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BASIC SCIENCES: Contrasting Perspectives in Exercise Science and Sports Medicine

Partial Weight Bearing Does Not Prevent Musculoskeletal Losses Associated with Disuse


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Medicine & Science in Sports & Exercise: November 2013 - Volume 45 - Issue 11 - p 2052-2060
doi: 10.1249/MSS.0b013e318299c614
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Significant decreases in mechanical loading due to clinical conditions associated with muscle paralysis or even prolonged bed rest are associated with bone loss (21). The unique weightless environment of low-earth orbit induces surprisingly rapid loss of muscle and bone mass even in robust, healthy individuals. Advances in noninvasive imaging of bone over the last 30 yr have confirmed that crew members lose on average 1% of baseline bone mineral density (BMD) per month in the spine and lower limb (19,20,39). This magnitude of BMD loss and attendant changes in bone geometry produces declines in estimated bone strength equivalent to the median lifetime loss in stance strength for Caucasian women, as modeled by patient-specific finite element analysis of crew member data (18). The effect of this dramatic loss in both bone mass and estimated strength during flight is heightened by data demonstrating the likelihood that it takes years of normal weight-bearing activity to recover (31). In addition to skeletal losses, extended duration in microgravity results in significant reductions in skeletal muscle volume and plantarflexor muscle strength (36). Moreover, it is unknown if ambulation at lunar (16%) or Mars (38%) gravitational levels during exploration class missions will dampen the accelerated bone loss observed during microgravity. In addition, the long-duration exploration class missions of near-Earth objects at less than one gravity (1G), combined with the long-duration round-trip travel may render complete recovery of the musculoskeletal system even more unlikely upon return to Earth.

As NASA develops plans for returning humans to the moon or an asteroid with the eventual goal of reaching Mars, it becomes imperative to understand whether graded weight bearing in these extreme environments can protect against the deleterious musculoskeletal effects of the prolonged disuse of spaceflight. Such information has significant relevance back here on Earth. Understanding how partial reductions in gravitational loading affect muscle and bone integrity in populations with asymmetric paralysis (i.e., hemiparetic stroke patients), during physical rehabilitation after trauma (i.e., spinal cord or head injury), or with chronic conditions (i.e., cerebral palsy) is important to functional recovery (16). The nature of bone loss in all of these populations experiencing extensive bed rest mimics that of prolonged microgravity exposure, whereby cancellous bone is predominantly affected and reductions in femoral neck (FN), lumbar spine, and lower body BMD correspond with decreased bone volume fraction (%BV/TV) and trabecular thickness (Tb.Th) (8,12,13,30,35).

Hindlimb unloading (HU) of rodents via tail suspension has been shown to effectively mimic the musculoskeletal effects of weightlessness associated with spaceflight (25,26). HU reduces bone mass and deleteriously alters trabecular microarchitecture and biomechanical properties because of the immediate enhancement of osteoclast activity (bone resorption) and later depressions in osteoblast activity (bone formation) (2,3,5). In addition, HU results in significant skeletal muscle atrophy and reduced functional properties that may remain depressed for some time after the resumption of normal gravitational weight bearing (10,11,34,42). Recently, a modified tail suspension mouse model has been developed for ground-based studies, allowing the precise gradations of weight bearing to all four limbs (41). Ambulating in simulated Martian gravity (∼38% Earth gravity) causes significant reductions in cancellous bone mass and diaphyseal cortical thickness (Ct.Th) relative to full weight-bearing controls (41). This new model effectively and reproducibly reduces ground reaction forces on all four murine limbs; however, it is not known how the partial weight bearing of Martian gravity compares with microgravity in terms of maintenance of musculoskeletal tissue integrity. Specifically, it is unknown whether chronically reduced weight bearing at gravitational loading at or below 38% body mass would result in similar musculoskeletal effects as does simulated microgravity (0G) (i.e., Do musculoskeletal effects of 0G = lunar gravity = Martian gravity?).

The aim of this experiment was to determine whether partial weight-bearing activity would provide some protection against the deleterious effects of simulated microgravity muscle mass and bone quantitative/qualitative measures. We hypothesized that mice exposed to one-sixth (G/6) and one-third (G/3) gravitational loading, roughly approximating lunar and Martian gravity, would experience reductions in skeletal muscle mass and metaphyseal bone mass and altered bone microarchitecture as compared with ambulatory control animals. Furthermore, we hypothesized that the magnitude of these changes in muscle and bone parameters would be less in G/6 and G/3 mice versus those exposed to 0G.



This study was approved by Texas A&M University’s Institutional Animal Care and Use Committee. Four-month-old female BALB/cByJ mice (Jackson Laboratories, Bar Harbor, Maine) were rank ordered by body weight to one of four groups: 1) normal ambulatory cage activity or one gravity group (1G, n = 11), 2) hindlimb unloaded or 0 gravity group to simulate microgravity (0G, n = 11), 3) one-sixth weight-bearing activity group to simulate lunar gravity (G/6, n = 11), and 4) one-third weight-bearing activity group to simulate approximate Martian gravity (G/3, n = 13). The mice were group housed and acclimated for 1 wk with standard conditions of temperature (23°C ± 2°C) and light-controlled environment (12-h light–12-h dark cycle). Thereafter, all mice were single housed for 2 wk before the initiation of the experiment in cages with removable polypropylene perforated floors. Mice were fed standard rodent chow, Harlan Teklad 8604 (composed of 24% protein, 4.7% fat, 40% carbohydrate, 7.4% ash, 4% fiber, 1.4% calcium, 1.1% phosphorus, and vitamins) and provided water ad libitum. All animals were monitored for health and body weight was recorded daily.

Four-month-old BALB/cByJ female mice were used in this investigation for two main reasons. First, we sought to make direct comparisons to a previous investigation that developed and validated the murine partial weight-bearing model used in our study (41). In addition, peak bone volume in BALB/cByJ female mice occurs at this age, allowing for extrapolation of data from these mice to skeletally mature human astronauts (43).

On day 21, animals were anesthetized (ketamine 100 mg·kg−1 + xylazine 10 mg·kg−1) before being euthanized; all suspended animals (0G, G/6, G/3) were anesthetized before removal from suspension apparatus to prevent any additional weight bearing by the limbs. Animals were euthanized by decapitation, and both femora and tibiae were harvested, stripped of soft tissue, and stored in either 70% ethanol at 4°C (for micro–computed tomography [μCT] and histomorphometric analyses) or wrapped in gauze soaked with phosphate-buffered saline and immediately and stored at −30°C (for mechanical testing). The ankle plantarflexor muscles (gastrocnemius, plantaris, and soleus) of the left and right hindlimbs were collected and weighed.

Hindlimb unloading

Simulated microgravity (0G), via HU, was achieved by tail suspension as previously described (26), which permits movement with the forelimbs while fully unloading the hindquarters. The height of the animal’s hindquarters was adjusted to prevent any contact of the hindlimbs with the cage floor, resulting in an approximately 30° head-down tilt. Suspended mice were single housed in custom-built cages (13 × 13 × 13 inches) with clear polycarbonate walls and removable polypropylene perforated floors. A stainless steel rod spanned the top of the cage to support the animal and the suspension system. The same custom-built cages were used for both 0G and partial weight-bearing activity groups.

Partial weight-bearing activity

Weight titration to simulate the moon’s partial gravity (G/6) and Martian gravity (G/3) was achieved using a previous model (see reference #37 for a picture representation of the model) (41). This model unloads the forelimbs and hindlimbs equally, so the animals remained horizontal (no head-down tilt). To effect this, support was provided to the forelimbs through a moleskin jacket around the shoulders and to the hindlimbs through a tape wrap at the base of the tail. The daily weighing of suspended animals was accomplished by using electronic scale (Ohaus Corp., Pine Brook, NJ), customized weight, and titration columns. Full body masses were first obtained by placing the weight column on the scale and briefly hanging the animal over the scale, so as to avoid full weight bearing. Then, the titrated weight was calculated and recorded. The titration column, which was built with the same height (floor to center of cross rod) as the suspension cage, was then placed around the scale, and each animal’s weight was titrated using its own suspension mechanism. Adjustments to spring tension were made as necessary to accommodate changes in body mass. Animals in the G/6 and G/3 groups had their weight bearing titrated back to approximately one-sixth and one-third, respectively, of their daily body mass. Mice in the G/3 and G/6 groups were observed and weighed twice daily (early morning and late afternoon) to determine whether they were ambulating equally on all four limbs (i.e., not retracting any of their limbs resulting in unequal loading patterns) and the degree to which the desired gravitational loading was still. Any mice that did not remain in their partial weight-bearing harness (or were found out of their harness) or retracted their limbs during an observation period were removed from the study. The coefficient of variation for daily titration efficiency in G/6 and G/3 mice was 0.7% and 1.2%, respectively.

Peripheral quantitative computed tomography

At baseline (day 0) and on day 21, mice were anesthetized (isoflurane, ∼2.5% mixed with oxygen) and scanned using an XCT Research-M device (Stratec Corp., Norland, Fort Atkinson, WI). Machine calibration was performed using a hydroxyapatite cone phantom. Scans were performed on the left tibia at a scan speed of 2.5 mm·s−1 with a voxel size of 100 μm and a scanning beam thickness of 500 μm. Three slices, 1 mm apart, were scanned at the proximal metaphysis of the tibia, and two slices were scanned at the middiaphysis of each bone (50% of the total bone length ± 0.50 mm). Standardized analyses of proximal metaphysis bone (outer threshold of 400 mg·cm−3, inner threshold of 750 mg·cm−3) or diaphyseal bone (threshold of 750 mg·cm−3) were applied to each section. The total volumetric BMD (vBMD) for the proximal tibia metaphysis was calculated as the average of the three slices. At the diaphysis, the polar moment of inertia (J) was determined by averaging values for the two slices. The cross-sectional moment of inertia was estimated as half of the polar moment of inertia.

Micro–computed tomography

μCT (SkyScan 1172; SkyScan, Kontich, Belgium) was used to quantify two- and three-dimensional microarchitecture. Ex vivo scans of right distal femora were conducted using an x-ray source set at 60 kV over an angular range of 180° with rotational steps of 0.70°. Projection images were attained at 6-μm resolution and then reconstructed and analyzed using manufacturer-provided software (NRecon and CTAn; SkyScan). For trabecular bone analyses, a 1-mm segment of the distal metaphysis secondary spongiosa was defined and the trabecular region within the segment was manually traced. A threshold was applied to separate bone from soft tissue (range, 100–255), and then the specimen was analyzed in three dimensions for bone volume/trabecular volume (BV/TV), trabecular number (Tb.N), Tb.Th, and tissue mineral density (TMD). Ct.Th was also assessed in the metaphyseal region. For cortical bone parameters, a single slice located 3 mm proximal to the most distal end of the metaphyseal segment was analyzed to determine cortical bone area (Ct.Ar), polar moment of inertia, Ct.Th, and TMD. All nomenclature follows currently accepted guidelines for the micro–computed tomographic evaluation of bone (6).

Histomorphometry analysis

Fluorochrome labeling was performed by intraperitoneal injection of calcein (15 mg·kg−1 body weight; Sigma Chemical, St. Louis, MO) 7 and 2 d before euthanasia. Undemineralized right distal femora were subjected to serial dehydration and embedded in methylmethacrylate. Serial frontal sections were cut 8 μm thick and left unstained for fluorochrome label measurements of cancellous bone (distal femur). Histomorphometry analyses were performed using the OsteoMeasure Analysis System, Version 1.3 (OsteoMetrics, Atlanta, GA). A defined region of interest in distal femur metaphysis was established approximately 0.5 mm from the growth plate and within the endocortical edges encompassing 4.5 mm2 at ×400 magnification. Mineral apposition rate (MAR; μm·d−1), mineralizing surface/bone surface (MS/BS; %), and bone formation rate (BFR; mm3·mm−2·yr−1) were derived from two sections of each specimen using standard equations. For cortical specimens, serial sections (120–150 μm) of tibia diaphysis were cut using an Isomet diamond wafer low-speed saw (Buehler, Lake Bluff, IL) approximately 1 mm proximal from the tibiofibular junction. Similar to cancellous tissue, MAR, MS/BS, and BFR were calculated for both endocortical and periosteal surfaces from two sections of each specimen. All nomenclature for histomorphometry follows standard usage (28).

Mechanical testing

Bones used for mechanical testing were thawed to room temperature and tested on an Instron 3345 machine (Norwood, MA; 100 N load cell; Bluehill version 2.14.582 for data acquisition and analysis). The FN region was tested to failure as previously described (33). In brief, FN testing was performed by placing the proximal half of the femur upright with the diaphysis portion of the bone supported in a metal plate. A quasistatic load was applied to the femoral head in displacement control at 1.27 mm·min−1. The maximum value of the load during the test was designated the ultimate load (N) and recorded as the main outcome variable.

The diaphysis of the tibia was tested to failure by three-point bending as previously described (15). Each tibia was placed lateral side down on metal pin supports with a span S of 10 mm. Quasistatic loading was applied at 2.54 mm·min−1 through a metal pin contacting the medial surface at middiaphysis (50% of length) until fracture occurred. Load and displacement were recorded and analyzed using Matlab (The Mathworks, Inc., Natick, MA). The ultimate load (N) was the maximum load during the test, and the stiffness k (N·mm−1) was determined as the slope of the linear portion of the load versus displacement curve in the elastic region. Material properties, ultimate stress and elastic modulus, were calculated using standard equations (37).

Statistical analysis

All data are presented as group mean ± SD and evaluated using the Statistical Package for the Social Sciences (version 15; SPSS Inc., Chicago, IL). All end point data (i.e., μCT, muscle masses, histomorphometry, mechanical testing) were analyzed using ANOVA to compare group differences between experimental groups, and when appropriate, Fisher’s least significant difference post hoc analyses were performed for pairwise comparisons. In vivo peripheral quantitative computed tomography (pQCT) data (total vBMD) were presented as a change value (post − pre) and analyzed using ANOVA. ANOVA tests were used to compare the before and after values of pQCT data to determine whether the change values presented in graph represented a significant difference between day 0 and day 21 values. For all data, statistical significance was accepted at P ≤ 0.05.


Partial weight-bearing activity attenuates 0G-associated reductions in body and muscle mass

Simulated 0G resulted in significant reductions in body mass by day 14 (−9% vs day 0); body mass reductions were sustained through day 21 (−7.6% vs day 0; Fig. 1A). Partial weight bearing did not result in significant declines in total body mass during the 21 d but did impair the weight gain observed in ambulatory controls (1G).

Effects of altered gravitational loading on total body mass (A) and plantarflexor muscle masses (B–E). B, Gastrocnemius muscle mass. C, Plantaris muscle mass. D, Soleus muscle mass. E, Total plantarflexor muscle mass. The values are given as group mean ± SD. aSignificantly different versus 1G (P < 0.05). bSignificantly different versus 0G (P < 0.05). cSignificantly different versus G/3 (P < 0.05). dSignificantly different versus all other groups (P < 0.05). *Significantly different versus day 0.

Gastrocnemius, plantaris, and soleus muscle masses (−18% to −46% vs 1G controls; Figs. 1B–1D) were reduced after 0G. These declines as well as total ankle plantarflexor muscle mass were attenuated in the G/3 and G/6 groups, but in a graded fashion proportional to the imposed gravitational loading.

Partial weight bearing attenuates 0G-associated reductions in proximal tibia total vBMD

Simulated 0G and partial weight bearing (G/3, G/6) resulted in significant reductions in proximal tibia total vBMD after 21 d (Fig. 2). Total vBMD in the 0G group decreased by 20% (day 21 vs day 0). However, total vBMD declined significantly less (−14%) in the partial weight-bearing G/3 and G/6 groups as compared with 0G mice.

Effects of altered gravitational loading on changes in total vBMD of the proximal tibia metaphysis as measured by in vivo pQCT scans taken on days 0 and 21. The values are given as group mean ± SD. aSignificantly different versus 1G (P < 0.05). bSignificantly different versus 0G (P < 0.05). dSignificantly different versus all other groups (P < 0.05). *Significantly different versus prevalue (day 0).

G/6 partial weight bearing is not sufficient to mitigate deleterious 0G-induced changes in cancellous microarchitecture and cortical bone geometry

Distal femur BV/TV was significantly lower in the 0G (−13%), G/3 (−9%), and G/6 (−11%) mice versus 1G mice at day 21 (Table 1), with no significant differences among mean values for the three reduced weight-bearing groups. Tb.Th was significantly lower in all reduced weight-bearing groups (0G [−14%], G/3 [−10%], and G/6 [−12%]), whereas Tb.N was significantly lower only in G/6 mice as compared with 1G. Distal femur Ct.Th was significantly lower only in 0G (−10%) and G/6 (−9%) compared with 1G mice. Similarly, distal femur TMD was significantly lower in the 0G and G/6 groups (−3% and −2%, respectively), but not in the G/3 group as compared with 1G mice.

Effects of reduced gravitational loading on bone architecture and geometry.

Simulated 0G and G/3 partial weight-bearing activity did not produce any significant deleterious effects on diaphyseal cortical bone (Table 1). Reductions in Ct.Th and Ct.Ar of the diaphysis did reach statistical significance after 21 d in the G/6 group (−5% and −6%, respectively, vs 1G controls).

Partial weight-bearing activity at any level suppresses bone formation

The 0G group had significantly lower distal femur cancellous MS/BS (−47%) and MAR (−21%), resulting in a 57% lower BFR versus 1G controls (Figs. 3A–3C). Partial weight-bearing activity resulted in similar reductions in cancellous BFR (−46% and −49% in G/3 and G/6 mice, respectively) compared with 1G controls, although this was attributed to lower % MS/BS, whereas MAR was not significantly reduced.

Effects of altered gravitational loading on cancellous (A–C) and cortical (D–F) bone dynamic histomorphometry analyses measured at the distal femur and femur middiaphysis. Distal femur (A–C). A, Mineralizing surface (%MS/BS). B, Mineral apposition rate (MAR). C, Bone formation rate (BFR). Femur middiaphysis (D–F). D, Mineralizing surface (%MS/BS). E, Mineral apposition rate (MAR). F, Bone formation rate (BFR). The values are given as group mean ± SD. aSignificantly different versus 1G (P < 0.05).

Cortical bone formation at the tibia middiaphysis was significantly reduced during all levels of unloading on both periosteal and endocortical surfaces of midshaft femur as compared with 1G controls (1G; Figs. 3D–3F). Partial weight-bearing mice demonstrated similar deficits in endocortical BFR (−64% to −76%) and periosteal BFR (−76% to −85%) versus 1G mice; neither group’s mean values were significantly different from the 0G group. Changes on both surfaces were driven by altered %MS/BS, with no effect of partial weight-bearing or simulated 0G on MAR.

Partial weight-bearing activity at 1/3, but not at 1/6, body mass prevents 0G-induced reductions in mechanical properties

Simulated 0G and G/6 partial weight-bearing activity resulted in significantly lower FN ultimate load (−19% and −14% vs 1G controls, respectively; Table 2), whereas G/3 weight-bearing mice exhibited a milder (−8%) and nonsignificant reduction in FN strength.

Effects of altered gravitational loading on mechanical properties of mixed bone (cancellous and cortical) at the FN and of cortical bone at the tibia middiaphysis.

Three-point bending to failure of middiaphyseal tibia did not reveal any changes in structural or material mechanical properties with 0G and with G/3 weight bearing (Table 2). In contrast, tibial stiffness and ultimate load were lower in the G/6 group (−20%, vs 1G controls), although the corresponding material properties, modulus and ultimate stress, were not statistically different compared with 1G controls.


Simulated microgravity resulted in an 8% reduction in body mass by day 21, whereas G/3 and G/6 mice experienced only half that amount (Fig. 1A). In our study, the consistent body mass of the G/6 and G/3 groups, albeit lower than the 1G group, suggests that this partial gravity treatment was well tolerated. The lower body weight in the partial weight bearing and 0G groups could be due to both a lack of appetite with less activity. Reductions in soleus, gastrocnemius, and total ankle plantarflexor muscle masses evidenced during simulated 0G were mitigated by partial weight-bearing activity at 16% (G/6) and 33% (G/3) (Figs. 1B–1E), in agreement with our hypothesis. These findings are consistent with a previous investigation, which reported 23% lower gastrocnemius muscle mass in mice subjected to 38% gravitational weight bearing (i.e., Martian gravity) as compared with controls (41). In addition, previous reports involving simulated microgravity in this strain of mouse demonstrated similar reductions in soleus mass (14,24).

Our data demonstrate that partial weight-bearing activity as high as 33% of body mass does not inhibit or mitigate most of the deleterious effects of full unloading on bone, and the effects are bone site specific. Similar to 0G, G/6 mice demonstrated lower distal femur BV/TV and deleterious effects on microarchitecture, reduced FN strength, and lower cortical and cancellous bone formation compared with 1G controls. Surprisingly, one-sixth partial weight-bearing activity seemed to be more detrimental to cortical bone than full unloading (0G) in some cases. Mice maintained at G/6 had lower Ct.Ar, Ct.Th, and biomechanical properties at the femur middiaphyis (Table 2) versus 1G mice, whereas 0G mice did not experience reductions in any of these parameters. Contrary to these data at G/6, there were no deleterious effects of 33% partial weight-bearing activity on cortical bone geometry or strength. However, G/3 mice did experience lowered endocortical and periosteal bone formation, similar to that of both G/6 and 0G mice, which could result in altered properties with longer duration unloading.

An additional objective of this study was to determine whether both skeletal compartments (cortical and cancellous) were negatively affected by partial weight bearing, or whether cancellous bone is preferentially lost as observed with simulated 0G. Consistent with data from astronauts (19), full unloading targets cancellous regions in mice (17,23,32). BALB/c mice, the strain used in these experiments, demonstrate high sensitivity to mechanical disuse in the metaphyseal trabecular bone region (17), which was confirmed in our study. Alterations in trabecular bone volume in 0G mice were confirmed at 21 d, resulting in a reduction in bone volume (−13% vs group control) and deleterious alterations to microarchitectural parameters (Tb.Th. −14%, Tb.N. −13% vs 1G; Table 1). However, cortical and cancellous bone compartments were not affected to a similar degree with partial weight-bearing activity. The effect of partial weight-bearing activity on cortical bone near the femur middiaphysis (Ct.Ar −3% to −6%, Ct.Th −2% to −5% vs 1G; Table 1) was significantly less than that on cancellous bone (BV/TV −20% to −26% vs 1G; Table 1). The greater metabolic activity of the metaphyseal region of tibiae and femora, primarily composed of cancellous bone, likely directed greater percentage of bone loss in this region as compared with cortical bone in the diaphysis during partial weight-bearing activity and simulated 0G. Most importantly, data from this investigation demonstrate that partial weight-bearing activity at one-sixth and one-third of normal gravitational loading does not diminish 0G-induced bone loss in the cancellous compartment.

We observed a site-specific response to mechanical disuse. Previously published data demonstrate a twofold greater loss of cortical bone loss in the femur metaphysis than in diaphysis with simulated 0G (17). Although one-sixth partial weight bearing demonstrated similar reductions in Ct.Th at the distal femur metaphysis as unloaded animals (−9% to −10% vs 1G), 33% gravitational loading spared these declines (Table 1). A similar pattern emerged at the cortitpinscal diaphysis. These data demonstrate that, in contrast to cancellous bone, the cortical compartment (at least in metaphyseal compartments) seems to demonstrate an attenuated response with partial weight-bearing activity equivalent to one-third body mass.

Wagner et al. (41) developed and validated the model used in our study and tested the efficacy of Martian gravity on musculoskeletal parameters. Our cancellous microarchitecture data of the partial weight-bearing groups herein are in general agreement with this previous study, which demonstrated that 38% weight bearing resulted in approximately 24% reduction of trabecular BV/TV compared with unloaded controls (41). However, Wagner et al. did not demonstrate lower cancellous bone formation with partial weight-bearing activity. This previous investigation may have lacked appropriate power (five mice in each group for histomorphometric analyses) to determine this effect. Most importantly, their study did not determine the relative efficacy of partial weight bearing on musculoskeletal system, as the investigation did not include a fully unloaded (0G) group.

Although one-sixth and one-third partial weight bearing attenuated reductions in muscle mass, they did not protect against reductions in either cortical or cancellous bone formation. However, there was no negative effect of partial weight bearing (G/6 or G/3) on mineral apposition (intensity of osteoblast activity), although the present study and numerous previous studies demonstrate reduced MAR after a period of simulated 0G (22,38). Lowered MAR seems to be transient with the major decrement during the first week (40). We measured MAR during the final week of the protocol, and at this later time point, MAR may have reached a new steady-state level. This provides one a potential explanation for a lack of difference between our experimental groups (i.e., G/3, G/6, and 0G). We find a similar suppressive effect of reduced weight bearing (G/3, G/6) and 0G on MS/BS. Therefore, our investigation did not reveal a protective effect of partial weight-bearing activity on 0G-induced reductions in endocortical or periosteal bone formation activity.

Unloading has recently been characterized as stimulating initial increases in osteocyte and osteoblast apoptosis, preceding diminished cancellous bone mass and strength, and followed thereby with increased osteoclast number (1,9). In addition, mechanical loading has been shown to attenuate increases in osteocyte apoptosis due to complete unloading (4,27). We speculate that partial gravitation weight bearing, at 33% or lower, is unable to inhibit early osteocyte apoptosis, thus allowing for later increases in osteoclast number and activity. Alternatively, similar to unloading, partial gravity may enhance sclerostin expression and down-regulate canonical Wnt signaling, leading to decreased bone mass and formation. Future mechanistically driven investigations are necessary to outline the effects of partial gravitational weight bearing on these signaling pathways so that targeted countermeasures targeting can be established.

Some limitations should be noted in the present study. First, we did not precisely mimic Martian gravity (38% of Earth). We approximated Martian gravity and used 33% gravitational weight bearing for this investigation, as we did not expect any significant differences in variables between 33% and 38% gravitational weight-bearing activity. Second, hindlimb suspension induces a head-down position that generates the cephalic fluid shift as occurs in humans during head-down bed rest and space flight (7,25,29), whereas the horizontal posture of the partial weight-bearing model does not produce a cephalic fluid shift. Therefore, those deleterious effects observed using the partial weight-bearing model are likely independent of cephalic fluid shift-associated changes. Mice using the partial weight-bearing harness did not lose as much body mass as hindlimb unloaded animals (Fig. 1A), indicative of less stress than the amount encountered during hindlimb suspension as previously suggested by Wagner et al. (41). Finally, although we did not monitor food intake in any of the treatment groups and cannot decisively determine whether suppressed appetite was a contributing factor to weight loss in partial weight-bearing or suspended mice, it is likely that food intake in G/3 and G/6 mice were suppressed during the initial days or week of the investigation as previously demonstrated in the initial study validating this model (41).

Although there are some limitations, there are several strengths to highlight in the present study. This study provides critical evidence by comparing partial weight bearing to the traditional hindlimb disuse model. The traditional hindlimb disuse model was developed by NASA in the 1970s and today is considered the gold standard for investigation of disuse effects on bone and muscle. Therefore, the present study provides important comparator data for future partial weight-bearing studies of different murine strains, ages, and sex. Moreover, the present study provides mechanical strength data at tibia middiaphysis and FN not previously measured during partial weight bearing. Our measurement of a diverse set of bone sites highlights the bone site–specific adaptations that occur during partial weight bearing. In addition, the present study provides measurements of both cortical and cancellous bone formation and makes comparisons between partial weight bearing and disuse groups. Understanding the bone sites most affected may help aid clinical rehabilitation programs for both astronauts and Earth-bound patients.

In conclusion, our study demonstrates the diverse response of musculoskeletal tissue to partial weight bearing and disuse. Although reduced gravitational weight-bearing activity does not protect lower limb bone or muscle tissue from atrophy, as little as one-sixth partial weight-bearing activity does mitigate some disuse-induced musculoskeletal alterations. However, weight-bearing activity at 33% of daily body mass demonstrates greater ability to attenuate, but not inhibit, reductions in cancellous bone microarchitecture and biomechanical properties associated with zero weight bearing. This evidence is critical to the future of sustained manned space exploration on terrestrial surfaces with gravitational loading below our Earth’s 1G. Our data suggest that if a minimum mechanical stimulus threshold is present in bone (and muscle) to maintain these tissues, the level of gravitational loading that is necessary to reach this point is greater than one-third of the body mass.

These studies were funded through the NASA Cooperative Agreement NCC 9-58 with the National Space Biomedical Research Institute (SAB). JMS and BRM were supported by a National Space Biomedical Research Institute Graduate Training Fellowship NSBRI-RFP-05-02. The μCT scanner used in this study was purchased by an equipment grant from the National Institutes of Health (grant no. S10-RR023710). The authors gratefully acknowledge Sarah Luna for assistance with animal care and Lynette Jackson for assistance with μCT data collection. Furthermore, the authors sincerely thank the invaluable assistance of Dr. Erika Wagner (MIT) with constructing the partial gravity model used.

The views, opinions, and findings contained herein are those of the authors and do not necessarily reflect official policy or positions of the Department of the Navy, the Department of Defense, or the U.S. government.

JMS, FL, MRA, HAH, and SAB designed and developed the research study. JMS, FL, ESG, and BRM completed all the animal work for the investigation. JMS, FL, ESG, BRM, JSK, YSF, MRA, HAH, and SAB assisted with the data collection, analysis, production of graphs and tables, manuscript preparation, and manuscript review.

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

The authors have no conflicts of interest.

The results of the present study do not constitute endorsement by the American College of Sports Medicine.


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