**File Name: E194.DOC**

Bone, Calcium and Space Flight

Emily R. Morey-Holton1, Christopher E. Cann2, Stephen B. Doty3,

W. Eugene Roberts4, and Arthur C. Vailas5

Ames Research Center, Moffett Field, CA1, University of California, San
Francisco, CA2, Hospital for Special Surgery, New York City, NY3,
Indiana University School of Dentistry, Indianapolis, IN4, and
University of Wisconsin, Madison, WI5

**ABSTRACT**

The hypotheses tested in this spaceflight experiment were 1) the type of
housing (group vs individual) will influence the bone response to
spaceflight and the recovery from spaceflight and 2) the response of
bone to spaceflight will be localized and will differ not only from bone
to bone but also at different sites within the same bone. Growing male
rats were flown on Space Life Science 1 (SLS1) on the shuttle Columbia.
The rats were housed in groups of five on the shuttle middeck or
individually in the spacelab. The flight lasted 9d. Half the animals
were euthanized at the end of the flight period and the other half was
allowed to readapt to Earth for 9d following flight. The results suggest
that housing affects the response to spaceflight. The singly housed
animals showed greater inflight changes and a slower recovery from
spaceflight than the group housed rats. These differences occurred in
bone mineralization rates, mechanical properties, and enzyme
histochemistry. Also, not all regions of all bones or all bones were
affected by flight; in the long bones, the periosteal surface showed
suppression of formation while the endosteal surface showed little
change, and no changes were noted in the ribs, calvaria, vertebra or
maxilla suggesting that the response to spaceflight is not uniform
throughout the skeleton.

**INTRODUCTION**

During growth, the force imposed by gravity causes bone to increase
simultaneously in mass and strength; as a result, larger animals usually
have larger and stronger bones; stronger bones are the result of higher
skeletal forces from increased body weight and inertial forces.

Previous studies of bone changes during spaceflight have shown a
reduction in periosteal bone formation rather than the loss of bone by
resorption or degradation (Morey and Baylink, 1978; Wronski and Morey,
1983; Vico, et al, 1988). Bones grow about 40% slower in diameter during
spaceflight than on earth (Morey and Baylink, 1978; Shaw et al., 1988;
Wronski and Morey, 1983) even though body mass gain is comparable and
net bone resorption is essentially unchanged compared to ground controls
(Cann and Adachi, 1983). Precursor cells which become osteoblasts may
differentiate at a slower rate during spaceflight (Roberts et al, 1987)
and fewer osteoblasts would

form less bone. In addition, the vascular supply within the bone matrix
may affect the reduced osteoblastic activity, since the blood vessels
not only provide nutrients to these cells, but the vessels are contained
in channels lined with osteoblasts and pre-osteoblasts (Doty, et al.,
1992). Focal vascular occlusion just beneath the outer surface of bone
has been noted both following flight and in unloaded bones on earth, and
could contribute to localized bone changes (Doty et al, 1990). Changes
in mineralization rates and bone turnover, maturation, and progenitor
populations have been reported in the maxilla and mandible (Simmons et
al., 1983; Roberts et al, 1987; Jackson et al., 1988) as well as the
long bones. A possible increases in matrix in the calvaria of Cosmos
animals has been reported (Lorenz et al, in press). An increase in
mineral in the skull has been reported in head-down rats (Roer and
Dillaman, 1990) and humans (Arnaud et al., 1989; LeBlanc et al., 1990)
and in cosmonauts on Mir (Schneider et al., 1992) suggesting a possible
role of fluid shifts or perfusion pressure on bone formation.

In addition to the decreased bone formation, collagen concentration was
decreased immediately after flight (Yamauchi et al., in press), but
increased within 2-days postflight (Patterson-Buckendahl et al, 1987).
Cytoskeletal elements in the osteoblasts, aligned to orient collagen
vesicles during secretion (Doty, 1988), may be disrupted in space; this
malfunction could alter the ability of collagen to form a stable
structure (Turner et al., 1985). Lack of an organized structure could
partially explain the smaller bone crystals found in flight rats
(Simmons et al, 1983 and 1986) as well as the lack of increase in the
bone strength during growth in microgravity. The amount of mineral added
per unit mass of tissue is the same as in growing bones on earth and few
changes in density have been reported. These results implicate the
osteoblast as the responder to gravity or non-weightbearing.

The mechanical changes in bone occurring during spaceflight have been
examined on several Cosmos biosatellite missions and also on NASA space
shuttle missions. All experiments have used the rat; however, the
results cannot always be compared directly because the bone examined and
tests performed have varied between experiments; even when the same test
is performed, the parameters reported may differ. In general,
investigators have found that mechanical strength, compared to ground
controls of the same age, is lower in skeletal tissue following
spaceflight, but the extent of the change depended on the site measured,
the animals\' age, and the length of time elapsing between return of the
spacecraft and obtaining samples (Spengler et al, 1983; Shaw et al.,
1988; Vailas et al., 1990, 1992; Zernicke et al., 1990).

Our understanding of the response of growing bone to spaceflight has
increased significantly in the past ten years, but many questions still
remain unanswered. The importance of cage size, configuration, and
animal housing (single vs group) has not been investigated. Whether
total skeletal turnover is altered during flight or the effect of
spaceflight on the skeleton is limited to specific locations in specific
bones is not known. The purpose of this experiment was to delineate the
early changes that occur in both weightbearing and non-weightbearing
bone tissues in growing animals in

different cage configurations during spaceflight, and to relate these
changes to alterations in cell proliferation and mineralization, bone
subcellular characteristics, and bone biomechanics.

**EXPERIMENT MATERIALS AND METHODS**

Specific pathogen free, male, Sprague-Dawley derived rats from Taconic
Farms, Germantown, NY were used. All animals were given food and water
*ad libitum* throughout the experiment. Upon arrival at Kennedy Space
Center on L-28d, the rats were given Wayne rat chow. At L-13, rat diet
was changed to Teklad NASA Experimental Rodent Diet #TD 88179 extruded
into food bars. The food bars were dipped in 15% sorbate to retard mold
growth, radiation sterilized, sealed in polyethylene bags, and stored at
4C. The light/dark cycle was 12light:12dark (9AM to 9PM, EST). The
ambient temperature for rats preflight and postflight was 23±1C.
Inflight the AEM was kept between 27-32C while the RAHF was maintained
at 25±2C. The animals, food, and water were weighed every third day
except that weights were also taken the morning before and at loading
into the cages for flight. The animals were not weighed during the
flight period, but food and water were monitored in both the flight and
ground control cages. During flight, the water calculation for inflight
consumption on the research animal holding facility (RAHF) suggested
that the water supply might be insufficient to support the rats for two
contingency days after Spacelab deactivation; thus two to three packs of
gelatin containing 30ml of 1% agar/pack were placed in the RAHF flight
and singly housed ground control cages on the eighth day of flight.

The rats were randomly divided into groups 13 days before launch (L-13).
The groups for the flight experiment included: 1) animal enclosure
module or group housed controls (AEM-C), 2) AEM flight groups (AEM-F),
3) vivarium or singly housed controls (VIV-S) , 4) research animal
holding facility or individually housed flight rats (RAHF-F) and 5)
basal or time 0 controls. A total of 29 rats was flown (10 group-housed
and 19 individually housed). The AEM animals were housed five rats per
cage in standard, clear plastic, rat colony cages with corn cob bedding
preflight and postflight. During the flight period, the AEM groups were
housed in AEM flight units. VIV-S animals were housed one rat per cage
in standard, clear plastic, rat colony cages with corn cob bedding
throughout the experiment. RAHF rats were housed one per cage in
standard, clear plastic, rat colony cages with corn cob bedding both
preflight and postflight; during the flight period, the RAHF animals
were in RAHF cages. The rats were loaded into the flight units
approximately 33-hours before launch. Each AEM unit supported five
group-housed rats while the RAHF had one rat per cage. Two AEM units (10
rats) were flown on SLS1. The RAHF holds 12 housing units; each unit has
two cages arranged in tandem. Ten RAHF housing units contained 19
individually-housed rats; one cage was left empty due to a malfunction
in the water leak alarm. The remaining RAHF units were reserved for the
particulate containment demonstration test.

The shuttle was launched from the Kennedy Space Center in Florida on
June 5, 1991. On the day of launch, tissues were taken from a basal or
time zero individually housed control group. Two days later, control
animals were transported to Dryden Flight Research Facility in Edwards,
CA. The shuttle landed at Dryden after a nine day mission. The AEM
flight rats were received within two hours of landing and the RAHF
flight animals were received within four hours. Euthanization was by
guillotining. Bones were processed within 20 minutes of decapitation.
All flight rats were processed within 6.5 hours of landing. The AEM
control group was processed shortly after the shuttle touched down.
Within 10 hours, all R+0 animals had been processed. Half the animals in
each group were euthanized at the end of the flight period (R+0), while
the remaining animals were allowed to recover for nine days (R+ML). The
R+ML rats were weighed daily for nine days. During the postflight
period, all animals received injections and were bleed from the tail for
other experiments.

The bone markers used for this experiment included calcein
(15mg/kg)(Sigma Chemicals, St. Louis, MO), demeclocycline hydrochloride
(15mg/kg) (Sigma Chemicals, St. Louis, MO), and tritiated proline
(3mCi/kg)(Amersham Corporation, Arlington Heights, IL). Calcein was
given subcutaneously 13d before launch. Demeclocycline was given
subcutaneously two days prelaunch. Proline and calcein were given
intraperitoneally into the recovery animals on the day of landing (R+0).

The right tibia, femur, and humerus from RAHF and VIV animals and the
left tibia from the AEM rats were cleaned of muscle tissue and divided
into three pieces (shaft and ends containing epiphysis and metaphysis)
using anatomical landmarks. The proximal tibia, distal femur, and distal
humerus was immersed in a mixture of 2% paraformaldehyde plus 0.5%
glutaraldehyde in 0.05 M cacodylate buffer, pH 7.2, for 18 hours at 4°C.
Following fixation, tissues were stored in 0.05 M cacodylate buffer, pH
7.2 containing 0.01% sodium azide. For histochemistry, vibrotome
sections were collected or frozen sections taken following an overnight
cryoprotection procedure using 20% sucrose plus 5% glycerol solution.
Bones for demineralization were immersed in 10% EDTA in 0.1M Tris
buffer, pH 7.2, at 4°C, then processed for electronmicroscopy as
previously described (Doty and Schofield, 1990). The humeral head plus
about 2-3 mm of attached shaft was embedded in JB-4 plastic which
polymerizes at -20C (to prevent heat denaturation of proteins) and
alkaline phosphatase could be localized on these sections of mineralized
bone. Alkaline phosphatase activity of the osteoblasts and perivascular
cells was visualized by Burstone's azo dye method or by a lead capture
process (Doty and Schofield, 1990). NADPase activity was used to monitor
the Golgi activity of these same cell populations (Doty and Schofield,
1990). Tartrate resistant acid phosphatase (TRAP) activity was
determined on frozen sections from aldehyde-fixed demineralized
metaphyseal bone from femurs using an azo dye method (Doty and
Schofield, 1990). The bone shafts were placed in acetone and processed
for bone mineralization rates as previously described (Hill et al., in
press). The maxillae were cleaned of muscle tissue and immersed in cold
(2-4C) 10% neutral pH formaldehyde solution buffered with dibasic and
monobasic phosphate (PBF) for 7d, then changed to

70% alcohol for storage. Prior to embedding, tissues were immersed in a
decalcification solution of 10% EDTA/2% PBF at pH 7.4, embedded in
plastic, sectioned at 4 µm in the midsagittal plane of the mesial root
of the first molar and stained with hematoxylin and eosin. Only samples
with a resorbing or resting bone margin were analyzed. Nuclear volume
for each of 100 cells samples was calculated using the formula for a
prolate spheroid: V=4/3πab2 where a and b are the radii of the major and
minor nuclear dimensions respectively. Each cell was classified as type
A+A\', B, C, or D according to the following categories: 40-79, 80-119,
120-169, and ≥170µm3, respectively and data were expressed at group mean
%±SEM (Garetto et al., 1992; Roberts et al., 1987).

The calvaria was taken from the R+ML rats because the brain dissectors
reported that the calvaria in the flight animals seemed more difficult
to cut than calvaria from controls. It was frozen at -20C for transport;
it was bisected anterior-posterior and half the bone processed for
mineral apposition rates and the other half analyzed for calcium. The
left femur was cleaned of adjacent muscle tissue and shipped in medium
for removal of the marrow; the bone was then frozen for calcium
analysis. The first lumbar vertebra (L1) was also processed for calcium
analysis as previously described (Globus et al., 1986).

The left humerus, left tibia (RAHF and VIV only), and L4 vertebra were
wrapped in individual foil squares without removing adjacent muscle mass
and frozen in liquid nitrogen. These bones were processed for density,
calcium, collagen parameters, and mechanical properties (Vailas et al.,
1988; Salem et al., 1989; Shaw et al., 1988). Due to freeze fracture of
L4, it was only processed for matrix components. The long-bones were
placed into a physiological medium that was equilibrated at 37°C.
Subsequently, the bone samples were placed upon into a three-point
bending chamber that was equilibrated in the physiological fluid and
tested until failure. All bones samples failed at mid diaphysis.
Parameters calculated included: 1) flexural rigidity, 2)bone strength,
3) failure energy, 4) maximum linear stress and (5) linear energy
storage. Bone fragments from the biomechanical experiments were frozen
at -70°C and placed into a lyophilizing chamber. Samples were dried to a
constant weight and density was measured by water displacement.
Subsequently, bone samples were hydrolyzed in 6M HCI in sealed ampules
equilibrated at 110°C for 24 hours. After acid hydrolysis, the samples
were cooled to room temperature. An aliquot from each sample was dried
by evaporation and derivitized by an Edman Reagent
(phenylisothiocynate). Derivitized samples were prepared for amino acid
analysis using a C18 reverse phase column (Waters Pico-Tag) HPLC method
for separating proline and hydroxyproline. Eluent from the column was
passed through a UV detector (absorbence = 254nm) and then into a
fraction collector. Radioactive proline and hydroxyproline were measured
in the fractions. Absorbence measurements were integrated for
determining the quantity of unlabeled proline and hydroxyproline.
Specific activities for proline and hydroxyproline were derived from UV
absorbence and scintillation counting and expressed as DPM/µM of
hydroxyproline. Hydroxyproline values were used to quantify the collagen
content based

on a collagen molar calculation assuming that hydroxyproline constitutes
14% of collagen. The molar value was used to normalize the
hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) mature collagen
crosslinks. Analyses of HP and LP required drying, reconstituting an
hydrolyzed aliquot with buffer and acetonitrile, and separating the
crosslinks with 35cm reverse phase C18 HPLC column (Beckman) using
isocratic conditions (16% acetonitrile). Both pyridoxamine and purified
HP (99% purity Bovine tendon) were used to identify the mature collagen
crosslinks. Values were expressed as mole of crosslink to mole collagen.

Data were analyzed with the SuperANOVA program (Abacus Concepts, Inc.,
Berkeley, CA) using a 1 factor ANOVA and Student-Newman-Keuls as the
posthoc test. Significance was set at the 95% probability level
(P≤0.05).

**RESULTS**

**Body/bone mass**

The rats (n=74) were 65d old at launch and weighed an average of
285±16gm (mean±SD) two days before launch; the day of launch the basal
controls (n=15) weighed an average of 295±12gm. At recovery the rats
weighed 331±19gm (n=59) and by R+ML the average weight was 342±20gms
(n=29). No significant difference in mean body weights was noted among
any of the groups on the same day, only the weight gain reflected
postflight differences (Figures 1, 2). Flight rats and ground controls
gained an average of 57±8g during the preflight period and 46±0.8g
during the flight period. During the post-flight period, VIV-S gained
weight faster than the other groups (VIV-S gained 21±9g or 2.3±1.0gm/d,
AEM-C gained 12±8g or 1.4±0.9g/d, AEM-F gained 6±2gm or 0.7±0.3g/d, and
RAHF-F gained 4±7g or 0.5±0.8g/d during the postflight period)(Figure
2). VIV-S was significantly different from the flight groups in weight
gained during the recovery period, but AEM-C was not different from any
group at R+ML. If one AEM-C rat that lost weight during the postflight
period was eliminated (16±3g or 1.8±0.3g/d), then both control groups
gained significantly more weigh than both flight groups in the
postflight period. Although differences in growth rates between flight
and control rats occurred when the gain was calculated for the entire
postflight period, growth in the flight groups paralleled the control
groups after the third postflight day (Figure 1). The transient
postflight decrease in AEM-C weight may reflect a transition from
housing in the flight-type unit to vivarium cages. The tibia, femur, and
humerus exhibited significant growth in length between launch and R+ML
(Figure 3), but bone length was similar in all groups at R+0 or R+ML
(Figure 3). L1 and rib masses were similar at R+0 and at R+ML in all
groups (Figure 3), and the bones exhibited normal age-related increases
in mass. The femur mass in the RAHF-F was smaller than VIV-S at R+0 but
was not significantly different until R+ML while the AEM-F was very
similar to AEM-C femur mass at all time points (Figure 3).

**Bone Mineralization Rates**

Bone mineralization decreased with age (Figure 4) similar to rate of
weight gain (Figure 3). At the periosteal surface of the tibia at the
tibiofibular junction, the control groups showed a 13% age-related
decrease in TFJ periosteal mineralization during the flight period and
about a 26% decrease during the postflight period for an overall
decrease of about 34% during the experimental period. Bone markers were
given preflight and postflight, so that each rat could serve as his own
control; thus, statistics were based on % change from preflight or
flight rates in addition to comparing mean values. If mean values were
used, no difference in TFJ periosteal mineralization was found between
AEM-C and AEM-F (-8%) during the flight period while the RAHF-F still
were significantly lower than VIV-S (-27%) (Figure 4). When expressed as
a % change from preflight values, periosteal mineralization decreased
significantly during the flight period in AEM-F (-28±8%, p\<0.05) and
RAHF-F (-37±7%, p\<0.05) rats compared to controls (-13±6% for AEM-C and
-13±10% for VIV-S). During the postflight period, the flight rats tended
to show less age-related decrease in TFJ mineralization rather than an
increase in mineralization compared to the controls, but no R+ML
differences were noted between any groups (Figure 4). Interestingly,
tibial periosteal mineralization rate correlated significantly with the
rate of weight gain (R=0.714, p\<0.0001). Like the periosteal surface,
the endosteal surface at the midshaft of the tibia showed age-related
decreased formation, but unlike the periosteal surface, the
mineralization rate was very similar in all groups at either L+0, R+0,
or R+ML (Figure 4). At the tibial endosteum, the average % decrease from
the preflight period was 45% while in the postflight period it was about
25% in the AEM groups and VIV-S. RAHF-F showed little change in
corticoendosteal mineralization rate between the flight and postflight
periods. If only the R+ML rats (n=8) are used in calculating the RAHF-F
R+0 and R+ML values, then a -6% change is noted rather than the implied
8% increase at R+ML when all rats (n=18) are used at the R+0 time point
(Figure 4). The mineralization rates at both the periosteum and
corticoendosteum were very similar and the ordinates in Figure 4 for
these surfaces are identical in each bone (tibia = 0.02-0.07 while
humerus = 0.005-0.055) for a range of 0.05 mm2/d in both bones at both
surfaces. In the midshaft of the humerus, no difference between flight
and control occurred during the preflight period, but during the flight
and postflight periods, the flight rats showed significantly retarded
periosteal mineralization whether the mean or the % change was compared.
The control rats showed an age-related 29±9% decrease in periosteal
mineralization rates in the humerus from preflight to R+0 and a 29±6%
decrease at R+ML compared to R+0; the RAHF-F rats were significantly
lower than VIV-S with a -44±11% change at R+0 compared to preflight and
a -53±7% change at R+ML vs R+0. At the periosteal surface of the
humerus, the mineralization rates (rather than % change) in RAHF-F were
not different from mean VIV-S values during the preflight period (-9%)
but were significantly lower than VIV-S at both R+0 (-28%) and R+ML
(-50%)(Figure 4). At the corticoendosteum in the humerus, mineralization
rate for RAHF-F animals was a nonsignificant \~20% lower than VIV-S at
all time periods; both groups showed an age-related % change from the
previous period of about 44% in the

flight and postflight periods with an overall change of \~-60% when R+ML
is compared to the preflight period. (Figure 4)

Mineral apposition rate in the calvaria from the R+ML animals was
similar among groups both preflight and at R+0 (Figure 5).

**Calcium Concentration/Density**

Calcium concentration was measured in the left femur, humerus, L1, rib,
calvaria (R+ML only) of RAHF, VIV-S, and AEM rats, and in L4 of RAHF and
VIV-S rats. No significant differences in any parameters were noted
between any groups at the same time period. Calcium correlated very
significantly with the bone mass (e.g., femur, R=0.934 and p\<0.0001;
L1, R=0.934, p\<0.0001) suggesting any change in calcium content would
reflect changes in the size of the bone. Tibia and humerus densities
(\~1.65mg/mm3 and \~2mg/mm3, respectively) were the same at R+0 and R+ML
in all groups.

**Matrix Parameters**

Tibia and humeri of flight animals contained significantly less new
collagen than controls only in AEM-humerus and RAHF-tibia (Figure 6).
However, the humeri and vertebrae of RAHF rodents showed no significant
differences between flight and ground. Stable collagen crosslink content
(hydroxylysylpyridinoline and lysylpyridinoline) and hydroxyproline
concentration in the bones were similar in all animals (data not shown).

**Structural properties**

No differences in structural properties were noted in the humerus of
AEM-F vs AEM-C rats at either R+0 or R+ML. During the flight period no
differences in biomechanical properties in the tibia of RAHF rats were
noted. However, in the postflight period RAHF-F tibiae did not show the
increases in stiffness and strength noted in the controls (Figure 7).
Although highest value for stiffness in the humerus of VIV-S rats
occurred at R+ML (Figure 7), it was only equivalent to AEM values at R+0
and less than AEM values at R+ML (Figure 8) suggesting that stiffness
was lower in the humerus of the singly housed animals. No differences in
stiffness were noted in the group housed animals at any time (Figure 8).
Failure energy and deformation to failure showed similar trends.
Material analyses were conducted on RAHF groups only; flight had no
significant effect on humerus or tibia elastic modulus (\~2250N/mm2 and
\~3700N/mm2, respectively).

**Progenitor Populations**

Distribution of progenitor populations in AEM, RAHF, and VIV rats was
not similar to recent Cosmos and ground-based studies (Figure 9). No
difference in progenitor populations were noted in any groups at any
time periods, but the A+A\' population was depressed to about 15% of
normal and the C+D fraction is raised to 25-30% of expected values in
this experiment. The data suggest that the rats may have been stressed
prior to euthanasia.

**Enzyme Histochemistry**

Alkaline phosphatase activity was demonstrated along osteoblast-lined
surfaces of new bone. In the AEM R+0 group, no significant difference in
alkaline phosphatase activity was noted between control and flight
animals. In the humerus of the RAHF R+0 group, the controls showed more
enzyme activity than the flight group. The same sections were scanned
with a backscatter electron detector to study the mineralization pattern
and content of the trabecular bone, but no differences were noted
between control and flight bones. In the tibial corticoendosteum, the
RAHF-F R+ML group showed much greater enzyme activity compared to the
controls and the R+0 RAHF-F showed slightly greater activity than R+0
VIV-S. Normal alkaline phosphatase localization is on the external cell
membrane of the osteoblast, but in these RAHF animals the stromal cells
adjacent to the endosteal osteoblasts and not the osteoblasts showed
alkaline phosphatase activity (Figure 10).

Using frozen sections of aldehyde fixed demineralized tibial cortex, the
Golgi activity of the endosteal osteoblasts was determined by the
NADPase reaction. In the AEM R+0 group, the flight animals and controls
showed similar activity in the osteoblasts. Golgi activity at this site
was suppressed immediately after flight in RAHF-F. An increase in the
numbers of granules was noted in osteoblasts in the RAHF R+ML flight
group compared to the RAHF R+0 flight animals (Figure 11).

Tartrate resistant acid phosphatase (TRAP) activity is a specific
indicator of osteoclasts in bone. RAHF-F showed more activity at R+0
than at R+ML. The R+0 AEM-F showed similar, if not slightly greater,
activity than the control group.

Decalcified vibrotome sections from the distal ends of the tibia samples
(AEM and RAHF animals) were analyzed for acid and alkaline phosphatase
activity. The alkaline phosphatase was quite strong among the endosteal
osteoblasts and the perivascular cells of the vessels within the bone.
The reticular component of the marrow, immediately adjacent to the
endosteal osteoblasts, was also strongly reactive. The enzyme reactivity
among the subperiosteal vessels was less than the vascular reaction near
the endosteum, however no consistent pattern of change between flight
and control animals was found relative to these vascular reactions.

**discussion**

The rats grew linearly from receipt through the flight period. In the
postflight period, the growth rate was slower in all groups and the rate
of weight gain was significantly lower in the flight groups compared to
VIV-S or AEM-C (when n=4) (Figures 1, 2). The decreased growth rate in
all groups in the postflight period probably reflects animal
manipulations for other experiments. Growth rate (body mass) is a very
important determinant of bone mass as evidenced by the strong
correlation between rate of weight gain and mineralization rate. Also,
calcium content was significantly correlated to bone size, and any
differences in calcium could be attributed to differences in bone mass.
No difference in calcium concentration was found between groups in any
bones. Since only about 10% of the body mass of the animals was formed
during the flight period and only 3% of the body mass was added during
the postflight period, most of the bone mass was probably present at

the beginning of the experiment and whole bone analyses may not be
sufficiently sensitive to dissect out changes in a small portion of that
bone. In fact, cross sectional analysis showed that less than 15% of the
bone in tibia (TFJ) and humerus (midshaft) cross-sections was formed
during the flight period. In the early Cosmos missions that lasted close
to 3wk and the young animals on the Shuttle Spacelab 3, the rats gained
around 20% of their body mass during the flight period and more dramatic
bone changes were noted (Morey and Baylink, 1978, Spengler et al., 1983;
Wronski and Morey, 1983, Shaw et al., 1988) suggesting that growth rate
and flight duration are important determinants of detectable changes in
bone structure.

Housing and loading did not seem to affect bone elongation. In
controlled experiments, no definitive effects of loading on bone
elongation have been reported (Biewener and Bertram, 1993). On the other
hand, bone mass did appear to be housing and load sensitive. The femurs
were significantly smaller than similarly housed ground controls and
were slightly, but not significantly, smaller than the group housed
rats. Group housed animals showed no differences in bone mass whether on
Earth or in space. The data suggest that singly-housed rats show a
decrement in accumulation of bone mass during flight that becomes
significant during the postflight period compared to similarly housed
controls

Bone mineralization rates showed significant suppression at the
periosteal, but not corticoendosteal, surface during flight whether the
animals were singly or group-housed. However, the singly-housed flight
rats showed a greater suppression of tibial periosteal mineralization
during flight than the group-housed flight animals. The tibial defect
was corrected postflight by slowing the age-related decrease in
formation rather than increasing the formation rate over the postflight
period (Figure 3). The suppression of periosteal mineralization in the
humerus appeared to continue into the postflight period. Regional
changes, periosteal vs endosteal, have been reported with loading. In
young rats, treadmill exercise increased cortical bone area by
increasing periosteal formation without changes to the endosteal surface
(Yeh et al, 1993). At increased load levels, the rat bending model
resulted in explosive periosteal woven bone formation with no response
on the endosteum (Hagino et al., 1993). Although increases in head
density of cosmonauts (Schneider et al., 1992) and head calcium
concentration in suspended weanling rats (Roer and Dillaman, 1990) have
been reported, no change in calvarial mineral apposition rate, mass, or
calcium concentration was noted in this experiment.

One of the unique aspects of the SLS-1 mission was a high dose of
labeled proline that was injected into the R+ML rats immediately
postflight. The label was incorporated into newly formed extracellular
collagen as hydroxyproline and served as an indicator of matrix
synthesis since hydroxyproline is exclusive to collagen. Collagen
secretion is an essential function in the process of biomineralization
and bone growth. Vertebra had a higher specific activity than either the
tibia or humerus indicating a higher collagen synthesis rate in this
primarily cancellous bone (Figure 6), but

showed no differences between flight and control animals. The humerus
from the AEM-F, but not RAHF-F, showed a lower activity than AEM-C, yet
the mineralization rate appeared impaired in RAHF-F humerus during the
postflight period. Interestingly, neither the AEM-F or AEM-C humerus
specific activity are different from the RAHF-C or RAHF-F; the AEM-C is
somewhat higher and the AEM-F activity is somewhat lower than the RAHF
groups (Figure 6). The tibia of RAHF-F showed significantly lower
activity than RAHF-C, yet the tibial mineralization rates in both groups
were similar during the postflight period. The lack of differences in
hydroxyproline concentration and crosslink content are probably
reflective of the age and, hence, bone turnover rate of the rats similar
to the lack of change in calcium concentration.

The structural properties indicated that the flight had little effect on
the humerus of either singly or group-housed rats. The tibia of singly
housed flight rats was not different from controls immediately
postflight, but it did not show the age-related increase in structural
properties noted during the nine day recovery period in the singly
housed controls (Figure 7). Elastic modulus, which incorporates size and
geometry of cortical bone, showed no differences in the long bones
suggesting minimal changes in size and geometry in the flight animals
compared to controls. No difference in bone density was noted.

Several interesting histochemistry observations were made in the
singly-housed, but not group-housed rats. First, bone formation
(alkaline phosphatase activity) and bone resorption (TRAP activity)
showed inverse relationships\--in the flight animals, after nine days in
flight, formation in the corticoendosteum was reduced whereas the
trabecular bone resorption was increased. Second, the increase in
alkaline phosphatase activity of the stromal cells, as seen by electron
microscopy, may be another response to short term spaceflight.
Typically, the well differentiated osteoblast shows a reduction in
alkaline phosphatase activity during flight, however the stromal cells
have not previously been studied. An increase in activity immediately
following spaceflight may reflect a change of activity
(differentiation?) in these undifferentiated cells which may be
precursors to bone cells. Third, procollagen containing granules in the
osteoblast's cytoplasm reflect the "secretory" activity of the
osteoblast and this function may be very sensitive to the presence or
absence of gravity. The "synthesis" of the procollagen products via the
Golgi apparatus may be responsive to spaceflight or non-weight bearing
but is more difficult to quantify by morphological parameters. Thus the
increase in these granules in the R+ML group and their reduction in the
R+0 group accurately reflects a highly specific gravitational effect on
the osteoblast function. At the EM level, singly housed animals showed
an accumulation of golgi-derived granules in osteoblasts during the
postflight as compared to the flight period. Procollagen containing
granules, derived from the Golgi, are transported to the osteoblast cell
membrane adjacent to the bone matrix. These granules normally are
exocytosed and the collagenous products become incorporated into the
newly forming osteoid.

The hypotheses tested in this experiment: 1) the type of housing (group
vs individual) will influence the bone response to spaceflight and the
recovery from spaceflight and 2) the response of bone to spaceflight
will be localized and will differ not only from bone to bone but also at
different sites within the same bone, appear to be valid.

**ACKNOWLEDGMENTS**

We would like to acknowledge the help and support of many people. Dr.
Gary Jahns and Howard Schneider were the project and program scientists
respectively,. Dr. Lawrence P. Garetto, Dr. Daniel Martinez, Charlotte
Cone, Patricia Currier, Sharon Tanner, and Katherine Bock provided
technical support, Paula Dumars was the experiment support scientist, Jo
Ann Williams provided logistics support, and Bill Munsey and the Hangar
L team (Ramona Bober, John Carver, Mimi Shao, Yasmin Tirado, and Debbie
Vordermark) provided laboratory and logistics support at KSC. We also
want to thank the crew and many other people associated with the shuttle
mission without whom the experiment would never have flown.

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**FIGURE LEGENDS**

1\. The upper panel depicts rat growth during the Spacelab Life Sciences
1 experiment. The lower panel expands the postflight growth patterns and
shows the initial suppression of growth in the flight animals
immediately after the flight. Data are plotted as mean±SEM.

2\. The growth rate of animals during the experiment. Growth rates are
very similar preflight and inflight, but the flight animals showed a
decreased gain compared to the ground controls. The RAHF and AEM-F rats
were significantly lower than the VIV group during the postflight
period. This difference appears to be due to the initial decrease in
weight in the flight animals immediately after flight (see Figure 1).
Data are plotted as mean±SEM.

3\. Bone accretion during the flight period. No differences were noted
in length of any of the long bones during this flight. Bone mass in the
vertebra and rib did not appear affected by this flight. However, the
femur in the individually housed flight rats (RAHF) was significantly
smaller than its control (VIV-S) after 9d recovery. Data are plotted as
mean±SEM.

4\. Bone mineralization rates at the periosteal and endosteal surfaces
of the tibia and humerus. Significant decreases were noted at the
tibiofibular junction of animals during spaceflight compared to controls
and between singly housed (RAHF) and group housed (AEM) rats during the
flight period. Postflight the defect was corrected. The significant
decrease in mineralization rate in the humerus during flight did not
appear to be corrected postflight. No difference in mineralization due
to flight were noted at the endosteal surface of either the tibia or
humerus. Data are plotted as mean±SEM.

5\. Calvarial periosteal apposition rate from the recovery rats. Since
bone markers were given to all animals preflight, the preflight and
inflight mineral apposition rates could be determined in the rats that
recovered following flight. No difference in apposition rate was noted
either preflight or during flight. Also, no change in calvarial weight
or calcium concentration was obvious between control and experimental
groups. Data are plotted as mean±SEM.

6\. Hydroxyproline specific activity during the recovery period. Rats
received 3H-proline shortly after the shuttle returned to Earth and the
incorporation was measured during the nine day recovery period. The
tibia from the singly housed rats and the humerus from the group housed
rats showed significantly lower activity than the ground controls.
Interestingly, the activity in the humerus of the singly housed animals
was not different from either AEM group. \* indicates a value that is
significantly different from the corresponding control. Data are plotted
as mean±SEM.

7\. Mechanical properties of singly housed rats. Tibia stiffness was
significantly less in the basal than all other groups (\*). The R+0
flight rat tibia was significantly less stiff than the R+ML control (=)
and the R+ML control was significantly greater than all other groups
(¶). Tibia energy to failure was significantly less in the basal than
all other groups (\*), while the R+ML flight rats were stronger than the
basal, but weaker than the R+ML control (=) and the R+ML control was
significantly greater than

the R+ML flight (¶). Humerus stiffness was significantly greater in the
R+ML rats compared to the R+0 animals (\*), but no differences were
noted at the same time point. Humerus energy to failure was not
different between groups. Data are plotted as mean±SEM.

8\. Humerus stiffness in group housed animals. No differences were found
between groups. However, the values at R+0 were significantly higher
than the values for the individually housed rats at that time period and
the R+ML AEM flight rat humerus was significantly stiffer than the R+ML
RAHF flight humerus (Figure 7).

9\. Progenitor populations in the periodontal ligament. Distribution of
progenitor populations in AEM, RAHF, and VIV rats was not similar to
Cosmos 2044 and ground-based studies (Garetto et al., 1992). No
difference in progenitor populations were noted in any groups at any
time periods, but the A+A\' population was depressed to about 15% of
normal and the C+D fraction is raised to 25-30% of expected values in
this experiment. The data suggest that the rats may have been stressed
prior to euthanasia.

10\. The top panel shows normal alkaline phosphatase activity associated
with the cell membrane (arrows) of an osteoblast in a control animal
(X11,500). The bottom panel is from a singly housed flight animal at R+0
and shows alkaline phosphatase activity (arrows) is associated with the
adjacent stromal cells (SC) rather than the osteoblast (OB) cell
membrane (x15,000). N=nucleus.

11\. The top panel is an electron micrograph of an osteoblast from an
individually housed flight rat at R+0. The osteoblast (N=nucleus) is
aligned along the bone surface (COL=collagen) and no dense cytoplasmic
granules are present. The bottom micrograph shows osteoblasts adjacent
to bone matrix, but these cells from a RAHF-F R+ML rat contain numerous
dense granules (arrows) whose contents are about to be secreted onto the
bone surface (X15,000).

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