additional 12% weight-bearing level, bone formation was promoted and cancellous bone microstructure was significantly improved . The choice of age and gender has an important impact on the experimental results. Previous study showed that treadmill running had different adaptability to bone structure, biomechanical properties and molecular signals between 12-week-old male and female rats . Compared to female rats, male rats reduced estrogen intervention and were more suitable for treadmill running. Adult male rat reaches peak bone mass, and it can reduce the effect of age on bone mass in the experiment. Therefore, 5-month-old male rats were used to study the effects of different intensities treadmill running on bone mass and muscle biomechanical properties in adult male rats. The study of bones and muscles can provide a more comprehensive experimental basis for the selection of exercise intensity in adult male rats.
In this study, treadmill running at different intensity levels was carried out, in which multi-level test and analyses, including serum analysis, mechanical tensile test of soleus muscle, three-point bending test, micro-CT scanning, and nanoindentation test, were performed to investigate the effects of different levels of mechanical stimuli on the long bone quality and muscle performance of adult male rats, thereby providing feasible suggestions for selecting a proper exercise intensity for adult males.
Mechanical properties and weight of lower soleus muscle
The ultimate load and ultimate displacement values of soleus muscle obtained from tensile test, mean ± SD
Ultimate load (N)
Ultimate displacement (mm)
2.82 ± 0.38
7.75 ± 1.70
3.51 ± 0.55a
9.45 ± 2.13a
3.36 ± 0.46a
9.77 ± 1.77a
3.33 ± 0.53a
9.92 ± 2.10a
Activity evaluation of osteoblasts and osteoclasts by the serum analysis
The failure load and elastic modulus of the femur evaluated by the three-point bending mechanical test
The failure load and elastic modulus values of femurs obtained from three-point bending mechanical test, mean ± SD
Failure load (N)
Elastic modulus (GPa)
124.52 ± 30.50
6.85 ± 2.72
155.62 ± 20.77a,b
7.39 ± 1.32
142.21 ± 33.71
7.24 ± 1.95
122.49 ± 24.17
8.00 ± 2.76
Microarchitectural evaluation of the left femur by micro-CT scanning
Microarchitecture parameters of trabecular bone in the distal femur evaluated by micro-CT, mean ± SD
44.70 ± 6.24
0.53 ± 0.06
0.14 ± 0.01
3.32 ± 0.57
0.38 ± 0.10
0.69 ± 0.41
53.12 ± 8.09a,c,d
0.61 ± 0.07a,c,d
0.16 ± 0.02a,c,d
3.60 ± 0.40
0.30 ± 0.08
1.14 ± 0.73
45.89 ± 6.98
0.55 ± 0.06
0.14 ± 0.01
3.37 ± 0.50
0.30 ± 0.07a
0.60 ± 0.39b
42.09 ± 7.67
0.51 ± 0.06
0.12 ± 0.02a,c
3.42 ± 0.52
0.32 ± 0.10
0.58 ± 0.50
Microarchitecture parameters of cortical bone in the femur diaphysis evaluated by micro-CT scanning, mean ± SD
1.67 ± 0.03
2.51 ± 0.60
32.40 ± 3.32
1.71 ± 0.03a,c
2.25 ± 0.45
35.23 ± 2.97a,d
1.67 ± 0.05
3.17 ± 0.71a,b
34.41 ± 4.48
1.68 ± 0.03
2.67 ± 0.59
32.40 ± 2.50
Elastic modulus and hardness measured by nanoindentation test
E, H and E/H ratio of cortical bone obtained by nanoindentation tests, mean ± SD
23.57 ± 3.85
24.15 ± 3.85
25.04 ± 3.84
20.71 ± 3.03a,b,c
1.01 ± 0.21d
1.27 ± 0.49a,c,d
1.01 ± 0.17
0.92 ± 0.08
24.10 ± 4.67b
21.62 ± 7.30
25.03 ± 3.26b
22.62 ± 3.01
19.69 ± 2.97
21.56 ± 2.29a,d
20.97 ± 1.83a,d
18.74 ± 1.67
0.88 ± 0.20c,d
0.89 ± 0.24d
0.97 ± 0.19d
0.74 ± 0.08
22.87 ± 2.88b,d
25.91 ± 6.83
22.40 ± 4.31b
22.55 ± 3.31
In this study, forty-eight 5-month-old male SD rats were divided into three intensity exercise groups and one control group. Macroscopic, microscopic, and nanoscopic perspectives were used to investigate the effects of different treadmill exercise intensities on rat femur. Three-point bending test, micro-CT scanning, and nanoindentation test were performed to study bone quality from multiple perspectives. Moreover, since the mechanical stimulus generated during treadmill exercise is derived not only from gravity, but also from muscle contraction, the mechanical properties of the soleus muscle were also investigated. It was found that exercise at a speed of 12 m/min could significantly increase the long bone quality and increase the ultimate load carried by the soleus muscle of rats.
In recent years, female rats were used in most of the research on the effect of treadmill running on bone mass and strength, which were in the growing period or elderly; while there were relatively few studies on the effect of treadmill running on bone mass in male rats, 4- to 10-week-old growing period [28, 29] or 15- to 23-month-old [30, 31] rats were predominant. From the growth phase to the adult phase and the elderly phase, bone mass changes significantly, so the study for each phase is essential. Previous studies have only observed the effects of treadmill running on bone  or muscle , and rarely combine together. Adult male rats were used in this study, and the best form of exercise to improve bone quality in adulthood was explored by combining bone and muscle investigations. In addition, previous studies on skeletal muscles in the lower limb were usually focused on the gastrocnemius muscle, and relatively less attention was paid to the soleus muscle. These research protocols focused on the muscle fiber mass  and functional characteristics . Studying the mechanical properties of soleus muscle is important for the treatment of muscle atrophy.
Mechanical stimuli play an important role in bone strength . Exercise with different intensity, frequency, and duration produces different mechanical stimuli, which has different effects . In this study, the EX12 group had the maximum failure load, which was significantly greater than those of the SED and the EX20 groups (p < 0.05); while the failure load in the EX20 group was smaller than that of the SED group. Bone is a living organ with functional adaptability. It can adjust itself according to the surrounding mechanical environment, developing the optimal structure able to bear the applied loads. Bone can achieve the maximum structural strength with the least materials . Appropriate mechanical stimuli can increase bone quality and improve bone structure; while excessive mechanical stimuli may cause bone tissue deterioration in quality and microstructure, and decrease the mechanical properties . Previous studies on 5-week-old male rats also revealed that, after high-intensity treadmill training, both tibial strength and proximal bone mineral density (BMD) reduced, while the trabeculae in the epiphysis thinned . The treadmill running with 20 m/min speed in this study caused excessive mechanical stimulus to rat femur, which resulted in the decrease in failure load.
The mechanical properties of bone are closely related to its microstructure. Bone strength can be effectively predicted by combining three-point bending test and micro-CT scanning [38, 39]. Compared to the microstructure parameters of the trabecular bone in other groups, the EX12 group demonstrated higher BV/TV, Tb.BMD, and Tb.Th (p < 0.05). The EX12 group significantly improved the trabecular structure, increased bone density, and tightened trabecular arrangement as compared to the SED group. These results are consistent with the results from the previous studies, which indicated that exercise can improve the trabecular bone structure in rats . For cortical bone, the EX12 group had higher Ct.BMD and Ct.Th compared to the SED group (p < 0.05); while Ct.P was the minimum among all groups. This indicated that applying a mechanical stimulus with a velocity of 12 m/min can increase bone density and cortical bone thickness.
It has been reported in the literature that exercise can increase the levels of growth hormone (GH), prostaglandin E2 (PGE2), parathyroid hormone (PTH), and thyroid hormone (TH) . Among them, PGE2 can stimulate mesenchymal stem cells to differentiate into osteoblasts . ALP is an indicator of bone formation, and is closely related to the activity of osteoblasts. Analysis of ALP in serum can effectively reveal the effect of treadmill running on the active of osteoblasts . Recent studies showed that physical exercise has impacts on the amount and osteogenic differentiation potential of mesenchymal stem cells. After running on the treadmill for 5 weeks, the mesenchymal stem cells of 4-week-old male C57BI/6 mice significantly increased, and the ALP activity also increased . In the present study, no significant difference was found in the serum TRACP concentration among groups. The ALP concentrations in the exercise groups were higher than that in the SED group; while the ALP concentration of the EX12 group was significantly higher than that of the SED group (p < 0.05). The results suggested that treadmill exercise with a 12 m/min speed contributed to increasing bone formation, but did not remarkably suppress bone resorption, which was consistent with the increase in bone density in the EX12 group, as observed by micro-CT evaluation. Previous studies have shown different results on whether treadmill running can reduce TRACP concentration. A study on the treadmill running in rats also found that rats increased bone formation after 8 weeks of running without affecting bone resorption . This is consistent with our current conclusion. Other researchers found that treadmill running not only increases bone formation but also inhibits bone resorption . This may be related to different animal species, age and experimental design. In this study, however, the EX16 and SED groups demonstrated similar TRACP concentrations, which should be further investigated.
Nanoindentation is an effective method for measuring nanomechanical properties, and has been widely used in studying the mechanical properties of bone tissue . In the present study, the longitudinal hardness (HL) and the transverse elastic modulus (ET) of the EX12 group were both significantly improved compared to the SED group (p < 0.05), indicating that the nanomechanical properties in the EX12 group were superior to those in other groups. The E/H ratio describes the material deformation during the indentation process, which is proportional to the fracture toughness; thus, it is a crucial indicator for the toughness of materials . The ET/HT ratio of the EX12 group was significantly greater than that of the SED group (p < 0.05), suggesting that the EX12 group had a high transverse fracture resistance. In addition, we also observed that the variation tendencies in elastic modulus and hardness were consistent.
In human activities, most of the loads acting on bones are produced by externally exerted forces and skeletal muscle contraction . In this study, the ultimate load and the ultimate displacement values of the soleus muscle in the exercise groups were significantly higher than those in the SED group (p < 0.05). The EX12 group demonstrated the maximum ultimate load and the soleus muscle weight. Mechanical environment is a key factor in maintaining musculoskeletal functions . Exercise increases the muscle myosin and actin contents ; and during exercise, muscle contraction increases the number of cells in the muscle tendon, thus improving the load-bearing capacity of the muscle . The increase in the soleus muscle weight in the exercise groups may be due to the apparent “demand” for soleus muscle in rats from the treadmill running. With long-term running, muscle contraction increases the number and percentage of muscle fibers, as well as muscle mass. This is in accordance with the results of a previous study, in which the effects of treadmill training on the soleus muscles were explored in rats after complete spinal cord transection at T8–T9. It was found that 9 weeks of treadmill running increased soleus muscle mass and cross-sectional area of muscle fibers in rats .
There were some limitations in the present study. Firstly, findings obtained from 5-month-old male SD rats may not be applicable to female rats. A recent study showed that loading of a 1-month-old female rat with a weight-bearing and moderate-intensity treadmill can promote bone formation and improve trabecular microstructure . Future work will include the effects of treadmill exercise with different intensities on bone quality of female SD rats with the same age to obtain more comprehensive results and conclusions. Secondly, three exercise groups with different intensities (EX12, EX16 and EX20 groups) were used, and the total distance per day for each group was not equal, which may possibly affect the changes in bone quality. However, the exercise time for different groups was kept the same. Thus, it is meaningful to study the effects of different exercise intensities on bone quality. Future work can specify the total distance traveled and explore the effects of treadmill running on the bone of the small mammalian. Finally, the effects of different exercise intensities on nearly one single intact bone (femur) were investigated, which may not be applicable to other bones. However, considering that femur is load-bearing bone prone to fracture, this study is still meaningful.
The results of this study demonstrated that after 4 weeks of treadmill running, among the three exercise intensities, the speed of 12 m/min could significantly affect the microstructure and mechanical properties of bone and improve the ultimate load of the soleus muscle. The treadmill running established in this study is effective and provides a reference for the selection of proper exercise intensity levels for adult males.
This study was approved by the Medical Ethics Committee of the First Hospital of Jilin University (No. 2018-238). This study was in strict accordance with the requirement of the Laboratory Animal Standardization Committee. And all efforts were made to alleviate suffering of animals.
Sixty 5-month-old male Sprague–Dawley rats were procured from the Animal Experimental Center of Jilin University. Animals were housed in groups of 4 (57 × 39 × 20 cm3), in laboratory cages under controlled laboratory conditions, a temperature of 23 ± 2 °C, and a relative humidity of 55 ± 5%, under a 12-h dark/light cycle. Rats were given freedom of movement in their cages, and provided ad libitum access to standard rodent food pellets (autoclaved diet National Institutes of Health-31 with 6% fat; 18% protein; Ca:P = 1:1; and supplemented vitamins and fortified minerals) and tap water. All the rats were healthy during the experimental period.
48 h after the final exercise session, the rats were anesthetized with isoflurane, and then the blood sample with no less than 5 ml was obtained through the abdominal aorta of each rat. Serum was separated by centrifugation at 3000 rpm/min for 15 min. The obtained serum samples were then immediately stored in a refrigerator at − 20 °C until further analysis.
Soleus tensile test
Alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRACP) were quantitatively analyzed by enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay assay kit, NanJing, China) using ELIASA (CLARIOstar, BMG LABTECH, Germany), and bone formation and absorption rates were measured.
Three-point bending mechanical test
Left femurs were initially fixed with 80% ethanol (EtOH). Subsequently, femur samples were scanned using a benchtop micro-CT system (Skyscan 1076, Bruker-MicroCT, Belgium) at 18-μm voxel image resolution with 70 kV, 100 μA, and a 1.0-mm aluminum filter, to obtain three-dimensional (3D) microstructure parameters of trabecular bone and cortical bone. The projection data were then reconstructed with NRecon (Skyscan, Belgium) to create 3D images. The region of interest (ROI) was manually selected for the analysis of micro-CT images. The entire trabecular bone of the femoral lateral condyle was selected as trabecular ROI, and 5-mm cortical bone from femoral shaft to the proximal was selected as cortical ROI. Image analysis was performed with CTAn analysis software (CTAn, Skyscan, Belgium). The 3D morphometrical parameters of femoral head trabecular bone were measured, including bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp). The 3D microstructure parameters of femoral shaft cortical bone were also measured, including cortical bone mineral density (Ct.BMD), cortical bone thickness (Ct.Th) and cortical bone porosity (Ct.P).
A Berkovich diamond indenter was used for the measurements. Nanoindentation tests were performed with the indenter speed of 750 μN/s and the indentation depth of 1000 nm, holding this load for 10 s, and finally unloading to 15% of the peak load at a rate equal to half that used during loading . E and H were calculated using the method described by Oliver and Pharr .
All statistical analyses were performed using SPSS 19.0 software. The mean value of each parameter of each group was calculated. All data obtained were analyzed using one-way analysis of variance (ANOVA) for differences among all groups. If significant difference was observed, the least significant difference test was used for post hoc comparison to compare the difference between every two groups. The significance level of p was chosen to be 0.05.
The work was supported by the National Natural Science Foundation of China (Nos. 11702110, 11872095 and 11432016) and the Natural Science Foundation of Jilin Province (Nos. 20170519008JH and 20170520093JH).
ZHL, JZG and HG conceived and designed the experiment, analyzed and interpreted the data; ZHL performed treadmill running and the statistical analysis; ZHL, JZG and HG performed the micro-CT scanning, three point bending mechanical test, serum analysis, soleus tensile test and nanoindentation test. ZHL, JZG and HG wrote the paper. All authors participated in the trial design, provided feedback on drafts of the manuscript. All authors read and approved the final manuscript.
The work was supported by the National Natural Science Foundation of China (Nos. 11702110, 11872095 and 11432016) and the Natural Science Foundation of Jilin Province (Nos. 20170519008JH and 20170520093JH).
Ethics approval and consent to participate
This study was approved by the Medical Ethics Committee of the First Hospital of Jilin University (No. 2018-238). This study was in strict accordance with the requirement of the Laboratory Animal Standardization Committee.
Consent for publication
The authors declare that they have no competing interests.
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