JBMR

ORIGINAL ARTICLE

Trabecular and Cortical Bone Density and Architecture in Women After 60 Days of Bed Rest Using High-Resolution pQCT: WISE 2005 Gabriele Armbrecht , 1 Daniel Ludovic Belavy´ , 1 Magdalena Backstro¨m , 1 Gisela Beller , 1 Christian Alexandre , 2 Rene Rizzoli , 3 and Dieter Felsenberg 1 1

Charite´ Universita¨tsmedizin Berlin, Center for Muscle and Bone Research, Berlin, Germany INSERM Unit 1059, University-Hospital, St-Etienne, France 3 Division of Bone Diseases, Department of Rehabilitation and Geriatrics, Geneva University Hospitals and Faculty of Medicine, Geneva, Switzerland 2

ABSTRACT Prolonged bed rest is used to simulate the effects of spaceflight and causes disuse-related loss of bone. While bone density changes during bed rest have been described, there are no data on changes in bone microstructure. Twenty-four healthy women aged 25 to 40 years participated in 60 days of strict 6-degree head-down tilt bed rest (WISE 2005). Subjects were assigned to either a control group (CON, n ¼ 8), which performed no countermeasures; an exercise group (EXE, n ¼ 8), which undertook a combination of resistive and endurance training; or a nutrition group (NUT, n ¼ 8), which received a high-protein diet. Density and structural parameters of the distal tibia and radius were measured at baseline, during, and up to 1 year after bed rest by high-resolution peripheral quantitative computed tomography (HR-pQCT). Bed rest was associated with reductions in all distal tibial density parameters (p < 0.001), whereas only distal radius trabecular density decreased. Trabecular separation increased at both the distal tibia and distal radius (p < 0.001), but these effects were first significant after bed rest. Reduction in trabecular number was similar in magnitude at the distal radius (p ¼ 0.021) and distal tibia (p < 0.001). Cortical thickness decreased at the distal tibia only (p < 0.001). There were no significant effects on bone structure or density of the countermeasures ( p
0.057). As measured with HR-pQCT, it is concluded that deterioration in bone microstructure and density occur in women during and after prolonged bed rest. The exercise and nutrition countermeasures were ineffective in preventing these changes. ß 2011 American Society for Bone and Mineral Research. KEY WORDS: MICROGRAVITY; OSTEOPOROSIS; BONE STRUCTURE; HIGH-RESOLUTION PQCT; 3D PQCT

Introduction

I

nformation about structural and material properties is mandatory to calculate the strength of bone, and therefore, specific measurements have been recommended for some time. The World Health Organization (WHO) noted the importance of deterioration of bone microarchitecture several years ago by adding it to the definition of osteoporosis.(1) In clinical trials on the efficacy of osteoporosis treatments, it has been demonstrated that fracture risk is influenced not only by loss of bone mass but also by changes in bone architecture and geometry.(2) Unfortunately, the measurement of trabecular architecture has in the past not been possible in vivo because no system could perform high-resolution images. With the development of a

high-resolution peripheral quantitative computed tomography (HR-pQCT) device specifically designed for this task, in vivo trabecular 3D structure can be visualized and analyzed quantitatively.(3–9) Using a resolution of 82 mm, HR-pQCT enables the measurement of both density and structural parameters and their changes under intervention. One modality where bone remodeling can be studied in a controlled fashion is experimental bed rest. Bed rest typically is used as a ground-based model for simulating the effects of spaceflight on the human body(10,11) and leads to losses of bone in the load-bearing regions of the body.(12) When compared with longitudinal studies in older populations,(13–15) the rates of bone loss during bed rest(12,16) typically are a number of orders of magnitude greater than that seen as part of the aging process.

Received in original form December 28, 2010; revised form May 7, 2011; accepted July 26, 2011. Published online August 2, 2011. Address correspondence to: Gabriele Armbrecht, MD, PhD, Charite´ Universita¨tsmedizin Berlin, Centre of Muscle and Bone Research, Hindenburgdamm 30, 12200 Berlin, Germany. E-mail: [email protected] These data have been presented in part at the Osteologie-Kongress 2007 in Vienna, Austria, February 28 to March 3, 2007. Journal of Bone and Mineral Research, Vol. 26, No. 10, October 2011, pp 2399–2410 DOI: 10.1002/jbmr.482 ß 2011 American Society for Bone and Mineral Research

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Bed rest also offers the opportunity to assess the effectiveness of countermeasures against disuse-related bone loss. Previous studies on bone metabolism under bed-rest conditions typically have been performed with male subjects.(17–27) While some studies have included female subjects,(28) given the higher prevalence of osteoporosis and osteopenia in women,(29) it is nonetheless relevant to expand our knowledge base on the effect of prolonged inactivity on women. In the disuse mode, bone remodeling will be activated and bone resorption increased.(30,31) Presumably, changes in bone structure should occur owing to bed rest, but this has not yet been assessed. The Women International Space Simulation for Exploration bed rest study (WISE 2005) offered the opportunity not only to measure the effectiveness of exercise and nutrition countermeasures concerning bone loss used in women but also to investigate changes in bone structure with HR-pQCT during bed rest. We hypothesized that changes in bone structure and density would be greater at the distal tibia than at the distal radius and that the exercise countermeasure performed in the WISE study would attenuate or prevent these changes.

Materials and Methods Bed-rest protocol and subjects The WISE 2005 study was supported by the European, French, Canadian, German, and North American space agencies (ESA, CNES, CSA, DLR, and 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 three groups: 8 control subjects (CON) without countermeasures 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). Only subjects with a regular menstrual cycle were included in the study, and a total-hip and lumbar dual-energy X-ray absorptiometry (DXA) score of more than 1.5 SD (as measured on a Hologic QDR 4500W running software Version 11.2; Hologic, Bedford, MA, USA). For the spine, Hologic reference values and for the total hip the National Health and Nutrition Examination Survey (NHANES) references values (Hologic version) were used. Other relevant exclusion criteria included use of hormonal contraceptives in a period of 2 months or less before the start of the experiment, current breast-feeding

or cessation of breast-feeding within 2 months prior to the start of the study, postpartum or postabortion within a period of 3 months prior to the start of the study, and use of an intrauterine device at the beginning of the study. The study was performed in two sessions with 12 volunteers each. The experimental protocol lasted 100 days: a 20-day period (baseline data collection [BDC]) prior to 60 days of 6-degree head-down tilt bed rest (BR) with 20 days of post–bed rest recovery data collection (RDC). 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.(32) Resistance training was performed on an inertial ergometer (flywheel exercise device, described elsewhere(33–35)) every 2 to 3 days. Force and flywheel rotational velocity were measured, and work and power were calculated throughout each repetition.(33–35) This exercise protocol was similar to a previous 90-day study conducted in males(33,36) and involved supine squat exercises (four sets of 7 maximal repetitions, 2 minutes between sets) and then calf press exercises (four sets of 14 maximal repetitions, 2 minutes between sets). The aerobic countermeasure was done on a supine exercise treadmill within a lower body negative-pressure (LBNP) system every 3 to 4 days. Each subject performed 40 minutes of treadmill exercise, followed by 10 minutes of resting LBNP. The exercise device used was similar to that used in previous 5-day,(37) 15-day,(38) and 30-day bed rest studies.(39) More details on the exercise protocols can be found elsewhere.(32) The participants of all three groups received meals with a controlled amount of macronutrients and energy. In accordance with standard nutrition guidelines, the EXE and CTR groups received approximately 1.0 g/kg of body weight of protein and free leucine per day during bed rest. The NUT group received approximately 1.6 g/kg of body weight per day. The rationale for this countermeasure was that results of a prior study(40) showed a reduction in protein synthesis after meals during bed rest, and it was thought that by increasing protein intake, this could counteract protein breakdown and hence loss of muscle. Effects on bone were not expected. A detailed description of exercise and nutritional countermeasures has been published

Table 1. Subject Characteristics at Baseline Group CON EXE NUT

Age (years)

Height (cm)

Weight (kg)

BMI

DXA T-score spine

DXA T-score hip

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)

0.50 (0.57) þ0.06 (0.70) 0.50 (0.92)

0.43 (0.50) þ0.08 (0.56) þ0.03 (0.63)

Note: Values are mean (SD). BMI ¼ body mass index, T-score spine ¼ L1–L4 assessed by DXA. None of these measures was significantly different between groups.

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ARMBRECHT ET AL.

elsewhere(32) (see also www.spaceflight.esa.int/wise/; select ‘‘experiment protocols’’ and then ‘‘nutritional and exercise countermeasures’’).

High-resolution peripheral computed tomography The left distal forearms and left distal tibias of the volunteers were measured with a high-resolution (HR) pQCT system (XtremeCT 3306, Scanco Medical AG, Bassersdorf, Switzerland)(3–9) to assess density and microarchitecture of the bone tissue. If any conditions (eg, prior fracture) were found that precluded a valid measurement, the right side was measured for the duration of the study. Measurements were performed by the same operator 9 days prior to the bed rest (BDC 9), on day 15 (HDT 15) and day 43 (HDT 43) during the bed rest phase, and days 3, 90, 180, and 360 in the post–bed rest recovery phase (R þ 3, R þ 90, R þ 180, and R þ 360). A total scan length of 9.02 mm in the axial direction divided into 110 slices was measured simultaneously with a nominal isotropic resolution of 82 mm over a scan time of 2.8 minutes. The standard patient settings were applied concerning effective energy, X-ray tube current, and matrix size (60 kVp, 95 mA, and 1536  1536 pixels, respectively). Total effective dose was less than 3 mSv per scan. In order to achieve a high reproducibility, the subjects were positioned carefully in a formed carbon-fiber cast and fixated in the gantry. Measurements were performed in the supine position. An anteroposterior scout view was used to determine the start position of the measurement (Fig. 1). A reference line was positioned at a certain landmark in the joint space, and the measurement started 9.5 and 22.5 mm proximal to this line in the radius and tibia, respectively. A number of criteria were used to determine whether a measurement was valid or if it had to be repeated directly. In some cases, a certain measurement day could not be included in further analysis because no valid measurement was achieved. If a subject moved during image acquisition the segmentation of bone and soft tissue could be affected. Therefore, the measurements were validated and classified as follows: 1. 2. 3. 4.

The measurement shows no signs of blurriness. A minimal nonsharpness of the contours can be indicated. The measurement is blurred with small visible artificial lines inside the image. The contours of the cortical structures are discontinued and broken. The results of the measurement are not valid.

Only images with grades of 3 or less were considered valid and hence used in the final analyses. Another reason for an invalid measurement was that there had to be at least an 80% overlap in the length of the region scanned in follow-up measurements compared with that of the baseline measurement (common region). For evaluation, the bone volume is separated from the surrounding soft tissue by a threshold-based contour algorithm. The extracted bone volume thereafter is divided into cortical and trabecular volumes. Density measurements were carried out to determine the density of the entire volume (Dtot) as well as of the cortical (Dcort) and trabecular regions (Dtrab). The trabecular HR-PQCT IN WISE 2005

subregion was further divided to calculate the density in the outer (Dmeta) and inner (Dinn) regions of the trabecular network by peeling voxel by voxel the total trabecular area concentrically until less than 60% of the original area remains. The inner 60% defines Dinn, and the outer 40% defines the area for Dmeta.(41) The high isotropic resolution of HR-pQCT permits direct 3D assessment of intertrabecular distances and calculation of trabecular number (Tb.N).(42) Since the trabecular thickness (Tb.Th) is on average only 100 to 150 mm, and with the resolution of 82 mm used, only one to two voxels wide, a direct 3D measurement is not possible owing to partial-volume effect. Therefore, trabecular bone volume to tissue volume (BV/TV) was derived from trabecular volumetric BMD assuming a fixed mineralization of 1200 mg of hydroxyapatite per cubic centimeter for compact bone (BV/TV ¼ Dtrab/1200). From the densitometrically derived BV/TV and direct measure of Tb.N, trabecular thickness (Tb.Th) and trabecular separation (Th.Sp) are derived assuming plate-model geometry.(43) Additionally, the SD of the trabecular separation (Tb.Sp.SD, a measure of trabecular network heterogeneity) was calculated within the entire trabecular region. The thickness of the cortical layer (Ct.Th) also was measured.

Reproducibility measurements To estimate the short-term coefficient of variation (CV) of the method (both operator and system), two consecutive measurements with full repositioning of the volunteer was carried for the radius (n ¼ 18) and tibia (n ¼ 16) on the same day.

Statistical analysis Statistical analysis was based on results from the common region of all valid measurements. The CVs and their 95% CIs were calculated as described in prior work.(44) Linearity of the relationships was checked graphically and is not shown here. To examine baseline differences between groups, analysis of variance (ANOVA) evaluated BDC 9 data. To evaluate the effect of bed rest, recovery, and the impact of countermeasures, linear mixed-effects models(45) were used. Analysis first was conducted on absolute values with ‘‘group’’ (CON, EXE, and NUT) and ‘‘study date’’ (BDC 9, HDT 15, HDT 43, R þ 3, R þ 90, R þ 180, and R þ 360) 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 of less than 0.05, further testing was done: (1) The models were repeated using data on percentage change compared with baseline to try to rule out the potential effect of subtle differences in baseline data between groups, and then, (2) if the effect still persisted, subsequent two-group models (ie, CON versus EXE, CON versus NUT, and EXE versus NUT) using percentage-change data were conducted to examine which group(s) could have been responsible for the effect. Furthermore, a priori contrasts were done comparing days HDT 15 and beyond with baseline within each group as well as 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 Journal of Bone and Mineral Research

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Fig. 1. Sample images from the radius (left) and tibia (right). (Above) Scout views. (Center) Valid measurements. (Below) Invalid measurements. Arrows indicate movement artifacts.

these model terms that were less than 0.05 but greater than 0.01 were considered trends. For analysis of changes during and after bed rest, since 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 covariates and hence were 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; The R Foundation for Statistical Computing, Vienna, Austria).

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Results Two subjects from the EXE group did not attend R þ 90 testing, and one subject from the same group could not be measured on R þ 360; otherwise, all subjects attended testing. In addition, valid measurements could not be obtained on all measurement days (owing to movement artifacts or insufficient common region), despite repeated attempts. In the CON group, valid HRpQCT data could not be obtained from one subject each on HDT 15, HDT 43, and R þ 90 at the radius and from one subject on HDT 43. In the EXE group, valid data could not be obtained from the radius of one subject on R þ 90. In the NUT group, ARMBRECHT ET AL.

0.5%

Change compared to baseline (BDC)

Distal tibia

a

0.0%

Distal radius

Table 2. Short-Term Reproducibility as Coefficient of Variation CV (%)

a b

-0.5%

Radius (n ¼ 18)

a c

Parameter

-1.0% -1.5%

b

c

c

-2.0%

c

-2.5%

c -3.0%

0

15 43 3 30 60Recovery 90 Bed-rest

90 120

150

180 180

210

b Change compared to baseline (BDC)

Distal tibia 0.5% 0.0%

240

360 270

300

330

360

390

420

450

Distal radius

b c

-1.5%

c c

-2.0% -2.5% -3.0%

0

15 43 3 30 60Recovery 90 Bed-rest

90 120

150

180 180

210

240

(0.59, (0.39, (0.26, (0.55, (0.40, (0.32, (2.19, (2.52, (2.30, (1.41, (0.71,

1.15) 0.77) 0.52) 1.08) 0.78) 0.64) 4.29) 4.92) 4.51) 2.75) 1.39)

CV

CV 95% CI a

0.35 0.48 0.25a 0.85 0.16a 0.45 1.06a 1.35a 1.05a 0.77a 0.52a

(0.26, (0.36, (0.18, (0.63, (0.12, (0.33, (0.79, (1.00, (0.78, (0.58, (0.39,

0.53) 0.74) 0.38) 1.29) 0.24) 0.68) 1.62) 2.05) 1.60) 1.18) 0.79)

360 270

300

330

360

390

420

450

Fig. 2. Effect of 60 days of bed rest and recovery on trabecular density (above) and cortical thickness (below). Values are mean (SEM) percentage change compared with baseline. Significance indicated by ap < 0.05; b p < 0.01; cp < 0.001. Since there were no differences between groups, all subjects have been pooled (see text and Tables 3 and 4 for more detail).

valid measurements could not be obtained from one subject each on HDT 15 and HDT 43. Overall, five distal radius measurements but only one distal tibia measurement of a total of 165 were invalid. By design, the common region could not be less than 80% (see ‘‘Methods’’), and the mean (SD) common region was 88.5% (3.9%) at the distal radius and 90.7% (4.7%) at the distal tibia and did not differ between subject groups ( p
0.40).

Measurement precision Short-term reproducibility (CV) of density (total, trabecular, and cortical) parameters ranged from 0.36% to 1.02% (Table 2). The structural parameters (trabecular number Tb.N, trabecular thickness Tb.Th, cortical thickness Ct.Th) showed a reproducibility ranging from 0.52% to 3.19%. The CVs of Dtot, Dcomp, Tb.N, Tb.Th, Ct.Th, and TbSp were significantly better in the distal tibia than in the radius on a 0.05 level (data not shown).

Baseline data A significant difference in structural parameters between the radius and tibia at baseline was found only for cortical thickness. For comparisons between groups, for all parameters at the HR-PQCT IN WISE 2005

0.78 0.52 0.35 0.73 0.52 0.43 2.90 3.33 3.05 1.86 0.94

CV 95% CI

Dtot ¼ total density; Dtrab ¼ trabecular density; Dmeta ¼outer trabecular density; Dinn ¼inner trabecular density; Dcomp ¼ cortical density; BV/ TV ¼ bone volume-to-tissue volume; Tb.N ¼ trabecular number; Tb.Th ¼ trabecular thickness; TbSp ¼ trabecular separation; Tb.Sp. SD ¼ standard deviation of trabecular separation, a measure of trabecular network heterogeneity; Ct.Th ¼ cortical thickness; CI ¼ confidence interval a Significant difference between radius and tibia at a 0.05 level.

-0.5% -1.0%

Dtot Dtrab Dmeta Dinn Dcomp BV/TV Tb.N Tb.Th TbSp Tb.Sp.SD Ct.Th

CV

Tibia (n ¼ 16)

distal tibia, with the exception of Tb.Th and Tb.Sp. SD, there was some evidence for differences between the groups at baseline ( p  0.085). At the distal radius, for Dtot (group main effect on ANOVA of baseline data, p ¼ 0.016), Dmeta (p ¼ 0.099), Dcomp (p ¼ 0.012), and Ct.Th (p ¼ 0.021), there was some evidence for a difference between the groups at baseline. Table 5 presents the baseline data with all subjects pooled together, and Tables 3 and 4 present the data from the distal radius and distal tibia, respectively, in each group.

Effect of countermeasures Only for cortical thickness (Ct.Th) at the distal tibia and inner trabecular density (Dinn) at the distal radius was there some evidence (group  study-date interaction on ANOVA, p ¼ 0.019 for Ct.Th and p ¼ 0.025 for Dinn; Tables 3 and 4) for a different response between the groups. Analysis of data on percentage change compared with baseline (in an attempt to control for baseline differences between groups) showed persistence of this effect (group  study-date interaction on ANOVA, p  0.036 and p ¼ 0.031 respectively), with further two-group ANOVAs of the percentage-change data suggesting a greater loss in distal tibia cortical thickness (group  study-date interaction on two-group ANOVA, p  0.013) in the NUT group than in both the CON and EXE groups. For distal radius inner trabecular density, there was only weak evidence of a difference in the response of the EXE and NUT groups (group  study-date interaction on two-group ANOVA, p ¼ 0.051), with no clear pattern of difference in response in these two groups (Table 3). For all other parameters, there was no evidence of an impact of countermeasures on changes in HR-pQCT measures during and after bed rest (group  study-date interaction on ANOVA, p
0.057). Journal of Bone and Mineral Research

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Table 3. Effect of Bed Rest and Recovery on HR-pQCT Measures at the Distal Radius Study date Group

BDC 9

HDT 15

HDT 43

Rþ3

R þ 90

Dtot (mg/cm ), BDC 9: p ¼ 0.016; group  date: p ¼ 0.442 0.2 (0.5)% 0.0 (0.5)% 0.4 (0.4)% 0.1 (0.4)% 0.6 (0.3)%a 0.0 (0.3)% 0.4 (0.3)% 0.4 (0.5)% 0.2 (0.8)% 0.6 (0.7)% 0.7 (0.6)% 0.2 (0.7)% Dtrab (mg/cm3), BDC 9: p ¼ 0.254; group  date: p ¼ 0.107 0.0 (0.4)% 0.1 (0.4)% 0.5 (0.3)%a 0.3 (0.5)% a 0.9 (0.3)% 0.7 (0.4)% 0.8 (0.4)%a 0.2 (0.3)% 0.6 (0.5)% 1.2 (0.4)%b 1.1 (0.4)%b 0.5 (0.4)% Dmeta (mg/cm3), BDC 9: p ¼ 0.099; group  date: p ¼ 0.512 0.1 (0.4)% 0.2 (0.3)% 0.5 (0.4)% 0.1 (0.6)% 0.4 (0.3)% 0.0 (0.4)% 0.3 (0.3)% 0.2 (0.4)% 0.1 (0.4)% 0.4 (0.4)% 0.4 (0.5)% 0.1 (0.4)% Dinn (mg/cm3), BDC 9: p ¼ 0.392; group  date: p ¼ 0.025 0.3 (0.7)% 0.2 (0.7)% 0.5 (0.6)% 0.2 (0.8)% 1.7 (0.6)%b 1.3 (0.6)%a 1.4 (0.4)%b 0.7 (0.4)% 1.2 (0.6)%a 2.0 (0.5)%c 1.6 (0.5)%b 0.9 (0.5)% Dcomp (mg/cm3), BDC 9: p ¼ 0.012; group  date: p ¼ 0.319 0.1 (0.3)% 0.1 (0.3)% 0.2 (0.3)% 0.1 (0.2)% 0.3 (0.2)% 0.0 (0.2)% 0.2 (0.2)% 0.2 (0.3)% 0.4 (0.5)% 0.0 (0.4)% 0.0 (0.3)% 0.2 (0.3)% BV/TV (%), BDC 9: p ¼ 0.254; group  date: p ¼ 0.246 0.02 (0.05) 0.04 (0.04) 0.08 (0.05) 0.04 (0.08) 0.10 (0.05)a 0.09 (0.06) 0.10 (0.05)a 0.02 (0.05) 0.08 (0.06) 0.13 (0.05)a 0.13 (0.05)a 0.05 (0.05) Tb.N (mm1), BDC 9: p ¼ 1.000; group  date: p ¼ 0.739 1.2 (1.0)% 2.3 (1.5)% 2.8 (2.2)% 1.6 (0.8)% 0.9 (1.6)% 0.3 (1.6)% 1.2 (1.7)% 1.1 (2.0)% 0.1 (2.0)% 1.4 (1.6)% 0.1 (1.3)% 1.0 (0.8)% Tb.Th (mm), BDC 9: p ¼ 0.200; group  date: p ¼ 0.888 1.0 (1.1)% 2.3 (1.3)% 2.1 (2.3)% 0.8 (1.1)% 1.6 (1.5)% 0.5 (1.5)% 0.2 (1.8)% 1.3 (2.0)% 1.5 (1.8)% 0.1 (1.5)% 0.9 (1.2)% 0.4 (1.0)% TbSp (mm), BDC 9: p ¼ 0.966; group  date: p ¼ 0.833 1.2 (1.2)% 2.5 (1.5)% 2.7 (2.1)% 1.6 (0.9)% 0.8 (1.3)% 0.2 (1.5)% 0.8 (1.7)% 1.9 (2.1)% 0.0 (1.6)% 1.1 (1.9)% 0.1 (1.5)% 1.2 (0.8)% Tb.Sp SD (mm), BDC 9: p ¼ 0.501; group  date: p ¼ 0.927 0.5 (0.9)% 0.5 (1.1)% 1.4 (1.3)% 0.4 (1.1)% 0.5 (0.9)% 0.1 (0.8)% 0.3 (1.0)% 1.6 (1.2)% 0.3 (1.1)% 0.2 (0.7)% 0.8 (0.5)% 0.7 (0.5)% Ct.Th (mm), BDC 9: p ¼ 0.021; group  date: p ¼ 0.753 0.3 (0.8)% 0.1 (0.7)% 0.3 (0.6)% 0.0 (0.7)% 0.6 (0.4)% 0.3 (0.4)% 0.0 (0.4)% 0.5 (0.8)% 0.4 (1.7)% 0.6 (1.5)% 0.7 (1.3)% 0.2 (1.2)%

R þ 180

R þ 360

3

CON EXE NUT

345.5 (15.2) 333.1 (15.1) 283.8 (15.2)

CON EXE NUT

161.6 (9.7) 169.9 (9.7) 149.4 (9.7)

CON EXE NUT

229.1 (9.1) 233.1 (9.1) 210.3 (9.1)

CON EXE NUT

114.8 (10.3) 126.0 (10.3) 107.3 (10.3)

CON EXE NUT

914.0 (11.9) 897.4 (11.8) 856.4 (12.0)

CON EXE NUT

13.5 (0.8) 14.2 (0.8) 12.5 (0.8)

CON EXE NUT

1.770 (0.067) 1.77 0(0.070) 1.769 (0.068)

CON EXE NUT

0.076 (0.004) 0.080 (0.004) 0.071 (0.004)

CON EXE NUT

0.494 (0.022) 0.493 (0.023) 0.500 (0.023)

CON EXE NUT

0.208 (0.012) 0.198 (0.012) 0.192 (0.012)

CON EXE NUT

0.879 (0.045) 0.824 (0.045) 0.694 (0.045)

0.0 (0.5)% 0.3 (0.3)% 0.4 (0.6)%

0.4 (0.4)% 0.5 (0.4)% 0.2 (0.6)%

0.4 (0.5)% 0.9 (0.3)%a 0.3 (0.4)%

0.4 (0.5)% 1.2 (0.6)% 0.7 (0.6)%

0.4 (0.5)% 0.5 (0.3)% 0.1 (0.4)%

0.4 (0.4)% 0.6 (0.4)% 0.2 (0.5)%

0.4 (0.8)% 1.8 (0.4)%c 0.8 (0.5)%

0.3 (0.8)% 1.8 (1.0)% 1.3 (0.8)%

0.3 (0.3)% 0.0 (0.2)% 0.3 (0.3)%

0.2 (0.3)% 0.3 (0.3)% 0.5 (0.3)%

0.06 (0.06) 0.14 (0.04)b 0.06 (0.05)

0.06 (0.07) 0.16 (0.09) 0.09 (0.07)

2.7 (1.4)% 1.5 (1.3)% 1.2 (1.3)%

3.0 (1.5)% 1.6 (1.4)% 0.7 (0.9)%

2.1 (1.3)% 0.5 (1.3)% 0.5 (1.3)%

2.3 (1.8)% 0.7 (1.7)% 0.2 (1.4)%

2.6 (1.2)%a 1.4 (1.1)% 1.4 (1.3)%

3.1 (1.5)%a 1.5 (1.2)% 1.2 (1.0)%

0.0 (1.1)% 0.3 (0.9)% 0.7 (0.6)%

1.4 (0.9)% 0.3 (0.9)% 1.0 (0.6)%

0.4 (0.7)% 0.3 (0.4)% 0.5 (1.3)%

0.4 (0.7)% 0.2 (0.5)% 0.4 (1.3)%

Note: Values at baseline (BDC 9) are mean (SEM) in absolute values. With the exception of BV/TV, beyond this time point (at days 15 and 43 of bed rest [HDT] and days 3, 90, 180, and 360 of post–bed rest recovery [R þ]), values are mean (SEM) percentage difference to baseline value. For BV/TV, HDT 13 and beyond are mean (SEM) change in BV/TV. Dtot ¼ total density; Dtrab ¼ trabecular density; Dmeta ¼ outer trabecular density; Dinn ¼ inner trabecular density; Dcomp ¼ cortical density; BV/TV ¼ bone volume/total volume; Tb.N ¼ trabecular number; Tb.Th ¼ trabecular thickness; TbSp ¼ trabecular separation; Tb.Sp SD ¼ standard deviation of trabecular separation (indicator of matrix homogeneity); Ct.Th ¼ cortical thickness. a p < 0.05. b p < 0.01 c p < 0.001. These values indicate significance change on a given study date compared with baseline within each group. p Values next to parameter title indicate significance between groups at baseline (BDC 9) and whether evidence was present for an impact of the countermeasures on bone changes (group  date).

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Journal of Bone and Mineral Research

ARMBRECHT ET AL.

Table 4. Effect of Bed Rest and Recovery on HR-pQCT Measures at the Distal Tibia Study date Group

BDC 9

HDT 15

HDT 43

Rþ3

R þ 90

R þ 180

Dtot (mg/cm ), BDC 9: p ¼ 0.093; group  date: p ¼ 0.647 1.3 (0.8)% 2.5 (0.8)%b 1.8 (0.8)%a 1.5 (0.8)% b c 1.0 (0.3)% 1.5 (0.4)% 0.9 (0.4)%b 0.8 (0.3)%b c c c 1.3 (0.4)% 2.3 (0.4)% 1.3 (0.3)% 1.1 (0.4)%b 3 Dtrab (mg/cm ), BDC 9: p ¼ 0.045; group  date: p ¼ 0.433 0.6 (2.0)% 1.8 (1.5)% 3.8 (1.5)%a 2.7 (1.4)% 2.5 (1.5)% a b 1.0 (0.5)% 1.6 (0.6)% 2.4 (0.6)%c 1.3 (0.4)%b 1.4 (0.4)%b b 0.3 (0.7)% 1.0 (0.6)% 1.8 (0.6)% 1.0 (0.5)% 0.7 (0.6)% Dmeta (mg/cm3), BDC 9: p ¼ 0.085; group  date: p ¼ 0.330 0.3 (1.5)% 1.5 (1.2)% 2.9 (1.1)%b 2.5 (1.1)%a 2.0 (1.1)% a c 0.5 (0.3)% 1.1 (0.4)% 1.8 (0.5)% 0.9 (0.3)%c 0.8 (0.3)%b c a 0.3 (0.5)% 0.8 (0.4)% 1.5 (0.3)% 0.8 (0.3)% 0.8 (0.4)%a Dinn (mg/cm3), BDC 9: p ¼ 0.043; group  date: p ¼ 0.753 0.7 (2.7)% 2.2 (2.0)% 4.9 (2.0)%a 3.1 (2.0)% 3.0 (2.0)% a 1.4 (0.9)% 2.2 (0.9)% 3.0 (0.9)%b 1.6 (0.8)%a 1.8 (0.7)%a a 0.4 (1.1)% 1.3 (0.9)% 2.0 (1.0)% 1.1 (0.8)% 0.8 (0.8)% Dcomp (mg/cm3), BDC 9: p ¼ 0.048; group  date: p ¼ 0.057 0.0 (0.3)% 0.2 (0.3)% 0.9 (0.3)%b 0.6 (0.2)%a 0.3 (0.2)% b 0.1 (0.1)% 0.1 (0.1)% 0.4 (0.1)% 0.4 (0.2)%a 0.3 (0.1)%b a 0.1 (0.2)% 0.2 (0.2)% 0.4 (0.2)% 0.3 (0.1)% 0.4 (0.2)%a BV/TV (%), BDC 9: p ¼ 0.040; group  date: p ¼ 0.642 0.04 (0.24) 0.20 (0.18) 0.45 (0.17)a 0.34 (0.17) 0.29 (0.17) a a 0.15 (0.07) 0.24 (0.10) 0.38 (0.10)c 0.18 (0.06)b 0.19 (0.06)b b 0.05 (0.11) 0.14 (0.08) 0.25 (0.08) 0.14 (0.08) 0.10 (0.09) Tb.N (mm1), BDC 9: p ¼ 0.061; group  date: p ¼ 0.740 0.8 (1.5)% 1.4 (1.5)% 1.5 (1.5)% 1.0 (1.4)% 1.8 (1.3)% 0.2 (1.0)% 1.0 (0.9)% 0.8 (0.9)% 0.6 (1.6)% 1.6 (1.1)% 0.4 (1.9)% 0.6 (1.8)% 1.2 (1.8)% 1.6 (1.8)% 0.3 (2.0)% Tb.Th (mm), BDC 9: p ¼ 0.572; group  date: p ¼ 0.241 0.8 (1.6)% 0.3 (1.7)% 2.2 (1.5)% 1.2 (1.5)% 0.8 (1.9)% 1.5 (0.9)% 1.0 (0.9)% 1.9 (0.9)%a 0.8 (1.7)% 0.3 (1.2)% 1.2 (1.6)% 0.5 (1.4)% 0.8 (1.1)% 0.5 (1.2)% 0.8 (1.5)% TbSp (mm), BDC 9: p ¼ 0.041; group  date: p ¼ 0.523 0.9 (1.7)% 1.8 (1.6)% 2.0 (1.6)% 1.5 (1.6)% 2.0 (1.6)% 0.1 (1.0)% 1.3 (1.0)% 1.2 (1.1)% 0.8 (0.9)% 2.1 (0.7)%b 0.8 (1.7)% 0.4 (1.6)% 1.0 (1.5)% 1.4 (1.6)% 0.3 (1.6)% Tb.Sp SD (mm), BDC 9: p ¼ 0.103; group  date: p ¼ 0.374 0.0 (0.9)% 0.1 (0.9)% 0.9 (0.8)% 0.8 (0.8)% 0.4 (0.7)% 0.1 (0.6)% 0.7 (0.6)% 0.2 (0.6)% 0.1 (0.7)% 1.1 (0.8)% 0.1 (1.0)% 0.2 (1.4)% 0.5 (1.0)% 1.4 (1.0)% 0.2 (1.2)% Ct.Th (mm), BDC 9: p ¼ 0.021; group  date: p ¼ 0.019 2.0 (0.7)%b 1.4 (0.4)%c 0.8 (0.4)%a 0.2 (0.5)% 1.5 (0.5)%b a b a 0.2 (0.2)% 0.6 (0.2)% 0.9 (0.3)% 0.7 (0.4)% 0.3 (0.3)% 0.4 (0.7)% 2.2 (0.5)%c 3.8 (0.6)%c 1.8 (0.6)%b 1.4 (0.6)%a

R þ 360

3

CON EXE NUT

299.5 (13.8) 326.5 (13.6) 282.9 (13.6)

CON EXE NUT

154.0 (7.8) 178.9 (7.5) 170.4 (7.6)

CON EXE NUT

220.6 (8.1) 245.1 (7.8) 232.1 (7.8)

CON EXE NUT

108.6 (8.2) 133.6 (8.0) 128.4 (8.0)

CON EXE NUT

923.3 (12.1) 916.4 (11.9) 876.8 (11.9)

CON EXE NUT

12.8 (0.7) 14.9 (0.6) 14.2 (0.6)

CON EXE NUT

1.594 (0.066) 1.759 (0.065) 1.773 (0.070)

CON EXE NUT

0.081 (0.004) 0.085 (0.004) 0.081 (0.004)

CON EXE NUT

0.551 (0.022) 0.488 (0.021) 0.495 (0.022)

CON EXE NUT

0.243 (0.013) 0.209 (0.013) 0.209 (0.013)

CON EXE NUT

1.189 (0.062) 1.300 (0.062) 1.041 (0.062)

0.3 (1.0)% 0.6 (0.3)%a 0.4 (0.5)%

1.7 (0.8)%a 0.6 (0.4)% 1.0 (0.3)%b 3.7 (1.5)%a 1.5 (0.5)%b 1.3 (0.5)%a 2.9 (1.1)%b 0.9 (0.4)%b 1.0 (0.4)%a 4.4 (2.0)%a 2.1 (0.9)%a 1.8 (0.9)%a 0.3 (0.3)% 0.0 (0.1)% 0.1 (0.2)% 0.41 (0.18)a 0.21 (0.07)b 0.19 (0.09)a 3.1 (1.2)%b 1.5 (1.2)% 1.4 (1.7)% 0.0 (1.7)% 0.3 (1.3)% 0.3 (1.2)% 3.7 (1.4)%b 1.8 (0.8)%a 1.5 (1.5)% 1.7 (0.6)%b 1.1 (0.8)% 1.3 (0.9)% 0.6 (0.5)% 0.1 (0.3)% 0.6 (0.7)%

Note: Values at baseline (BDC 9) are mean (SEM) in absolute values. With the exception of BV/TV, beyond this time point (at days 15 and 43 of bed rest [HDT] and days 3, 90, 180, and 360 of post–bed rest recovery [R þ]) values are mean (SEM) percentage difference to baseline value. For BV/TV, HDT 13 and beyond are mean (SEM) change in BV/TV. Dtot ¼ total density; Dtrab ¼ trabecular density; Dmeta ¼ outer trabecular density; Dinn ¼ inner trabecular density; Dcomp ¼ cortical density; BV/TV ¼ bone volume/total volume; Tb.N ¼ trabecular number; Tb.Th ¼ trabecular thickness; TbSp ¼ trabecular separation; Tb.Sp SD ¼ standard deviation of trabecular separation (indicator of matrix homogeneity); Ct.Th ¼ cortical thickness. a p < 0.05. b p < 0.01. c p < 0.001. These values indicate significance change on a given study date compared with baseline within each group. p Values next to parameter title indicate significance between groups at baseline (BDC 9) and whether evidence was present for an impact of the countermeasures on bone changes (group  date).

HR-PQCT IN WISE 2005

Journal of Bone and Mineral Research

2405

Table 5. Effect of Bed Rest and Recovery on HR-pQCT Parameters at the Distal Radius and Distal Tibia (All Subjects Pooled) Study date Parameter

HDT 43

Rþ3

R þ 90

R þ 180

R þ 360

0.6 (0.2)%a

0.0 (0.2)%

0.4 (0.2)%

0.2 (0.2)%

0.3 (0.2)%

0.3 (0.2)%

0.5 (0.2)%a

0.7 (0.2)%b

0.8 (0.2)%c

0.2 (0.2)%

0.4 (0.2)%

0.7 (0.3)%a

BDC 9

HDT 15

Dtot (mg/cm3) p ¼ 0.002

320.779 (10.057)

Dtrab (mg/cm ) p < 0.001

160.258 (5.641)

Distal radius 3

Dmeta (mg/cm ) p ¼ 0.068

224.174 (5.436)

0.2 (0.2)%

0.3 (0.2)%

0.3 (0.2)%

0.0 (0.2)%

0.3 (0.2)%

0.4 (0.2)%

Dinn (mg/cm3) p < 0.001

116.237 (5.908)

1.0 (0.3)%b

1.5 (0.3)%c

1.4 (0.3)%c

0.7 (0.3)%a

1.2 (0.3)%c

1.2 (0.5)%b

Dcomp (mg/cm ) p ¼ 0.054

889.542 (8.171)

3

3

BV/TV (%) p < 0.001

13.4 (0.5)

0.0 (0.2)%

0.0 (0.1)%

0.2 (0.1)%

0.1 (0.1)%

0.1 (0.1)%

0.0 (0.2)%

0.04 (0.03)

0.09 (0.03)b

0.11 (0.03)c

0.04 (0.03)

0.07 (0.03)a

0.10 (0.04)a

Tb.N (mm1] p ¼ 0.021 Tb.Th (mm) p ¼ 0.050

1.773 (0.038) 0.075 (0.002)

0.2 (0.7)% 0.4 (0.7)%

1.3 (0.8)% 0.8 (0.8)%

1.3 (0.8)% 0.2 (0.8)%

1.5 (0.5)%b 0.7 (0.7)%

1.9 (0.6)%b 1.4 (0.6)%a

1.6 (0.6)%b 1.2 (0.8)%

TbSp (mm) p < 0.001

0.494 (0.012)

0.3 (0.7)%

1.3 (0.8)%

1.2 (0.9)%

1.7 (0.5)%c

2.2 (0.6)%c

2.1 (0.6)%c

Tb.Sp SD (mm) p ¼ 0.273

0.199 (0.007)

0.1 (0.5)%

0.4 (0.4)%

0.7 (0.4)%

0.4 (0.4)%

0.6 (0.4)%

0.9 (0.4)%a

Ct.Th (mm) p ¼ 0.672

0.798 (0.030)

0.4 (0.4)%

0.1 (0.4)%

0.1 (0.3)%

0.1 (0.3)%

0.0 (0.3)%

0.1 (0.4)%

Distal tibia Dtot (mg/cm3) p < 0.001

302.514 (8.409)

0.6 (0.2)%a

1.1 (0.2)%c

2.0 (0.2)%c

1.2 (0.2)%c

1.0 (0.2)%c

0.9 (0.2)%c

Dtrab (mg/cm3) p < 0.001

167.260 (4.787)

0.8 (0.4)%

1.2 (0.4)%b

2.3 (0.4)%c

1.3 (0.3)%c

1.2 (0.3)%c

2.0 (0.3)%c

Dmeta (mg/cm3) p < 0.001 Dinn (mg/cm3) p < 0.001

231.779 (4.904) 123.256 (5.029)

0.5 (0.3)% 1.0 (0.7)%

0.9 (0.3)%b 1.4 (0.6)%a

1.6 (0.2)%c 3.2 (0.6)%c

1.0 (0.2)%c 1.6 (0.5)%b

0.8 (0.2)%c 1.6 (0.5)%b

1.3 (0.2)%c 2.4 (0.6)%c

Dcomp (mg/cm3) p < 0.001

905.287 (7.802)

0.0 (0.1)%

0.1 (0.1)%a

0.5 (0.1)%c

0.3 (0.1)%c

0.3 (0.1)%c

0.1 (0.1)%

0.11 (0.06)

0.17 (0.05)b

0.31 (0.05)c

0.18 (0.04)c

0.16 (0.05)c

0.24 (0.05)c

0.0 (0.7)%

1.2 (0.6)%

1.2 (0.6)%

1.3 (0.8)%

1.4 (0.7)%

1.8 (0.6)%b

0.5 (0.8)%

0.2 (0.7)%

BV/TV (%) p < 0.001 Tb.N (mm1) p ¼ 0.001

13.9 (0.4) 1.709 (0.041)

Tb.Th (mm) p ¼ 0.011

0.082 (0.002)

1.0 (0.7)%

0.7 (0.6)%

1.5 (0.6)%a

0.6 (0.7)%

TbSp (mm) p < 0.001

0.511 (0.014)

0.1 (0.7)%

1.1 (0.7)%

1.5 (0.6)%a

1.3 (0.7)%

1.6 (0.6)%b

2.1 (0.6)%c

Tb.Sp SD (mm) p < 0.001

0.220 (0.008)

0.1 (0.4)%

0.8 (0.4)%

0.4 (0.4)%

0.7 (0.5)%

0.3 (0.5)%

1.5 (0.4)%c

Ct.Th (mm) p < 0.001

1.174 (0.041)

0.4 (0.2)%

1.0 (0.2)%c

1.4 (0.3)%c

1.1 (0.2)%c

0.6 (0.2)%b

0.1 (0.3)%

Note: Values at baseline (BDC 9) are mean (SEM) in absolute values. Beyond this time point (at days 15 and 43 of bed rest [HDT] and days 3, 90, 180, and 360 of post–bed rest recovery [R þ]) values are mean (SEM) percentage difference to baseline value. Dtot ¼ total density; Dtrab ¼ trabecular density; Dmeta ¼ outer trabecular density; Dinn ¼ inner trabecular density; Dcomp ¼ cortical density; BV/TV ¼ bone volume/total volume; Tb.N ¼ trabecular number; Tb.Th ¼ trabecular thickness; TbSp ¼ trabecular separation; Ct.Th ¼ cortical thickness. Only for Ct.Th at the distal tibia was there some evidence for a different response between the groups (see Table 4). For the distal radius at HDT 15, data were missing from two subjects, and excluding this time point from modeling results in insignificance of the study date effect on ANOVA for Dtot (p ¼ 0.11) but with the effect remaining for Dtrab and Dinn (p < 0.003). a p < 0.05. b p < 0.01. c p < 0.001. These values indicate significant change on a given study date compared with baseline. p Values next to parameter name indicate significance from ANOVA of changes over the course of the study with all groups pooled.

Effect of bed rest and recovery At the distal radius, strong effects (study-date main effect on ANOVA, p < 0.001) were seen for overall trabecular density (Dtrab), with the effect concentrated mainly in the inner region of the trabecular structure (Dinn) but not in the outer trabecular region (Dmeta, p ¼ 0.068). Both Dtrab and Dinn decreased during bed rest, with reductions persisting up to 1 year after bed rest (R þ 360; Table 5). Since BV/TV is derived from Dtrab, similar results were seen for this parameter. Strong effects (study date, p < 0.001) also were seen for an increase in trabecular separation (Tb.Sp) and a trend (study date, p ¼ 0.011) for a decrease in trabecular number (Tb.N), with both these effects first reaching significance on a priori comparisons with baseline 90 days after bed rest (R þ 90) and persisting up to R þ 360. At the distal tibia, strong effects (study-date main effect on ANOVA, p < 0.001) were seen for changes in all parameters

2406

Journal of Bone and Mineral Research

during and after bed rest, with the exception of trabecular thickness (Tb.Th; p ¼ 0.011; Table 5). Reductions in bone density (Dtot, Dtrab, Dmeta, Dinn, and Dcomp) were seen during bed rest, with this persisting up to 1 year after bed rest in some parameters. Cortical thickness (Ct.Th) decreased during bed rest and remained reduced up to 180 days after bed rest. Trabecular number decreased and trabecular separation increased (first significant on R þ 3), with these effects persisting up to 1 year after bed rest. Trabecular network heterogeneity (Tb.Sp. SD) also increased, but this first reached significance 1 year after bed rest. Similar to the radius, since BV/TV is derived from Dtrab, similar results were seen for both these variables.

Discussion In this study we evaluated the changes in bone density and microarchitecture during long-term bed rest using an HR-pQCT ARMBRECHT ET AL.

system. In terms of the impact of the countermeasures, there was only a trend for a greater loss in cortical thickness at the distal tibia in the nutrition group than in the other two groups, and it is unclear what impact a thinner cortex at baseline in this group may have had. There was little evidence for an impact of the exercise or nutrition countermeasures on the remaining bone density and bone structure parameters. In terms of the effect of bed rest and recovery as a whole, statistically significant effects were seen in density parameters at the distal tibia. At the distal radius, reductions in trabecular density, particularly in the innermost trabeculae, were seen, with no significant changes in other density parameters. Changes in bone structure parameters were seen, with trabecular separation increasing at both the distal tibia and distal radius, but these effects were first significant after bed rest. Reduction in trabecular number was similar in magnitude at the distal radius and distal tibia, but the effect was stronger, statistically, at the distal tibia. Cortical thickness decreased at the distal tibia only, with this effect reaching significance during bed rest. Although a number of effects were similar in magnitude, such as the magnitude of density and structure parameter changes or changes in trabecular number at the radius and tibia, the coefficients of variation from repeated measurements typically were higher at the radius than at the tibia, and structure parameters typically were less repeatable than density parameters. This may be part of the explanation why similar magnitudes of change were seen for a number of parameters, but some of these changes reached statistical significance at a later time point. The results showed that there was little effect of the countermeasures on the bone changes at the distal tibia and distal radius during and after bed rest. Only a trend was seen for a greater loss in cortical thickness at the distal tibia in the nutrition group, with no evidence for an impact of the exercise countermeasure on any parameters. Comparison of the current findings with other studies is difficult because only one other study has examined the effects of countermeasures on bone during bed rest in women, and in that study, DXA measurements were performed.(28) It may be possible to argue that the low numbers of subjects in each group, coupled with measurement repeatability, may have been part of the explanation for the nonsignificant impact of countermeasures. While this is conceivable, there was no consistency across parameters in, for example, losses in bone density or changes in bone structure in the exercise group that may suggest an (albeit nonsignificant) impact of the exercises. Also, examinations on the same subjects using standard pQCT did not show any impact of countermeasures in these same subjects,(46) whereas other exercise approaches(16,19,20) can have an impact on such measures of bone loss during and after bed rest. Hence we would argue that the exercise countermeasures actually were ineffective for preventing bone loss at the distal tibia and distal radius rather than issues of measurement error or low subject numbers playing a role. Typically, bone is better stimulated by dynamic loads that cause larger bone strains(47,48) and faster rates of bone deformation,(49–52) with certain minimum strain(53–56) and strain-rate(57) thresholds needing to be exceeded, although the number of loading cycles, among other factors, is important.(48,58–60) The conduct of comparatively low-load HR-PQCT IN WISE 2005

exercise (such as with treadmill running during lower body negative pressure in this study) typically is less effective for maintenance of bone during weightlessness(12,61,62) and bed rest,(63) with high-load resistive exercise, as performed on the infrequent schedule in this study, also generally less effective than other approaches during bed rest.(16) This study is the first to date to examine changes in bone density and structure using HR-pQCT during and after prolonged bed rest, a model of disuse. While it was possible to detect changes in bone structure, particularly at the distal tibia, these effects typically reached statistical significance later in the course of the study than the density parameters. One may be tempted to hypothesize, therefore, that first bone density reduces during bed rest, with later changes in bone structure. While this is possible, it is important to note that this study (consistent with prior work(64)) found the reproducibility for density parameters to be better than for trabecular structural parameters. When measurement variability is greater (eg, for trabecular structure parameters), this requires a larger effect size before an effect is statistically significant. Hence it is likely that changes in bone structure occurred on a similar time course to losses in bone density, not earlier or later, and the effects became statistically significant only at a later time point owing to greater measurement error. It is worthy to note that some of the structural changes first reach statistical significance after bed rest. In this regard, prior work has shown that bone loss continues for a number of weeks after bed rest, with the extent of bone loss becoming greater in the immediate post–bed rest period.(16,20,65) Thus, while the continued alteration of bone structure in the weeks after bed rest is a novel finding, it is not surprising. Also, contrary to what may be expected from prior findings in men during prolonged bed rest,(19,20) changes at the distal radius in women also occurred during bed rest. Although lesser in magnitude than those at the distal tibia, significant reductions in distal radius trabecular bone density, being greatest in the center of the trabecular structure, and increases in trabecular separation were seen. This suggests that changes at the distal radius do indeed occur in women during prolonged bed rest. In terms of the recovery of distal tibia and radius bone density and structure changes, a number of effects persisted until the final measurement date 1 year after bed rest. At the distal radius, significant reductions in trabecular density and number and increases in trabecular separation persisted. At the distal tibia, significant reductions in all bone density parameters and significant increases in trabecular separation persisted. This incomplete recovery of bone structure and density at 1 year after bed rest is understandable given that duration of time required for the recovery of bone is many orders of magnitude longer than the actual duration of exposure to inactivity or weightlessness.(66) The protein-leucine nutritional countermeasure also had no positive impact on the bone measures, with a trend for a greater loss in distal tibial cortical thickness in the nutrition group. From other nutritional studies, the effect of protein intake on bone metabolism is not clear. Some studies have shown an anabolic effect of higher protein intake on bone,(67) whereas others have shown a negative(68) or equivocal effect(69) on calcium Journal of Bone and Mineral Research

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metabolism. Furthermore, while leucine intake is important for muscle homeostasis,(70) there are no data available on the effect of leucine intake on bone or calcium metabolism. In any case, the high protein and leucine nutritional countermeasure, as performed, did not have any protective effects on changes in bone density and structure at the distal tibia and radius in during 60 days of bed rest in women. Some mention of the measurement methodology is appropriate. Aside from Tb.N, all other structural parameters are density-based. BV/TV in particular gives no additional information at all to Dtrab mainly because BV/TV is derived directly from Dtrab (BV/TV ¼ Dtrab/1200). The reader should note that since BV/TV is already a percent value, we chose to present the changes in BV/TV during bed rest as absolute change in BV/TV rather than calculate a percent change of a percentage value. For the remaining variables, including Dtrab, data are presented as percentage change compared with baseline. There are several potential concerns with the approach of measuring or calculating cortical and trabecular parameters in longitudinal studies. Since it is difficult to always examine the same bone region, the manufacturer provides software that automatically matches slices based on cross-sectional area and limits the analyzed region to the slices common to baseline and all follow-ups. This could lead to mismatching in longitudinal studies where periosteal apposition and changes in crosssectional area could occur. Alternatively, an improved short- and medium-term reproducibility can be achieved using a 3D image registration technique compared with the default slicematching approach.(71) A second concern applies to the segmentation and assessment of cortical bone. Segmentation of cortical and trabecular compartments is performed using an edge-enhancement and threshold procedure; this segmentation provides the basis for the subsequent morphometric and densitometric analyses. This segmentation procedure may fail to capture the cortical structure in subjects with very porous or thin cortexes.(72,73) Additionally, the cortical thickness is not measured directly but rather is calculated from cortical area and perimeter. More sophisticated approaches to segmentation of cortical bone have been proposed,(74) including a direct 3D assessment of cortical thickness and quantification of intracortical porosity and canal diameter.(75,76) In conclusion, this study showed that changes in bone structure were measurable in women during and after prolonged bed rest using HR-pQCT and occurred concurrent with changes in bone density. The changes in bone structure appeared to occur on a similar time course as the changes in bone density but, owing to greater structure-measure variability, reached statistical significance at a later time point. While losses in bone density generally were greater in magnitude at the distal tibia than at the distal radius, reductions in trabecular thickness and increases in trabecular separation were similar in magnitude at both the radius and distal tibia. Cortical thinning was significant only at the distal tibia. The findings of this study also suggest that the exercise and nutrition countermeasures, as performed, were ineffective in preventing bone loss at the distal tibia and distal radius in women after 60 days of bed rest, as measured with HRpQCT.

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Disclosures All the authors state that they have no conflicts of interest.

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’s International Investigation for Space Exploration) was sponsored by the European Space Agency (ESA), the US National Aeronautics and Space Administration (NASA), the Canadian Space Agency (CSA), 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. Authors’ roles: GA: Co-leader of project, contributions to conception and design, interpretation of data, drafting of manuscript. DLB: Statistical analysis, generation of tables/figures, drafting of manuscript with GA, responsible for statistical analysis. MB: Acquisition and analysis of data during bed rest, quality assurance of data. GB: Acquisition and analysis of data during recovery phase, quality assurance of data. CA: Co-investigator, critical review of manuscript. RR: Co-investigator, critical review of manuscript. DF: Leader of project, secured funding, critical review of manuscript.

References 1. Consensus development conference: diagnosis, prophylaxis, and treatment of osteoporosis. Am J Med. 1993;94(6):646–50. 2. Faulkner KG. Bone matters: are density increases necessary to reduce fracture risk? J Bone Miner Res. 2000;15(2):183–7. 3. Hildebrand T, Ru¨egsegger P. Quantification of bone microarchitecture with the structure model index. Comput Methods Biomech Biomed Engin. 1997;1(1):15–23. 4. Laib A, Ha¨uselmann HJ, Ru¨egsegger P. In vivo high resolution 3D-QCT of the human forearm. Technol Health Care. 1998;6(5–6):329–37. 5. Laib A, Hildebrand T, Ha¨uselmann HJ, Ru¨egsegger P. Ridge number density: a new parameter for in vivo bone structure analysis. Bone. 1997;21(6):541–6. 6. Laib A, Newitt DC, Lu Y, Majumdar S. New model-independent measures of trabecular bone structure applied to in vivo highresolution MR images. Osteoporos Int. 2002;13(2):130–6. 7. Laib A, Ru¨egsegger P. Comparison of structure extraction methods for in vivo trabecular bone measurements. Comput Med Imaging Graph. 1999;23(2):69–74. 8. Laib A, Ru¨egsegger P. Calibration of trabecular bone structure measurements of in vivo three-dimensional peripheral quantitative computed tomography with 28-microm-resolution microcomputed tomography. Bone. 1999;24(1):35–9. 9. Parfitt AM, Mathews CH, Villanueva AR, Kleerekoper M, Frame B, Rao DS. Relationships between surface, volume, and thickness of iliac trabecular bone in aging and in osteoporosis. Implications for the microanatomic and cellular mechanisms of bone loss. J Clin Invest. 1983;72(4):1396–409.

ARMBRECHT ET AL.

10. Pavy-Le Traon A, Heer M, Narici MV, Rittweger J, Vernikos J. From space to Earth: advances in human physiology from 20 years of bed rest studies (1986-2006). Eur J Appl Physiol. 2007;101(2):143–94. 11. Nicogossian AE, Dietlein LF. Microgravity Simulation and Analogues. In: Nicogossian AE (ed.) Space physiology and Medicine, 1982; vol. Lea & Febiger, Philadelphia, pp 240–248. 12. Le Blanc A, Schneider V, Shackelford L, West S, Oganov V, Bakulin A, Voronin L. Bone mineral and lean tissue loss after long duration space flight. J Musculoskelet Neuronal Interact. 2000;1(2):157–60. 13. Greenspan SL, Maitland LA, Myers ER, Krasnow MB, Kido TH. Femoral bone loss progresses with age: a longitudinal study in women over age 65. J Bone Miner Res. 1994;9(12):1959–65. 14. Reid IR, Ames RW, Evans MC, Gamble GD, Sharpe SJ. Effect of calcium supplementation on bone loss in postmenopausal women. N Engl J Med. 1993;328(7):460–4. 15. Ito M, Nakamura T, Tsurusaki K, Uetani M, Hayashi K. Effects of menopause on age-dependent bone loss in the axial and appendicular skeletons in healthy Japanese women. Osteoporos Int. 1999; 10(5):377–83. 16. Belavy´ DL, Beller G, Armbrecht G, Perschel FH, Fitzner R, Bock O, Bo¨rst H, Degner C, Gast G, Felsenberg D. Evidence for an additional effect of whole-body vibration above resistive exercise alone in preventing bone loss during prolonged bed-rest. Osteoporosis Int. 2011;22(5): 1581–91. 17. Inoue M, Tanaka H, Moriwake T, Oka M, Sekiguchi C, Seino Y. Altered biochemical markers of bone turnover in humans during 120 days of bed rest. Bone. 2000;26(3):281–6. 18. Rittweger J, Felsenberg D. Patterns of bone loss in bed-ridden healthy young male subjects: results from the Long Term Bed Rest Study in Toulouse. J Musculoskelet Neuronal Interact. 2003;3(4):290–1 discussion 292-4. 19. Rittweger J, Frost HM, Schiessl H, Ohshima H, Alkner B, Tesch P, Felsenberg D. Muscle atrophy and bone loss after 90 days bed rest and the effects of flywheel resistive exercise and pamidronate: results from the LTBR study. Bone. 2005;36(6):1019–29. 20. Rittweger J, Beller G, Armbrecht G, Mulder E, Buehring B, Gast U, Dimeo F, Schubert H, de Haan A, Stegeman DF, Schiessl H, Felsenberg D. Prevention of bone loss during 56 days of strict bed rest by sidealternating resistive vibration exercise. Bone. 2010;46(1):137–47. 21. Van der Wiel HE, Lips P, Nauta J, Kwakkel G, Hazenberg G, Netelenbos JC, Van der Vijgh WJF. Intranasal calcitonin suppresses increased bone resorption during short-term immobilization: a double-blind study of the effects of intranasal calcitonin on biochemical parameters of bone turnover. J Bone Miner Res. 1993;8(12):1459–65. 22. Zerwekh JE, Ruml LA, Gottschalk F, Pak CYC. The effects of twelve weeks of bed rest on bone histology, biochemical markers of bone turnover, and calcium homeostasis in eleven normal subjects. J Bone Miner Res. 1998;13(10):1594–1601. 23. Heer M, Kamps N, Biener C, Korr C, Boerger A, Zittermann A, Stehle P, Drummer C. Calcium metabolism in microgravity. Eur J Med Res. 1999;4(9):357–60. 24. Scheld K, Zittermann A, Heer M, Herzog B, Mika C, Drummer C, Stehle P. Nitrogen metabolism and bone metabolism markers in healthy adults during 16 weeks of bed rest. Clin Chem. 2001;47(9):1688–95. 25. Smith SM, Heer M. Calcium and bone metabolism during space flight. Nutrition. 2002;18(10):849–52. 26. Smith SM, Nillen JL, Leblanc A, Lipton A, Demers LM, Lane HW, Leach CS. Collagen cross-link excretion during space flight and bed rest. J Clin Endocrinol Metab. 1998;83(10):3584–91.

measure to disuse-induced bone loss. J Appl Physiol. 2004;97(1): 119–29. 29. Lips P. Epidemiology and predictors of fractures associated with osteoporosis. Am J Med. 1997;103(2A):3S–8S discussion 8S-11S. 30. Frost HM. From Wolff’s law to the Utah paradigm: insights about bone physiology and its clinical applications. Anat Rec. 2001;262: 398–419. 31. Armbrecht G, Belavy´ DL, Gast G, Bongrazio M, Touby F, Beller G, Roth HJ, Perschel FH, Rittweger J, Felsenberg D. Resistive vibration exercise attenuates bone and muscle atrophy in 56 days of bed rest: biochemical markers of bone metabolism. Osteoporosis Int. 2010; 21(4):597–607. 32. Trappe TA, Burd NA, Louis ES, Lee GA, Trappe SW. Influence of concurrent exercise or nutrition countermeasures on thigh and calf muscle size and function during 60 days of bed rest in women. Acta Physiol (Oxf). 2007;191(2):147–59. 33. Alkner BA, Tesch PA. Knee extensor and plantar flexor muscle size and function following 90 days of bed rest with or without resistance exercise. Eur J Appl Physiol. 2004;93(3):294–305. 34. Alkner BA, Tesch PA. Efficacy of a gravity-independent resistance exercise device as a countermeasure to muscle atrophy during 29day bed rest. Acta Physiol Scand. 2004;181(3):345–57. 35. Berg HE, Tesch A. A gravity-independent ergometer to be used for resistance training in space. Aviat Space Environ Med. 1994;65(8): 752–6. 36. Trappe S, Trappe T, Gallagher P, Harber M, Alkner B, Tesch P. Human single muscle fibre function with 84 day bed-rest and resistance exercise. J Physiol. 2004;557(Pt 2):501–13. 37. Lee SM, Bennett BS, Hargens AR, Watenpaugh DE, Ballard RE, Murthy G, Ford SR, Fortney SM. Upright exercise or supine lower body negative pressure exercise maintains exercise responses after bed rest. Med Sci Sports Exerc. 1997;29(7):892–900. 38. Watenpaugh DE, Ballard RE, Schneider SM, Lee SM, Ertl AC, William JM, Boda WL, Hutchinson KJ, Hargens AR. Supine lower body negative pressure exercise during bed rest maintains upright exercise capacity. J Appl Physiol. 2000;89(1):218–27. 39. Cao P, Kimura S, Macias BR, Ueno T, Watenpaugh DE, Hargens AR. Exercise within lower body negative pressure partially counteracts lumbar spine deconditioning associated with 28-day bed rest. J Appl Physiol. 2005;99(1):39–44. 40. Biolo G, Ciocchi B, Stulle M, Bosutti A, Barazzoni R, Zanetti M, Antonione R, Lebenstedt M, Platen P, Heer M, Guarnieri G. Calorie restriction accelerates the catabolism of lean body mass during 2 wk of bed rest. Am J Clin Nutr. 2007;86(2):366–72. 41. Dalzell N, Kaptoge S, Morris N, Berthier A, Koller B, Braak L, van Rietbergen B, Reeve J. Bone micro-architecture and determinants of strength in the radius and tibia: age-related changes in a populationbased study of normal adults measured with high-resolution pQCT. Osteoporos Int. 2009;20(10):1683–94. 42. Hildebrand T, Ru¨egsegger P. A new method for the model-independent assessment of thickness in three-dimensional images. J Microsc. 2003;185(1):67–75. 43. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, Recker RR. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res. 1987;2(6): 595–610. 44. Glu¨er CC, Blake G, Lu Y, Blunt BA, Jergas M, Genant HK. Accurate assessment of precision errors: how to measure the reproducibility of bone densitometry techniques. Osteoporos Int. 1995;5(4):262–70.

27. Vico L, Hinsenkamp M, Jones D, Marie PJ, Zallone A, Cancedda R. Osteobiology, strain, and microgravity. Part II: studies at the tissue level. Calcif Tissue Int. 2001;68(1):1–10.

45. Pinheiro JC, Bates DM. Mixed-effects models in S and S-PLUS. 1st ed. Berlin: Springer; 2000.

28. Shackelford LC, LeBlanc AD, Driscoll TB, Evans HJ, Rianon NJ, Smith SM, Spector E, Feeback DL, Lai D. Resistance exercise as a counter-

46. Beller G, Belavy´ DL, Sun L, Armbrecht G, Alexandre C, Felsenberg D. WISE-2005: Bed-rest induced changes in bone mineral density in

HR-PQCT IN WISE 2005

Journal of Bone and Mineral Research

2409

women during 60 days simulated microgravity. Bone. 2011. DOI: 10.1016/j.bone.2011.06.021. 47. Rubin CT, Lanyon LE. Regulation of bone mass by mechanical strain magnitude. Calcif Tissue Int. 1985;37(4):411–7. 48. Srinivasan S, Ausk BJ, Poliachik SL, Warner SE, Richardson TS, Gross TS. Rest-inserted loading rapidly amplifies the response of bone to small increases in strain and load cycles. J Appl Physiol. 2007; 102(5):1945–52. 49. Mosley JR, Lanyon LE. Strain rate as a controlling influence on adaptive modeling in response to dynamic loading of the ulna in growing male rats. Bone. 1998;23(4):313–8. 50. O’Connor JA, Lanyon LE, MacFie H. The influence of strain rate on adaptive bone remodelling. J Biomech. 1982;15(10):767–81. 51. Turner CH, Owan I, Takano Y. Mechanotransduction in bone: role of strain rate. Am J Physiol. 1995;269 (3 Pt 1): E438–42. 52. Rubin C, Turner AS, Bain S, Mallinckrodt C, McLeod K. Anabolism. Low mechanical signals strengthen long bones. Nature. 2001;412(6847): 603–4. 53. Turner CH, Forwood MR, Rho JY, Yoshikawa T. Mechanical loading thresholds for lamellar and woven bone formation. J Bone Miner Res. 1994;9(1):87–97. 54. Hsieh YF, Robling AG, Ambrosius WT, Burr DB, Turner CH. Mechanical loading of diaphyseal bone in vivo: the strain threshold for an osteogenic response varies with location. J Bone Miner Res. 2001; 16(12):2291–7. 55. Hsieh YF, Turner CH. Effects of loading frequency on mechanically induced bone formation. J Bone Miner Res. 2001;16(5):918–24. 56. De Souza RL, Matsuura M, Eckstein F, Rawlinson SC, Lanyon LE, Pitsillides AA. Non-invasive axial loading of mouse tibiae increases cortical bone formation and modifies trabecular organization: a new model to study cortical and cancellous compartments in a single loaded element. Bone. 2005;37(6):810–8. 57. Turner CH, Forwood MR, Otter MW. Mechanotransduction in bone: do bone cells act as sensors of fluid flow? FASEB J. 1994;8(11):875–8. 58. Rubin CT, Lanyon LE. Regulation of bone formation by applied dynamic loads. J Bone Joint Surg Am. 1984;66(3):397–402. 59. Cullen DM, Smith RT, Akhter MP. Bone-loading response varies with strain magnitude and cycle number. J Appl Physiol. 2001; 91(5):1971–6. 60. Umemura Y, Ishiko T, Yamauchi T, Kurono M, Mashiko S. Five jumps per day increase bone mass and breaking force in rats. J Bone Miner Res. 1997;12(9):1480–5. 61. Oganov VS, Grigoriev AI, Voronin LI, Rakmanov AS, Bakulin AV, Schneider V, LeBlanc A. Bone mineral density in cosmonauts after 4.5-6 month long flights aboard orbital station MIR. Aerospace Environ. Med. 1992;26(5–6):20–24. 62. Le Blanc A, Lin C, Shackelford L, Sinitsyn V, Evans H, Belichenko O, Schenkman B, Kozlovskaya I, Oganov V, Bakulin A, Hedrick T, Feeback D. Muscle volume, MRI relaxation times (T2), and body composition after spaceflight. J Appl Physiol. 2000;89(6):2158–64.

2410

Journal of Bone and Mineral Research

63. Smith SM, Zwart SR, Heer MA, Baecker N, Evans HJ, Feiveson AH, Shackelford LC, Leblanc AD. Effects of artificial gravity during bed rest on bone metabolism in humans. J Appl Physiol. 2009;107(1):47–53. 64. Boutroy S, Bouxsein ML, Munoz F, Delmas PD. In vivo assessment of trabecular bone microarchitecture by high-resolution peripheral quantitative computed tomography. J Clin Endocrinol Metab. 2005; 90(12):6508–15. 65. Rittweger J, Felsenberg D. Recovery of muscle atrophy and bone loss from 90 days bed rest: results from a one-year follow-up. Bone. 2009;44(2):214–24. 66. Sibonga JD, Evans HJ, Sung HG, Spector ER, Lang TF, Oganov VS, Bakulin AV, Shackelford LC, LeBlanc AD. Recovery of spaceflightinduced bone loss: bone mineral density after long-duration missions as fitted with an exponential function. Bone. 2007;41(6):973–8. 67. Dawson-Hughes B, Harris SS. Calcium intake influences the association of protein intake with rates of bone loss in elderly men and women. Am J Clin Nutr. 2002;75(4):773–9. 68. Lutz J. Calcium balance and acid-base status of women as affected by increased protein intake and by sodium bicarbonate ingestion. Am J Clin Nutr. 1984;39(2):281–8. 69. Pannemans DL, Schaafsma G, Westerterp KR. Calcium excretion, apparent calcium absorption and calcium balance in young and elderly subjects: influence of protein intake. Br J Nutr. 1997;77(5): 721–9. 70. Rennie MJ. Exercise- and nutrient-controlled mechanisms involved in maintenance of the musculoskeletal mass. Biochem Soc Trans. 2007;35(Pt 5):1302–5. 71. MacNeil JA, Boyd SK. Improved reproducibility of high-resolution peripheral quantitative computed tomography for measurement of bone quality. Med Eng Phys. 2008;30(6):792–9. 72. Davis KA, Burghardt AJ, Link TM, Majumdar S. The effects of geometric and threshold definitions on cortical bone metrics assessed by in vivo high-resolution peripheral quantitative computed tomography. Calcif Tissue Int. 2007;81(5):364–71. 73. Kazakia GJ, Hyun B, Burghardt AJ, Krug R, Newitt DC, de Papp AE, Link TM, Majumdar S. In vivo determination of bone structure in postmenopausal women: a comparison of HR-pQCT and high-field MR imaging. J Bone Miner Res. 2008;23(4):463–74. 74. Buie HR, Campbell GM, Klinck RJ, MacNeil JA, Boyd SK. Automatic segmentation of cortical and trabecular compartments based on a dual threshold technique for in vivo micro-CT bone analysis. Bone. 2007;41(4):505–15. 75. Burghardt AJ, Kazakia GJ, Ramachandran S, Link TM, Majumdar S. Age- and gender-related differences in the geometric properties and biomechanical significance of intracortical porosity in the distal radius and tibia. J Bone Miner Res. 2010;25(5):983–93. 76. Nishiyama KK, Macdonald HM, Buie HR, Hanley DA, Boyd SK. Postmenopausal women with osteopenia have higher cortical porosity and thinner cortices at the distal radius and tibia than women with normal aBMD: an in vivo HR-pQCT study. J Bone Miner Res. 2010; 25(4):882–90.

ARMBRECHT ET AL.