Effects of artificial gravity during bed rest on bone metabolism in humans

Abstract

We report results from a study designed to explore the utility of artificial gravity (AG) as a countermeasure to bone loss induced by microgravity simulation. After baseline testing, 15 male subjects underwent 21 days of 6° head-down bed rest to simulate the deconditioning associated with spaceflight. Eight of the subjects underwent 1 h of centrifugation (AG; 1 G_z at the heart, 2.5 G_z at the feet) each day for 21 days, whereas seven of the subjects served as untreated controls (Con). Blood and urine were collected before, during, and after bed rest for bone marker determinations. Bone mineral density (BMD) and bone mineral content (BMC) were determined by dual-energy X-ray absorptiometry and peripheral quantitative computerized tomography before and after bed rest. Urinary excretion of bone resorption markers increased during bed rest, but the AG and Con groups did not differ significantly. The same was true for serum C-telopeptide. During bed rest, bone alkaline phosphatase (ALP) and total ALP tended to be lower in the AG group (P = 0.08, P = 0.09). Neither BMC nor BMD changed significantly from the pre-bed rest period in AG or Con groups, and the two groups were not significantly different. However, when AG and Con data were combined, there was a significant (P < 0.05) effect of time for whole body total BMC and total hip and trochanter BMD. These data failed to demonstrate efficacy of this AG prescription to prevent the changes in bone metabolism observed during 3 wk of bed rest.

on earth, bone homeostasis depends a great deal on mechanical loading. The mechanical loading provided by gravity is coupled with bone remodeling, as shown by cellular responses to partial gravity or hypergravity, but the precise mechanism of this coupling is unclear (25, 32, 44, 51). Spaceflight and bed rest studies produce disuse or decreased mechanical loading of muscles, which has a negative effect on bone (18, 21, 30, 36, 42).

On 4- to 6-mo spaceflight missions, it is estimated that the rate of bone mineral loss ranges from 0.5 to 1.5% per month (18, 19, 47) and that over 90% of crew members on these missions lose 5% or more of their bone mineral density (BMD) in at least one site (21). Skylab, Mir, and International Space Station (ISS) data indicate that the degree of change in bone metabolism is related to mission duration (35, 40, 50), but it is unknown whether bone loss will continue with longer exploration missions (29). These data also show that bone mineral is not uniformly lost from all parts of the skeleton or type of bone during spaceflight (21). For example, in the hip, it is estimated that 90% of mineral loss from bone of ISS crew members is from cortical bone (18).

Bone resorption increases in the first 2 wk of spaceflight, as evidenced by increased urinary concentrations of N-telopeptide and pyridinium cross-links (6, 7, 38, 40, 41). Bone formation is unchanged or decreases during spaceflight (7, 40, 41, 47). Calcium absorption in astronauts has been observed to decrease (40, 53) with increased calcium excretion (40, 49, 50, 53). Together, increased resorption and decreased or unchanged bone formation, with decreased calcium absorption and increased calcium excretion, yield an overall negative calcium balance during long-duration spaceflights.

Minimizing the risk of fracture and maximizing the ability of crew members to perform critical tasks when they return to a nominal gravity or reduced gravity environment will be important on long-duration missions, and maintaining bone will aid in both of these actions. To date, bone loss during spaceflight has not been effectively mitigated by countermeasures (21, 42).

In studies of humans and some animals (15, 23, 24, 27, 45), artificial gravity (AG) or hypergravity has been shown to positively affect bone (or related markers), and for this reason, among other benefits, it has been brought to the attention of the National Aeronautics and Space Administration (NASA). Studies in trainee fighter pilots exposed to positive sustained accelerative forces (G_z) over a period of 8–12 mo have shown that positive G_z-induced loading has an osteogenic effect on bone in a site-specific manner. Regions that were maximally strained (head and spine due to helmet and mask assembly) had increased BMD and bone mineral content (BMC) (23, 24). Studies of mature animals show that centrifugation can similarly increase bone density in a site-specific manner (27). Furthermore, Vernikos et al. (45) reported that intermittent exposure to 1 G_z (by standing or walking) during a 4-day head-down tilt bed rest was effective in preventing elevated urinary calcium that typically occurs during bed rest. The optimal AG prescription for bone, including dose, duration, and frequency of centrifugation, is unknown. One goal of the NASA Artificial Gravity Pilot Study was to determine the effectiveness of one AG prescription to mitigate bone loss during bed rest. Our primary hypothesis was that AG would result in a mitigation of the increase in bone resorption associated with bed rest, as indicated by biochemical markers of bone metabolism.

METHODS

Design.

This study consisted of a 21-day period of 6° head-down tilt (HDT) bed rest, with a 12-day period of data collection before bed rest and a 7-day data collection period after bed rest. The bed rest and AG protocols for this study are described in detail in a companion to this article (54). Briefly, during the bed rest phase of the study, subjects were confined to strict 6° HDT bed rest. Subject monitors outside each room ensured round-the-clock compliance. A 6° HDT gurney was used for transport to the centrifuge facility and the shower.

Diets were controlled and were matched for all subjects by relative study day (that is, on any given day, all subjects consumed the same menu). Food quantities for each individual were adjusted to maintain energy intake at a level that would maintain body mass. Details and nutritional aspects of the study have been published elsewhere (58). Blood and urine samples were collected before, during, and after bed rest for analysis of markers of bone metabolism.

Subjects.

A total of 15 healthy male subjects participated in this study. The mean (±SD) age was 27 ± 2 yr for the control (Con) group (n = 7) and 31 ± 3 yr for the artificial gravity (AG) group (n = 8). The Con and AG subjects had an average height of 176 ± 7 and 175 ± 6 cm, respectively. Con subjects weighed 82 ± 8 kg, and AG subjects weighed 81 ± 9 kg.

Treatments.

During the bed rest phase, Con and AG subjects were transported daily on a 6° HDT gurney to the centrifuge facility, where they were transferred to the centrifuge arm (at 6° HDT) and monitored. AG subjects received 1 h of centrifugation, whereas Con subjects did not. Artificial gravity was produced by rotating the subjects on a centrifuge arm with a 3.0-m radius (54). The subjects were allowed to perform antiorthostatic maneuvers (heel raises and shallow knee bends) while spinning but were otherwise passive.

All study protocols were approved by the Johnson Space Center Committee for the Protection of Human Subjects, the University of Texas Medical Branch (UTMB) Institutional Review Board, and the UTMB General Clinical Research Center Advisory Committee. Subjects provided written, informed consent before they were enrolled in the study.

Biological sample collection and processing.

Pre-bed rest blood samples were collected 9 days before bed rest and immediately before bed rest began. Blood was also collected on bed rest days (BR)8, BR15, and BR21 (the last day of bed rest) and again 8 days after bed rest ended (BR+8). Urine was collected throughout the study, but key analyses were performed only on days BR−9, BR−8, BR−4, BR−3, BR−2, and BR−1 before bed rest, on days BR8, BR9, BR15, BR16, BR20, and BR21 during bed rest, and days BR0, BR+1, BR+6, and BR+7 after bed rest. Subjects were fasting for 8 h before each blood draw.

Blood samples were collected into appropriate tubes and processed to yield serum. All single-void samples of urine were collected in individual bottles and stored in coolers until they were processed (within 24 h). Twenty-four-hour urine pools were created, pH was measured, and aliquots were prepared and frozen for analysis. All samples were stored at −80°C until analysis. Fecal samples were collected into individual containers and frozen at −80°C until they were processed. Processing of these samples consisted of homogenization and microwave digestion in nitric acid, as previously reported (39, 40).

Biochemical analyses.

Most analyses were performed using standard commercially available techniques, as described previously (40, 41, 55, 56). Serum, urinary, and fecal calcium concentrations were measured using atomic absorption spectrometry. Ionized calcium was determined using ion-sensitive electrode techniques (i-STAT; Princeton, NJ). Serum intact parathyroid hormone (PTH), receptor activator for nuclear factor-κB ligand (RANKL), and osteoprotegerin (OPG) were measured using immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA, for PTH; Alpco Diagnostics, Salem, NH, for RANKL and OPG). The vitamin D metabolites 25-hydroxycholecalciferol (25-OH vitamin D) and 1,25-dihydroxycholecalciferol [1,25-(OH)₂ vitamin D] were also determined using commercially available RIA kits (DiaSorin, Stillwater, MN). Bone alkaline phosphatase (ALP) was measured using ELISA (Quidel, Santa Clara, CA), total ALP was analyzed using a Beckman SYNCHRON CX7 automated clinical chemistry system (Beckman Coulter, Brea, CA), and serum osteocalcin was measured using a commercial RIA kit (Biomedical Technologies, Stoughton, MA). Another bone formation marker, procollagen type I N propeptide (PINP), was analyzed using commercially available RIA kits (Orion Diagnostica, Espoo, Finland).

Bone resorption markers were also determined. In serum, tartrate-resistant acid phosphatase (TRAP-C) was analyzed using commercially available ELISA (SBA Sciences BioCity, Turku, Finland), as was COOH-terminal cross-linking telopeptide of type I collagen (C-telopeptide; CrossLaps ELISA, Nordic Bioscience, Herlev, Denmark). Urinary collagen cross-links were determined: NH₂-terminal cross-linking telopeptide of type I collagen (N-telopeptide; Osteomark, Ostex International, Seattle, WA), and pyridinoline and deoxypyridinoline (PYD and DPD, respectively; Pyrilinks, Quidel). Cross-link data were expressed as nanomoles of excretion per day, because we have shown that this reduces within-subject variability (37).

Bone imaging analyses.

Bone density was determined before and after bed rest. All scans were acquired by the same operator to ensure consistency of positioning and were analyzed by Johnson Space Center Bone and Mineral Laboratory personnel.

BMD was measured by dual-energy X-ray absorptiometry (DXA Hologic Discovery W) before bed rest and after 21 days of bed rest. Scans of each volunteer's whole body, pelvis, heel, spine, and proximal femur were acquired in triplicate before and after bed rest. Triplicate measures were averaged before statistical analysis. In accordance with procedures used for analyzing and reporting earlier spaceflight and bed rest data (19, 20, 34), the hip region was manually defined, with the lateral margin placed adjacent to the greater trochanter lateral cortex and the distal border placed relative to the distal margin of the lesser trochanter. Precision of the manual procedure is equivalent to that of the automated procedures available on current DXA analysis software. The highest coefficient of variation (CV) for the DXA procedure in our laboratory is 2.0% for the pelvis and <1.2% for all other regions.

The peripheral quantitative computerized tomography (pQCT) device used was an XCT 3000 (Stratec Medizintechnik). At the time of the first test scan, the length of the subject's tibia was measured. Two scan slices were obtained at 5 and 50% (1 slice at each site) of the tibia length proximal to the distal end plate of the tibia. The slice thickness was 2.4 mm, and the pixel size was 0.4 × 0.4 mm. In addition, three scan slices measured at the insertion of the patellar tendon were obtained. This position was determined by taking a lateral scout scan (starting at the middle of the patella and moving distally) and projecting the intersection of the patellar tendon and tibia. Three adjacent slices were taken at this position, which was referenced to the proximal end plate of the tibia and labeled “proximal 1–3.” To increase the measurement precision, this five-slice sequence was measured in triplicate at each session, and then an average of the three scans was determined. All repeat measurements were referenced to the end plate of the tibia using the same separation measured in the first test. The analysis was performed using the software provided by the manufacturer. The polar strength-strain index (SSI), related to polar moment of inertia, was calculated for each pQCT site and time point. CVs for pQCT at total density ranged (across 2 distal and 3 proximal slices) from 0.4 to 1.6%; for cortical density, from 0.5 to 4.0%; for trabecular density, from 0.7 to 1.5%; and for SSI, from 1.0 to 5.0%.

Statistical analysis.

Statistical analyses were performed on the outcome measures shown in Tables 1–3 (concentrations of the analytes sampled) with the data in their original form (neither transformed nor expressed as percent change). Data from the pre-bed rest days were averaged and used as the pre-bed rest data point. In an exploratory analysis phase, each outcome measure was the dependent variable in a separate repeated-measures ANOVA, with time as the repeated factor and group as a grouping factor. The exploratory phase was designed to flag possible changes in any of the mean outcome measures throughout the study period (before, during, and after bed rest), both overall (“time” main effect) and differentially by treatment group (time × group interaction). After each ANOVA, post hoc Bonferroni t-tests were performed to assess specific differences between pre-bed rest and subsequent time periods, adjusting for multiple comparisons across time; however, no adjustment was made for multiple testing of all outcome measures.

Table 1.

Blood and urinary markers of vitamin D, calcium, and bone metabolism before, during, and after 21 days of bed rest

	Con											AG
		Pre	BR8/9	BR15/16	BR20/21	BR+0/+1	BR+6/+7	Pre	BR8/9	BR15/16	BR20/21	BR+0/+1	BR+6/+7
25-OH vit D, nmol/l	55±16	49±15	54±14	56±17		54±15	63±17	65±17	65±24	65±15		66±14
1,25-(OH)2 vit D, pmol/l	167±63	147±74	162±93	163±89		187±96	145±57	113±34	118±36	114±40		136±39
PTH,b pg/ml	50±27	41±20	35±12c	39±17c		39±17c	50±15	46±12	40±7c	41±6c		41±6c
Ionized calcium, nmol/l	1.21±0.02	1.21±0.02	1.24±0.02	1.22±0.03		1.22±0.03	1.22±0.02	1.22±0.02	1.22±0.03	1.22±0.03		1.22±0.05
Serum calcium, mmol/l	2.299±0.083	2.327±0.099	2.337±0.092	2.277±0.119	2.291±0.095	2.274±0.141	2.273±0.058	2.314±0.062	2.339±0.092	2.285±0.073	2.265±0.091	2.318±0.076
Urinary calcium, mmol/day	6.5±1.4	5.9±2.5	6.7±1.2	7.1±1.4	7.1±1.2	6.1±1.3	5.5±2	6.3±1.3	6.0±2.2	6.2±2.1	6.1±2.4	5.4±1.9
N-telopeptide,b nmol/day	541±194	673±189c	788±160c	781±146c	770±205c	668±165c	555±189	815±204c	775±225c	661±343c	844±284c	812±183c
Serum C-telopeptide,b mmol/l	0.9±0.2	1.1±0.3c	1.2±0.2c	1.2±0.3c		1.0±0.1	0.9±0.2	1.0±0.2c	1.2±0.3c	1.1±0.2c		0.9±0.2
Helical peptide,b mg/day	772±194	802±144c	926±265c	1193±347c	1048±317c	903±233	826±297	1075±385c	1062±411c	1176±495c	1082±382c	953±260
Deoxypyridinoline,b nmol/day	40±18	55±20c	52±21c	62±25c	50±16c	54±17	54±17	77±23c	70±20c	78±23c	82±16c	60±11
Pyridinium cross-links, nmol/day	223±41	285±83	260±98	319±59	308±100	248±67	259±101	285±92	299±112	321±69	285±80	268±56
Bone alkaline phosphatase, U/l	31±5	32±8	32±6	32±4		30±5	30±7	28±7	27±6	26±5		27±5
Total alkaline phosphatase, U/l	61±8	64±9	69±11	67±10		64±7	61±12	61±15	61±13	59±11		62±12
PINP,b ng/ml	59±15	58±16	59±13	57±12		68±15c	68±15	62±12	61±16	60±10		72±20c
Osteocalcin,a ng/ml	23±4	22±5	24±5	22±4c		23±4	26±4	24±4	26±5	23±4c		25±5
Osteoprotegerin,a pmol/l	4.6±1.3	4.3±1.5	4.4±1.3	4.6±1.5		4.7±1.7c	4.4±1.2	4.3±1.1	3.9±0.8	4.5±1.2		5.8±1.9c
TRAP-C,a ng/ml	3.0±0.7	3.3±1.0	2.9±0.9	3.5±0.9		3.7±0.9c	3.2±0.7	3.4±0.9	3.5±0.7	3.7±0.8		3.8±1.1c

Values are means ± SD; n = 7 for the control (Con) group and n = 8 for the artificial gravity (AG) group. For urinary variables, the means of individual values from the 2 days at each time point were used to determine the group mean ± SD. 25-OH vit D, 25-hydroxyvitamin D; 1,25-(OH)₂ vit D, 1,25-dihydroxyvitamin D; PINP, procollagen type I N propeptide; PTH, parathyroid hormone; TRAP-C, tartrate-resistant acid phosphatase; Pre, pre-bed rest ambulatory period; BR, bed rest days; BR+0/+1 and BR+6/+7, 0 to 1 and 6 to 7 days after bed rest.

^aP < 0.01;

^bP < 0.001, significant main effect of time with groups combined.

^cP < 0.05 vs. Pre (determined by a post hoc Bonferroni t-test in the exploratory ANOVA).

Table 3.

Bone density of the tibia determined by pQCT before and after bed rest

	Con			AG
		Pre	Post	Pre	Post
Total density (distal), mg/cm3	386±35	389±36	374±52	374±55
Cortical region density (distal), mg/cm3	492±37	498±40	493±88	493±94
Trabecular region density (distal), mg/cm3	257±43	256±44	229±23	228±22
SSI P (distal), mg/cm3	1,864±297	1,893±297	1,886±561	1,887±578
Total density (mid), mg/cm3	875±58	872±59	853±64	853±68
Cortical region density (mid), mg/cm3	1,177±23	1,170±28	1,155±31	1,159±37
Trabecular region density (mid), mg/cm3	505±122	509±119	482±117	480±121
SSI P (mid), mg/cm3	2,598±249	2,605±259	2,859±366	2,852±385
Total density (proximal), mg/cm3	311±35	312±38	308±24	308±26
Cortical region density (proximal), mg/cm3	432±53	433±58	421±34	421±38
Trabecular region density (proximal), mg/cm3	163±19	163±17	170±21	170±21
SSI P (proximal), mg/cm3	4,503±937	4,556±1079	3,985±817	3,956±943

Values are means ± SD; n = 7 for the Con group and n = 8 for the AG group. Proximal means were calculated from the average of 3 slices per session and 3 sessions per time point for each subject; distal and midshaft data were calculated from the average of 3 sessions per time point (1 slice per session) for each subject. pQCT, peripheral quantitative computerized tomography; SSI P, polar strength-strain index.

In a second analysis phase, for the pre-bed rest and in-bed rest periods only, time was treated as a continuous covariate with the aim of identifying possible linear trends, either overall or by group, within the bed rest period for the 17 bone marker variables listed in Table 1. In this analysis phase, P values were adjusted for multiple testing of the bone marker variables, using the free-step-down method of Westfall and Young (48) to control the family-wise type I error rate to 0.05. For BMC and the seven measurements of BMD listed in Table 2, which were made only before and after bed rest, P values from the repeated-measures ANOVA were similarly adjusted for multiple testing. No adjustment was made in analysis of pQCT, because no significant changes were observed for these measurements.

Table 2.

Bone mineral density determined by DXA before and after bed rest

	Con			AG
		Pre	Post	Pre	Post
Whole body total BMC,a g	2,759±269	2,728±250	2,914±223	2,894±213
Whole body total BMD, g/cm2	1.204±0.059	1.200±0.060	1.252±0.080	1.252±0.080
Pelvis BMD, g/cm2	1.193±0.096	1.180±0.087	1.241±0.129	1.233±0.136
Heel BMD, g/cm2	0.711±0.084	0.715±0.084	0.696±0.063	0.695±0.066
Spine total BMD, g/cm2	1.021±0.064	1.015±0.068	1.065±0.094	1.063±0.095
Trochanter BMD,b g/cm2	0.779±0.094	0.772±0.095	0.829±0.129	0.823±0.126
Femoral neck BMD, g/cm2	0.902±0.065	0.901±0.067	0.950±0.129	0.952±0.129
Total hip BMD,a g/cm2	1.058±0.081	1.050±0.081	1.099±0.115	1.093±0.116

Values are means ± SD; n = 7 for the Con group and n = 8 for the AG group. BMC, bone mineral content; BMD, bone mineral density; DXA, dual-energy X-ray absorptiometry; Pre, ambulatory period before bed rest; Post, ambulatory period after bed rest. Individual Pre and Post values are the averages of 3 consecutive scans.

^aP < 0.05;

^bP < 0.001, significant effect of time for combined data before adjustment for multiple testing.

Statistical analyses were performed using SigmaStat software version 3.01a (SPSS, Chicago, IL) and Stata statistical software version 10.0 (College Station, TX), and P < 0.05 was the level of significance. Data are means ± SD.

RESULTS

Blood and urinary markers of vitamin D and calcium metabolism and of bone cellular activity are presented by group and time period in Table 1. Neither the exploratory analysis nor the more focused trend analysis showed a change in 25-OH vitamin D or 1,25-(OH)₂ vitamin D during bed rest in either group. Similarly, neither blood ionized calcium nor serum calcium changed significantly during bed rest.

Cumulative calcium balance (Fig. 1), determined by subtracting daily fecal and urinary calcium from dietary calcium for each phase of the study, decreased in both groups during bed rest (P < 0.001). Note that because the cumulative balance is “reset” at the beginning of each phase, and the phases have different durations, absolute numbers should not be compared between phases. The data suggest that the subjects tended to be in negative calcium balance even during recovery, but a longer recovery phase would be needed to draw any conclusions about how long it would take to recover to pre-bed rest levels.

Fig. 1.

Cumulative calcium balance of control (Con) and artificial gravity (AG) subjects before, during, and after 21 days of bed rest. This was calculated for each individual as the difference between dietary intake of calcium and the combined urinary and fecal excretion of calcium. Cumulative balance was “restarted” with each phase of the study (before, during, and after bed rest; break points are indicated by vertical dotted lines), so that balance calculations are cumulative for days BR−9 through BR1 (before), BR2 through BR21 (during), and BR+0 through BR+7 (after bed rest). Values are means ± SD; n = 6 Con subjects and 8 AG subjects. Dietary intake methodology is described in a companion article (54). There were no differences between the 2 groups, but time had a significant effect. Calcium balance for BR11 through BR21 was significantly more negative than before bed rest (P < 0.001).

Bone resorption was assessed by several markers (Table 1). The exploratory analysis showed that serum intact PTH was significantly lower, but serum C-telopeptide and urinary excretion levels of N-telopeptide, helical peptide, and DPD were significantly greater during bed rest than before bed rest, and the changes did not depend on group (Table 1). All of the increases were corroborated with the trend analysis, even after adjusting for multiple testing of 17 bone markers. Pyridinium cross-link excretion did not change significantly.

With all time points taken into consideration, the exploratory analysis indicated that blood concentrations of bone ALP and total ALP, markers of bone formation, tended to change differently over time in the AG group compared with the Con group (P = 0.08 and P = 0.09, respectively; Table 1). The trend analysis further revealed that concentrations of these markers decreased significantly more in the AG group during bed rest than in the Con group (P = 0.0077 and P = 0.0028 for bone ALP and total ALP, respectively). After adjustment for multiple testing, the group effect on the slope of total ALP during bed rest was still significant (adjusted P = 0.035), but not the effect for bone ALP (adjusted P = 0.087). When the two groups were combined, the serum concentration of PINP, another marker of bone formation, was significantly greater during recovery than before bed rest (P < 0.001), but there was no differential group effect (Table 1). Serum concentrations of osteocalcin showed a significant (P < 0.01) effect of time (lower concentrations on BR20/21 than before bed rest, as shown by the post hoc Bonferroni t-test), but no group effect was found.

Using the exploratory ANOVA, we found that the osteoclastogenesis inhibitory factor OPG was significantly elevated during the recovery phase (BR+7, P < 0.01), and the AG subjects tended to have higher blood concentrations at that time than did the Con subjects (P = 0.054). Concentrations of the OPG ligand RANKL were below the detectable limit of the assay for most subjects; therefore, statistical analyses were not performed. TRAP-C was significantly greater during recovery in both groups than it was before bed rest, indicating that osteoclast activity was higher at that time.

AG had no significant effect on the change over time in whole body total BMC or BMD of the bone regions studied. However, when the two groups were combined, the whole body total BMC and trochanter and total hip BMD were significantly less after bed rest (P = 0.003, 0.014, and 0.0003 respectively; Table 2). After adjustment for multiple testing, the above changes in BMD were still significant (adjusted P < 0.05), but not the whole body total BMC. Bone density of the tibia determined by pQCT was not different after bed rest in any region examined, and it was not different between groups (Table 3).

DISCUSSION

Despite decades of research, it has proven difficult to mitigate weightlessness-induced bone loss. Bone loss as well as other negative physiological effects of weightlessness would be expected to be mitigated by AG. The aim of this project was to determine whether AG would protect multiple physiological systems during unloading; bone, in particular, was thought to be responsive to AG during bed rest.

We hypothesized that AG would protect bone from demineralization during bed rest and that this protection would be evident from changes in markers of bone formation and resorption. The rationale was that the loading produced by AG would stimulate bone formation and decrease bone resorption through a mechanotransduction process by which the mechanical load on bone is transduced into biochemical signals (9, 26). During mechanotransduction, the function of bone cells can change and induce changes in bone structures to adapt to the load.

In the present study, an AG prescription of 1 h/day was used, but it clearly did not protect against the increase in bone resorption that in the past 10 years has become a hallmark of bed rest (3, 14, 21, 30, 34, 36, 46). No evidence was found that bone was protected from demineralization associated with bed rest. Although we did not expect to find any changes in bone density during this relatively short 21-day study, we fully expected that if AG had had a protective effect on bone, we would have observed differences in the biochemical markers of bone resorption and formation.

Several parameters of the AG prescription must be considered to make it optimally effective. Some of these are rate of centrifugation, acceleration, position of the subject on the centrifuge, body mass, timing of exposure, and duration of exposure. The 1 h/day duration of centrifugation (the G_z force) simply may not be long enough to have a protective effect on bone. Vernikos et al. (45) mitigated a bed rest-induced increase in urinary calcium by having subjects walk for 2 or 4 h/day, but standing for 2 or 4 h/day was not enough. Standing for 2 h/day during bed rest was enough to mitigate symptoms of orthostatic intolerance. Minimum requirements for gravity exposure also varied greatly for different physiological systems in an animal study in which rat hindlimbs were unloaded for 28 days (52). The investigators found that the cardiovascular system responded when the rats were exposed to as little as 1 h/day of gravity (1 G_z by standing). They also found that bone was the most resistant system and required up to 4 h/day of standing or 1 h/day of centrifugation (1.5× gravity) to only partially mitigate femur deconditioning (52). No change or a decrease in bone mass was also found after prolonged hypergravity (12, 16, 22). Potential explanations for these disparate results include the age of the animals and the dose of the gravity prescription.

In addition to duration of the force, the G_z force itself that was used in our experiment may not have been great enough to stimulate an effect on bone, or perhaps it could have been enhanced by combining it with exercise. In one 20-day bed rest study in which AG (a maximum of 2 G_z) was combined with ergometric exercise, a protective effect was found on bone as evidenced by a decrease in the urinary excretion of the collagen cross-link DPD (15). This was the only such marker reported, however. Similar effects on other markers might strengthen confidence in this finding.

In another bed rest study, bone loss associated with disuse was effectively mitigated by lower body negative pressure (LBNP) combined with treadmill exercise (36, 56). The LBNP in that study allowed an average ground reaction force (GRF) of 120% of body weight on the treadmill. The GRF provided by the AG prescription in the experiment reported in this study ranged from 112 to 128% of body weight (120 ± 6%). One difference between the force in the AG study and that in the LBNP study is that the force in the AG study was fairly constant over a 1-h period each day. The 120% of body weight average GRF in the LBNP study was intermittent with each step while subjects ran on the treadmill. Several studies suggest that interruption of loading by rest periods amplifies the bone formation response (4, 13, 43). Interestingly, the GRF for AG subjects in this study was positively correlated with their change in heel BMD (r = 0.80, P < 0.01).

Beyond GRF, the other stimulus provided by LBNP is an increase in blood flow and fluid shifting to the lower extremities, with an increase in interstitial fluid pressures. These also have been implicated in bone changes in unloading and spaceflight (5, 8) and may have provided some of the resorption mitigation offered by LBNP combined with treadmill exercise. Centrifugation, as utilized in this study, also results in a fluid shift to the lower body, but at the duration and intensity used, it failed to affect bone metabolism.

Bone ALP of AG subjects decreased during bed rest, and this change was positively correlated with the change in PINP (r = 0.33, P < 0.01). This decrease in bone formation markers of AG subjects during bed rest was not expected, but the mechanism may be related to AG-induced fluid shifts. Several in vitro studies suggest that the flow of interstitial fluid can directly affect bone formation (11, 26). This may explain why, in 6° HDT bed rest and rat hindlimb suspension models, bone mass in the skull and mandible is increased after several weeks (1, 31). Other studies show that osteoblasts are not stimulated very much by mechanotransduction of static loads but that bone formation depends more on strain rate, amplitude, and duration of loading and is threshold driven (11, 17). Perhaps the threshold required to stimulate bone formation was not exceeded by the duration or force of 1 h/day of centrifugation. The fact that the bone formation markers ALP, total ALP, and PINP are not site specific must also be considered. For instance, if bone formation increased to a certain extent in regions that were maximally loaded but decreased to a greater extent in regions that were not loaded, an overall negative change from the pre-bed rest period would result, and we would have seen only the overall negative change.

Urinary calcium did not increase during bed rest as much as we would have expected, although a negative calcium balance during bed rest was observed for both groups. The negative calcium balance resulted from the combination of a decrease in calcium intake (P < 0.05) and an increase in fecal calcium (P < 0.05) during bed rest compared with before bed rest. Urinary calcium excretion, although often considered a hallmark of bed rest, is not always elevated. This can be related to a number of factors, including general variability between subjects. Furthermore, dietary variation between subjects, days, and studies also may contribute to this. Specifically, dietary calcium, sodium, protein (total, or animal vs. vegetable), potassium, and other nutrients can all have an effect on urinary calcium (2, 10, 33, 57).

We know from the data we have presented that 1 h/day of passive centrifugation at 2.5 G_z at the feet is not enough to mitigate bone loss associated with disuse during 21 days of bed rest in men. It is clear that, at some point, bone loss associated with disuse will be mitigated by AG, since it is known that musculoskeletal loading in a 1-G environment protects against such loss. Whether the response of women would differ is also unknown. Further studies with an AG protocol should aim to combine exercise with AG and perhaps include several shorter AG sessions instead of one long session each day.

GRANTS

This work was funded by the NASA Human Research Program and was conducted at the National Institutes of Health-funded (M01 RR 0073) General Clinical Research Center at the University of Texas Medical Branch, Galveston, TX.