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The impact of microgravity on bone in humans

Daniela Grimm, Jirka Grosse, Markus Wehland, Vivek Mann, JanneElin Reseland, Alamelu Sundaresan, Thomas Juhl Corydon

PII: S8756-3282(16)30078-3DOI: doi: 10.1016/j.bone.2015.12.057Reference: BON 10996

To appear in: Bone

Received date: 25 June 2015Revised date: 17 November 2015Accepted date: 18 December 2015

Please cite this article as: Grimm Daniela, Grosse Jirka, Wehland Markus, Mann Vivek,Reseland Janne Elin, Sundaresan Alamelu, Corydon Thomas Juhl, The impact of micro-gravity on bone in humans, Bone (2016), doi: 10.1016/j.bone.2015.12.057

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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THE IMPACT OF MICROGRAVITY ON BONE IN HUMANS

Daniela Grimm*1, Jirka Grosse

+, Markus Wehland

±, Vivek Mann

§, Janne Elin

Reseland#, Alamelu Sundaresan

§, Thomas Juhl Corydon*

*Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark

+Department of Nuclear Medicine Germany, University of Regensburg, D-93042 Regensburg,

Germany

±Clinic for Plastic, Aesthetic and Hand Surgery, Otto-von-Guericke University, D-39120

Magdeburg, Germany

§Department of Biology, Texas Southern University, 3100 Cleburne, Houston, TX 77004,

USA

#Department of Biomaterials, Faculty of Dentistry, University of Oslo, N-0317 Oslo, Norway

Running Title: Microgravity and bone

Word count: 14009

Tables: 2

Figures: 2

1Correspondence: Department of Biomedicine, Aarhus University, Wilhelm Meyers Allé 4,

DK-8000 Aarhus C, Denmark, E-mail: dgg@biomed.au.dk

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THE IMPACT OF MICROGRAVITY ON BONE IN HUMANS

Daniela Grimm*1, Jirka Grosse

+, Markus Wehland

±, Vivek Mann

§, Janne Elin

Reseland#, Alamelu Sundaresan

§, Thomas Juhl Corydon*

*Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark

+Department of Nuclear Medicine Germany, University of Regensburg, D-93042 Regensburg,

Germany

±Clinic for Plastic, Aesthetic and Hand Surgery, Otto-von-Guericke University, D-39120

Magdeburg, Germany

§Department of Biology, Texas Southern University, 3100 Cleburne, Houston, TX 77004,

USA

#Department of Biomaterials, Faculty of Dentistry, University of Oslo, N-0317 Oslo, Norway

Running Title: Microgravity and bone

Word count: 14009

Tables: 2

Figures: 2

1Correspondence: Department of Biomedicine, Aarhus University, Wilhelm Meyers Allé 4,

DK-8000 Aarhus C, Denmark, E-mail: dgg@biomed.au.dk

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ABSTRACT

Experiencing real weightlessness in space is a dream for many of us who are interested in

space research. Although space traveling fascinates us, it can cause both short-term and long-

term health problems. Microgravity is the most important influence on the human organism in

space. The human body undergoes dramatic changes during a long-term spaceflight. In this

review, we will mainly focus on changes in calcium, sodium and bone metabolism of space

travelers. Moreover, we report on the current knowledge on the mechanisms of bone loss in

space, available models to simulate the effects of microgravity on bone on Earth as well as the

combined effects of microgravity and cosmic radiation on bone. The available

countermeasures applied in space will also be evaluated.

Words: 122

Keywords: Microgravity, metabolism, calcium, bone loss, countermeasures

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BACKGROUND

Since the first manned spaceflight in 1961 (Vostok 1), astronauts, cosmonauts, and taikonauts

have been spending increasingly longer time periods on board orbiting spaceships. In that

time, the health and safety of humans has been extensively monitored before, during and after

spaceflight. These medical examinations have revealed several health problems for space

travelers, including bone and muscle loss, cardiovascular dysfunction, and reduced immune

function [1-5]. Consequently, the field of space medicine developed as an addendum to space

missions. From the beginning, the success of the missions depended on a sometimes difficult,

but close collaboration between medicine and engineering [6]. The designs of the early

vehicles only allowed for a more general systemic space medicine investigation of the flight

crew, whereas subsequent space stations facilitated more sophisticated research, including

animal and in vitro studies.

The International Space Station (ISS) and the Shenzhou programs are the currently important

research platforms for experimental cell [7-9] and space medicine research. They are used for

studies on the health of astronauts (e.g. bone and muscle loss, the cardiovascular system,

immune system, radiation studies, and more) [5], in the field of gravitational biology on cells

as well as for investigations on mechanisms for control of cell growth related to cancer

research on Earth [10].

Nowadays, researchers aim to investigate the mechanisms of the physiological alterations that

occur when the human body is exposed to microgravity during spaceflight and to develop

countermeasures accordingly. Hence, space exploration is linked to considerable efforts in

aerospace medical research. Studies on human physiology and health in space require

integrated and synergistic expertise and resources from many disciplines. Therefore, a careful

and comprehensive preparation of each flight experiment is indispensable.

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Long-term spaceflights can dramatically influence the health of humans in space. This is due

to the absence of gravity. In fact, experiencing weightlessness triggers a shift in body fluids,

particularly the redistribution of blood and lymph towards the head, which results in a

syndrome called “puffy face-bird legs” [11].

In addition, space motion sickness is very common. Even though this can be a difficult

experience, space motion sickness lasts only a few days [12]. Another problem is the post-

flight orthostatic intolerance and reduced exercise capacity, which are well substantiated by

spaceflight and ground experiments [13-15]. Another major system that is altered by a

microgravity environment is the immune system. Immunosuppression occurs in space [5, 16].

The majority of immune cell types are hampered and the secretion of immunologic proteins,

such as cytokines, is changed. Changes in T lymphocytes in vitro have been extensively

investigated by a variety of researchers [3, 17-19]. Due to the negation of weight, muscles

lose both mass and strength and the bone starts to demineralize, which can eventually result in

osteoporosis [20-22].

In this review, we present the current knowledge on sodium and calcium metabolism of

humans in space. Moreover, we will focus on bone metabolism, the mechanisms of bone loss

in space, the combined effects of radiation and microgravity on bone and available

countermeasures to protect crewmembers against bone loss.

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CALCIUM AND BONE METABOLISM OF HUMANS IN SPACE

Calcium metabolism in space

Astronauts have an increased risk of renal stone formation, largely because of elevated

calcium excretion secondary to bone loss [23] (Fig. 1). Bone mineral loss occurs as secondary

osteoporosis due to the unloading of weight-bearing bones, observed during both bed-rest

experiments and spaceflight.

Spaceflight or a condition requiring long-term bed-rest both increase bone resorption,

inducing the rapid decrease of bone minerals and osteoporosis [21, 24-27]. The lack of

gravitation and the resulting decreased mechanical load on the weight-bearing bones induced

during spaceflight results in an increase in bone resorption and a decrease in bone formation.

The longer the mission, generally the more bone and calcium is lost [28]. In addition to

mechanical stress, the diet or individual nutrients exert both positive (e.g. calcium, vitamin D,

vitamin K) and negative effects (for example sodium chloride, vitamin A overdose) on bone.

Complete calcium balance studies were performed during the Skylab missions in the early

1970s [29].

Smith et al. [30] showed that, during long-term spaceflight (115 days – MIR 18), calcium

intake and absorption decreased by up to 50%, urinary calcium excretion increased by up to

50%, and bone resorption (determined by kinetics or bone markers) increased by over 50%

[30]. The three male subjects lost 250 mg of bone calcium per day during flight and regained

bone calcium at a slower rate of 100 mg/day for up to three months after landing [30].

The loss of calcium is a recurrent observation on spaceflights, mainly via urinary and fecal

excretion [20, 31-37], which gives rise to an increased risk of renal stone formation during

and after the mission [34, 35, 38]. Nonetheless, the human body retains its ability to regulate

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circulating calcium levels. Highly increased urinary concentrations of uric acid, calcium

oxalate, brushite, and sodium urate have been observed on a multitude of space missions [39].

Additional factors that can promote the formation of kidney stones in space include reduced

fluid intake, hypercalciuria, and the presence of nanobacteria [40]. In a study comprising a

total of 42 astronauts (33 men and 9 women) on long-duration missions to the ISS, it was

shown that the effect of spaceflight on bone density in different exercise groups was the same.

The urinary supersaturation risk, as well as biochemical markers of bone formation and

resorption, did not differ significantly between men and women during spaceflight [23]. In

addition, this study showed that, although there are documented sex differences on Earth

regarding bone mineral density, content and renal stone risk, the response to spaceflight is

similar in men and women [23].

In real microgravity (spaceflight) and simulated microgravity (bed-rest models), changes in

bone biochemistry occur rapidly after humans enter the altered environment. As a reaction to

unloading, calcium is released from bone, which suppresses parathyroid hormone. This

induces a drop in circulating 1,25-dihydroxyvitamin D (1,25(OH)2D) and leads to a decrease

in calcium absorption [30, 37, 41-43] as indicated by increased fecal and urinary calcium

excretion during flight [33, 44, 45]. Bed-rest studies have shown effects similar to those

during spaceflight [46-53], albeit to a lesser extent.

Pre- and postflight data obtained during the Shuttle-Mir Science Program showed that, after

landing, bone resorption was increased. Moreover, urinary calcium and collagen cross-links

(N-telopeptide, pyridinoline, and deoxypyridinoline) were all increased above preflight levels

[43]. Smith et al. demonstrated in 2005 that 1,25(OH)2D levels were reduced early inflight

after 14 days in space, as were parathyroid hormone levels, which decreased during flight and

were elevated again after returning from the space mission (intact PTH was decreased during

the flight: preflight 26±10 pg/mL; early inflight (14 d): 16±4 pg/mL; late inflight 1: 16±8

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pg/mL; late inflight 2: 15±6 pg/mL and after return 0/1: 25±12 pg/mL) [43]. The authors

hypothesized that the decreases in circulating 1,25(OH)2D are related to lower PTH levels,

rather than to increased disposal of vitamin D metabolites. They concluded that calcium

kinetic studies indicated that bone loss in space is a consequence of elevated bone resorption

and reduced intestinal calcium absorption [43].

Bone metabolism in space

Humans in space are at high risk for bone loss. Living in microgravity results in elevated bone

resorption and reduced bone formation [5]. The impact of spaceflight on calcium and bone

metabolism is shown in Fig. 1.

Throughout life, bone remodeling or bone metabolism is an ongoing process where bone

resorption occurs and new bone tissue is formed. During adult life, bone structure and

function are maintained through a constant remodeling process [54, 55]. This active process

helps the bone to adapt to changing loads. In healthy people, there is a balance between bone

formation and bone resorption. Bone remodeling occurs through the interaction of two central

cell types: osteoblasts, responsible for bone matrix formation, and osteoclasts, which mediate

bone matrix resorption. The balance of bone metabolism at the cellular level is regulated by

molecular mechanisms. Cytokines, growth factors (IGF, FGF, TGF-1), hormones, age and

gender (estrogen deficiency), the extent of physical activity and some drugs play a key roles

in these processes. The Wnt family and bone morphogenetic proteins (BMP) are involved in

bone and chondrocyte remodeling; for example, when human chondrocytes are exposed to

short-term real microgravity [56]. In general, chondrocytes are very robust under conditions

of parabolic flight maneuvers [57].

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Bone cell culture experiments under conditions of microgravity have shown that cultures of

MC3T3-E1 bone cells undergo changes in the actin cytoskeleton [58]. Integrins bind to actin

to form a link between the cytoskeleton and the extracellular matrix (ECM). Thus, any

changes in actin can affect these relationships and influence bone cell function [59-62]. In

addition, changes in the ECM may be responsible for altered cell function. However, MC3T3-

E1 cell synthesis and the orientation of fibronectin are unaltered by microgravity [63]. These

findings suggest that changes in the ECM protein fibronectin are not responsible for

alterations in osteoblast cell function in microgravity. Furthermore, a recent study revealed

that the responsiveness of runt-related transcription factor (Runx2), a major transcription

factor involved in osteoblast differentiation, to bone morphogenetic protein 2 (BMP2)

stimulation is reduced under conditions of simulated microgravity due to the disruption of the

F-action microfilament network [64]. In a follow-up study, Dai et al. [65] also showed that

gravity- and IGF-1-dependent Runx2 transcription is regulated by integrin αvβ3. While

further investigation into the possible roles of other inter- and extracellular components in

altering bone cell function is warranted, it appears, at least, that changes in the cytoskeletal

structure of some components are central to the modification of bone cell function. Apart

from the role of the ECM and the cytoskeleton, cell culture experiments in simulated

microgravity have provided further insight into the possible mechanisms involved in bone

loss in space. In a recent study, Sun et al. observed that microgravity inhibits L-type voltage

sensitive calcium channel currents, which are central for the cellular responses of osteoblasts

to mechanical stimuli. In addition, they also reported a parallel down-regulation of Cav1.2

protein [66]. The authors concluded that these effects were triggered by an up-regulation of

the microRNA miR-103, offering a novel mechanism for bone formation dysregulation under

microgravity. Furthermore, it has been shown that preosteoblast mineralized nodule formation

is significantly suppressed under simulated microgravity using a random positioning machine

(RPM) [67]. Both alkaline phosphatase as well as extracellular signal-regulated kinases

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(ERK) signaling seemed to be involved in the down-regulation of osteocalcin, collagen I α1,

dentin matrix protein 1, and Runx2, which are all important markers and regulators of

osteoblast differentiation [67].

Bone loss in weight-bearing bone during spaceflights

The loss of normal weight-bearing activity occurs during bed-rest, limb immobilization, and

spaceflight. The underlying bone loss is detected first in weight-bearing bones and later in less

weight-bearing bones [68]. The result is a catabolic response in muscles and bones.

Significant losses of bone mass and bone mineral density in weight-bearing bone are mainly

observed in the spine and lower limbs. Early studies demonstrated mean losses in the spine,

femur neck, trochanter, and pelvis of about 1%-1.6%, with considerable variation between

individuals [69, 70]. These findings are a result of gravitational unloading in a microgravity

environment [68, 70]. The result is a condition that is suggested to be similar to disuse

osteoporosis or secondary osteoporosis, which result from a decrease in weight-bearing [71].

An increase in bone resorption and a decrease in bone formation have been found [71].

During ISS missions, seven of eight cosmonauts revealed a reduction in BMD (2.5-10.6%) in

the lumbar vertebrae, all eight showed decreased BMD in the femur (3-10%), and four of the

eight showed a 1.7-10.5% decrease in BMD in the femoral neck [72].

It has been shown that bone formation is reduced partly as a result of impaired osteoblast

function [73]. This deficit in bone mass can be replaced, but the time span for restoration

exceeds the period of unloading. During the post-flight period, the time required to recover

lost bone mass is greater than the mission length (by a factor of three or four times longer)

[74]. Thus, the post-flight period deserves attention and increased research effort.

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Determination of the areal bone mineral density (aBMD) is currently used to measure the

effects of spaceflight on bones. Clear declines in aBMD with losses >10% had been measured

in the hip and spine in humans following a typical six-month spaceflight [75]. Rates of bone

loss in astronauts exposed to a long-term space missions are about 10-fold greater than those

observed in postmenopausal women [76]. Possible mechanisms for bone loss in space have

been investigated received using the hindlimb unloading animal model.

Serotonin (5-hydroxytryptamine (5-HT) produced in the central nervous system and in the gut

is mainly known to be involved in the control of mood, sleep, and intestinal function, but

circulating serotonin may also have effects on bone metabolism [77-79]. The molecular

mechanisms of direct and/or indirect effects of serotonin on bone metabolism are still unclear,

and gender- and age-specific factors may play important roles in humans [80]. Yoashioka and

coworkers demonstrated that, during a parabolic flight experiment with mice, the serotonergic

system was affected, and presynaptic synthetic pathways were activated in the central nervous

system by microgravity-mediated stress [81]. In contrast, Popova and coworkers found no

effect of one month of spaceflight on the expression of genes encoding the main regulators of

serotonergic activity in mice [82]. Radiation has been shown to induce elevated serotonin

levels in the intestinal tissue in mice [83]. 5-hydroxy-tryptophan (5-HTP) levels were

increased in the intestinal tissue after γ radiation exposure, with long-term differential changes

in mouse intestinal metabolomics after γ and heavy ion radiation exposure [83]. Studies on the

effects of weightlessness or radiation alone or in combination on circulating serotonin or

serotonin receptors in bone has to our knowledge not been reported.

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Animal models to study the effects of microgravity on bone metabolism

Animal models are a relatively easily accessible means to study the effects of microgravity on

bone metabolism. The hind-limb unloading (HLU) technique has been used to mimic aspects

of microgravity, such as unloading of weight-bearing parts of the skeleton and fluid shifts.

Apart from these effects, it has been shown that this method does not exert any more stress on

the animals, as weight gain and eating habits remained normal [84]. Overall, HLU induces

bone loss similar to those effects observed during spaceflight and can be considered a

relatively accurate approximation of exposure to real weightlessness for the unloaded limbs

[71]. The rat tail suspension method was originally designed to examine the effects of skeletal

unloading and reloading occurring during and following spaceflight [84]. HLU mimics the

characteristics of microgravity by removing weight-bearing loads from the hindquarters and

inducing a cephalic fluid shift [84]. The method was reviewed in detail concerning the history

of the technique, comparison with spaceflight data, technical details, and extension of the

model to mice by Morey-Holton and Globus in 2002 [85]. The hindlimbs of rats or mice are

elevated to produce a 30˚ head-down tilt. The forelimbs remain loaded in the model and

provide a good internal control to distinguish between the local and systemic effects of HLU

[84]. The HLU rodent model is a good analog to examine the physiological, cellular or

molecular mechanisms of the skeletal response to weight-bearing loads, and has been shown

to be a model for simulating a spaceflight and its effects on tissues, like bone [84, 86]. Today

the model is widely used for different periods of unloading in rodents to study, for instance,

the molecular mechanisms of cortical bone loss [87] or to investigate the effects of vibration

on bone loss [88] as well as to examine the combined effects of radiation and microgravity

[89]. The HLU model also seems to provide new information not only on the molecular

mechanisms involved in bone loss, but also on potential countermeasures [90, 91].

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One of the more recent results from this kind of study was the finding that 1,25-

dihydroxyvitamin D3 supplementation may prevent the cellular processes of osteoporosis

induced by weightlessness [92]. Furthermore, HLU demonstrated that mechanical unloading

of the limbs resulted in a decrease in early B-cell differentiation, which showed similarities to

age-related modifications in B lymphopoiesis [93]. In hind-limb-suspended rats, localized

vibration of 35 Hz was able to counteract microgravity-induced musculoskeletal loss [88] and

a specially designed stepper-device preserved the bone and muscle structure [94].

Space Shuttle flights have also provided the possibility of expose rodents to real microgravity

for a limited period of time. Two recently published studies conducted during the STS-131

mission demonstrated that non-weight-bearing bones are also affected by microgravity,

probably induced by fluid shifts alone [95]. The pelvic and femoral regions in mice were the

most active sites of rapid bone loss, which was not limited to osteoclastic degeneration but

was also mediated by cell cycle inhibition in osteoblasts [96].

Human bed-rest studies as an experimental analog for spaceflight

In human test subjects bed-rest studies have been used to examine muscle and bone changes

due to simulated microgravity [97]. The ground-based analogue bed-rest is a good model of

microgravity-induced bone loss [49, 98], which occurs in space. In study participants, bed-rest

induces metabolic changes as well as bone loss comparable to event occurring during a stay in

orbit. An overview of the most recent bed-rest studies is presented in Table 1. The amount of

bone loss in bed-rest is less than that observed in astronauts [98, 99]. Related to bone

biochemistry, short-term bed-rest studies fulfill the requirements of simulating spaceflight

[49, 100]. A combined analysis of a total of five head-down-tilt bed-rest studies showed that

men and women do not have substantially different responses to skeletal unloading, but that

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these changes may predispose men to higher renal stone risk due to their elevated excretion of

bone resorption biomarkers and calcium [101]. Cervinka et al. found that the greatest mean

bone loss occurs in the cortical compartment, but only during the first 60 days of bed-rest.

Later, a trabecular loss was observed [97]. Continued disuse, i.e. longer than 2 months, would

result in bone loss in the trabecular compartment [97]. Therefore, more long-term bed-rest

studies should be performed for confirmation [97].

In a number of studies in animals, reduced bone formation and increased bone resorption have

been repeatedly demonstrated [102]. These findings have not been consistently shown in

humans. However, in general, bone formation decreases slightly during spaceflight and bone

resorption increases in humans. Frequently measured bone formation markers are bone-

specific alkaline phosphatase (BSAP) and osteocalcin [103, 104]. The rate of formation

cannot be quantified without proper bone morphometric analysis of biopsies, e.g. after

tetracycline administration. During bone resorption, elevated urinary calcium excretion is

often observed during and post-spaceflight [33]. Urine hydroxyproline is another marker of

bone resorption, but the metabolism of dietary collagen can also produce hydroxyproline [33].

In addition, collagen crosslinks, appearing in the urine because of collagen degradation, are

associated with bone resorption. Pyridinoline, deoxypyridinoline, N-telopeptide, and C-

telopeptide are formed only in mature collagen. The release of these compounds reflects the

breakdown of mature collagen, as they are not absorbed from the gut, and they are stable in

frozen urine samples [30, 103-105].

Hormones also have an influence on bone remodeling processes. The absence of ultraviolet

light and reduced dietary intake of vitamin D during a stay on the ISS can diminish vitamin D

pools in the body. Circulating parathyroid hormone concentrations decrease during flight,

albeit with some variability [30, 43, 45, 103]. A reduced concentration of 1,25-

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dihydroxyvitamin D is observed during spaceflight and seems to be related to decreased

production secondary to decreased parathyroid hormone concentrations.

In addition, several nutrients affect bone and calcium homeostasis, such as calcium, vitamin

D, vitamin K, protein, sodium, and phosphorus. Supplemental calcium is important in the

treatment of osteoporosis patients [106]. For periods when calcium intake is insufficient, it

does not correct the problem of bone loss during spaceflight. A high calcium diet (>1

mg/day), vitamin D, and alendronate together with exercise (advance resistance exercise

device, ARED) may be useful to prevent the decline in bone mass in astronauts during long-

term spaceflight [107, 108]. Alendronate Supplementation with calcium and vitamin D, as

well as agents with potent effects on cancellous and cortical bone, are important to stabilize

calcium balance and bone metabolism and prevent bone loss in astronauts during long-term

spaceflight [106].

SODIUM METABOLISM OF HUMANS IN SPACE

High sodium intake has bone-resorbing effects during periods of inactivity such as bed-rest.

When a very high NaCl intake is consumed during bed-rest, the increase in the excretion of

bone resorption markers is higher than it would have been because of immobility alone [109].

High NaCl intake has been shown to enhance the excretion of calcium, resulting in 43–50%

greater excretion of the bone resorption markers COOH- (CTX) and NH2- (NTX) terminal

telopeptide of type I collagen in the bed-rest group [109].

During several space missions, it was found that the sodium intake of astronauts was very

high. Prepackaged food for the ISS was originally high in sodium (5300 mg/d), but NASA

has reformulated 90 foods to reduce sodium intake to 3000 mg/d [110].

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Water-salt homeostasis plays an important role in the adaptation of the human body to

microgravity. Conditions of microgravity induce a negative balance of liquids and basic

electrolytes [111]. In the early periods of spaceflight, hypohydration of the body occurs as an

adaptive response. Directly after spaceflight, cosmonauts have shown reduced body weight

compared to the preflight value, but after the third post-flight day, their body weight was

normalized, indicating that this deficit may be due to hypohydration of the body [111].

In general, the adaptation to microgravity is known to occur in two phases. The initial

response phase takes place during the first 3-6 weeks of spaceflight, when gradual

readjustments occur in all body systems [112]. Changes in neurovestibular receptors,

sympathetic and parasympathetic function, fluid volume, metabolism, hemodynamics, and

endocrine regulation have been found [112]. In the subsequent time period, changes such as

regional bone and muscle loss occur, changes in neurotransmitter and receptor functions,

further dysfunction of the immune system, as well as alterations in the central nervous system,

which are known health problems of humans in space [5, 112, 113].

MOLECULAR MECHANISMS OF BONE LOSS IN SPACE

Bed-rest has a similar effect as spaceflight and microgravity on bone mineral density, bone

markers, and calcium balance and excretion, although of lower magnitude (close to two-fold

lower) [76, 99, 114]. A key role has been found for sclerostin in the regulation of the Wnt/β-

catenin signaling pathway and subsequent bone loss during disuse [76]. The Wnt/β-catenin

signaling pathway is an important regulator of osteoblastogenesis [115]. Canonical Wnt

signaling induces the differentiation of osteoblast precursors toward mature osteoblasts and

prevents osteoblast and osteocyte apoptosis [115]. The glycoproteins sclerostin and Dkk-1

inhibit the canonical Wnt cascade by binding to Wnt co-receptors, low-density lipoprotein

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receptor-related protein (LRP) 5 and 6 [116-119]. One of the mechanisms by which

microgravity depresses bone formation is through effects on the Wnt/β-catenin signaling

pathway. Recent studies have shown that conditions like microgravity increase the expression

of sclerostin and Dkk-1 [120].

The Wnt-signaling pathway not only increases osteoblastic cell differentiation and bone

formation, but also inhibits bone resorption by blocking the receptor activator of nuclear

factor-κB-ligand (RANKL)/RANK interaction [121]. Preosteoblasts secrete RANKL, so if

Wnt signaling is inhibited, there are more preosteoblasts and more RANKL, but less

osteoprotegerin, and fewer mature osteoblasts capable of refilling resorption cavities. A recent

histochemical study of human iliac crest biopsies has shown that the sclerostin is not present

in osteocytes near the bone surface, but only in the more mature osteocytes that lie deeper in

the bone [122]. Osteocytes are terminally differentiated osteoblasts and form a network in the

bone reminiscent of the neural network of the body. These osteocytes are able to detect

mechanical strain by sensing fluid movement within the canaliculi, and they also secrete

sclerostin. Osteocytes tonically suppress the lining cells via sclerostin and then stop secreting

it when the need arises to form new bone. When larger stresses are applied to bone, small

cracks appear, followed by bone resorption that removes the damaged bone. Although the

exact mechanism is not known, it seems possible that sclerostin plays a significant role in

skeletal adaptation to mechanical forces. In support of this notion, a recent study investigating

the effects of microgravity on osteoblasts and osteoclasts during a five-day spaceflight found

an increase in bone resorption by osteoclasts as well as a decrease in osteoblast cellular

integrity [123]. Interestingly, results from bed-rest studies, showed increases in sclerostin,

suggesting that the Wnt signaling pathway is involved in disuse-induced bone loss in humans

[124]; this is consistent with findings showing that long-term sclerostin deficiency inhibits

bone loss normally induced by decreased mechanical load [125]. A recent study by Spatz and

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coworkers investigated the expression sclerostin in osteocytes in vitro and showed that

sclerostin is upregulated by mechanical unloading, suggesting that mechanical loading

regulates intrinsic osteocyte responses [126]. Together, these findings support the importance

of the Wnt/beta-catenin pathway as a potential target for strategies to prevent bone loss in

space.

COMBINED EFFECTS OF RADIATION AND MICROGRAVITY

Besides the risks of spaceflight to bone health, ionizing radiation (IR) can also damage bone.

Galactic cosmic rays (containing heavy ions and protons) and solar particle events (protons)

are radiation sources that influence the health of astronauts, who may be exposed to 2 Gy of

radiation in space [127, 128]. Little is known about the combined effect of weightlessness and

IR. It is a challenge to simulate both conditions of microgravity and cosmic space radiation in

a laboratory, but ground-based facilities such as the random positioning machine (RPM) or

the rotating wall vessel (RWV) for the simulation of spaceflight conditions exist for in vitro

cell culture experiments [7, 129, 130]. Both devices can be used for microgravity experiments

on different cell types [131-133] and for tissue engineering of three-dimensional aggregates

(spheroids) [134], such as bone [135, 136] or cartilage [137].

Beck et al. assessed fetal mouse fibroblasts in the RPM with or without IR. Gene array data

revealed that a large number of genes altered in either RPM or IR were not changed following

combined treatment, indicating a complex interaction of radiation and microgravity on the

cellular level [138, 139]. It is known that the inflammatory cytokine IL-6 plays an important

role in different biological processes in microgravity [7, 129, 140, 141]. Alterations in

cytokine signaling have also been reported in different cell types after radiation exposure

[142, 143]. A recent paper showed that radiation in combination with microgravity using the

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NASA-developed rotary cell culture system increased IL-6 production in the MC3T3-E1

subclone 4 mouse pre-osteoblast cell line [144].

A widely used model for the simulation of microgravity is hindlimb unloading (HU) by tail

traction. The combination of HU and radiation has been shown to increase bone loss [89,

145]. Yumoto et al. [146] investigated mice using the HU model for seven days and exposed

them to 56

Fe radiation for three days, starting from day 4. HU and radiation caused a 126%

increase in tartrate-resistant acid phosphatase (TRAP) positive osteoclasts on the cancellous

bone surface. The combined effect of HU and IR led to greater suppression of

osteoblastogenesis ex vivo than either treatment alone [146]. In addition, in vitro heavy ion

irradiation acutely increased osteoclast numbers in cancellous tissue, and with

musculoskeletal disuse, it enhanced the sensitivity of ostoprogenitors [146].

In a further study, the effect of proton irradiation and hindlimb suspension on bone in mice

was studied [147]. IR exposure was performed at day 1 and a day later the hindlimb

suspension treatment started for 4 weeks. The authors found that radiation alone resulted in a

20% loss of trabecular bone volume (tibia, femur) and had no effect on the cortical bone

compartment. HU induced a greater loss of trabecular bone (60-70%) and cortical bone (20%)

in the tibia and femur [147]. Moreover, the combined effect appeared to be additive for

certain parameters, such as an increase in TRAP, reduction in osteocalcin, and decreased bone

volume fraction in the femur and tibia. The combination resulted in significantly greater

alterations in bone structural properties [147]. A recent study [148] investigated the effects of

4 Gy of X-ray radiation following HU (4 weeks) in rats. The combination induced a combined

effect on bone mass with a reduction of 64.8%. Radiation alone resulted in a 30.7%-reduction

and HU alone in a 56.6 reduction of bone mass [148]. Moreover, nuclear factor of activated

T-cells, cytoplasmic 1 (NFATC1) and RANKL were clearly increased in rats with combined

treatment compared with corresponding controls [148].

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Taking these findings together, the combination of HU and radiation causes cumulative,

adverse alterations in the structure and function of bone and results in severe bone loss. Future

studies are necessary to investigate the cellular and molecular mechanisms in more detail.

Such investigations should focus on osteoprogenitor cells and test whether, for example,

antioxidants can effectively protect these cells and thus influence the bone response to

radiation and microgravity.

BONE LOSS COUNTERMEASURES

Since the early days of manned spaceflight, the negative impact of microgravity on skeletal

health had been mentioned as a major concern. The recommendations from the NASA Bone

Summit have been thoroughly described in a recent paper by Orwoll and coworkers [24]. In

this section, we review the available physical countermeasures. The relevant papers are listed

and described in Table 2.

Bed-rest studies have included exercise, vibration, and centrifugation. Shackelford et al.

investigated nine subjects who participated in a supine maximal resistance exercise-training

program during bed-rest and concluded that resistance exercise had a positive treatment effect

and may be a useful countermeasure to prevent bone changes after long-term spaceflight

[149]. The training group was compared with 18 control subjects who followed the same bed-

rest protocol without exercise. Heavy resistance exercise induced increased bone formation,

but had little or no effect on resorption, while maintaining bone mineral density [51, 149].

The duration of the experiment was 17 weeks. These experiments indicated that resistance

exercise prevented bone mineral density loss and significantly increased bone metabolism

markers and net calcium balance, suggesting that exercise is a countermeasure against disuse-

induced bone loss [149]. The WISE study set out to investigate whether a regime of combined

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resistive and aerobic exercise during bed-rest would prevent bone loss. Sixteen women were

involved in a 60-day bed-rest study. Half of the participants performed training alternating

between supine treadmill exercise (3-4 times per week) and flywheel resistive exercise (2-3

times per week). The other half of the subjects employed no countermeasures. The data

showed that exercise treatment attenuated the loss of bone mineral density. The WISE study

also revealed that the combination of resistive and aerobic exercise did not prevent bone

resorption, but did promote bone formation [51]. In a parallel study, it was shown that a

resistive vibration exercise countermeasure program prevented bone loss more effectively

than resistive exercise alone [150]. The 20 male subjects in this study underwent 56 days of

bed-rest and were prescribed to either a countermeasure group performing high-load resistive

exercise with whole-body vibration or an inactive control group. The utilized exercise device

permitted exercise in the supine position during the bed-rest period, and targeted the lower leg

muscle groups with resistive loading and muscular stimuli. The resistive loading was applied

at the feet by whole-body vibrations. The exercise session was given twice per day, 5 days

every week. In total, 89 resistive vibration exercises were given to each of the involved

subjects.

Exercise as a countermeasure against bone loss had also been studied for the past four decades

of spaceflights [42]. Hypergravity resistance exercise represents an important countermeasure

to microgravity; although it has proven beneficial for the muscular and cardiovascular

systems [151], clear effects on bone have yet to be shown. Recently, Rittweger et al. [100]

scrutinized human short-arm centrifugation as a musculoskeletal countermeasure. Eleven

participants were subjected head-down tilt bed-rest (HDT) for five days. Bed-rest without

artificial gravity was used as the control condition, and artificial gravity provided by means of

centrifugation (1 g) once per day (30 min bouts or in 6 bouts of 5 min) was the experimental

condition. The study demonstrated clear catabolic effects upon muscle and bone metabolism,

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which were not prevented by human centrifugation. Apart from exercise, lower body negative

pressure (LBNP) has been found to prevent increased tibial microvascular flow induced by

head-down tilt; the authors of this study suggested that LBNP may be a suitable

countermeasure for dysregulated microvascular flow during spaceflight [152]. The group of

Professor Dieter Felsenberg from the Charité-University Hospital in Berlin, Germany, showed

in 2010 that high-load resistive exercise, with or without whole body vibration, performed

three days/week, can reduce lumbar muscle atrophy in bed-rest subjects [153]. The group

showed that combining whole-body vibration and high-load resistance exercise seems to be

more efficient than high-load resistive exercise alone in preventing bone loss at some skeletal

sites during and after prolonged bed-rest [154].

The effects of exercise during bed-rest impact upon bone recovery up to three months after

bed-rest [154]. Taken together, the results have shown that vibration of higher intensity or a

combination of vibration and resistance exercise delivered promising results for bone and

muscle [51, 149, 150, 153-156]. Artificial gravity itself (1 hour of centrifugation daily) did

not negatively affect nutritional status during a bed-rest study [157]. The authors

demonstrated that artificial gravity also had no protective effect on nutritional status during

bed-rest [48, 157].

Heavy resistance exercise plus good nutritional and vitamin D status have been demonstrated

to reduce the loss of bone mineral density on long-duration ISS missions [27]. Smith et al.

showed in 2012 [42] that resistance exercise coupled with adequate energy and vitamin D

intake could maintain bone mass in most regions during four- to six-month missions in

microgravity. In this study, involving 13 crewmembers on ISS missions from 2006 to 2009,

eight members had access to an interim resistive exercise device (iRED) and five had access

to an advanced resistive exercise device (ARED). All crewmembers were provided with a

specific program prescribing 2.5 hours of exercise per day (6 days per week), including

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aerobic training and resistance exercise. The diet was designed to provide adequate intake of

seven nutrients of interest (protein, water, energy, sodium, calcium, iron, and potassium).

Astronauts receiving vitamin D were provided with a daily supplement of 800 IU. The authors

concluded that improved nutrition and resistance exercise during spaceflight could attenuate

the expected BMD deficits observed on prolonged missions [42]. A recent study reviewed

space missions flown between 2000 and 2013 [23]. The authors analyzed data based on the

available resistance exercise equipment of 42 ISS astronauts. Astronauts who launched before

November 14, 2008 were trained with iRED and the other group who launched on November

14, 2008 or later performed their training with the ARED. Men and women lost proportional

amounts of bone under iRED, but the ARED program protected them against severe bone

loss. The current ISS exercise program (ARED) seems to be beneficial for astronaut bone

health [42]. An important concern in disfavor of ARED is the notion that it is unlikely that a

long-duration mission to Mars will have room for an ARED device.

An international study, as a collaboration between the space agencies NASA and JAXA, has

investigated the potential value of anti-resorptive agents like bisphosphonates (alendronate) to

mitigate the well-established bone changes associated with long-duration spaceflight. LeBlanc

et al. [107] reported in 2013 the results of alendronate ingestion plus exercise in preventing

the declines in bone mass and strength and elevated levels of urinary calcium and bone

resorption in astronauts during 5.5 months of spaceflight [107]. Ten crewmembers were

enrolled in the bisphosphonate study. Of these ten, seven completed the treatment. Subjects

were given an oral dose of 70 mg of alendronate weekly starting three weeks before lift-off

and continuing during the entire flight mission. The in-flight exercise program consisted of

2.5 h of training divided into 1.5 h of resistance training (iRED or ARED) and 1 h of aerobic

training, 6 days/week. Combination of exercise plus an anti-resorptive drug may be useful for

protecting bone health and preventing renal stone formation during long-duration spaceflight

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and long-term bed-rest [107]. In support of this study, a previous simulated microgravity

experiment showed that alendronate attenuated most of the changes in bone occurring during

bed-rest [158]. In a study by LeBlanc and coworkers, eight male subjects received a daily oral

dose of 10 mg alendronate during 17 weeks of bed-rest [158, 159]. However, findings from

another bed-rest study investigating the effects of flywheel resistive exercise and pamidronate

(a nitrogen-containing bisphosphonate) on muscle atrophy and bone loss suggested that both

countermeasures were only partial effective at preserving bone mineral content [160].

Twenty-five male subjects participated in the study and underwent strict bed-rest with head-

down tilt for 90 days. Participants were divided into three groups: An exercise group that

practiced resistive exercise with a flywheel device every second or third day, a pamidronate

group that received i.v. injections of 60 mg pamidronate two weeks before bed-rest, and a

control group that received no countermeasures [160].

Several pharmacological studies focusing on bisphosphonates have been conducted in bed-

rest studies [158, 160, 161], animal studies [162], and also in studies on quadriplegic or

paraplegic subjects with anti-resorptive drugs [163]. Schapiro et al. [163] demonstrated that a

single administration of zoledronic acid could ameliorate bone loss and maintain parameters

of bone strength at three proximal femur sites for six months and at femur intertrochanteric

and shaft sites for twelve months in patients with acute spinal cord injury [163].

DISCUSSION

Today, humans in space have to undergo daily time-consuming workouts using special

equipment during their stay in orbit on the ISS. The aim of this training is to reduce the

detrimental effects of microgravity. However, the crewmembers will return to Earth with an

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average overall bone mass loss of one to two percent for each month spent on station. It is

important to say that exercise is beneficial for numerous body systems, but crewmembers

need to have additional options available to them to prevent bone loss on long-duration

flights. Bisphosphonates are such an option [107]. This is very important when an astronaut

has an injury and cannot perform the recommended exercise program. Another point is that

resistance exercise has risks. It has been shown that a large number of minor back injuries had

been experienced in the course of using exercise equipment on the ISS [164]. The majority of

the musculoskeletal injuries occurring in space were attributable to the use aerobic and/or

resistance exercise equipment [164].

Astronauts have a higher post-flight risk for disk herniation, and high resistance exercise

loading could exacerbate this problem during a spaceflight [165]. Despite intensive research,

calcium and bone metabolism as well as muscle atrophy remain key health problems for

astronauts. Physical, pharmacological, and nutritional means have been used to counteract

these changes. Uncertainty still continues to exist as to whether the bone is as strong after

spaceflight as it was before spaceflight and whether nutritional and exercise prescriptions can

be optimized during spaceflight. The findings from the studies reviewed here will not only

help future space explorers but will also broaden our understanding of the regulation of bone

and calcium homeostasis and muscle atrophy on Earth.

Recommendations for important research challenges for the field of bone loss during

spaceflight

Summarizing the available data, the following are our recommendations for research

challenges to address in the field of counteracting bone loss.

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The bone density changes in the hip and back during spaceflight are comparable to

osteoporosis seen in women after menopause. Hip and spine fractures can occur similar to

what is observed in patients suffering from osteoporosis. Studies on teriparatide would be

interesting in astronauts. Teriparatide is an anabolic bone-growing drug and is indicated for

use in postmenopausal women with osteoporosis at a high risk of fracture or with a history of

osteoporotic fracture, in patients with multiple risk factors for fracture, and for patients who

have failed or are intolerant to other available osteoporosis therapies. Because of these

anabolic properties, teriparatide should be studied in future spaceflights.

During the NASA Bone Summit [24], the experts recommended that quantitative computed

tomography (QCT) and hip strength assessments pre- and post-flight should be performed in

order to detect changes in bone density and strength. In addition, dual-energy X-ray

absorptiometry (DXA) should be applied to analyze the bone density of astronauts. Risk

factors for osteoporosis such as physical activity and nutrition should be optimized in

astronauts and drug interventions with bisphosphonates should be evaluated in future studies.

Bisphosphonates are applied in the treatment of osteoporosis and have proven to be effective

in increasing bone mass and in reducing the occurrence of bone fractures. During spaceflight,

the combination of exercise plus an antiresoptive drug is useful for protecting bone health

during long-duration spaceflight [107].

It is suggested that astronauts can reduce their risk of bone loss and renal stones with the

proper intake of appropriate nutrients, such as calcium and vitamin D, an effective exercise

program, and minimal amounts of medication. The drug alendronate was used weekly in the

study by Leblanc and coworkers. Future studies should be performed using other

bisphosphonates such as ibandronate as a monthly treatment regimen [166]. Because

spaceflights are rare, bed-rest studies may be good models to test the drugs ibandronate and

teriparatide in advance.

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As already mentioned, it is important to study the combined effects of cosmic radiation and

microgravity. This is very difficult as only cellular models and animal experiments are

currently available, and only few reports can be found today. Therefore, future studies are

necessary to test the effects of radiation and microgravity on osteoblasts, osteoclasts and

progenitor cells in vitro, but also in vivo using the HLU model. Furthermore, it has to be taken

into account that animals do not have a similar load on their limbs as humans, and that cell

studies in vitro are not as complex as in vivo studies concerning the interpretations of

mechanisms in tissue and in the organism concerning communication between tissues.

Therefore, it would be of interest to use the model of three-dimensional spheroids. These 3D

aggregates are very similar to tissues and are closer to the in vivo situation, as has already

been shown in space [8, 131, 136].

Tissue engineering of bone is currently a hot topic. Preosteoblasts cultured in a RWV could

be engineered into osseous-like tissue [167, 168]. These bone tissue pieces may be useful for

testing the combined effects of radiation and microgravity using the RWV. A similar

technical approach using 3D bone constructs was recently published by Beck et al. in 2014

[138], and is demonstrated in Fig. 2.

Besides exercise and dietary supplementation, several regulatory pathways are targets for

therapeutic strategies to combat against bone loss (e.g. Wnt pathway, estrogen, RANKL).

MicroRNAs regulate the expression of specific mRNA species and consequently the

concentration of encoded proteins, and miRNA-based therapeutic strategies might have

potential in various tissues, including bone [169, 170].

In a recent paper Smith and coworkers investigated the growth factor IGF-1 during

spaceflights [171]. The level of IGF-1 was increased in astronauts having access to restrictive

exercise countermeasure hardware. These finding is in support of the growing body of

evidence showing that exercise, together with nutritional supplementation, increase bone

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formation. However, the consequence of exercise-induced changes in bone metabolism on

bone strength as well as fracture risk still needs to be determined.

Osteoprotegerin-Fc (OPG-Fc) is a decoy receptor for receptor activation of RANKL and it has

been shown that this monoclonal antibody prevent loss of bone mass [172]. To investigate

whether RANKL inhibitors like OPG-Fc should be used to mitigate bone loss in astronauts,

mice were flown for a 12-day mission with and without OPG-Fc treatment. Using this set up

Lloyd and colleagues found that a single prophylactic injection of OPG-Fc effectively could

prevent adverse effects on mouse bone related to spaceflight [173], thereby paving the way

for using osteoprotegerin as a countermeasure for bone loss in future spaceflights.

Another recent study has investigated the effect of spaceflight on non-weight bearing bones of

the head [174]. In this study Ghosh and coworkers demonstrated that during spaceflight

mandibular bones experience skeletal alterations, suggesting that non-weight bearing bones

are altered in a weightless environment [174]. Notably, they showed that the adaptation of

mandible to spaceflight to some extent is different to that of the cranium.

CONCLUSIONS

The flight of STS-135 was the final mission of the American Space Shuttle program [16].

Currently, the SpaceX Falcon rocket and its capsule, Dragon, are delivering supplies and

experiments to the ISS. Researchers are able to send their experiments with the Dragon to the

ISS. We had this opportunity last year with the SpaceX CRS-3 mission [9]. The same

hardware suitable for cell experiments in space was flown earlier during the Sino-German

Shenzhou-8/SIMBOX spaceflight in 2011 [175]. Future studies on spaceflight-induced

osteoporosis may well yield new information and pharmaceutical targets to treat Earth-bound

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human diseases [16].

Today, we know more about the unique differences between humans because of advances in

omics technologies like proteomics, genomics, transcriptomics, and metabolomics [165],

which can be used in spaceflight to develop a personalized countermeasure package for each

space traveler. For instance, applying pharmacogenomics and targeted metabolomics to

personalize drug prescribing is one preventive method to improve the safety and efficacy of

drugs used in space [165]. The development of a comprehensive omics-based countermeasure

repertoire may help to protect astronaut health in the future. Since omics investigations have

revealed that unique differences exist between individuals, personalized medicine may

become a standard of care for astronauts in space.

ACKNOWLEDGMENTS

The authors would like to thank the German Space Agency (DLR; (DG) BMWi projects

50WB1124 and 50WB1524) and Aarhus University, Denmark (DG, TJC) for funding our

research. Proof-Reading-Service (PRS, Hertfordshire, UK) is thanked for professionally

proofreading of the paper.

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FIGURE LEGENDS

Figure 1: The most important physical health problems of humans in space.

Examples of the most critical health concerns for space travelers, including reduced tissue

load, radiation, space motion sickness, increased protein turnover, reduced immune response,

disruption of vision and taste, cardiovascular problems, orthostatic intolerance, disturbance of

the biological clock and intestinal fluid shifts. In astronauts, bone loss in response to

microgravity causes the release of calcium from bone (1), which suppresses parathyroid

hormone (2). This suppression of parathyroid hormone in turn is associated with a drop in

circulating 1,25-dihydroxyvitamin D and a decrease in calcium absorption (3).

Figure 2: Combined radiation and simulated microgravity in vitro.

Radiation can be combined with microgravity using primary human cells (mesenchymal stem

cells, osteoblasts, osteoclasts and others) cultured in 3D or in bone-like structures (e.g.

titanium dioxide scaffolds; www.biomaterials.no) (A), placed on a desktop random

positioning machine (RPM) (manufactured by Airbus, Defense and Space (ADS), former

Dutch Space, Leiden, NL) (B), or in a rotating wall vessel (purchased from Cellon,

Bascharage, Luxembourg) (C).

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Table 1: Recent bed-rest studies investigating the influence of simulated microgravity on bone

Type of bed-rest Duration Observations Reference

HDT with or without

exercise

5 d Bone resorption increased during BR, locomotion replacement training or 25 min of upright

standing had no effect

(176)

HDT with or without

resistive vibration exercise

or resistive exercise

60 d Increases of sclerostin and dickkopf-1 in all groups, no evidence for an influence of exercise on the

rise in serum sclerostin and dickkopf-1 levels

(177)

HDT with or without

resistive vibration exercise

60 d Serum osteocalcin was significantly associated with serum insulin and leptin (increased during BR

in both groups)

(178)

HDT 35 d Increased bone demineralization, increased urinary calcium and decreased aquaporin-2 excretion (179)

HDT with or without 30

min centrifugation (1g at

center of mass)

5 d Serum sCD200 levels fall and sCD200R1 levels rise (the author propose them as useful surrogate

markers for bone loss). Centrifugation abolished or attenuated these changes.

(180)

HDT 14 and 21 d The Wnt-pathway is involved in bone loss under microgravity. Sclerostin levels rose during BR and

declined at the ends of the studies. Bone formation marker PINP decreased and bone resorption

marker NTx increased during BR

(124)

HDT 30 d Urinary markers of bone resorption increased, and serum parathyroid hormone decreased. Urinary

oxalate excretion decreased and correlated inversely with urinary calcium

(49)

HDT 90 d Bone mineral density declined significantly, Serum sclerostin was elevated. Serum PTH levels were

reduced, urinary bone resorption markers and calcium were significantly elevated

(181)

HDT with or without

vibration training

14 d Increase in bone resorption, no effect of vibration on bone resorption markers, bone formation

markers, and calcium excretion.

(182)

HDT with or without

exercise or high-protein

60 d Deterioration of bone microstructure and density, no effect of exercise and nutrition. (183)

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nutrition

HDT with or without

exercise or high-protein

nutrition

60 d Regional differences in bone loss in women with incomplete recovery one-year after bed-rest. No

effects of exercise or nutriton

(184)

HDT 21-90 d No changes in phylloquinone, urinary γ-carboxyglutamic acid, or undercarboxylated osteocalcin,

comparable to spaceflights,indicating vitamin K supplemetation in microgravity is not needed to

counteract bone loss

(185)

HDT with or without

resistive vibration exercise

or resistive exercise

60 d Reductions in cortical area, cortical thickness and bone density at the distal tibia, but increases in

periosteal perimeter and trabecular area. Recovery within 180 d after BR. At the distal radius,

persistent increases in cortical area, cortical thickness, cortical density and total density and

decreases in trabecular area. Only on the cortical area at the distal tibia resistive vibration exercise

had a significant effect.

(186)

BR= bed-rest, HDT = head down tilt, d = days, NTx = amino-terminal collagen crosslinks, PINP = Procollagen type I N-terminal propeptide,

sCD200 = soluble CD200, cCD200R1 = soluble CD200R1

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Table 2: Overview of the bone loss countermeasures used in real and simulated microgravity

Countermeasure Microgravity

stimulus

Duration Observations Reference

iRED Real (ISS) 6 months No effects on bone loss (42)

ARED Real (ISS) 6 months Helps maintaining bone mass when combined with adequate energy intake (42)

70 mg alendronate

once/week + iRED or

ARED

Real (ISS) 5.5

months

High variability of data, hints towards superiority of combination vs.

training alone

(107)

HEM (resistance exercise

training)

Simulated

(horizontal bed-rest)

17 weeks Prevention of BMD loss in total hip, calcaneus, pelvis and total body,

significantly increased bone metabolism markers and net calcium balance

(149)

Resistive exercise ±

vibration

Simulated (HDT

bed-rest)

60 d The combination of vibration and resistive exercise prevents bone loss at

the tibial diaphysis and proximal femur more efficiently than resistive

exercise alone

(150)

Supine treadmill exercise within LBNP/ flywheel

resistive exercise

Simulated (HDT

bed-rest)

60 d Exercise treatment significantly attenuated loss of hip and leg bone mineral

density

(51)

Artificial gravity (1g at

center of mass)

Simulated (HDT

bed-rest)

3x5 d No protection by artificial gravity (100)

Alendronate (10 mg/d) Simulated

(horizontal bed-rest)

17 weeks Alendronate attenuated most of the changes in bone occurring during bed

rest

(158)

EHDP (5 or 2 x 20 mg/d) Simulated

(horizontal bed-rest)

20 weeks Only minor effects, no change in skeletal mineral loss (161)

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Flywheel resistance

training + 1 x 60 mg

pamidronate 14 d before

start of bed-rest

Simulated (HDT

bed-rest)

90 d No effect of pamidronate on bone metabolism (160)

HEM = Horizontal Exercise Machine, BMD = Bone Mineral Density, iRED = Interim Resistive Device, ARED = Advanced Resistive Exercise

Device, HDT = Head Down Tilt, d = days, LBNP = Lower Body Negative Pressure, EHDP = disodium ethane-1-hydroxy-1, 1-diphosphonate

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Figure 1

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Figure 2

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Highlights

Current status of bone loss research from both actual spaceflights and analog models

Bone loss of astronauts is a well-known health concern of long-term spaceflight

Calcium release from the skeleton of crewmembers elevates the risk of renal stone

development

Wnt-ß-catenin signaling pathway is involved in bone loss in space

Combination of exercise (ARED), nutrition, calcium, vitamin D and bisphosphonates are

useful to protect bone health