Models for Investigating Bone Loss Mechanisms
and Countermeasures
Sara Arnaud, M.D., NASA Ames Research Center
Adrian LeBlanc, Ph.D., Baylor College of Medicine
The potential for significant and possibly irreversible loss of bone is one of the most
important medical concerns for long-duration manned space flight. NASA's ability to
assure the health of the astronauts during long-duration space station missions or travel to
Mars is compromised without an effective countermeasure to disuse bone loss. Attempts
to understand and prevent the bone loss associated with space flight have implications not
only for NASA's manned space program, but for the community at large. Development
of an effective countermeasure for disuse bone loss could lead to a better understanding
of other forms of osteoporosis, and perhaps the development of new and effective ways
to prevent or treat osteoporosis in the general population. Proposed potential
countermeasures include exercise, nutritional and pharmacological strategies. In order to
investigate the effectiveness of these various proposed countermeasures, ground-based
animal and human models that simulate some of the effects of weightlessness on the
skeletal system have been employed.
Animal Models
Most of the ground-based data about the effects of space flight on the musculoskeletal
system have been generated by experiments that use animal models. Information from
models in the rat and the monkey can be compared to information acquired before and
after exposure to the same species in space. Newer models are being developed in the
mouse, especially those with genetic defects, but there are not yet enough flight missions
using this species for comparison of results of experiments on Earth and after space
flight. Obviously, extrapolation of data from a quadriped nocturnal rodent to the human
is out of the question, but there are a number of similarities in the physiology and
responses of the skeleton and calcium metabolism in these two species to support the
large amount of work in rats in a biomedical research program. If it were possible for the
non-human primate to travel in space unrestrained, the monkey would be a very good
animal model for human bone loss. This species, no longer approved by NASA as a
flight animal, has contributed important information from two week unmanned space
flights in Russian biosatellites.
The best known animal model for space flight is one developed at NASA Ames Research
Center in the rat (Wronski and Holton, 1987). It incorporates the two features of space
flight that affect bone: unloading of weight bearing bones and the cephalad fluid shift.
Orthopedic tape is firmly attached to the sides of the tail and to an overhead pulley
system that raises the hind limbs off the cage floor and maintains the body of the animal,
tail-up, at an angle of about 30 degrees. The forelimbs are not overloaded. The overhead
pulley system has a swivel that allows the animal free movement about the cage. After a
few days adaptation when the appetite is suppressed and weight gain is poor, tailsuspended animals are active and eat and drink normally. They show few signs of
distress for periods of time ranging from two to five weeks. This model avoids the
problems of nerve resection (Yeh, 1989), overloading of the contralateral leg (Jee, 1999)
and other techniques (Musacchia, 1988) used to study unloaded bone or muscle atrophy.
The relatively benign technique, its relatively rapid and accurate simulation of the
changes occurring during space flight and more recently, a cage modification that allows
metabolic collections, i.e. urine and feces (Harper et al, 1994) probably all account for its
frequent use.
A medline search using the term "tail-suspension" recently listed well over 200
investigations. Most of these reports concern muscle atrophy that precedes the loss of
bone. Neither the communication system between muscle and bone nor the precise
mechanism of bone loss is understood. Characterization of the response to skeletal
unloading at the tissue and cellular level is one of the major contributions of the use of
the rat model. The most commonly used experimental subject is the juvenile male rat.
Morphologic studies reveal a decrease in the rate of bone formation (Morey-Holton and
Globus, 1998). Femurs and tibias have predictably less mineral than weighted control
limbs after two to four weeks and curiously, the skull mineral content increases in the
growing rat (Roer & Dillaman). There are fewer investigations in the mature than
juvenile rat, but loss, rather than depressed growth of bone, can be demonstrated after 4
weeks in the femur (Arnaud et al., 1995). Changes in calcium balance and in the calcium
endocrine system show decreases in intestinal absorption of calcium, and in the
circulating levels of the hormones which regulate this process. The response to skeletal
unloading in bone appears to be an adaptation to disuse, remarkably similar to
observations in the human.
Monkey Models
A great deal of attention was directed to the development of a ground-based model for
space flight in macaques (Young et al., 1983). Young's model was a chair-restraint system
that was effective in inducing bone changes expected from space flight (Young et al.,
1986), and was applied to the development of a non-invasive instrument to monitor bone
strength. The ground-based models in the monkey were similar to the human bed rest
model (Sandler H., 1979). This early work anticipated the participation of this species in
the Cosmos or Bion Program carried out by the Russians (Ballard and Conolly, 1990).
While only two monkeys were flown, scientists conducted the same experiments in both
control and flight animals on the ground during or after the flights (Koslovskaya et al.,
2000). Bone mass was estimated from the tibia of juveniles to be 10% lower than
preflight bone density, consistent with morphologic changes in the iliac crest that showed
depressed bone formation (Zerath et al., 1996). Morphologic results were reproducible in
the most recent Bion 11 mission and differed from the results in ground-based controls
(Zerath et al., 2000). Of interest is data from the most recent mission that showed
remarkable similarity of the changes in calcium endocrine system in ground controls and
flight monkeys, an indication that the adaptive mechanisms for disuse proceed normally in
microgravity. The data in flight animals was distinguished from that in the ground based
controls by body weight losses, decreases in total body water and all fluid compartments,
and increases in serum calcium, total proteins and cortisol, all suggesting the importance
of fluid and electrolytes on bone integrity (Arnaud et al., 2000). Although the last Bion
mission yielded an impressive amount of data on the response of the primate to
microgravity, flights for this species and for its model have ended.
Human Models
Human bed rest has been commonly used as a ground-based model to test the effects of
weightlessness and proposed countermeasures upon the musculoskeletal system. In this
model research subjects are required to remain in bed either horizontal and at 6 degree
head-down tilt for lengths of time from weeks to several months. Mineral losses during
bed rest and space flight have been found to be similar in magnitude (Oganov, 1992;
LeBlanc, 1990; Schneider, 1989). Loss of bone during disuse (bed rest or weightlessness)
is a regional phenomenon, with losses averaging approximately 0.5-1.5% per month in
such regions as the lumbar spine and hip. This loss of skeletal mass may prove
hazardous to astronauts on flights of long duration because hypercalciuria might lead to
the formation of renal calculi during flight and weakened bones may be more susceptible
to skeletal fractures upon return to gravity.
Exercise Countermeasures
Evidence from bed rest studies and space flight suggests that bone loss is a regional
phenomenon in which the bone areas with the greatest decrease in load, lose the most
bone (Oganov, 1992; LeBlanc, 1990). Skylab astronauts averaged 0.5% per month total
body calcium loss despite exercising a number of hours a day using a resistive device for
the arms, a treadmill, and a bicycle ergometer (Johnston, 1977). During long duration
missions the cosmonauts are required to maintain physical fitness through a series of
exercises consisting of bungee cords for resistive exercises, bicycle ergometer exercise,
and walking on a treadmill (Nicogossian, 1994). Exercise schedules require three hours
of exercise daily. However, cosmonauts continue to lose bone selectively from the spine
and lower extremities while maintaining upper body bone mineral density (Oganov,
1992). Similar losses also occur in bed rest subjects not performing exercise (LeBlanc,
1990). Previous bed rest studies of exercise as a countermeasure showed no protection
from use of an 8-lb resistance pulley device, standing 3 hours a day, or standing 3 hours
and bicycle ergometry exercise 20 minutes a day (Schneider, 1984, 1993). Walking for 1
hour at 3 miles per hour four times a day and supine bed rest for 20 hours per day
maintained skeletal calcium (Schneider, unpublished). A current study, conducted at
Baylor College of Medicine in collaboration with the Johnson Space Center, is
investigating a protocol involving heavy resistive exercise to prevent bone loss during
long duration bed rest. To date 8 controls and 9 subjects performing exercise 6 days per
week have completed the study. The results indicate that the exercise group appears to
maintain or increase BMD in the lumbar spine, femoral neck, pelvis and the calcaneus
during 17 weeks of bed rest compared to pre bed rest values. The trochanter continues to
show losses similar to bed rest without countermeasure.
Nutritional Countermeasures
It is generally recognized that the maintenance of bone mass is dependent on a level of
nutrition sufficient to maintain body weight. One might speculate that caloric intakes
required to maintain body weight in a weightless environment would be less than on
Earth. Actually, there is a mandatory exercise program during space flight designed to
maintain muscle and bone during flight. This exercise schedule seems to have created an
energy deficit and negative nitrogen balance that varies significantly with the mission
(Stein, 1996 and 1999). During the Skylab mission, when calcium and nitrogen balances
were conducted, the energy deficit was less but as might be expected these balances
reflecting bone and muscle tissue, paralleled one another. Calcium loss in the calcaneus
was related to the balance. In addition to caloric intakes, protein and calcium, other
nutrients that are associated with bone metabolism, phosphorus, sodium, potassium and
magnesium have no limits or requirements specific for the microgravity environment.
Nutritional recommendations for space flight have not differed from the recommendations
of the National Research Council for life on Earth. There is a substantial amount of
knowledge yet to be acquired in this area (McCormick D. B., 2000).
Pharmacological Countermeasures
Five biochemical regimens have been studied previously: 1) synthetic salmon calcitonin
(Hantman, 1973), a hormone which inhibits bone resorption (100 MRC U), was given
daily by injection during 8 weeks of bed rest; 2) phosphate supplements were given in
divided doses as a neutral potassium salt (Hulley, 1971); 3) oral calcium and phosphate
were given in divided doses (Hantman, 1973); 4) etidronate either as a 5 mg/kg/day dose
or as a 20 mg/kg/day dose (Lockwood, 1975); and 5) clodronate, 1600 mg/day
Schneider, 1981). Little or no protection was afforded by the first three methods during
long-term bed rest. Low dose etidronate showed no beneficial effect. The high dose
etidronate appeared to have a protective effect starting in the 16th of 20 weeks of bed
rest. During the first 17 weeks of bed rest, the subjects lost significant amounts of
mineral from the calcaneus. During the last 3 weeks of bed rest, the usual progression of
calcaneal mineral loss was no longer observed. Calcium kinetic studies revealed that
bone accretion and resorption rates fell progressively and in parallel fashion to levels
50% below baseline by the end of bed rest. Etidronate, however, has been associated with
an accumulation of osteoid tissue both in animals and man when given at the antiresorptive dose for extended periods of time (Fleisch, 1969; Meunier, 1987). Clodronate
was tested in a bed rest study in which Ca balance decreased to baseline by week 6 and
remained at neutral balance for the completion of the 17 weeks of bed rest. CT
densitometry of the spine in the 9 treated subjects showed essentially no change and
approached statistical significance in preventing lumbar spine bone loss compared to the
5 controls. Single photon absorptiometry of the calcaneus showed no statistically
significant difference between the two groups; one treated test subject showed severe
calcaneal density loss while maintaining normal calcium balance [Schneider, unpub.].
Clodronate was withdrawn from clinical investigation in the United States due to a
potential serious adverse reaction.
New bisphosphonates are being tested for treating global bone loss diseases such as postmenopausal osteoporosis. Recently a new bisphosphonate, alendronate, has been
approved by the FDA for the treatment of post-menopausal osteoporosis and Paget's
disease of bone [Adami, 1994; McCarthy, 1995]. Alendronate (FOSAMAX, Merck,
Inc.) is available in tablet form in doses of 10 mg and 40 mg. Alendronate is structurally
similar to the bisphosphonate etidronate, but has different antiresorptive and bone
mineralization effects. Alendronate was effective in preventing hypercalciuria in eight
men who participated in a 3-week bed rest study in which 20 mg per day of alendronate
was given for 2 weeks prior to and throughout the bed rest period [Ruml, 1995].
Alendronate is currently being tested in a 4-month bed rest study at Baylor College of
Medicine in collaboration with the Johnson Space Center. To date 16 subjects, 8 controls
and 8 subjects taking 10 mg/day-have completed the study. Results indicate that
alendronate treated subjects maintained or increased bone mineral density (BMD) in the
femoral neck, femoral trochanter, spine, and pelvis. Moderate loss of BMD occurred in
the calcaneus, but the loss was significantly less than in the control group. Other newer
bisphophonates are likely to be even more effective.
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