Dong-Ho Park and Changsun Kim contributed equally to this work and should be considered co-corresponding authors.
Aging leads to significant bone loss and elevated osteoporosis risk. Exercise slows age-related bone loss; however, the effects of various moderate-intensity exercise training volumes on bone metabolism remain unclear. This study aimed to determine the degree to which different volumes of moderate-intensity aerobic exercise training influence bone mineral density (BMD), bone mineral content (BMC), femoral trabecular bone microarchitecture, and cortical bone in middle-aged mice.
Twenty middle-aged male C57BL/6 mice were randomly assigned 8 weeks of either (1) non-exercise (CON); (2) moderate-intensity with high-volume exercise (EX_MHV); or (3) moderate-intensity with low-volume exercise (EX_MLV) (N=6–7, respectively). Femoral BMD and BMC were evaluated using dual energy X-ray absorptiometry, and trabecular and cortical bone were measured using micro-computed tomography.
Femoral BMD in EX_MHV but not EX_MLV was significantly higher (
Long-term moderate-intensity aerobic exercise with low to high volumes can be thought to have a positive effect on hindlimb BMD and attenuate age-associated trabecular bone loss in the femur. Moderate-intensity aerobic exercise may be an effective and applicable exercise regimen to prevent age-related loss of BMD and BV.
Age-related alterations occur in the skeletal system associated with bone loss and increased fracture risks, leading to a myriad of pathological developments such as bone diseases like osteopenia and osteoporosis.[
Maintenance of a healthy lifestyle including habitual exercise with loading is a renowned non-pharmaceutical intervention that effectively improves bone metabolism by stimulating bone adaptation to mechanical forces.[
To achieve this aim, we tested the hypothesis that a higher volume of long-term moderate-intensity aerobic exercise would result in greater increases in femoral BMD, bone mineral content (BMC), trabecular bone microarchitecture, and cortical bone strength parameters compared to lower volume moderate-intensity exercise and no exercise. To test this hypothesis, middle-aged mice (14-months old) were randomly assigned to 8-weeks of either (1) non-exercise (CON; N=6); (2) moderate-intensity with high-volume exercise (EX_MHV; N=7); (3) moderate-intensity with low-volume exercise (EX_MLV; N=7).
The animal experimental protocol was approved by Institutional Animal Care and Use Committee (IACUC) of the Sungkyunkwan University School of Medicine (Approval no. SKKUIACUC 18-5-24-3) and followed the AAALAC International Guidelines for animal experiments. A total of 20 male C57BL/6 mice aged 14 months were purchased from Daehan Biolink Co. Ltd. (Daejeon, Korea). All mice were housed in standard cages at a pathogen-free animal care facility in a temperature- (22±2°C), humidity (50%), and light (12:12-hr light-dark) controlled room. Tap water and standard chow (Purina Mills, St Louis, MO, USA) were given ad libitum. All mice were weighed weekly.
An overview of the experimental exercise protocol is shown in (
After completion of the exercise protocol, all mice were anesthetized by a mixture of zoletil and rompun, and were sacrificed via myocardial removal. Right femora were harvested at sacrifice and placed in 4% paraformaldehyde at 4°C for 24 hr. Femora was subsequently stored in 70% ethanol in −20°C freezer until getting scanned by dual energy X-ray absorptiometry (DXA) and micro-computed tomography (μCT). Heart, epididymal fat, and skeletal muscles, including gastrocnemius and soleus, were also harvested and weighed at a sacrifice to show the effectiveness of the current exercise protocol.
UltraFocus DXA by Faxitron (UltraFocus DXA by Faxitron; Faxitron Bioptics, LLC, Tucson, AZ, USA) with a small animal-specific software was utilized to perform DXA scan following the tissue harvest. Whole femora were scanned and analyzed to measure the BMD and BMC.
The femur was dissected free of skin and muscle and evaluated using a SkyScan1172 high-resolution μCT imaging system (Bruker, Kontich, Belgium). Scans were obtained at an image resolution of 10 μm, with the following settings: 1 mm aluminum filter, 65 kVP X-ray voltage, 153 μA anode current, 65 ms exposure time, 5 frames averaging, and 0.3 degrees rotation step. Trabecular bone microarchitecture parameters in the distal femoral metaphysis were determined as previously described.[
One-way ANOVA was utilized for parametric data analyses to examine differences among groups, followed by a post-hoc Tukey’s test. Experimental sample sizes presented in
Pre- and post-exercise body masses and tissues weights following 8 weeks of exercise training were examined (
To examine the role of various volumes of moderate-intensity exercise programs in the prevention of age-related BMD and BMC loss, we first determined the BMD and BMC of femora in middle-aged mice subjected to EX_MHV and EX_MLV programs over 8 weeks (
Next, we set out to determine the effectiveness of long-term moderate-intensity aerobic exercise in the prevention of age-associated bone loss. Meaningful differences in distal femoral trabecular bone microarchitecture were observed in the EX_MHV and EX_MLV groups compared with CON. The 3D μCT reconstructions of trabecular bone microarchitecture in the secondary spongiosa of the distal femoral metaphysis (
We next determined the effect of various volumes of moderate-intensity aerobic exercise on mid-diaphysis cortical bone parameters (i.e., Ct.BV, Ct.Ar, cross-sectional Thickness, and pMOI) in femora. A significant difference in pMOI between groups was observed, whereby EX_MLV showed significantly higher (
Current data indicate that long-term moderate-intensity aerobic exercise training, overall, beneficially preserves BMD and attenuates age-associated trabecular bone loss in the distal femora of middle-aged male mice. Moreover, we show that 8-weeks of moderate-intensity exercise training increased soleus and gastrocnemius muscle mass and reduced epididymal fat. These hypotheses generating data highlight the potential of moderate-intensity exercise training as a robust non-pharmaceutical therapy for the prevention of osteoporosis.
Osteoporosis is a common but destructive age-related metabolic disease caused by low BMD and decreasing BV.[
Various durations and intensities of aerobic exercise training (i.e., treadmill running, swimming, etc.) for 8 to 14 weeks have been shown to have differential outcomes in bone quality, bone mass, and BV in rodents.[
The protective role of long-term exercise against chronic diseases such as osteoporosis, diabetes, and cardiovascular diseases, as well as beneficial outcomes of long-term exercise on disease conditions have been documented, thereby showing improvement in health status.[
In the current investigation, we hypothesized that moderate-intensity aerobic exercise for 8 weeks would improve cortical bone parameters in the mid-diaphysis of long bones, allowing for augmenting the strength of long bones as a training effect. However, contrary to our hypothesis, cortical bone parameters, including cortical volume, area, cross-sectional thickness, and pMOI following moderate-intensity aerobic exercise, did not differ from the non-exercise group. Discrepancies may relate to the intensity and volume of aerobic exercise. Cortical bone strength gain may be more susceptible to weight-bearing exercises such as jumping and landing instead of treadmill aerobic exercise.[
The current study has some limitations worthy of discussion. Firstly, the omission of a young control group limits our ability to strictly describe whether moderate-intensity exercise attenuated age-associated bone loss in the distal femoral trabecular bone. However, in comparing non-exercise mice with mice that performed exercise, the same species, strain, and age offset the confounding variables, which support the notion that moderate-intensity exercise improved femoral BMD and trabecular bone microarchitecture. Second, due to the limitations of the methodology applied herein, we were not able to assess the mechanical force-derived bone cellular activity and recruitment, including osteoblast and osteoclast activity and recruitment. Future work should include bone histomorphometry analysis with Goldner’s trichrome and immunohistochemical analysis of ALP or OCN to confirm the osteoblast activity and recruitment following moderate-intensity exercise.
In conclusion, the current results demonstrate that femoral BMD is beneficially preserved following 8 weeks of high volume moderate-intensity but not the low volume of moderate-intensity aerobic exercise training. Age-related trabecular bone loss was mitigated by various volumes of moderate-intensity exercise, which is presumably stimulated by increased mechanical force-derived osteoblast activity and recruitment in the distal femur. This investigation highlights the clinical impact of long-term moderate-intensity treadmill exercise as a non-pharmacological therapy for preventing bone loss and osteoporosis progression in the elderly.
The abstract of this study has been presented as the Annual Meeting of the American Society of Bone and Mineral Research (ASBMR) (1–4 Oct. 2021).
All animal studies were approved by the Institutional Animal Care and Use Committee at Sungkyunkwan University School of Medicine (SKKUIACUC 18-5-24-3) and followed the AAALAC International Guidelines for animal experiments.
No potential conflict of interest relevant to this article was reported.
Schematic overview of study design and exercise protocol. (A) C57BL/6 mice underwent 1 week of familiarization with the exercise protocol at 14-months old followed by 8 weeks of treadmill exercise training 5 days/week for exercise groups or no exercise for the non-exercise (CON) group. (B) Exercise protocol for moderate-intensity with high-volume exercise (EX_MHV) and moderate-intensity with low-volume exercise (EX_MLV) groups which consists of a warm-up, main exercise protocol, and cool-down.
Long-term moderate-intensity aerobic exercise preserves bone mineral density (BMD) in femora of middle-aged mice. (A, B) Quantification of BMD and bone mineral content (BMC) of femora. (A) Femoral BMD were significantly higher (
Age-associated distal femoral trabecular bone loss is attenuated by long-term moderate-intensity aerobic exercise. (A–C) Representative images of distal femoral trabecular bone microarchitecture in (A) non-exercise (CON), (B) moderate-intensity with high-volume exercise (EX_MHV), and (C) moderate-intensity with low-volume exercise (EX_MLV) mice. (D) Femoral bone volume (BV) tended to be higher (
Pre- and post-exercise body masses, heart, muscle, and epididymal fat weight of animals following moderate-intensity aerobic exercise program
CON | EX_MHV | EX_MLV | |
---|---|---|---|
Pre-exercise body mass (g) | 33.8±1.6 | 33.3±0.8 | 32.8±1.3 |
Post-exercise body mass (g) | 35.2±2.7 | 30.3±1.1 |
30.8±1.3 |
Heart (mg) | 130.7±8.6 | 124.7±9.4 | 127.7±9.6 |
Gastrocnemius (mg) | 126.9±9.8 | 140.4±9.2 |
140.5±6.6 |
Soleus (mg) | 8.7±0.9 | 11.1±1.2 |
11.0±1.1 |
Epididymal fat (mg) | 1117.2±200.0 | 695.1±165.7 |
822.95±140.5 |
The data is presented as mean±standard deviation.
Indicates difference from CON (
Indicates difference from CON (
Indicates trending toward difference from CON (
CON, non-exercise; EX, exercise; MHV, moderate-intensity with high-volume exercise; MLV, moderate-intensity with low-volume exercise.
Femoral mid-diaphysis cortical bone properties following moderate-intensity aerobic exercise program
CON | EX_MHV | EX_MLV | |
---|---|---|---|
Ct.BV (mm3) | 0.595±0.017 | 0.937±0.057 | 0.996±0.030 |
Ct.Ar (mm2) | 0.973±0.027 | 0.937±0.057 | 0.996±0.030 |
Cross-sectional Thickness (mm) | 0.181±0.003 | 0.176±0.008 | 0.183±0.007 |
pMOI (mm4) | 0.601±0.057 | 0.544±0.058 | 0.633±0.033 |
The data is presented as mean±standard deviation.
Indicates difference from EX_MHV (
Indicates trending toward difference from EX_MHV (
Ct.BV, cortical bone volume; Ct.Ar, cortical bone area; pMOI, polar moment of inertia; CON, non-exercise; EX, exercise; MHV, moderate-intensity with high-volume exercise; MLV, moderate-intensity with low-volume exercise.