AHLES, CAMMIE P.1; SINGH, HARPREET1; JOO, WOOJIN1; LEE, YVONNE1; Lee, LUCY C.1; COLAZAS, WILLIAM1; PIERCE, R. ANDER1; PRAKASH, ANURADHA1; JAQUE, S. VICTORIA2; SUMIDA, KEN D.1
An elevation in peak bone mass has been advocated as a prophylactic against osteoporosis (10,30). Given that the hormonal milieu associated with the maturation process stimulates bone modeling, the growth period would be an optimal time to promote an even greater increase in peak bone mass. In support, exercise intervention studies in children appear to be more effective for bone formation compared with the elderly where the impact of exercise helps to prevent and/or to minimize bone loss (26). Elevating peak bone mass can be accomplished with resistance exercise that has been promoted as an effective method to stimulate bone formation (12,26). Thus, incorporating resistance exercise during the growth period would be beneficial in maximizing peak bone mass with the potential to attenuate the bone loss associated with advanced age. However, the amount of resistance exercise to implement during the growth period for maximal stimulation of bone accrual remains to be determined.
Turner and Robling (27) first described an exercise protocol with the potential to augment bone formation via interrupted exercise bouts throughout a training day rather than continuous exercise performed in a single training bout. Specifically, partitioning the exercise into multiple bouts allows the “mechanosensors” to reset and become restimulated, resulting in an even greater bone formation response (27). However, these initial studies were performed in anesthetized adult rats using a bone loading protocol for 16 wk (27). In a previous study, we sought to test this hypothesis by providing an additional stimulus for bone formation via interrupted resistance training during the growth period in conscious rats. We first attempted three bouts of exercise separated by 4 h (6), then two bouts of exercise separated by 12 h (5), and finally a single bout separated by 24 h (11). Given the relatively short training duration (i.e., 6 wk) and high volume of work performed, we chose to lengthen the time between each interrupted exercise bout to assist in the recovery of the mechanosensors. In all these previous studies, the total volume of work was equivalent between the interrupted and the continuous bouts of exercise (5,6,11). Despite our previous attempts to further augment bone formation (5,6,11), we found that the interrupted bouts of exercise were just as effective as the continuous resistance training regimen in elevating bone mineral density (BMD). In support, Umemura et al. (28) observed equivalent training-induced elevations in bone mass between an interrupted jumping protocol (2 × 10 jumps) compared with a continuous jumping protocol (1 × 20 jumps) in young rats. Thus, we suspected that during the growth period, a threshold existed whereby any additional exercise stimulus (resistance training or high impact) would be ineffective because a maximal capacity for bone formation had been reached. This was confirmed by our recent investigation where one group of growing rats performed a lower volume of resistance exercise per training session yet demonstrated the same elevation in BMD as animals that exercised with a greater volume of resistance training (20). However, the minimum amount of resistance exercise that could still elicit a bone formation response remains unresolved.
Therefore, the purpose of the current study was to determine the minimum amount of resistance exercise (via our training protocol) that would be just as effective as higher volumes of resistance exercise for stimulating increases in BMD during the growth period in rats. Further, if low volumes of resistance exercise stimulate bone formation to the same extent as high volumes of resistance exercise, this would indirectly support the existence of an exercise threshold whereby any additional work would not result in further increases in BMD. Thus, we compared four resistance training protocols where each exercised group performed different volumes of work per training session. The resistance exercise protocol used has previously been reported by our laboratory to stimulate bone formation (5,6,11,20,25). As in our previous work, we also assessed bone strength via three-point bending tests. We hypothesized that a low amount of resistance exercise performed during the growth period would be just as effective as greater volumes of resistance training in stimulating elevations in BMD and bone strength.
The experimental protocol for this study was preapproved by the Chapman University Institutional Review Board and in accord with both the American College of Sports Medicine standards for animal care and use for research as well as the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Forty male Sprague–Dawley rats (initially ∼175 g, ∼6 wk old) obtained from Charles River Laboratories (Wilmington, MA) were housed individually and maintained on a reverse 12-h light–12-h dark cycle. Food and water were provided ad libitum throughout the experimental period. The animals were acclimated to their living conditions for 5 d before random separation into a control group (Con, n = 8), which remained sedentary, or one of four resistance-trained groups where the animals performed one ladder climb per exercise session (1LC, n = 8), two ladder climbs per exercise session (2LC, n = 8), three ladder climbs per exercise session (3LC, n = 8), or four ladder climbs per exercise session (4LC, n = 8).
The strength training regimen, via ladder climbing, has previously been described (5,6,11,20,25). Briefly, the animals were required to climb a vertical ladder with weights appended to their tail. The animals were positioned to ensure that they performed each sequential step, where one repetition along the 1-m ladder required 26 total lifts by the animal (or 13 lifts per limb). Resistance-trained animals exercised 3 d·wk−1 for a total of 6 wk. The control animals were handled on the same days and times as the trained groups to minimize any stress attributable to handling. Thus, the only difference between the control animals and the exercised animals involved the resistance training. Animals were weighed at the beginning of each week to monitor weight gains and, for the resistance-trained animals, to help determine the amount of weight to append to their tail for the remainder of the week. All resistance-trained animals started with 30% body mass (BM) appended to their tail. Every week, the carrying weight was elevated by 30% BM for the next 4 wk (i.e., 30%, 60%, 90%, and 120%) until the beginning of week 5. At week 5, they carried 135% BM, and at week 6, they carried 150% BM. The vertical ladder climbing task and carrying weight during the 6-wk training period has consistently been demonstrated to be an effective stimulus for bone formation during the growth period in rats (5,6,11,20,25).
Collection of samples
Animals were killed 48 h after their final training session to minimize any residual effect of the last training bout. The flexor hallucis longus (FHL) was rapidly dissected from the right hindlimb, weighed, and then immediately frozen in liquid nitrogen for the subsequent determination of protein content. We chose the FHL because ladder climbing has previously been observed to elicit an elevation in FHL protein content providing support of a training effect (5,6,8,11,20,25). All the remaining soft tissues were removed from the right tibia and right femur, and the bones were submerged in a scintillation vial filled with an ethanol–saline (50/50) solution, capped, and kept at room temperature. Bone strength was assessed from the right tibia and right femur within 1 wk after dissection. The left hindlimb was rapidly amputated, positioned, and frozen in liquid nitrogen for the assessment of BMD of the tibia and femur. Blood samples were collected, allowed to clot, and centrifuged, and the serum was frozen for the subsequent measurement of serum osteocalcin (OC) and serum pyridinoline cross-links (PYD). The FHL, left hindlimb, and serum were kept at −80°C until their analyses.
Protein concentration in the FHL was assessed (14) as an indirect indicator of training (i.e., muscle hypertrophy). A sandwich enzyme-linked immunosorbent assay (Biomedical Technologies, Inc., Stoughton, MA) was used to determine serum osteocalcin levels (an indicator of osteoblast activity). The intra-assay variation and the interassay variation was <4%. Serum pyridinoline cross-links (an indicator of osteoclast activity) was measured using a competitive enzyme immunoassay (Quidel Corp., San Diego, CA). The intra-assay variation and the interassay variation was <5%. A microplate reader (MaxLine; Molecular Devices Corp., Sunnyvale, CA) was used with the absorbance set at 450 nm for the enzyme-linked immunosorbent assay and 405 nm for the enzyme immunoassay. A standard curve was generated for all chemical analyses, and controls were run to ensure quality. For all standard curves, the correlation coefficient (Pearson product for linear curves, i.e., protein) or coefficient of determination for nonlinear curves (i.e., OC and PYD) was greater than 0.99.
Bone mineral density
A dual-energy x-ray absorptiometer (DXA; GE Lunar Prodigy, Chicago, IL) using the small animal software module (version 6.81) was used to assess the BMD of the entire left tibia and left femur. Briefly, the left hindlimb was thawed and positioned, and the entire tibia and femur were scanned. Condyle and malleolus curvatures of the tibia were used as anatomical markers to ensure proper positioning and to prevent twisting so that the curvatures were not exaggerated or obliterated. Trochanter and condyle curvatures of the femur were used as anatomical markers and situated to ensure proper positioning. For each scan, care was taken to ensure the proper orientation of the hindlimb by an experienced technician so that the geometry would be similar. Three consecutive measurements were performed with the hindlimb repositioned between each scan. The reported BMD was the average of three scans for each bone, and the coefficient of variation for repeated scans (mean ± SE) that included hindlimbs from all animals was 1.49 ± 0.24% for the tibia and 1.36 ± 0.13% for the femur.
Three-point bending test
The mechanical properties of bone were measured at room temperature using a three-point bending rig placed onto the stage of a texture analyzer instrument (TA-XT2; Texture Technologies, Ramona, CA). Before testing, the right tibia and femur were rinsed in saline and then submerged in saline for 24 h at room temperature. On the day of testing, the texture analyzer was calibrated using a standard weight, and then the tibia or femur was patted dry and secured to the rig. The span of the two support points was 15.0 mm. The deformation rate was set at 0.9 mm·s−1 for all groups. A medial to lateral force was applied to the midshaft of the bone to allow the force arm to break a flat portion of the bone and to minimize differences due to bone curvatures or processes between animals. Three parameters of bone strength were measured: maximal load to failure (Fmax, N), energy to failure (EF, determined from the area under the load-deformation curve to the fracture point, N·mm), and bone stiffness (slope of the linear portion from the load-deformation curve, N·mm−1). All measurements were assessed using Texture Expert (version 1.22; Stable Micro Systems Ltd., Surrey, England, UK).
Calculations and statistics
Work (i.e., training volume) was calculated as the product of the total weight lifted by the animal (BM plus the amount of weight appended to the tail), the acceleration due to gravity, and the distance covered. The total training volume (i.e., work) per exercise session for all resistance-trained groups was expressed in joules. The total protein in the FHL was calculated as the product of protein concentration and muscle mass. An ANOVA was used for all comparisons, and when a significant F-ratio was identified, a Fisher PLSD post hoc test was used. The level of significance set was at P< 0.05 for all statistical comparisons, and the results were expressed as mean ± SE.
The initial and the final BM (i.e., after the 6-wk training program) was not significantly different between groups (Table 1). By design, the total training volume was significantly different between resistance-trained groups throughout the 6-wk period (Fig. 1). After the 6-wk exercise program, the FHL mass and the FHL total protein content were significantly greater for the 2LC, 3LC, and 4LC groups compared with Con (Table 1), indirectly supporting a training effect. There was no significant difference between the 2LC, the 3LC, and the 4LC groups.
The BMD from the entire left tibia was significantly greater for 2LC (i.e., 6.4% greater), 3LC (i.e., 7.3% greater), and 4LC (i.e., 6.9% greater) compared with the Con group (Fig. 2A). The BMD from the whole femur was similarly greater for 2LC (i.e., 9.0% greater), 3LC (i.e., 13.1% greater), and 4LC (i.e., 10.7% greater) compared with Con (Fig. 2B). The femur BMD was also significantly greater for the 3LC (i.e., 9.3% greater) and 4LC groups (i.e., 7.3% greater) compared with the 1LC group. For both the tibia and the femur, the training-induced augmentation in BMD was not significantly different between the 2LC, the 3LC, and the 4LC groups.
Training-induced elevations were also observed for bone strength parameters. Fmax, EF, and bone stiffness from the tibia were significantly greater for 2LC (Fmax = 24.6% greater, EF = 23.4% greater, stiffness = 22.0% greater), 3LC (Fmax = 28.0% greater, EF = 31.8% greater, stiffness = 25.6% greater), and 4LC (Fmax = 28.9% greater, EF = 39.4% greater, stiffness = 36.9% greater) compared with Con (Table 2). Further, bone stiffness of the tibia was also greater for 3LC and 4LC compared with the 1LC group (Table 2). In like manner, Fmax, EF, and bone stiffness from the femur were significantly greater for 2LC (Fmax = 39.8% greater, EF = 44.8% greater, stiffness = 48.2% greater), 3LC (Fmax = 44.3% greater, EF = 38.7% greater, stiffness = 49.2% greater), and 4LC (Fmax = 39.2% greater, EF = 35.5% greater, stiffness = 52.2% greater) compared with Con (Table 3). Further, these bone strength parameters were also greater for 2LC, 3LC, and 4LC compared with the 1LC group (Table 3).
There were also training-induced alterations in the serum biomarkers for osteoclast activity. Serum pyridinoline cross-links (PYD) were significantly lower for 2LC, 3LC, and 4LC groups compared with both the 1LC and the Con groups (Table 4). There were no significant differences in serum PYD between 2LC, 3LC, and 4LC groups. Serum OC did not differ between Con 1LC, 2LC, 3LC, or 4LC groups (Table 4).
Training-induced elevations in BMD for the left tibia and left femur were observed for the 2LC, 3LC, and 4LC groups compared with Con. In addition, the 2LC, 3LC, and 4LC groups demonstrated significantly greater bone strength parameters for both the right tibia and right femur compared with Con. Although training-induced alterations were observed for BMD, bone strength, and serum PYD for the 2LC, 3LC, and 4LCgroups compared with Con, there were no significant differences between the 2LC, the 3LC, and the 4LC groups. Taken together, the results support our hypothesis that a low amount of resistance training (i.e., two ladder climbs) was just as effective as greater volumes of resistance exercise for stimulating more BMD and enhanced bone strength during the growth period in rats.
Elevating peak bone mass during the growth period may help to prevent the onset or reduce the severity of osteoporosis (10,30). Although pharmacological treatment strategies are available to help slow or prevent the rate of bone degradation, currently there is no cure for osteoporosis. In the absence of a cure, prevention becomes of utmost importance where high-impact activities (e.g., jumping) or resistance exercise (e.g., strength training) have been advocated as effective activities to use to increase peak bone mass that could assist in reducing the risk of osteoporosis (12,26). Although numerous human studies (1,4,13,15,24,29,30) have supported the effectiveness of high-impact or resistance activities for increasing bone formation, these investigations often use cross-sectional comparisons or prospective research designs where heredity, diet, growth rate, and lean mass (to name a few) could be contributing factors for skeletal differences rather than the exercise intervention. In contrast, the use of an animal model minimizes many of the confounding variables associated with human studies. However, most of the previous animal studies investigated the impact of treadmill running (i.e., weight bearing) on the bone (9,18). The challenge of getting animals to lift a heavy mass has limited a thorough examination of the impact of resistance training on the skeletal system. To mimic resistance training, we used a modified version of a ladder climbing task for rats that was first described by Hornberger and Farrar (8). Our current observations of training-induced elevations in BMD for the tibia and femur, using a strength training model in rats, were consistent with previous animal reports on the effectiveness of incorporating exercise for stimulating bone for mation during the growth period in animals (5,6,11,17–20,23,25,28,31). Nevertheless, although physical activity has consistently been demonstrated to stimulate bone formation, as supported by both human and animal studies, the amount of exercise to maximally elevate peak bone mass during the growth period has been unresolved.
Using adult anesthetized animals and a bone loading protocol, Turner and Robling (27) hypothesized that mechanosensors within the bone can reset after a bout of exercise. As such, partitioning the exercise into multiple bouts throughout a training day would provide more stimulation for bone formation, yielding greater elevations in peak bone mass (21,27). Thus, this interrupted exercise strategy could potentially maximize BMD during the growth period. Using this premise, we made several attempts to support the enhanced effectiveness of interrupted exercise whereby bone formation could be maximally augmented. However, we observed equivalent training-induced elevations in BMD whether the resistance exercise was interrupted using various recovery times or the resistance exercise was continuous within a training day in young, growing male rats (5,6,11). Similarly, Umemura et al. (28) demonstrated equivalent osteogenic responses between an interrupted jumping protocol (2 × 10 jumps) compared with a continuous jumping protocol (1 × 20 jumps) in young female rats. Potential factors for the discrepancy between the results of Turner and Robling (27) compared with the results of Umemura et al. (28) and our findings (5,6,11) include the following: the age of the animal (i.e., adult vs young), the osteogenic stimulus (i.e., bone loading vs exercise), and the experimental protocol (i.e., anesthetized vs conscious animals). Alternatively, the inability to support the hypothesis submitted by Turner and Robling (27) led us to speculate that during the growth period there may be a threshold for bone formation.
The loading studies of Rubin and Lanyon (22,23) were among the first to demonstrate the existence of an exercise threshold whereby any additional increases in the number of load cycles failed to result in further changes in avian bone mineral content. These initial studies were performed on anesthetized animals where the advantage of using a bone loading protocol includes a quantification of load, cycles, and compressive strains. However, caution should be used in the interpretation when applied to dynamic exercise that can create unique load and strain distribution patterns. Alternatively, the use of conscious animals engaged in exercise allows for the full impact of load and compressive strains but does not allow for a quantification of the load and compressive strains imposed on the bone. Despite the limitations with use of conscious animals, our previous work and current results support the contention of an exercise threshold as originally proposed by Rubin and Lanyon (23). First, we recently reported equivalent training-induced elevations in BMD in two groups of resistance-exercised animals despite significant differences in the volume of work performed each training day between groups (20). By using the identical training protocol as described in the current investigation, we reported that young animals climbing the ladder three times per training session (with weights appended to the tail) demonstrated the same elevation in BMD as growing animals climbing the ladder six times per exercise session (with weights appended to the tail) (20). Further, we note that in all our previous studies (5,6,11,25), the average training-induced augmentation in tibia BMD from animals climbing the ladder six times per training session was approximately 9% greater than corresponding controls, similar to the elevation in the current investigation although the animals performed approximately 33%–66% less work per training bout. Next, in a previous study, we demonstrated equivalent elevations in tibia BMD with three interrupted exercise bouts (separated by 3–4 h) where growing animals engaged in two ladder climbs per session compared with six continuous ladder climbs on a given training day (6). As such, our previous study (6) lends support to our current contention regarding the effectiveness of two ladder climbs as a volume of work (using this training regimen) sufficient for maximal bone modeling during the growth period. Collectively, our current findings and previous reports (5,6,11,25) support the contention of the existence of an exercise threshold for stimulating bone formation as initially proposed by Rubin and Lanyon (22,23) who used a bone loading protocol in anesthetized animals. We now extend this presumption to conscious animals engaged in resistance training during the growth period.
Although training-induced elevations in BMD are important, the salient feature in the prevention of fractures is bone strength. The opportunity to measure bone strength with use of an animal model is a significant advantage over human studies. However, factors that can contribute to differences in bone strength between animal studies include specimen storage, bone hydration, the temperature at which the bones are broken, the site at which the bones are broken, the direction of the applied force, and so on. Thus, we recognize that interpretations of bone strength data represent relative changes compared with a corresponding Con rather than absolute changes. In the current report, when compared with control animals, we observed a training-induced average augmentation of 27%, 32%, and 28% in Fmax, EF, and bone stiffness, respectively, for the tibia. We also observed an exercise-induced average elevation of 41%, 40%, and 50% (compared with Con) in Fmax, EF, and bone stiffness, respectively, for the femur. The absence of any significant difference between 2LC, 3LC, and 4LC groups suggests equivalent training-induced elevations in bone strength. In addition, our results were consistent with previous animal reports (5,6,11,20,27,28), demonstrating that the concomitant elevations in bone strength after training are much greater than the elevations in BMD.
In the present investigation, we note that the training-induced elevations in BMD and bone strength were greater for the femur compared with the tibia. We have no objective measurements to explain the apparent bone-specific (i.e., femur vs tibia) response to our ladder climbing exercise. On the basis of subjective interpretations, we offer the following explanations. First, the vertical ladder climbs might place more mechanical stress on the femur compared with the tibia that would account for the greater BMD. Next, we determined the BMD of the entire femur and tibia without regard to geometric changes at specific bone sites. Thus, the mechanical stress of the ladder climbs might stimulate bone formation at specific skeletal sites that are distinct for the femur compared with the tibia that contribute to differences in bone strength. Irrespective of the mechanism for the apparent difference in bone-specific response, the ladder climbing training program appears to be an effective stimulus for bone adaptation where more exercise does not result in greater bone mass.
Contributing factors for an elevation in BMD would be the relative rate of bone deposition compared with bone degradation. Ostensibly, net bone formation can be attributable to an elevation in osteoblast activity, a decline in osteoclast activity, or a combination of both. Although most studies suggest that the training-induced elevation in BMD can be attributable to an increase in osteoblast activity (3,4,5,11,16,20,25,31), only a few reports, including our current results, have demonstrated a decline in osteoclast activity (6,32). We have no explanation for the apparent discrepancy, but we note that blood withdrawn during animal sacrifice only represents a snapshot at a single time point and not the sustained activity of osteoblasts and/or osteoclasts throughout the training period. In support, several studies have demonstrated training-induced elevations in BMD in the absence of any alterations in serum biomarkers (7,19). Therefore, we recognize the significant limitation of extrapolating on bone cell activity from a single serum sample taken at one time point. Despite the drawbacks and inconsistent results from previous studies (including our own), we note that the decline in osteoclast activity (as measured by serum PYD) still provides suggestive evidence of net bone formation and corroborates the training-induced elevation in BMD and bone strength.
Finally, we acknowledge several limitations in the current study. First, the epiphyseal plates in rats do not close. This would favor a bone formation response and limit an extrapolation of the results to adult humans. As in our previous reports (5,6,11,20,25), we chose to examine the growth period in rats where the findings using this animal model might be more comparable with growing humans. Next, we recognize the limitations in the use of the DXA for the assessment of BMD where the units are expressed as mass per area rather than mass per volume. Further, a DXA is unable to discriminate between cortical and trabecular bone. In addition, differences in bone size can also lead to misinterpretations with the use of the DXA (2). Nevertheless, the DXA was used to measure BMD from all animals of equivalent body weight, minimizing the potential for confounding factors related to bone size providing relative comparisons between groups. In addition, the training-induced augmentations in BMD were supported by corresponding increases in bone strength. Despite all of these limitations, to the extent that our findings in rats can be applied to humans, our results would suggest that in children, incorporating a low amount of resistance exercise would be just as effective as higher volumes of resistance training for maximal bone formation.
In summary, our results suggest that a low volume of resistance training was just as effective as higher amounts of resistance training for maximal stimulation of bone formation during the growth period. This was supported by the equivalent elevations in BMD for the 2LC, 3LC, and 4LC groups compared with Con, despite the significant difference in work performed by each resistance-trained group. Compared with the Con group, there were also training-induced alterations in bone mechanical properties and serum PYD for the 2LC, 3LC, and 4LC groups. There were no significant differences between the 2LC, the 3LC, or the 4LC groups for BMD, bone strength, or serum PYD. Therefore, our results support the contention of an exercise threshold whereby increases in work per exercise session did not yield further elevations in bone modeling.
This study was supported by a Chapman University Faculty Research Grant.
The authors do not have any conflicts of interest or professional relationships with companies or manufacturers who would benefit from the results of this study.
The results of this study do not constitute endorsement by the American College of Sports Medicine.
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