Bone 49 (2011) 858–866

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Bone j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b o n e

WISE-2005: Bed-rest induced changes in bone mineral density in women during 60 days simulated microgravity☆ Gisela Beller a,⁎, Daniel L. Belavý a, Lianwen Sun a, b, Gabriele Armbrecht a, Christian Alexandre c, Dieter Felsenberg a a b c

Charité Universitätsmedizin Berlin, Centre of Muscle and Bone Research, Berlin, Germany School of Biological Science and Medical Engineering, BeiHang University, China Inserm Unit 1059. University-Hospital, St-Etienne, France

a r t i c l e

i n f o

Article history: Received 5 April 2011 Revised 19 May 2011 Accepted 17 June 2011 Available online 24 June 2011 Edited by: Rene Rizzoli Keywords: Microgravity Spaceflight pQCT DXA Bone mineral density

a b s t r a c t To better understand the effects of prolonged bed-rest in women, 24 healthy women aged 25 to 40 years participated in 60-days of strict 6° head-down tilt bed-rest (WISE-2005). Subjects were assigned to either a control group (CON, n = 8) which performed no countermeasure, an exercise group (EXE, n = 8) undertaking a combination of resistive and endurance training or a nutrition group (NUT, n = 8), which received a high protein diet. Using peripheral quantitative computed tomography (pQCT) and dual X-ray absorptiometry (DXA), bone mineral density (BMD) changes at various sites, body-composition and lower-leg and forearm muscle cross-sectional area were measured up to 1-year after bed-rest. Bone loss was greatest at the distal tibia and proximal femur, though losses in trabecular density at the distal radius were also seen. Some of these bone losses remained statistically significant one-year after bed-rest. There was no statistically significant impediment of bone loss by either countermeasure in comparison to the control-group. The exercise countermeasure did, however, reduce muscle cross-sectional area and lean mass loss in the lower-limb and also resulted in a greater loss of fat mass whereas the nutrition countermeasure had no impact on these parameters. The findings suggest that regional differences in bone loss occur in women during prolonged bedrest with incomplete recovery of this loss one-year after bed-rest. The countermeasures as implemented were not optimal in preventing bone loss during bed-rest and further development is required. © 2011 Elsevier Inc. All rights reserved.

Introduction In clinical routine the aspects and causes of bone loss are complex. In postmenopausal osteoporosis, for example, there are typically a number of factors contributing to the loss of bone. As such, bone loss causes health care systems worldwide tremendous effort and monetary costs every year, as progressive osteoporosis frequently leads to reduced bone strength, which, when accompanied by falls, can lead to fractures, subsequent immobilisation and further musculoskeletal deconditioning. As part of this, one aspect that can contribute to bone loss is physical inactivity. Bone loss occurs with stroke [1,2], spinal cord injury[3,4], and

☆ Funding sources: WISE: sponsored by the European Space Agency (ESA), the National Aeronautics and Space Administration of the USA (NASA), the Canadian Space Agency, and the French “Centre National d'Etudes Spatiales” (CNES). ADOQ: funded by European Commission under Contract QLK-CT-2002-02363, Key Action n°6: “The ageing population and disabilities”, the Swiss government and ESA. DLR: participation of ADOQ group in WISE was funded by the German AeroSpace Center under Contract 50 WB 0522. ⁎ Corresponding author at: Centre of Muscle and Bone Research, Charité-Campus Benjamin Franklin, Hindenburgdamm 30, 12203 Berlin, Germany. Fax: + 49 30 793 5918. E-mail address: [email protected] (G. Beller). 8756-3282/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2011.06.021

prolonged bed-rest [5], all of which involve some form of inactivity or reduced function. In manned space research bone loss has been detected to be a serious problem. This bone loss can be difficult to prevent with exercise modalities [6–8] and is presumed to occur as a result of the reduced loading of bone associated with the microgravity environment. Simulated weightlessness, as induced in prolonged bed-rest studies [5,9], is a valuable tool to evaluate the deconditioning bone occurring in clinical medicine as well as in the manned spaceflight and to define countermeasures to prevent and/or rehabilitate such changes. Bed-rest studies have to date typically been performed with male subjects. There is of course no guarantee that countermeasures which may, or may not, work for male subjects would necessarily have the same effect on women. The common effects of bed-rest on bone, typically in male subjects, include an increase in bone resorption [10–13], no significant change [5,14,15] or marginal reduction [11,16–21] of bone formation, increased calcium excretion [5,11,13], and ultimately loss of bone mass and density, predominately from the load-bearing regions of the body [8,19,22]. The “Women International Space Simulation for Exploration” (WISE-2005) prolonged bed-rest study was initiated to improve our understanding of the adaptation of female physiology to spaceflight simulation (bed-rest) and to trial countermeasures against the expected deconditioning associated with this simulation in women.

G. Beller et al. / Bone 49 (2011) 858–866

In the WISE-2005 study, in addition to female subjects undergoing strict bed-rest, an exercise countermeasure comprising resistance training and aerobic exercise as well as a high protein and leucine nutrition countermeasure were trialled. As part of our involvement in the WISE-2005 study, we aimed to examine the changes in bone mass at the lower leg (tibia), forearm (radius), hip and lumbar spine and also the effects of the exercise and nutrition countermeasures against these changes. Materials and methods

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received an additional amount of protein and free leucine (0.6 g/kg body weight/day increase in protein intake compared to the other groups). The main aim of this countermeasure was to assess whether muscle loss can be prevented via this nutrition countermeasure, and effects on bone were not expected. Detailed description of exercise and nutritional countermeasures has been published elsewhere [23] (see also: www. spaceflight.esa.int/wise/, select “experiment protocols” and then “nutritional and exercise countermeasures”). Peripheral quantitative computed tomography (pQCT)

Bed-rest protocol and subjects The WISE-2005 study was supported by the European, French, Canadian, German and North American Space agencies (ESA, CNES, Canadian Space Agency, DLR, NASA) and was conducted at the Medes Institute for Space Medicine and Physiology at the Rangueil University Hospital in Toulouse, France (www.spaceflight.esa.int/wise/). Twenty four healthy female volunteers, 25 to 40 years old participated in this study (Table 1). They were matched according to pre-bed-rest aerobic fitness levels and then randomly allocated in 3 groups: 8 control subjects (CON) without countermeasure during bed-rest, 8 subjects with regular muscular exercise (EXE) that consisted of both aerobic and resistive elements and 8 subjects with a specific dietary protein supplementation (NUT). The study was performed in two campaigns with 12 volunteers each. The experimental campaigns lasted 100 days: a 20-day period (baseline data collection; BDC) prior to 60-days of 6° head-down tilt bed-rest (HDT), with 20-days of post-bed-rest recovery data collection (R+). All participants gave written informed consent prior to the study. All study procedures complied with the declaration of Helsinki and were approved by the local Ethics Committee in Toulouse. Countermeasures The effect of two different countermeasures was evaluated: physical exercise in one group and specific nutrition in another group. The exercise countermeasure (EXE) consisted of a combination of a resistance and aerobic training protocol with the primary aim to impede muscle atrophy and impairment of muscle function [23]. Resistance training was performed on an inertial ergometer (Flywheel Exercise Device, described elsewhere [24–26]) every 2–3 days. Force and flywheel rotational velocity were measured, and work and power were calculated throughout each repetition [24–26]. This exercise protocol was similar to a previous 90-day study conducted in males [24,27] and involved supine squat exercises (4 sets of 7 maximal repetitions, 2 min between sets) and then calf press exercises (4 sets of 14 maximal repetitions, 2 min between sets). The aerobic countermeasure was done on a supine treadmill exercise within lower-body negative pressure (LBNP) system every 3–4 days. Each subject performed 40 min of treadmill exercise, followed by 10 min of resting LBNP. The exercise device used was similar to that used in previous 5-day [28], 15-day [29], and 30-day bed-rest studies [30]. More details on the exercise protocols can be found elsewhere [23]. The participants of all three groups received meals with a controlled amount of macronutrients and energy. The nutrition group (NUT) Table 1 Subject characteristics at baseline. Group

Age (years)

Height (cm)

Weight (kg)

BMI

CON EXE NUT

34.4 (3.8) 32.8 (3.4) 29.4 (3.5)

162.8 (6.2) 164.8 (7.0) 170.2 (5.4)

56.5 (3.3) 59.5 (5.7) 61.6 (4.8)

21.4 (1.5) 21.9 (1.4) 21.3 (1.2)

Values are mean (SD). No significant difference between groups. CON: inactive control group, EXE: exercise countermeasure group, and NUT: nutrition countermeasure group. Eight subjects in each group.

An XCT 2000 (Stratec Medizintechnik, Pforzheim, Germany) was used to obtain pQCT scans from the left tibia and radius as described previously [31,32]. Scout-views were generated in the frontal plane to identify the tibio-talar (tibia measures) and radio-carpal (radius measures) cleft to position the reference line. Sectional images were then obtained from the tibia and radius at 4% (distal epiphysis) and 66% (diaphysis) of its length. Measurements were performed prior to bed-rest (BDC; double measurement and average of both measurements used in further analyses), during bed-rest on HDT15, HDT43 and after bed-rest on R + 3, R + 90, R + 180 and R + 360. The coefficient of variation [33] for distal tibia total BMD was 0.37% and 1.92% for distal radius total BMD (data from double baseline measurements in current study with complete repositioning between each measurement; n = 24). The integrated XCT 2000 software (version 5.50D) was used to analyse the pQCT images. The total and trabecular bone mineral density (BMD) was determined using a detection threshold of 180 mg/cm 3 (contour mode 1, peel mode 1, trabecular area 45%) at the epiphysis. Cortical BMD was assessed with a threshold of 710 mg/cm3 (cortical mode 1). As we expected intracortical remodelling which can be better assessed with a cortical bone measure and as, in contrast to the distal tibia, at the tibial diaphysis (66% position) there is very little trabecular bone a cortical bone measure is more appropriate. Thus, cortical BMD was assessed at the 66% position (tibial diaphysis) in preference to total BMD. Gross anatomical muscle cross-sectional area (CSA) was obtained from the scans taken at 66% of the tibia and radius length (diaphysis) and was calculated as total bone area (detection threshold of 280 mg/ cm3; contour mode 1, peel mode 1, filters 2: F03) subtracted from the combined muscle and bone area (detection threshold of 45 mg/cm3; contour mode 3, peel mode 1, filters 2: F03F05). Dual X-ray absorptiometry (DXA) Bone mineral content (BMD in g/cm 2) of the lumbar spine (L1–L4 in anteroposterior projection), the proximal femur (total) and wholebody were measured using dual x-ray absorptiometry (DXA) with a Hologic QDR 4500 W, Software Version 11.1 (Hologic, Bedford, MA). Hip and lumbar scans were performed according to the standard manufacturer DXA protocol prior to the start of bed-rest (BDC; triple measurement), on head-down tilt day HDT13, HDT31, HDT43 and on post-bed-rest recovery (R+) days R + 3, R + 45, R + 90, R + 180 and R + 360. DXA measurements were performed in triplicate before and after HDT (BDC and R + 3) and then the results were averaged. On the remaining measurement dates, single measurements were performed. The co-efficient of variation [33] for proximal femur (total) BMD was 0.71% and 0.93% for lumbar spine (L1–L4) BMD (data from triple baseline measurements in current study with complete repositioning between each measurement; n = 24). Whole-body scans were performed up until R + 3 only. Data on the head, arm, trunk and leg sub-regions were derived from the whole body scan. All scanning and analyses were performed by the same operator to ensure consistency and followed standard quality control procedures. Body composition (lean mass and fat mass in kilogrammes) data were also calculated from whole-body scans.

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Statistical analysis To evaluate the effect of bed-rest, recovery and the impact of countermeasures, linear-mixed effects models [34] were used. Analysis was first conducted on absolute-values with ‘group’ (CON, EXE and NUT) and ‘study-date’ main effects and their interaction. Allowances were made for heterogeneity of variance for study-date and group with random effects for each subject. Where there the group× study-date interaction showed a p-value less than 0.05, further testing was done: (a) the models were repeated using data on percentage change compared to baseline to try to rule out potential effect of subtle differences in baseline data between groups and, then, (b) if the effect still persisted, subsequent two-group models (i.e. CON vs. EXE, CON vs. NUT and EXE vs. NUT) using absolute and percentage change data were conducted to examine which group(s) could have been responsible for the effect. Furthermore, apriori contrasts comparing each measurement day to baseline (BDC) measures within each group and also for all groups pooled. An alpha level of 0.01 was used for statistical significance for the ‘study-date’ main-effect and ‘group× study-date’ interaction on ANOVA, and p-values for these model terms being less than 0.05 but greater than 0.01 were considered trends. For analysis of changes during and after bed-rest, as multiple measurement sessions were undertaken on the same subjects, a Bonferroni adjustment was not performed, rather we looked for consistent “significant” differences across time points. Subject age, height and weight had little influence on the findings if they were incorporated in the models as linear co-variates

and were hence excluded from the analyses presented here. The statistical analyses were performed with the “R” environment for statistical computing and graphics version 2.10.1 (www.r-project.org). Results Except for data being unavailable from two subjects in the exercise group at R + 90 and one exercise subject at R + 360, all subjects were measured at all planned points. With the exception of leg sub-region BMD (p = 0.009; whole-body DXA) there was no evidence for differences between groups at baseline for any of the BMD variables (p ≥ 0.10; see Tables 3–5). Data on changes in bone mineral content (BMC) are presented in online supplementary material (ASM Tables 1–3). The results for BMC are similar to BMD for pQCT measures but for DXA measures there were some differences in the changes over the course of bed-rest, presumably due to differences in subject positioning and hence the bone orientation during DXA scanning, but the main outcomes of the study are the same for BMC data. Effect of bed-rest and recovery When considering all subjects pooled together, strongly significant effects of bed-rest and recovery were seen on tibia (4% and 66%) BMD, total hip BMD, lumbar spine BMD, whole-body trunk sub-region BMD, whole-body leg sub-region BMD, calf muscle CSA and forearm muscle CSA (study-date: p ≤ 0.002; Table 2). Reductions in distal tibia BMD,

Table 2 Effect of bed-rest (all subjects pooled) on bone mineral density (BMD). Parameter

Study-date BDC

Peripheral quantitative computed Radius 4% total 342.0 (7.3) p = 0.044 183.3 (6.1) Radius 4% trabecular p = 0.046 1189.7 (4.2) Radius 66% cortical p = 0.235 Tibia 4% total 302.2 (6.3) p b .001 230.6 (5.8) Tibia 4% trabecular p b .001 1161.3 (4.0) Tibia 66% cortical p b .001 Dual X-ray absorptiometry (DXA) Total hip 0.93 (0.01) p b .001 Total lumbar 1.01 (0.02) p b .001 1.14 (0.01) Whole-body: total p = 0.225 2.29 (0.05) Whole-body: head p = 0.620 Whole-body: 0.75 (0.01) arm p = 0.022 Whole-body: 0.90 (0.01) trunk p b .001 Whole-body: 1.17 (0.01) leg p = 0.002

HDT15/13

HDT31

tomography (pQCT) − 0.32 (0.52)%

HDT43

R+3

− 0.11 (0.46)%

− 0.18 (0.46)%

R + 45

− 0.09 (0.09)%

0.02 (0.10)%

0.14 (0.11)%

R + 180

R + 360

0.33 (0.51)%

− 1.02 (0.38)%b

− 0.86 (0.30)%b − 0.70 (0.42)%

− 0.74 (0.37)%a

− 0.09 (0.14)%

− 0.12 (0.10)%

− 0.17 (0.10)%

− 0.77 (0.32)%a − 1.06 (0.34)%b

− 0.19 (0.41)%

− 0.73 (0.30)%a − 0.98 (0.33)%b

− 0.33 (0.32)%

R + 90

− 0.59 (0.38)%

− 1.64 (0.36)%c

− 2.83 (0.38)%c

− 1.34 (0.32)%c

− 0.24 (0.28)%

− 1.54 (0.25)%c

− 2.77 (0.34)%c

− 0.89 (0.43)%a − 0.44 (0.36)%

− 0.76 (0.41)%

− 0.11 (0.09)%

− 0.40 (0.08)%c

− 0.33 (0.08)%c

− 0.15 (0.07)%a

− 0.79 (0.43)%

− 0.52 (0.40)%

0.16 (0.06)%b

0.10 (0.07)%

− 0.66 (0.26)%a − 2.00 (0.32)%c − 2.22 (0.31)%c

− 3.43 (0.27)%c

0.16 (0.30)%

1.29 (0.34)%c

− 0.27 (0.18)%

− 0.08 (0.12)%

− 0.54 (0.20)%

b

0.62 (0.58)%

0.13 (0.42)%

− 0.05 (0.41)%

0.00 (0.33)%

0.09 (0.33)%

0.30 (0.26)%

0.58 (0.32)%

− 0.41 (0.33)% − 0.52 (0.24)%

− 0.25 (0.27)% a

− 1.98 (0.45)%c 0.15 (0.33)%

− 2.41 (0.38)%c − 1.52 (0.36)%c 1.68 (0.33)%c

1.06 (0.39)%b

1.16 (0.35)%b

0.50 (0.32)%

1.08 (0.28)%c

− 1.37 (0.34)%c − 1.17 (0.37)%b − 1.52 (0.32)%c − 0.24 (0.26)%

− 0.55 (0.29)%

− 1.49 (0.22)%c

Values at baseline (BDC) are mean (SEM) in absolute values (pQCT: mg/cm³; DXA: g/cm2). Beyond this time point values are mean (SEM) percentage difference to baseline value. HDT: day of head-down tilt bed-rest (HDT15/13: pQCT performed on HDT15, DXA on HDT13); R+: day of post-bed-rest recovery. 4% and 66% indicate position along the length of the radius or tibia at which the measurement was done with 4% representing the distal tibia. a: p b 0.05; b: p b 0.01; c: p b 0.001 and indicate significance change on a given study-date compared to baseline. p-Values next to parameter name indicate significance from ANOVA of changes over the course of the study with all groups pooled. Only for whole-body leg sub-region BMD was there evidence for a different response between the three groups: see Tables 3 and 4.

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total hip BMD, trunk sub-region BMD, leg sub-region BMD, calf muscle CSA and forearm muscle CSA were seen during bed-rest, with some of the BMD changes persisting up to 1-year after bed-rest. Tibia diaphysis (66% pQCT) cortical BMD increased slightly (and significantly) initially during bed-rest with significant reductions seen first late into the recovery phase. Lumbar BMD did not reduce significantly during bed-rest and was actually significantly increased on day-3 after bed-rest and remained so up to 6 months after bed-rest (R + 180). Whole-body total BMD was decreased during and immediately after bed-rest, arm sub-region BMD was increased after bed-rest, and distal radius trabecular BMD was decreased during and after bed-rest, but the strength of these effects on ANOVA were weak (study-date: p ≥ 0.022). ANOVA provided limited evidence for changes in distal radius (4% pQCT) total BMD (study-date: p = 0.044) though this effect was mainly due to the final measurement time-point (R + 360; Table 2) with no significant changes on the other study dates. No evidence was present on ANOVA for significant changes in radius diaphysis (66% pQCT) cortical BMD or head sub-region BMD over the course of the study (study-date: p ≥ 0.24). Effect of countermeasures Whilst the exercise countermeasure strongly impacted upon calf muscle CSA loss (group × study-date: p b 0.001; Table 5; Fig. 1) being significantly different to both the control and nutrition groups, analysis of BMD data showed no significant reduction in BMD loss in any body-region (Tables 3 and 4). Given the contrast of these findings, using the standard operating procedure for the DXA device, to data published by another group on the DXA hip scans using their own manual measurements of the same images showing a significant impact of the exercise countermeasure on hip BMD, [35] data on hip scan sub-regions have been presented in online supplementary material (ASM Tables 4 and 5). No significant differences between groups for BMD were seen on ANOVA for any of the sub-regions (trochanter, intertrochanteric, femoral neck, ward's triangle; p ≥ 0.056). At the leg sub-region, re-analysis of data on percentage change in BMD compared to baseline showed a statistically significantly (p = 0.022) greater loss of BMD at HDT31 in the EXE group compared to CON, with the differences between the twogroups on the other days being non-significant (p ≥ 0.13). Changes in forearm muscle CSA also differed between groups (group × studydate: p = 0.008; Table 5), with the loss of forearm muscle CSA being greater in the EXE group than in the NUT group. There was, however, an impact of the exercise, but not the nutrition, countermeasure on lean mass and fat mass changes in the whole-body and also in sub-regions (Table 6). Generally, the exercise group showed less loss of lean mass, greater reduction of fat mass than the control and nutrition groups. The nutrition group was not significantly different to the control group for changes in body-composition.

Fig. 1. Percentage changes in distal tibia bone mineral density (top), total hip bone mineral density (middle) and calf muscle cross-sectional area (bottom). Values are mean (SEM) percentage change compared to baseline. Differences between groups in losses in distal tibia total bone mineral density (p = 0.074) and total hip bone mineral density (p = 0.073) did not reach significance although losses of muscle cross-sectional area did (p b 0.001). See Tables 3–5 for more details.

Discussion Contrary to our expectations, the current study found that in women undergoing 60-days bed-rest, an exercise countermeasure combining high-load resistive and aerobic exercise components did not significantly reduce bone mineral density losses. As expected, however, the nutrition countermeasure, which was mainly targeted at muscle metabolism, had no effect on BMD change compared to control. The nutrition countermeasure, however, had no effect on lean and fat mass changes or on muscle CSA at the calf and forearm. The exercise countermeasure did, in contrast, result in less loss of lean mass, greater reductions in fat mass and less loss of muscle CSA at the calf than in the other two groups. The current study is one of a limited number of studies examining regional bone changes in women in bed-rest. We observed the greatest losses of BMD at the distal tibia and at the hip and no change

in lumbar BMD during bed-rest but an increase in this parameter after bed-rest. Overall trunk BMD reduced and so too did leg DXA subregion BMD. Few significant changes in BMD were seen in the arm and head. At the distal radius, a greater magnitude of BMD loss was seen in trabecular bone than of overall (total) BMD and no change was seen in the radial diaphysis. To the best of our knowledge, two prior works [5,36] have also examined the effects of bed-rest on bone mass/ density in female subjects. One of these works [36] in 30-days bedrest using DXA measures found the greatest losses at the hip and femoral shaft. The other study [5], in 17 weeks bed-rest, also used DXA measures as in the current work and showed comparable losses of BMD at the hip, lumbar spine and total body. The current work is, to the best of our knowledge, the first to evaluate in pQCT measures of the tibia and radius in women or to present data from bone recovery in women after bed-rest.

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Table 3 Influence of countermeasures on bone mineral density (BMD) on peripheral quantitative computed tomography. Group

Study-date BDC

HDT15

HDT43

R+3

R + 90

R + 180

R + 360

Radius 4% total, group ⁎ study-date p = 0.908 CON 356.9 (12.1) 0.08 (1.14)% EXE 349.7 (11.9) − 0.82 (0.83)% NUT 319.6 (12.0) − 0.22 (0.75)%

− 0.23 (0.85)% − 0.49 (0.44)% 0.45 (0.89)%

− 0.07 (0.64)% − 1.06 (0.70)% 0.67 (0.87)%

− 0.53 (0.77)% − 0.47 (0.81)% 0.46 (0.60)%

− 0.28 (0.88)% 0.53 (1.01)% 0.78 (0.84)%

− 1.44 (0.71)%a − 1.19 (0.55)%a − 0.48 (0.71)%

Radius 4% trabecular, group ⁎ study-date p = 0.937 CON 187.0 (10.4) − 0.34 (0.80)% EXE 192.7 (10.4) − 0.52 (0.39)% NUT 170.4 (10.4) − 0.12 (0.43)%

− 1.16 (0.63)% − 0.48 (0.47)% − 0.55 (0.49)%

− 1.52 (0.62)%a − 0.86 (0.50)% − 0.53 (0.62)%

− 1.37 (0.64)%a − 0.54 (0.45)% − 0.55 (0.51)%

− 1.67 (0.87)% − 0.47 (0.48)% 0.12 (0.71)%

− 1.06 (0.82)% − 0.89 (0.59)% − 0.24 (0.55)%

Radius 66% cortical, group ⁎ study-date p = 0.577 CON 1195.2 (7.5) − 0.17 (0.12)% EXE 1186.5 (7.4) 0.00 (0.10)% NUT 1187.4 (7.6) − 0.11 (0.23)%

0.15 (0.10)% − 0.04 (0.22)% − 0.04 (0.20)%

− 0.10 (0.19)% 0.13 (0.12)% 0.38 (0.24)%

− 0.03 (0.23)% − 0.38 (0.21)% 0.06 (0.27)%

− 0.16 (0.17)% − 0.24 (0.13)% 0.04 (0.25)%

− 0.15 (0.18)% − 0.30 (0.15)%a − 0.09 (0.19)%

Tibia 4% total, group ⁎ study-date p = 0.073 CON 296.8 (10.8) − 0.67 (1.04)% EXE 317.3 (10.6) − 0.89 (0.38)%a NUT 292.5 (10.5) − 0.18 (0.26)%

− 1.87 (0.88)%a − 1.49 (0.41)%c − 1.57 (0.42)%c

− 3.13 (0.86)%c − 2.23 (0.54)%c − 3.20 (0.43)%c

− 1.89 (0.78)%a − 1.11 (0.40)%b − 1.09 (0.45)%a

− 1.65 (0.79)%a − 0.19 (0.44)% − 0.50 (0.43)%

− 1.81 (0.82)%a − 0.76 (0.45)% − 0.65 (0.51)%

Tibia 4% trabecular, group ⁎ study-date p = 0.629 CON 223.7 (10.1) − 0.23 (1.16)% EXE 240.5 (9.9) − 0.08 (0.26)% NUT 227.7 (10.0) − 0.41 (0.27)%

− 1.89 (0.98)% − 1.31 (0.39)%b − 1.45 (0.41)%c

− 2.78 (0.99)%b − 2.49 (0.60)%c − 3.06 (0.66)%c

− 1.81 (0.94)% − 0.47 (0.34)% − 0.32 (0.79)%

− 1.18 (0.92)% − 0.26 (0.35)% 0.09 (0.56)%

− 1.52 (0.97)% − 0.43 (0.49)% − 0.26 (0.60)%

− 0.01 (0.15)% − 0.10 (0.13)% − 0.23 (0.16)%

− 0.51 (0.13)%c − 0.38 (0.13)%b − 0.30 (0.17)%

− 0.36 (0.11)%b − 0.26 (0.14)% − 0.38 (0.16)%a

− 0.17 (0.08)%a − 0.19 (0.07)%a − 0.11 (0.17)%

Tibia 66% cortical, group ⁎ study-date p = 0.789 CON 1169.9 (6.8) 0.11 (0.10)% EXE 1160.9 (6.8) 0.14 (0.09)% NUT 1153.0 (6.9) 0.23 (0.13)%

0.04 (0.11)% 0.01 (0.08)% 0.26 (0.15)%

At baseline (BDC) there were no significant differences between groups for any of the variables (p ≥ 0.10). Values at baseline are mean (SEM) in mg/cm³. Beyond this time point values are mean (SEM) percentage difference to baseline value. a: p b 0.05; b: p b 0.01; c: p b 0.001 and indicate significance of change within each group compared to baseline. p-Values next to parameter title indicate whether evidence was present on ANOVA for an impact of the countermeasures (group × study-date interaction). No significant differences between groups for any of these parameters.

Whilst bone losses in the current study may at first glance seem negligible, it should be considered that the BMD loss at the proximal femur in elderly women is approximately 0.7–1.0% per year [37,38] and approximately 2–4% per year at the distal tibia in post-menopausal women [39]. In the current study, we observed a 3.4% loss of BMD at the proximal femur and 2.8% at the distal tibia after 60-days bed-rest in women. When compared to data from post-menopausal women, the rate of bone loss in young healthy women during bed-rest is approximately 20 times that seen in more elderly female populations. Based on these data, it is reasonable to hypothesise that given a long enough time span (such as during long-term spaceflight) these losses in bone could become clinically relevant. Our group has published similar pQCT and DXA data from a total of sixty-nine male subjects in long-term bed-rest [32,40,41]. The distribution of bone loss seen in the female subjects of the current study is more or less comparable to that of male subjects, although direct statistical comparisons have not been performed. One interesting difference is, however, that we detected that losses of BMD at the distal radius occurred predominately in the central trabecular region of the distal radius. In the male subjects we have examined, this did not occur [32,40] (also unpublished observations for evaluation of trabecular region). This is interesting in light of evidence suggesting a higher incidence of distal radius fractures seen in women than in men [42]. Whilst the reasons for such a higher incidence of distal radius fractures in women are no doubt multi-factorial, it is nonetheless interesting that distal radius trabecular BMD appears to be particularly affected in women during inactivity. The metabolic processes that may, or may not, underlie such potential inactivity related sexdifferences would need detailed examination, but may be a fruitful path for understanding wrist fracture incidence in women. The exercise and nutrition countermeasures implemented were largely ineffective for preventing bone loss in women during and after

60-days of bed-rest. Inspection of the data may suggest a general pattern of less bone loss in the exercise subjects in some regions (i.e. hip, distal tibia) during and after bed-rest, but this effect was not at all significant. The exercise countermeasure did, however, reduce loss of lean mass and muscle CSA compared to the other groups (with magnetic resonance imaging data published elsewhere [23]), which is in our view a hallmark of exercise programmes that incorporate highload resistive exercise [43]. The nutrition countermeasure which was designed to target muscle metabolism, had no effect on bone, lean mass, fat mass or muscle CSA parameters, however. In terms of optimal exercise countermeasure design to prevent bone loss in spaceflight and its simulation (bed-rest), whilst some groups [44] have stressed how difficult it is to prevent bone loss in bed-rest, there is however an improving data pool to help understand what may indeed work. Exercise programmes including high-load resistive either with [40,41] or without [5,32,41] whole-body vibration have been shown to reduce bone loss in bed-rest at some body regions, although the effects were not perfect in these studies. The addition of whole-body vibration to high-load resistance exercise appears to have an additional effect on preventing bone loss at some skeletal sites [41]. These findings fit in well with animal studies of the response of bone to mechanical stimuli. Firstly, bone loading/strain must reach certain minimal levels for there to be an effect on bone formation [45–48] and these loads need to be dynamic rather than static [49,50]. Low loads result in less bone strain than high load protocols [45,51,52]. Secondly, the rate of bone loading/strain is also important, with higher rates of bone strain resulting in a greater stimulus for bone formation [53–55] and evidence also suggests that the minimum magnitude of bone strain required to impact on bone formation is lower when this load is applied at faster rates [47]. Indeed, a given load may impact upon bone formation when applied at higher rates, but have no effect when applied at slower rates [53].

G. Beller et al. / Bone 49 (2011) 858–866

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Table 4 Influence of countermeasures on bone mineral density (BMD) on dual X-ray absorptiometry. Group

Study-date BDC

HDT13

HDT31

HDT43

R+3

R + 45

R + 90

R + 180

R + 360

Total hip, group ⁎ study-date p = 0.074 CON 0.89 (0.02) − 0.82 (0.44)% EXE 0.95 (0.02) − 1.04 (0.71)% NUT 0.95 (0.02) − 0.82 (0.50)%

− 2.07 (0.51)%c − 1.90 (0.70)%b − 2.40 (0.62)%c

− 2.88 (0.64)%c − 1.29 (0.50)%a − 2.69 (0.54)%c

− 3.50 (0.55)%c − 2.76 (0.42)%c − 3.91 (0.60)%c

− 2.80 (0.77)%c − 1.00 (0.53)% − 3.61 (0.57)%c

− 2.46 (0.66)%c − 0.85 (0.94)% − 1.51 (0.51)%b

− 2.03 (0.94)%a 0.20 (0.67)% − 1.07 (0.72)%

− 0.52 (0.65)% 0.03 (0.68)% − 1.25 (0.97)%

Total lumbar, group ⁎ study-date p = 0.774 CON 0.99 (0.03) − 0.68 (0.71)% EXE 1.05 (0.03) − 0.27 (0.77)% NUT 0.99 (0.03) − 0.68 (0.50)%

− 0.48 (0.65)% − 0.24 (0.65)% − 0.24 (0.41)%

− 0.25 (0.68)% 0.23 (0.74)% 0.22 (0.42)%

0.44 (0.85)% 1.13 (0.86)% 1.60 (0.44)%c

0.92 (0.82)% 2.56 (1.00)%a 1.81 (0.40)%c

0.14 (0.75)% 1.91 (1.02)% 1.48 (0.57)%a

Whole-body: total, group ⁎ study-date p = 0.170 CON 1.12 (0.02) − 0.55 (0.35)% 0.07 (0.35)% EXE 1.17 (0.02) − 0.44 (0.49)% − 0.55 (0.38)% NUT 1.13 (0.02) 0.39 (0.46)% − 0.43 (0.28)%

0.20 (0.60)% 0.00 (0.16)% − 0.34 (0.23)%

− 0.30 (0.43)% − 0.18 (0.34)% − 0.94 (0.29)%b

Whole-body: head, group ⁎ study-date p = 0.053 CON 2.37 (0.08) 1.75 (1.16)% 0.14 (0.58)% EXE 2.29 (0.08) − 0.75 (1.15)% 1.24 (0.99)% NUT 2.23 (0.08) 1.00 (0.97)% − 0.70 (0.73)%

0.38 (1.07)% − 0.30 (1.30)% − 0.09 (0.59)%

0.16 (0.61)% − 0.51 (0.87)% 0.03 (0.52)%

Whole-body: arm, group ⁎ study-date p = 0.195 CON 0.73 (0.02) − 0.04 (0.53)% 0.52 (0.41)% EXE 0.77 (0.02) − 0.25 (0.78)% − 0.13 (0.78)% NUT 0.75 (0.02) 0.84 (0.71)% 0.38 (0.45)%

− 0.04 (0.43)% 1.49 (0.79)% 0.70 (0.53)%

1.13 (0.63)% 0.88 (0.84)% 1.18 (0.42)%b

Whole-body: trunk, group ⁎ study-date p = 0.229 CON 0.89 (0.02) − 2.96 (0.49)%c − 1.78 (0.52)%c EXE 0.93 (0.02) − 0.67 (1.20)% − 1.39 (1.12)% NUT 0.89 (0.02) − 0.84 (0.79)% − 1.06 (0.54)%

− 1.66 (1.19)% − 1.45 (0.76)% − 0.76 (0.50)%

− 1.65 (0.80)%a − 1.11 (0.61)% − 2.00 (0.48)%c

Whole-body: leg, group ⁎ study-date p b .001d,f CON 1.12 (0.02) − 0.36 (0.55)% 0.21 (0.37)% EXE 1.22 (0.02) 0.08 (0.55)% − 1.15 (0.38)%b NUT 1.18 (0.02) 0.68 (0.77)% − 0.29 (0.56)%

0.33 (0.75)% − 0.86 (0.49)% − 0.78 (0.57)%

− 1.29 (0.42)%b − 0.34 (0.41)% − 1.71 (0.43)%c

0.74 (0.69)% 1.30 (0.93)% 1.31 (0.78)%

− 0.15 (0.77)% 0.48 (0.68)% 1.76 (0.87)%a

With the exception of leg sub-region BMD (p = 0.009) there was no evidence for differences between groups at baseline (p ≥ 0.13). Values at baseline (BDC) are mean (SEM) in g/ cm2. Beyond this time point values are mean (SEM) percentage difference to baseline value. a: p b 0.05; b: p b 0.01; c: p b 0.001 and indicate significance of change within each group compared to baseline. p-Values next to parameter title indicate whether evidence was present on ANOVA for an impact of the countermeasures (group × study-date interaction) and “d”, “e”, “f” indicate, respectively, significant (p b 0.05) differences on ANOVA on subsequent two-group comparisons between CTR vs. EXE, CTR vs. NUT and EXE vs. NUT. CON: inactive control group, EXE: exercise countermeasure group, and NUT: nutrition countermeasure group. When percentage change compared to baseline are analysed, the significant differences between CON vs. EXE and EXE vs. NUT persist for leg sub-region BMD.

The number of loading cycles [56–59] and duration of loading [56,58,59] are also important, though evidence [59] suggests that an appropriate loading protocol need not be performed for lengthy periods in order to have an effect on bone metabolism. The data at hand suggests that, in bed-rest, exercise protocols incorporating highload resistive exercise with superimposed impact loading (such as from “jumping” type protocols and/or whole-body vibration) would

be more effective countermeasures. A number of exercise prescription parameters (e.g. a direct comparison of high-load, low-load and impact-loading protocols, duration of exercise, frequency of exercise, types of exercise manoeuvres) still need detailed examination in bedrest however. Such investigations would not only be useful for spaceflight countermeasures, but also in defining exercise rehabilitation protocols for patients after immobilisation or inactivity.

Table 5 Influence of countermeasures on muscle cross-sectional area at the lower-leg and lower-arm. Group

Study-date BDC

HDT15

HDT43

Radius 66% muscle CSA, group × study-date p = 0.008f CON 23.2 (1.3) − 2.2 (0.7)%b EXE 25.6 (1.3) − 3.3 (1.1)%b NUT 23.8 (1.3) − 2.4 (0.6)%c

− 4.4 (0.8)%c − 6.3 (1.0)%c − 4.0 (0.7)%c

CSA, group × study-date p b .001d,f 63.2 (3.7) − 9.1 (1.4)%c 61.5 (3.7) − 5.9 (2.1)%b 62.6 (3.7) − 9.5 (1.3)%c

− 23.0 (1.0)%c − 8.9 (1.6)%c − 22.1 (1.7)%c

Tibia 66% muscle CON EXE NUT

R+3

R + 90

R + 180

R + 360

− 5.1 (0.8)%c − 6.0 (1.0)%c − 4.5 (1.0)%c

2.7 (1.0)%b 2.7 (0.9)%b 0.9 (1.3)%

2.7 (0.9)%b 2.4 (1.1)%a 2.1 (1.0)%a

4.4 (1.3)%c 4.4 (1.8)%a 1.9 (1.0)%

− 19.6 (1.4)%c − 3.2 (1.2)%b − 16.0 (1.8)%c

4.4 (1.5)%b 3.6 (1.3)%b 4.1 (1.7)%a

2.4 (1.3)% 4.1 (1.0)%c 4.3 (1.5)%b

3.4 (2.0)% 3.6 (0.7)%c 2.8 (1.7)%

Measures were taken at 66% of radius and tibia length on peripheral quantitative computed tomography measurements. At baseline (BDC) there were no significant differences between groups for any of the variables (p ≥ 0.50). Values at baseline are mean (SEM) in cm2. Beyond this time point values are mean ( SEM) percentage difference to baseline value. a: p b 0.05; b: p b 0.01; c: p b 0.001 and indicate significance of change within each group compared to baseline. p-Values next to parameter title indicate whether evidence was present on ANOVA for an impact of the countermeasures (group × study-date interaction) and “d”, “e”, “f” indicate, respectively, significant (p b 0.05) differences on ANOVA on subsequent two-group comparisons between CTR vs. EXE, CTR vs. NUT and EXE vs. NUT. CON: inactive control group, EXE: exercise countermeasure group, and NUT: nutrition countermeasure group.

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G. Beller et al. / Bone 49 (2011) 858–866

Table 6 Influence of countermeasures on body composition on whole-body dual X-ray absorptiometry. Variable

Group

Study-date BDC

d,f

Total: lean p b .001 ; fat p b .001 Lean mass CON EXE NUT Fat mass CON EXE NUT

HDT13

HDT31

HDT43

R+3

− 6.5 (0.4)%c − 2.7 (0.6)%c − 5.7 (0.9)%c − 0.9 (1.0)% − 9.5 (1.5)%c 2.5 (1.5)%

− 6.4 (0.4)%c − 3.1 (0.6)%c − 5.0 (0.9)%c − 3.4 (1.2)%b − 11.0 (1.6)%c − 1.1 (1.5)%

− 5.5 (0.5)%c − 2.2 (0.6)%c − 3.9 (1.2)%b − 4.3 (1.6)%b − 15.8 (2.0)%c 1.5 (1.7)%

1.6 (1.1)% 1.4 (1.6)% 3.9 (1.0)%c 1.5 (1.1)% 1.2 (1.4)% 3.8 (1.0)%c

2.5 (1.4)% 8.1 (1.8)%c 2.9 (1.0)%b 2.2 (1.3)% 7.1 (1.5)%c 2.7 (0.8)%b

2.7 (1.0)%a 5.3 (1.5)%c 2.9 (1.1)%a 2.5 (1.0)%a 4.5 (1.3)%c 2.7 (1.0)%b

d,f

− 3.7 (0.4)%c − 1.9 (0.8)%a − 2.5 (0.9)%a − 0.5 (1.0)% − 5.8 (1.7)%b 0.0 (1.9)%

38.59 41.57 42.76 14.81 14.48 16.10

(1.55) (1.56) (1.59) (1.13) (1.14) (1.14)

Head: lean p b .001d,f; fat p b .001f Lean mass CON EXE NUT Fat mass CON EXE NUT

3.14 3.10 3.39 0.81 0.80 0.87

(0.11) (0.11) (0.11) (0.03) (0.03) (0.03)

Arm: lean p = 0.518; fat p b .001d,f Lean mass CON EXE NUT Fat mass CON EXE NUT

2.06 2.24 2.27 1.03 0.98 1.12

(0.12) (0.12) (0.12) (0.10) (0.10) (0.10)

− 1.4 (2.0)% 0.1 (2.2)% 0.6 (2.0)% − 7.9 (4.6)% − 16.4 (3.6)%c − 4.4 (4.0)%

− 3.0 (1.6)% − 1.1 (1.8)% − 2.3 (1.9)% − 5.0 (4.9)% − 22.5 (3.3)%c − 4.5 (3.1)%

− 1.4 (1.4)% 0.3 (1.8)% 2.3 (2.1)% − 11.7 (5.6)%a − 21.5 (3.8)%c − 4.6 (3.4)%

− 4.6 (1.6)%b − 1.6 (1.6)% − 0.1 (2.0)% − 6.5 (4.9)% − 24.4 (3.6)%c 1.1 (4.0)%

Trunk: lean p = 0.224; fat p b .001f Lean mass CON EXE NUT Fat mass CON EXE NUT

19.46 21.03 21.51 5.59 5.12 5.74

(0.75) (0.76) (0.78) (0.56) (0.56) (0.56)

− 2.5 (0.6)%c − 2.0 (1.1)% − 0.5 (1.1)% 1.1 (2.5)% − 4.2 (2.1)%a − 2.0 (3.4)%

− 5.0 (0.8)%c − 1.9 (1.1)% − 3.3 (1.2)%b − 1.1 (2.2)% − 6.9 (2.1)%b 3.3 (1.5)%a

− 3.9 (0.7)%c − 3.6 (0.9)%c − 1.8 (1.2)% − 3.2 (1.4)%a − 8.1 (2.3)%c − 2.8 (1.9)%

− 3.3 (0.8)%c − 1.9 (0.9)%a − 1.8 (1.3)% − 3.7 (2.2)% − 14.1 (2.4)%c − 0.6 (2.2)%

5.94 6.48 6.66 3.18 3.31 3.63

(0.28) (0.28) (0.29) (0.27) (0.29) (0.28)

− 7.4 (0.8)%c − 3.5 (1.0)%b − 7.4 (1.4)%c 0.3 (0.7)% − 4.9 (3.2)% 2.8 (1.8)%

− 12.2 (0.5)%c − 5.5 (1.4)%c − 13.2 (1.2)%c 0.2 (1.1)% − 9.0 (2.9)%b 3.8 (1.9)%a

− 14.5 (0.9)%c − 6.1 (1.0)%c − 14.6 (1.4)%c − 1.7 (0.9)% − 12.3 (2.8)%c 0.8 (1.8)%

− 11.6 (1.5)%c − 4.7 (1.0)%c − 10.2 (1.7)%c − 5.0 (0.9)%c − 17.0 (3.0)%c 3.1 (1.6)%

Leg: lean p b .001d,f; fat p b .001d,f Lean mass CON EXE NUT Fat mass CON EXE NUT

0.7 (1.3)% 2.7 (1.8)% 0.4 (1.6)% 0.9 (1.1)% 2.2 (1.6)% 0.6 (1.5)%

At baseline (BDC) there were no significant differences between groups for any of the variables (p ≥ 0.13). Values at baseline are mean (SEM) in kilogrammes. Beyond this time point values are mean (SEM) percentage difference to baseline value. a: p b 0.05; b: p b 0.01; c: p b 0.001 and indicate significance of change within each group compared to baseline. p-Values next to parameter title indicate whether evidence was present on ANOVA for an impact of the countermeasures (group× study-date interaction) for lean mass and fat mass changes. “d”, “e”, “f” indicate, respectively, significant (p b 0.05) differences on ANOVA on subsequent two-group comparisons between CTR vs. EXE, CTR vs. NUT and EXE vs. NUT. Note that the CON and NUT groups were never significantly different from one another. CON: inactive control group, EXE: exercise countermeasure group, and NUT: nutrition countermeasure group.

The data we present here from the DXA measurements conflict somewhat with those published by another group [35] from the exact same subjects and DXA measurements. This group concluded that hip and leg sub-region BMD loss was significantly less in the exercising subjects. In the current work, we found no significant reduction of hip BMD loss in the exercise group, but a statistically significant greater loss of leg sub-region BMD in these subjects compared to control at midbed-rest with otherwise no significant differences between the two groups. These divergent findings seem quite surprising, given that the methodological differences solely related to DXA image analysis: we followed the Hologic manufacturer's standard analysis protocols whereas the other group used Hologic software but with approaches established by their own laboratory. Statistical methods also differed slightly, though this should not lead to such gross differences. To a certain extent it is possible to reconcile the differences in the hip BMD data in that the exercise subjects showed less loss of BMD in our data set as well, and also in some sub-regions of the hip, just that this effect did not reach statistical significance. Reconciling the findings of leg subregion BMD loss is more difficult, as whilst inspection of their data also shows a similar pattern (greater loss in EXE group at mid-bed-rest, less at day-3 after bed-rest), the statistical significance of these findings are contradictory between the two data sets (Smith et al.: insignificant at mid-bed-rest but significant after bed-rest; current study: significant at

mid-bed-rest but insignificant after bed-rest). It should be remembered that both publications showed the EXE group to have a significantly higher leg sub-region BMD at baseline. We repeated our analyses of these data based upon percentage change in BMD compared to baseline in an effort to control for these baseline differences, and the effects still remained significant. How the higher BMD at baseline in the EXE group may have influenced subsequent bone loss during bed-rest and impact of exercise is unclear. Irrespective of whether these DXA leg-sub-region effects are statistically significant or not, the inconsistency of some of the changes across time needs to be questioned: the DXA whole-body measurement is too gross to be able to reliably measure a 1.15% reduction in 30 days and then an increase in the following 30 days. The pQCT measurement is much more sensitive but does not show such effects in the same subjects. Hence, any conclusions from the current or other [35] publication about the exercise countermeasure based upon the leg sub-region BMD data should be, at best, treated with caution. The current study has some important limitations. Firstly, there were a limited number of subjects, a limitation common to bed-rest studies due to their cost and complexity. This may well have resulted in false negatives for some parameters. In fairness, also, the stated aims of the exercise and nutrition countermeasures were to target muscle loss. In this regard, the countermeasures were partially effective. Nonetheless, other studies have found significant impacts of countermeasure exercise

G. Beller et al. / Bone 49 (2011) 858–866

protocols on bone loss and bone metabolism [5,11,32,40,41] despite low subject numbers, adding weight to the argument that the effect size of the countermeasure protocols implemented in the WISE-2005 study could well have been augmented with alternate approaches. The low number of subjects will also have been an important contributor to some of the differences seen at baseline between groups, such as for the data from the leg sub-region of whole-body DXA scanning. Another important limitation of the current study is that the exercise countermeasure comprised a number of exercise components. In a broad sense, there was aerobic, cardiovascular, exercise and also highload resistive exercise. There was no additional exercise group that performed only one of these exercise forms. Hence, it is difficult to say, based upon the results of the current study alone, which of the exercise components resulted in the better retention of lean mass and greater loss of fat mass in the exercise subjects. Another issue to keep in mind when comparing finding on muscle CSA and lean mass changes from the current study, to that of other studies is that subjects were not positioned in the horizontal position for at least 2 h prior to scanning as in our more recent bed-rest study [41]. Changes in body posture and fluid shifts can influence muscle CSA measurements [60], and our experience has shown that this can influence muscle CSA on pQCT and lean mass on DXA measurements particularly in the early recovery phase. Furthermore, examinations on the same subjects showed an increased incidence of menstrual irregularity during bed-rest [61]. Such changes in the menstrual cycle could influence bone loss [62], though in our data set there was no particular subject that consistently showed the most amount of bone loss at all regions measured. Unfortunately, the data published in abstract form [61] on changes in menstrual cycles were not available for comparison with our data. Regardless, such alterations in the menstrual cycle could be an important factor in impacting upon sex-related differences in bone loss during bed-rest. Conclusions In conclusion, the current study examined regional bone loss in women during 60-days bed-rest. Bone loss was greatest at the distal tibia and proximal femur. The pattern of bone loss was similar to that seen in men in other studies in bed-rest, though the data provide some suggestions of sex-differences, for example at the distal radius. The exercise countermeasure (high-load resistive exercise on the flywheel device with lower-body negative pressure treadmill locomotion) implemented provided no significant impact on bone loss, but did reduce muscle CSA and lean mass loss particularly in the lower-limb and also resulted in a greater loss of fat mass. The nutrition countermeasure, targeted at muscle metabolism, had no effect on bone, lean mass, fat mass or muscle CSA parameters. Acknowledgments We thank the 24 women who volunteered for this bed-rest investigation as well as the nurses, staff, and entire research team at the MEDES Space Clinic (Toulouse Rangueil Hospital) for their exceptional care of the subjects during bed-rest and exercise. The study WISE-2005 (Women International Investigation for Space Exploration) was sponsored by the European Space Agency (ESA), the National Aeronautics and Space Administration of the USA (NASA), the Canadian Space Agency, and the French “Centre National d'Etudes Spatiales” (CNES), which has been the “Promoteur” of the study according to French law. The bed-rest study was performed by MEDES, Institute for Space Physiology and Medicine in Toulouse, France. Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10. 1016/j.bone.2011.06.021.

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