Journal of Applied Physiology

Simulated resistance training, but not alendronate, increases cortical bone formation and suppresses sclerostin during disuse

B. R. Macias, J. M. Swift, M. I. Nilsson, H. A. Hogan, S. D. Bouse, S. A. Bloomfield


Mechanical loading modulates the osteocyte-derived protein sclerostin, a potent inhibitor of bone formation. We hypothesized that simulated resistance training (SRT), combined with alendronate (ALEN) treatment, during hindlimb unloading (HU) would most effectively mitigate disuse-induced decrements in cortical bone geometry and formation rate (BFR). Sixty male, Sprague-Dawley rats (6-mo-old) were randomly assigned to either cage control (CC), HU, HU plus either ALEN (HU+ALEN), or SRT (HU+SRT), or combined ALEN and SRT (HU+SRT/ALEN) for 28 days. Computed tomography scans on days1 and 28 were taken at the middiaphyseal tibia. HU+SRT and HU+SRT/ALEN rats were subjected to muscle contractions once every 3 days during HU (4 sets of 5 repetitions; 1,000 ms isometric + 1,000 ms eccentric). The HU+ALEN and HU+SRT/ALEN rats received 10 μg/kg ALEN 3 times/wk. Compared with the CC animals, HU suppressed the normal slow growth-induced increases of cortical bone mineral content, cortical bone area, and polar cross-sectional moment of inertia; however, SRT during HU restored cortical bone growth. HU suppressed middiaphyseal tibia periosteal BFR by 56% vs. CC (P < 0.05). However, SRT during HU restored BFR at both periosteal (to 2.6-fold higher than CC) and endocortical (14-fold higher than CC) surfaces (P < 0.01). ALEN attenuated the SRT-induced BFR gains during HU. The proportion of sclerostin-positive osteocytes in cortical bone was significantly higher (+121% vs. CC) in the HU group; SRT during HU effectively suppressed the higher proportion of sclerostin-positive osteocytes. In conclusion, a minimum number of high-intensity muscle contractions, performed during disuse, restores cortical BFR and suppress unloading-induced increases in sclerostin-positive osteocytes.

  • resistance exercise
  • Wnt
  • hindlimb unloading
  • histomorphometry
  • load

the estimated lifetime risk of an osteoporotic fracture is ∼50% in women and ∼22% in men (14); annual direct-care costs attributable to osteoporotic fractures in the United States are estimated to be as high as $18 billion (38). Regular weight-bearing exercise can help preserve or add small amounts of bone mass in adult women and men (9). The development of bisphosphonate therapies has helped to reduce fracture risk. However, no medical therapy is yet available to prevent osteoporosis, characterized by aggressive bone resorption and unchanged or decreased bone formation. It is well established that the mechanical loading that occurs with weight-bearing exercise is a potent anabolic stimulus to bone (25). In fact, mechanical loading via the Wnt/β-catenin (canonical) signaling pathway in bone cells plays a pivotal role in translating the forces imposed on bone into upregulated bone cell activity, resulting in a net gain of bone mass (26). Therefore, the mechanical loading that occurs during weight-bearing activity is requisite to the maintenance of bone mass and quality.

Bone and muscle tissues do not have to resist the force of gravity when “ambulating” in space, and it is hypothesized that this lack of mechanical load results in lower body musculoskeletal deconditioning (21). Biomechanical loads of the lower body musculoskeletal tissues during Mir Missions were calculated to be 30–40% less than those present on Earth when using treadmill exercise with bungee cords (40). Somewhat higher loads were achieved with treadmills on Shuttle flights (36). In-shoe measures of ground reaction force during treadmill, ergometer, and resistance exercise of International Space Station (ISS) crewmembers were less than those forces for a similar activity on Earth (5, 12). Furthermore, recent studies performed aboard the ISS demonstrate the need for development of improved exercise countermeasures that involve higher loading profiles to weight-bearing bones and muscles (8, 16, 37).

The lack of sufficient mechanical stimuli to weight-bearing regions of the skeleton during spaceflight has been documented during recent studies of ISS crewmembers. Volumetric quantitative computed tomography (QCT) measures of cortical bone mineral content (BMC) at the hip in 14 crewmembers before and after ISS missions (4–6 mo) show rates of loss to be −1.6%/mo at both the femoral neck and trochanter (16). In another study, 16 crewmembers on 4.5- to 6-mo ISS missions lost, on average, 11.9% in femoral neck cortical bone mass (17). Moreover, bone biomarkers, calcium metabolism, and calcium kinetics data collected during long-duration ISS and Mir missions suggest that the resulting bone loss is primarily due to increased bone resorption and decreased intestinal calcium absorption (30). These studies, taken together, highlight the robust thinning of the femoral neck cortex from the inner margin and presumed robust resorptive activity of osteoclasts during extended durations of microgravity exposure.

The rodent hindlimb unloading (HU) model is a well-established ground-based model for investigating disuse effects on bone (20). Previously, our laboratory has demonstrated that high-intensity muscle contractions at 100% peak isometric torque (P0), produced during simulated resistance training (SRT) undertaken during a period of HU produced significant gains in middiaphyseal tibia cortical bone mineral density (BMD). These gains were associated with a fivefold greater periosteal bone formation rate (BFR) compared with control animals (32). Numerous studies have investigated the efficacy of a pharmacological treatment [alendronate (ALEN)] in inhibiting disuse-induced bone loss or in models of estrogen deficiency (1, 2, 28, 29). Bisphosphonate therapies potently suppress accelerated osteoclast-mediated bone resorption. Currently, ALEN, an anti-resorptive bisphosphonate, is being administered to some astronauts aboard the ISS to investigate the efficacy of this bone loss countermeasure strategy (17).

Our laboratory previously demonstrated that bisphosphonate treatment, when combined with moderate-intensity muscle contractions (75% P0) during disuse, significantly increases proximal tibia total BMC (33). However, this combined drug/exercise countermeasure attenuates the cancellous bone formation response compared with the exercise-only group (33). The cellular mechanism by which SRT restores bone gain during unloading or how bisphosphonate treatment impacts the anabolic response of cortical bone to SRT remains undefined. To our knowledge, these are the first studies to employ a resistive exercise modality during HU with bisphosphonate treatment. The aim of this present investigation was to test the anabolic effects of SRT in combination with the anti-resorptive effects of ALEN during 28 days of HU in adult rats on disuse-induced cortical bone decrements. We hypothesized that SRT, in combination with ALEN therapy during HU, would better mitigate deleterious changes in cortical bone mass, cortical bone geometry, cortical BFR, and that SRT but not ALEN would mitigate elevations of sclerostin-positive osteocytes.


Animals and experimental design.

Sixty male Sprague-Dawley rats were obtained from Harlan (Houston, TX) at 6 mo of age and allowed to acclimate for 14 days before initiation of the study. All animals were singly housed after arriving at our animal facility in a temperature-controlled (23 ± 2°C) room with a 12:12-h light-dark cycle in an American Association for Accreditation of Laboratory Animal Care-accredited animal care facility and were provided standard rodent chow (Harlan Teklad 8604) and water ad libitum. Animal care and all experimental procedures described in this investigation were approved by the Texas A&M University Laboratory Animal Care Committee. Previously, tissues from these same animals were utilized to assess the impact of ALEN and SRT on cancellous bone (34).

Five experimental groups were studied: 1) cage control (CC, n = 12); 2) HU (n = 12); 3) HU animals administered 10 μg/kg ALEN via subcutaneous injection 3 times/wk (HU+ALEN, n = 12); 4) HU subjected to SRT (HU+SRT, n = 12); and 5) HU rats subjected to both ALEN and SRT (HU+SRT/ALEN, n = 12). Animals in all groups except CC underwent 28 days of HU. HU+SRT and HU+ALEN/SRT animals underwent nine sessions of simulated resistive exercise conducted once every 3 days during the 28-day protocol. The HU and HU+ALEN groups were also administered the same regimen (frequency, dose, duration) of isoflurane anesthesia (Minrad, Bethlehem, PA) as used for SRT groups. The CC animals were allowed normal ambulatory cage activity, but not exposed to anesthesia.

Calcein injections (25 mg/kg body mass) were given subcutaneously 9 and 2 days before euthanasia to label mineralizing bone for histomorphometric analysis. HU animals were anesthetized before removal from tail suspension at the end of the study to prevent any weight bearing by the hindlimbs. At the end of the experiment, on day 28, all animals were anesthetized with 50 mg/kg body wt of ketamine (Fort Dodge Animal Health, Fort Dodge, IA) and 0.5 mg/kg body wt of medetomidine (Pfizer, New York, NY) and euthanized by decapitation. Distal left femora were fixed in formalin, decalcified, and stored at 4°C for immunohistochemistry analyses; proximal left tibiae were fixed in formalin and then stored in 70% ethanol at 4°C for histomorphometry analyses.


HU was achieved by tail suspension, as previously described (32). The height of the animal's hindquarters was adjusted to prevent any contact of the hindlimbs with the cage floor, resulting in approximately a 30° head-down tilt. The forelimbs of the animal maintained contact with the cage bottom, allowing the rat full access to the entire cage. All animals were monitored daily for health, including assessment of tail integrity, and body weights were measured weekly.

SRT paradigm.

SRT was completed as previously described (32). Briefly, left plantar flexor muscles from animals in the HU+SRT group were trained once every 3 days during 28-day HU using a custom-made rodent isokinetic dynamometer. Animals were anesthetized with isoflurane gas (∼2.5%; Minrad, Bethlehem, PA) mixed with oxygen before removal from tail suspension to prevent any weight bearing by the hindlimbs. Each rat was then placed in right lateral recumbency on a platform, the left foot was secured onto the foot pedal, and the left knee was clamped so that the lower leg was perpendicular to the foot and the femur and tibia were at right angles to each other. This was referred to as the resting, 0° position. For isometric contractions, the foot pedal was held fixed in this position. For all contractions, the footplate was rotated in synchrony with muscle stimulation by a Cambridge Technology lever system (model 6900) interfaced with a 80486 66-MHz personal computer using custom software written in TestPoint (version 4.0; Capital Equipment, Billerica, MA). Torque generated around the footplate pivot (at the rat's ankle joint) was measured by the lever system's servomotor. Plantar flexor muscle stimulation was performed with fine-wire electrodes consisting of insulated chromium nickel wire (Stablohm 800B, H-ML Size 003, California Fine Wire), inserted intramuscularly straddling the sciatic nerve in the proximal thigh region. The stimulation wires were then attached to the output poles of a Grass Instruments stimulus isolation unit (model SIU5; Astro-Med, W. Warwick, RI) interfaced with a stimulator (S88; Astro-Med), which delivered current to the sciatic nerve and induced muscle contraction.

Voltage optimization to achieve P0 and stimulation frequency optimization of the eccentric torque were performed at the beginning of each session, as described previously (32). The eccentric phase of the muscle contraction was titrated to equal ∼75% of each animal's daily P0. The HU+SRT and HU+SRT/ALEN animals completed a combined isometric + eccentric SRT exercise paradigm, consisting of four sets of five repetitions, once every 3 days during HU (n = 9 total exercise sessions). The training paradigm consisted of a 1,000-ms isometric contraction (75%), immediately followed by a 1,000-ms eccentric contraction (75% of the peak isometric contraction).

Bisphosphonate treatment.

Animals in the HU+ALEN and HU+SRT/ALEN groups were administered 10 μg/kg ALEN (Merck and Rathway) via subcutaneous injection 3 times/wk for the duration of the 28-day study. The ALEN dose of 30 μg·kg−1·wk−1 was the lowest dose (of three tested) that effectively mitigated reductions in cancellous volumetric BMD at the proximal tibia during 28 days of HU (unpublished data) and is similar to the 30 μg/kg ALEN (15 μg/kg, 2 times/wk) shown to maintain femur and lumbar spine bone mass and strength after ovariectomy (OVX) in rats (10). This ALEN dose of 30 μg·kg−1·wk−1 is, however, much lower than the dose (100 μg·kg−1·day−1) used in previously published clinical studies and in OVX rats, demonstrating pronounced increases in bone mass and strength (27). Rats in the CC, HU, and HU+SRT groups were administered an equal volume of vehicle (phosphate-buffered saline) by subcutaneous injection 3 times/wk.

Peripheral QCT.

On days1 and 28 of the study, peripheral QCT (pQCT) scans were performed in vivo at the tibia middiaphysis with a Stratec XCT Research-M device (Norland, Fort Atkinson, WI), using a voxel size of 100 μm and a scanning beam thickness of 500 μm. Two slices centered at 50% of the tibial total length (determined from a scout view on the CT scanner) were collected. Calibration of this machine was performed on each day of scanning with a hydroxyapatite standard cone phantom. A standardized analysis for diaphyseal bone (contour mode 1, peel mode 2, outer threshold of 0.650 g/cm3, inner threshold of 0 g/cm3) was applied to each section.

Values of cortical BMC, cortical bone area, and the polar cross-sectional moment of inertia (CSMI) were averaged across the two slices at the tibial middiaphysis to yield a mean value. Polar CSMI was based on geometry only and not weighted by density. Machine precision (based on manufacturer's data) is ±9 mg/cm3 for cortical bone. Reproducibility in our laboratory was determined from five repeat scans using an in vivo multiple-slice scanning method; resulting coefficients of variation for cortical tibia BMD was ±0.59%.

Dynamic histomorphometry analysis.

Un-demineralized distal left tibiae were subjected to serial dehydration and embedded in methylmethacrylate (Sigma-Aldrich M5, 590–9, St. Louis, MO). Serial cross sections (150–200 μm) of midshaft cortical bone were cut starting 2.5 mm proximal to the tibiofibular junction with an Isomet diamond wafer low-speed saw (Buehler, Lake Bluff, IL). Sections were ground to reduce thickness (<80 μm) before mounting on glass slides. The histomorphometric analyses were performed by using the OsteoMeasure Analysis System, version 1.3 (OsteoMetrics, Atlanta, GA). Measures of labeled surfaces and interlabel widths were obtained at ×100 magnification of up to two slides per animal. Periosteal and endocortical mineral apposition rates (MAR, μm/day) were calculated by dividing the average interlabel width by the time between labels (7 days) and mineralizing surface (MS) for both periosteal and endocortical bone surfaces (BS) using the formula %MS/BS = {[(single-labeled surface/2) + double-labeled surface]/surface perimeter} × 100. BFR was calculated as (MAR × MS/BS).

Sclerostin immunohistochemistry.

Distal left femora were fixed in 4% phosphate-buffered formalin for 48 h at 4°C, then decalcified in a sodium citrate-formic acid solution for 14 days, and stored in 70% EtOH. Following decalcification, the distal left femora were embedded in paraffin, and transverse sections at the midshaft were cut 10 μm thick and mounted on slides. Five slides, one slide from each group, were placed in a dry incubator at 60°C for 15 min to melt the paraffin. Slides were then washed in 70, 95, 100, and 100% ethanol solution for 3 min each. Slides were then washed in xylene twice for 5 min each. To quench endogenous hydrogen peroxides that may interfere with the horseradish peroxidase reaction, slides were soaked in a 0.3% hydrogen peroxide solution for 30 min. A hydrophobic perimeter was drawn around the tissue to maintain small-volume solutions above the tissue sample during incubation. Slides were washed twice in PBS for 5 min each, and then slides were washed in a PBS/0.5% Triton solution for 5 min. Slides were washed twice in PBS for 5 min each. Tissues were blocked with normal goat serum for 20 min, followed by a 5-min wash in PBS/2% normal goat serum solution. To reduce nonspecific binding, the tissue samples were treated with the avidin/biotin blocking kit per manufacturer's instructions (SP-2001, Vector Laboratories, Burlingame, CA), followed by three 5-min washes with PBS. Tissues were then placed on a wet-incubation tray, and each sample was loaded with the sclerostin primary antibody (1:250 dilution, R&D Systems, Minneapolis, MN) and incubated at 4°C overnight. Following overnight incubation, tissues were washed twice with a PBS/2% BSA/0.2% Tween solution, then washed twice with a PBS/2% BSA solution. The tissue samples were loaded with the biotinylated secondary antibody and incubated for 30 min (Vectastain Elite ABC Kit, Vector Laboratories, Burlingame, CA). Tissue was developed with a peroxidase substrate solution (NovaRED, Vector Laboratories, Burlingame, CA) for 10 min to stain sclerostin-positive osteocytes. Slides were washed quickly in PBS to terminate the reaction, taped dry, counterstained with hematoxylin, taped dry, coverslipped, and allowed to dry in the dark for 2 days. The region of interest for quantification of total (number of osteocytes) and sclerostin-positive osteocytes (SOST+ Ot) included the entire midshaft femur cross section using the OsteoMeasure Analysis System, version 1.3 (OsteoMetrics, Atlanta, GA). The percentage of sclerostin-positive osteocytes was calculated as (SOST+ Ot/total Ot) × 100.

Statistical analyses.

All data are expressed as means ± SE and evaluated using the statistical package SPSS (version 15; Chicago, IL). Histomorphometry and sclerostin assays were first analyzed using a two-factor ANOVA (exercise and ALEN) to compare group differences between HU groups (HU, HU+ALEN, HU+SRT, and HU+SRT/ALEN). A Tukey's post hoc test was used for pairwise comparisons. Subsequently, a one-factor ANOVA was used to compare HU groups' values vs. that of the comparator CC group (Tukey's post hoc test for pairwise comparisons). A one-factor ANOVA was employed to determine significant longitudinal pQCT variable changes within each treatment group (in vivo pQCT data only). For all data, statistical significance was accepted at P < 0.05.


SRT during disuse restores normal age-related cortical bone accrual.

HU for 28 days suppressed the normal growth gains in cortical BMC by 72%, compared with CC. The lack of BMC accrual in the HU+ALEN rodents (−0.44% change over 28 days) was similar to the HU-alone group (0.48%). However, when SRT was prescribed during 28 days of HU, normal growth gains in cortical BMC were restored (Table 1). ALEN administration did not appear to negatively affect the restoration of BMC by the mechanical load imposed by SRT (HU+SRT/ALEN group). The magnitude of changes in cortical area at the tibial middiaphysis was similar to those of changes in BMC. The SRT exercise provided a potent mechanical stimulus to restore the normal growth expansion of the cortical shell in both the HU+SRT (7.88%) and HU+SRT/ALEN (5.70%) groups, comparable to the gain in cortical area of the CC (5.61%) group. As expected, there was essentially no change in cortical bone area in the HU group. ALEN administration during HU did not rescue gains in cortical bone area and actually resulted in a slight reduction (−2.23%, P = 0.079). The increase in cortical bone area was accompanied by increased polar CSMI in the CC (14%), HU+SRT (13%), and HU+SRT/ALEN (10%) groups. Polar CSMI was significantly reduced in the HU+ALEN group by −5%. A similar decrease (−3%) was noted in the HU group; however, this change was not statistically significant. An increase in cortical volumetric BMD (0.3 to 1.8%) was observed among the groups; however, this change was not statistically significant.

View this table:
Table 1.

SRT during disuse normalized cortical BMC, cortical area, and polar CSMI

ALEN blunts the cortical bone formation response to SRT during disuse.

Markers of osteoblast activity on the periosteal surface of the tibia middiaphysis were substantially lower in the HU group vs. those in the CC group. Those animals given ALEN during HU show the lowest BFR, compared with all other groups. The application of SRT during HU produced significantly higher BFR on the periosteal surface at the tibia diaphysis (Fig. 1). The HU+SRT group demonstrated an eightfold higher BFR compared with HU. Moreover, when SRT was prescribed alone or in combination with ALEN treatment during HU, periosteal MS/BS was significantly higher than CC (+30 and 18%), HU (+130 and 109%), and HU+ALEN (+10-fold greater) groups. Periosteal MAR was lower in the HU+SRT/ALEN compared with HU+SRT; however, MAR of the HU+SRT/ALEN group remained nearly twofold higher than that of CC and HU.

Fig. 1.

Effects of hindlimb unloading (HU), with or without alendronate (ALEN) treatment and/or simulated resistance training (SRT), on periosteal and endocortical surface dynamic histomorphometric analyses measured at the tibia diaphysis. A: mineralizing surface [%MS/bone surface (BS)]. B: mineral apposition rate (MAR). C: bone formation rate (BFR). Vertical dashed line indicates separation of cage control (CC) from the experimental groups for preliminary ANOVA. Values are means ± SE. a,b,c,d HU groups not sharing the same letter for respective surface measures are significantly different from each other (P < 0.05). †Significantly different vs. CC (P < 0.05).

Mechanical loading paradigms typically do not show robust bone formation effects at the endocortical surface. However, when SRT was prescribed alone or in combination with ALEN treatment during HU, endocortical MS/BS was significantly higher than CC (+270 and 420%), HU (+280 and 440%), and HU+ALEN (+28- and 40-fold greater) groups (Fig. 1). SRT alone or in combination with ALEN administration during HU show significantly higher MAR and BFR at the endocortical surface. As expected, the BFR in the HU+ALEN was maximally suppressed and significantly lower than all other groups. The endocortical-BFR was significantly higher in the HU+SRT/ALEN group compared with CC (+7-fold), HU (+7-fold), and HU+ALEN (+167-fold); however, it was significantly less than the HU+SRT group (−56%). ALEN did dampen the robust endocortical bone formation (MS/BS, MAR, and BFR) to SRT during HU; however, HU+SRT/ALEN was not significantly less than the HU+SRT group. The impaired bone formation by ALEN administration can be seen visually by narrower widths of calcein labeling on the periosteal and endocortical surfaces when comparing the HU+SRT/ALEN with HU+SRT groups (Fig. 2).

Fig. 2.

Visual depiction (×100 magnification) of calcein labeling of the periosteal and endocortical surfaces of cortical bone at the tibia diaphysis. Note the extensive fluorochrome labeling in CC, HU with SRT (HU+SRT), and HU+SRT and ALEN (HU+SRT/ALEN) and large interlabel width (HU+SRT and HU+SRT/ALEN).

The proportion of sclerostin-positive cortical osteocytes is suppressed by muscle contractions, but not ALEN treatment.

Complete unloading of the hindlimb in the HU animals resulted in a significantly higher number of sclerostin-positive osteocytes vs. that observed in weight-bearing CC animals. ALEN treatment of HU animals had no effect on the proportion of sclerostin-positive osteocytes. By contrast, both groups subjected to SRT during unloading exhibited much lower prevalence of sclerostin-positive osteocytes, close to CC rat values (Fig. 3).

Fig. 3.

Effects of HU with or without ALEN treatment and/or SRT on cortical bone osteocyte expression of sclerostin measured at the tibia diaphysis (×100 magnification). A: HU shows significantly greater number of sclerostin-positive osteocytes, where SRT (HU+SRT) tends to suppress sclerostin-positive (SOST+) osteocytes, with no untoward effects of ALEN administration. Data are expressed as a percentage of sclerostin-positive osteocytes [(SOST+ osteocytes/total osteocytes) × 100]. a,b HU groups not sharing the same letter for respective surface measures are significantly different from each other (P < 0.05). †Significantly different vs. CC (P < 0.05). B: note higher intensity staining in the HU group of the tibial cross section. M, marrow space; P, periosteal surface.


Our major finding in these studies was that ALEN treatment blunted the BFR response to SRT during rodent HU. Our data did not support our hypothesis that the combination of ALEN and resistance exercise therapies would better ameliorate deleterious changes in cortical bone than with ALEN or SRT administration alone.

Previously, we demonstrated that a similar simulated resistive exercise training paradigm with isometric and eccentric components (both at 100% peak isometric strength) effectively mitigates losses in muscle strength and provides a potent stimulus to bone during prolonged disuse (32). In the present study, a similar resistance exercise paradigm was utilized; however, the force of contractions, isometric and eccentric components, was titrated down to 75% of peak isometric strength. Even with this 25% reduction in muscle contraction intensity, this exercise modality elicited a robust bone formation response and restoration of normal bone accrual during a period of disuse. The gains in midshaft cortical BMC and in cortical area with the reduced intensity protocol were only slightly smaller than those observed with the previous higher intensity regimen; the gain in polar CSMI, a key predictor of bending strength, was identical in both protocols. Furthermore, the prescription of SRT at 100% P0 or 75% P0 during HU elicits a similar periosteal and endocortical bone forming response (as measured by MS/BS, MAR, and BFR) of cortical bone. Interestingly, visual inspection of the data appear to show more single and double labeling at the posterior-lateral and anterior-medial surfaces than other cortical bone surfaces of the tibia. Taken together, these data suggest that a 25% reduction in SRT intensity may not elicit a proportional reduction in the positive responses observed in bone geometry and bone formation in the context of disuse. Using a training intensity commonly used by humans performing resistance training programs, the present training regimen effectively prevented the suppression of cortical bone gain observed with unloading.

ALEN treatment given during HU produced significant further reductions in actively mineralizing surfaces (%MS/BS) and MAR beyond that observed with unloading alone, suggesting that ALEN treatment reduces both the number of remodeling sites and vigor of the individual osteoblast units (2). When given to animals also receiving infrequent bouts of SRT during HU, ALEN attenuated the cortical bone formation response primarily via a blunting of MAR without any significant impact on %MS/BS. Interestingly, when another common bisphosphonate, risedronate, is administered along with mechanical loading of mouse tibiae, no negative effects are observed in cortical geometric bone gains (31), consistent with the present data that ALEN and mechanical loading can exert independent effects. It is highly likely that the decreased bone formation observed at the periosteal surfaces with ALEN treatment at 28 days of HU would eventually impact on cross-sectional geometry, and/or local BMC were the period of disuse prolonged, resulting in changes in bone density and mechanical properties that could not be detected with the present study's limited time frame.

In general, cancellous bone is more responsive to disuse than is cortical bone, exhibiting more rapid declines in bone formation activity and in bone volume (4). Even so, we observed that cortical bone formation was significantly impaired on the periosteal surface during 28 days of HU, contributing to an inhibition of the slow midshaft cortical bone growth observed in these male rats. This adaptation of cortical bone is consistent with previous studies of space-flown and HU rats that document a reduction in radial bone growth and bone strength (20, 21). In the present study, cortical BFR in unloaded animals was further suppressed by ALEN administration, which contrasts with our laboratory's previous observations of no additional effect of ALEN administration on disuse-induced reductions on cancellous BFR in the proximal tibia (33). The major impact of disuse on osteoblast activity in the cancellous compartment may have occurred early in the 28-day unloading period, whereas the slower response of cortical bone to disuse enabled the present study to detect ALEN effects on bone formation within the same period. However, when ALEN was given to HU animals experiencing intermittent mechanical loading (HU+SRT/ALEN), the resulting robust increases in bone formation were impaired at both cortical and cancellous (33) bone sites, as measured over the last 9 days of the experiment.

Bisphosphonates may directly affect osteoblast precursors and osteoblast activity. For example, Gasser et al. (11) demonstrates that ALEN administration before PTH administration reduces MAR, possibly by blunting the bone-lining cell transition to osteoblast. In addition, risedronate treatment causes osteoblasts to adopt a flatter cell morphology, a reminiscent cell shape of bone-lining cells, suggesting that risedronate retards normal osteoblast activity (22). Therefore, the present study demonstration of ALEN blunting of BFR on the periosteal surface is consistent with previous reports that ALEN may have direct effect on osteoblast precursor cells and activity.

There are data to suggest that exercise, in combination with anti-resorptive compounds, provides additive, and perhaps synergistic, benefits to bone mass and strength. More data are available testing bisphosphonates' impact on bone loss subsequent to estrogen deficiency and less documenting effects of exercise during disuse. For example, etidronate treatment and treadmill exercise show significantly higher BMD at the midshaft femur in an OVX rat bone loss model (34). Similarly, OVX rats treated with ALEN and treadmill exercise therapy show higher midshaft femur BMC and cortical thickness (10). Therefore, at some bone sites, combined bisphosphonate and exercise therapy show added and perhaps synergistic effects at cortical bone sites. However, zoledronic acid and treadmill exercise therapy of OVX rats show no additive effect on cortical BMC or BMD (18). In addition, when OVX rats are exposed to both external mechanical loading of the ulna and ALEN treatment, the bone formation response to mechanical loading is not impaired (7). Conversely, ALEN given to rapidly growing young rats during HU inhibits cortical BFR and MAR at the tibia diaphysis by ∼15% (3). A more mature (300 g) rat treated with ALEN during HU, but without exercise, shows higher cortical BMC (tibia and femur) compared with vehicle-treated animals (2). Collectively, these data suggest that certain bisphosphonate treatments, when given to an ambulatory full-weight-bearing rat, may prevent OVX-induced bone loss. However, in the context of reduced weight bearing, bisphosphonates may attenuate the bone formation response to mechanical loading.

The Wnt pathway coordinates communication between mechanosensing osteocytes (via secreted sclerostin) and bone-forming osteoblasts by the binding of sclerostin to lipoprotein receptor-related protein (Lrp) 4/5/6 (6, 9, 39). Loss-of-function mutation of the Lrp5 gene results in decreases in bone mass and osteoblast proliferation (13). Sclerostin disrupts the interaction of Wnt proteins with Lrp4/5/6 receptors, effectively reducing Wnt signal transduction and resulting osteogenesis (25). Interestingly, mechanical loading in vivo downregulates sclerostin-positive osteocytes, the gene SOST, and upregulates osteogenic gene expression (26). Moreover, when sclerostin-null mice are subjected to simulated microgravity, the Wnt pathway is unaffected, and bone loss does not occur (19). Therefore, the robust increase of sclerostin-positive osteocytes during disuse observed in the present study is consistent with previous work demonstrating higher Sost transcript levels at day 3 of HU and reduced numbers of sclerostin-positive osteocytes after 2 days of ulnar loading (without muscle contraction) (26).

In the present investigation, ALEN treatment did not significantly affect the number of sclerostin-positive osteocytes. Although ALEN is effective in preventing osteocyte apoptosis (23, 24, 33), it does not appear to alter the mechanosensing function of osteocytes to load or the proportion of sclerostin-positive osteocytes. This finding suggests that the modulation of osteoclast activity and apoptosis by ALEN may be independent of sclerostin control.

In conclusion, the present study demonstrates that moderate-intensity, low-volume loading of bone by active muscle contraction during a period of imposed disuse reduces sclerostin-positive osteocytes, coincident with increased bone formation activity to levels higher than those observed in weight-bearing control animals. These data extend previous work utilizing the SRT paradigm (low-volume/high-intensity exercise via physiological muscle contraction), which mitigates cancellous bone loss and prevents suppression of middiaphyseal periosteal apposition during unloading (1). Future studies should investigate the intracellular signaling pathway responsible for the dampening of the bone formation response to SRT by ALEN given during HU. In addition, the inclusion of a weight-bearing recovery component to the present study may help elucidate if bisphosphonate treatment impairs long-term bone formation capability.


These studies were funded through the National Aeronautics and Space Administration Cooperative Agreement NCC 9-58 with the National Space Biomedical Research Institute (S. A. Bloomfield). B. R. Macias and J. M. Swift were supported by National Space Biomedical Research Institute Graduate Training Fellowship NSBRI-RFP-05-02. B. R. Macias is a National Science Foundation Graduate Research Fellow.


The views, opinions, and findings contained herein are those of the author J. M. Swift and do not necessarily reflect official policy or positions of the Department of the Navy, Department of Defense, nor the United States Government.


No conflicts of interest, financial or otherwise, are declared by the author(s).


B.R.M., J.M.S., M.I.N., H.A.H., and S.A.B. conception and design of research; B.R.M., J.M.S., M.I.N., H.A.H., S.D.B., and S.A.B. performed experiments; B.R.M., J.M.S., M.I.N., H.A.H., S.D.B., and S.A.B. analyzed data; B.R.M., J.M.S., M.I.N., H.A.H., S.D.B., and S.A.B. interpreted results of experiments; B.R.M., J.M.S., M.I.N., H.A.H., and S.A.B. prepared figures; B.R.M., J.M.S., M.I.N., H.A.H., and S.A.B. drafted manuscript; B.R.M., J.M.S., M.I.N., H.A.H., S.D.B., and S.A.B. edited and revised manuscript; B.R.M., J.M.S., M.I.N., H.A.H., S.D.B., and S.A.B. approved final version of manuscript.


The authors gratefully acknowledge Janet Stallone for assistance with animal care, and Drs. Gordon Warren (Georgia State University) and Ken Baldwin (University of California, Irvine) for assistance with the simulated resistive exercise programming and procedures. Alendronate was generously provided by Merck Pharmaceuticals.

Present address of J. M. Swift: Radiation Combined Injury Program, Armed Forces Radiobiology Research Institute, Uniformed Services University of the Health Sciences, Bethesda, MD 20889-5603.


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