Key words : hind, limb, unweighing, weightlessness, osteoporosis , bone mineralisation
Weightlessness during space flight results in decrease in mineral
content of weight bearing bone and an increase in urinary and
fecal calcium loss (1, 2, 3). The possibility that these calcium
losses may not abate with time of exposure has raised concerns
of serious biomedical risk during prolonged exposure to hypogravic
environments. In rats flown aboard the
Cosmos 782 and 1129 biosatellite missions and on experiments
conducted aboard the space shuttle, there was a decrease in
periosteal bone formation in the long bones of the limbs (4)
and trabecular bone formation in the proximal tibial and humeral
metaphyses (1) and an accumulation of marrow fat (1). In rats
exposed to weightlessness bone resorption was not found altered
(1, 5), however decline in the metaphyseal osteoblast population
was seen (6). These findings suggest that weightlessness during
space flight results in bone loss, probably due to diminished
Different workers have attempted to prevent these physiological
alterations in space by using methods viz. diet supplementation,
drugs, exercise and lower body negative pressure but without
achieving any effective countermeasure (2, 7, 8). It has been
reported that only severe exercise regimens are effective in
preventing physiological deconditioning of cosmonauts in very
long duration spaceflights (9). However, as the time required
for such a programme is more and it leads to muscular pain,
it is not a suitable countermeasure for long term space flights
and undoubtly some form of artificial gravity, such as by rotating
space station or the use of human centrifuge in space
station will be required for such missions (9, 10). A study
has shown that, the increase in urinary calcium output in continuous
bed rest subjects were reduced to nearly pre bed rest levels
with three hours of daily standing at 1 G. But neither daily
supine bicycle ergometry up to 4 hours (h) per day, nor sitting
at 1 G for 8 h per day had any effect on increased urinary calcium
from bed rest (9).
Anti orthostatic hypokinetic posture by tail suspension in
rats is an accepted model for simulating effects of weightlessness
on skeletomuscular system (11, 12, 13). This study was under
taken to see the usefulness of daily 4 h weight support (WS)
during simulated weightless environment (S-W) as induced by
tail suspension in rats, in preventing the bone demineralisation.
Wistar strain of male albino rats, aged 4 months to 8 months
and weighing 150 - 210 gm, were used in this study (13, 14).
They were housed individually in 'Weightlessness Simulation
Cages' (WSC) with food (pelleted, Gold Mohur feed) and water
provided ad libitum. They were allowed to adapt in WSC
for 7 d and observed daily for their feed intake, weight (wt)
gain and for any other unusual signs of stress. Rats showing
any unusual signs during adaptation were discarded from the
study. After 7 d of adaptation to WSC and the feed they were
divided randomly in 3 groups. Group 1 (CON, n = 12) rats were
left in WSC for another 15 d without any treatment. Group 2
(HU, n=18) rats were given S-W by hind limb unweighing by tail
suspension (8) for 15 d. Group 3 (4HRWS, n = 11) rats were given
S-W for 15 d but released from tail suspension for 4 h daily
from 0800 h to 1200 h, to bear their own weight.
After 15 d of experimentation rats were anesthetized by pentobarbital
sodium (50 mg/kg body wt, ip) and hind limb muscles were studied
for their contractile properties. After this, rats were sacrificed
and their tibiae removed, stripped of' adhering muscle and connective
tissue and weighed to the nearest 0.1 mg for their wet bone
wt. Bones were dried in individual steel container at 100 °'C for 24 h in a forced air drying oven,
removed to a covered tray containing dessicant and were reweighed
to obtain their constant dry bone wt (12, 13, 14). Organic
matrix of the dry bone was removed by incinerating it and converting
it in to ash. Bones were ashed in individual crucibles for
24 h at 600 °C
in a muffle furnace and the ash wt determined (12, 13, 14).
Ash, consisting of only inorganic component, was then transferred
to a 100 ml flask and dissolved in 10ml of 2N HC1. The sample
was then diluted to 100 ml in double distilled water and its
Calcium concentration was measured by Cresolphthalein complexone
(CPC) method (Manual microdetermination by Baginski et al, 1973)
by using Spectronic-21 (15). Water content of the bone was
determined by subtracting dry bone wt from wet bone wt and organic
matrix component of bone was determined by subtracting ash wt
from dry bone wt. All these parameters of bone were expressed
as mg/100 gm body wt (mg/ 100 gm BW) (8, 12). Bone calcium
was also expressed as mg/100 mg dry bone (8, 13, 14).
Bone changes during S-W and the effects of periodic support
on these are presented in Table I.
| Table I
click to see full view
Table I: Effects of periodic weight support in a simulated
weightless environment (S-W) in preventing bone demineralisation
HU group showed reductions in the wt of wet bone by 20.9%,
water content by 35.8%, collagen matrix by 12.2%, total inorganic
content (ash wt) by 14.6% and calcium content by 33.4% of tibia,
when these parameters were expressed as mg/ 100 gm BW. The
bone calcium in dry bone was found to be reduced by 22.4% in
HU group. These findings are in agreement with the reports
of other workers (8, 12, 13).
4 HRWS group showed no significant difference in the water
content and organic matrix content of the bone when compared
with CON. Calcium content (mg/100 mg BW) of tibia in 4 HRWS
remained 15.2% less as compared to CON. However, calcium in
dry bone was found to be similar to CON.
Student's unpaired t test was used to compare various bone
parameters of HU and 4 HRWS groups with CON group. In all cases,
the level of significance was set as P<0.05.
Bone is a modified connective tissue consisting of living cells
(viz. osteoblast, osteoclast etc), organic intercellular matrix
(viz. collagen, mucopolysaccharides and lipids etc) and inorganic
(minerals viz. calcium, phosphorus etc) component. Results
of CON group show that wet tibia consists of 1/3 water while
2/3 of it is dry bone consisting of organic matrix (collagen,
lipids etc) and mineral content (calcium, phosphorus etc).
Analysis of dry bone revealed that 450/c of dry bone is organic
matrix and 55% of it is mineral component. These findings are
in agreement with the data available in literature (13, 16,
S-W by tail suspension in rats for 15 d resulted in reduction
of wet bone wt. This reduction in wet bone weight may be the
result of reduction in water/organic matrix/ mineral content
of bone. On comparing water content in tibia of HU group with
CON group it was found 35.8% reduced in HU group. Reduction
in the water content of bone may be due to reduction in collagen
matrix leading to less osmotic binding of water molecule or
as a part of reflex reduction in blood volume and body water
due to cephalad fluid shift as induced during S-W by HU (8,
17). The wt of organic matrix (collagen and lipids etc) was
found to be reduced by 12.2% in HU group. As majority of the
organic matrix in a bone is made up of collagen fibres (16,
17), reduction in organic matrix can be interpreted as reduction
in collagen fibres of the bone. Reduction in collagen matrix
of bone in space flight has also been reported by others (12).
It is also possible that some of the collagen fibres of bone
was replaced by lipid material (1). HU group also showed reduction
in total mineral content of bone as evident by the reduction
in ash bone wt of HU group by 14.6%. Reduction in mineral content
of bone during weightlessness has also been reported by others
(1). The reduction in mineral content of bone may be due to
reductions in calcium, phosphorus or any other mineral of the
bone. Calcium content of tibia was found 33.4% reduced in HU
group. Calcium in relation to dry bone and ash bone were also
reduced in HU group. Reduction in calcium in dry bone may be
the result of either less mineralisation of collagen fibres
of bone and/or replacement of some collagen fibres by lipid
materials in the bone (1). Lipid material of bone are normally
not calcified in contrast to collagen fibres which are calcified
as soon as these are formed by osteoblast (16, 17). A 33.4%
decrease in calcium of tibia as compared to 12.2% decrease in
organic matrix further indicate replacement of some of the collagen
fibres of bone by lipid materials during SW. Reduction in the
calcium content of the bone associated with increased calcium,
phosphorus and other mineral loss in urine during actual and
simulated weightlessness has been found by various other workers
(1, 2, 18). Work done in the field of bone deconditioning due
to weightlessness in human and animals has been extensively
reviewed by Russell T Turner during the year 2000 (19). He
observed that, it is still not clear whether the bone loss is
associated with increased bone remodeling, reduced bone remodeling,
or an uncoupling between bone formation and resorption, associated
with decreased mRNA levels for bone matrix proteins. The molecular
mechanism for bone deconditioning due to weightlessness is still
unknown, but there is evidence for changes in selected cytokines
(e.g., transforming growth factor-b and insulin-like growth factor I),
that have been implicated in the regulation of bone formation
(19). Irrespective of the mechanism involved in bone deconditioning,
it is clear through our study that S-W by tail suspension in
rats resulted in reduction of water, collagen and calcium content
of the wt bearing bone i.e. tibia.
4 h S during 15 d of S-W resulted in partial improvement of
wet tibia wt. On further analysis water content of tibia was
found to be improved. Although water Content in 4 HRWS was
still 14% less than CON but difference was not found significant.
Organic matrix content was also found to be improved and it
was not found to be significantly different in 4 HRWS when compared
to CON. Total mineral content of the tibia (ash wt) did not
improve at all and it was still 13.2% less in 4 HRWS as compared
to CON. It was still possible that organic matrix in bone of
4 HRWS group had less of collagen fibres and more of lipid material
(1) while ash bone of 4 HRWS group had more of calcium content
and less of other mineral. Improvement in the calcium content
with out any improvement in the total mineral content suggests
that bone calcium in 4 HRWS group improved at the cost of other
minerals of the bone.
Improvement in the bone calcium was not complete as it was
found still 15.21% less than CON group. As calcium in bone
gets deposited only on collagen fibres (16, 17), it is concluded
that collagen content of bone was also improved at the cost
of lipid material of bone. Calcium content in relation to dry
bone in 4 HRWS was not significantly different from CON group.
It is further suggestive of proportionate improvement in calcium
and collagen fibres of the bone in 4 HRWS group (16, 17). 4
h WS during S-W was not found sufficient in total prevention
of calcium loss of bone as calcium content of tibia were found
still 15.2% less than CON. A 10.8% reduction in the
wt of wet tibia, inspite of complete improvement in tibia water,
in 4HRWS group as compared to CON group, further suggests above
view point. In one of our earlier study conducted at IAM, effect
of 2 h daily weight support in preventing the atrophic changes
in tibia due to simulated weightlessness was found ineffective
in preventing reductions in organic matrix, bone minerals and
calcium (14). 4 h WS during S-W is found effective in
complete prevention of water loss and partial prevention of
bone demineralisation of tibia as induced by HU.
HU in rats simulates deconditioning effects of weightlessness
on weight bearing bone tibia resulting in reduction of water
content, organic matrix and calcium content of bone. 4 h WS
during S-W resulted in complete prevention of water loss and
partial prevention of demineralisation of' tibia as induced
WSS, Wronski TJ, Morey ER et al. Effects of spaceflight on
trabecular bone in rats. Am J Physiol 1983; 244: R310-R314.
GD, Lutwak L, Rambaut PC et al. Mineral and nitrogen balance
study observation : The second manned Skylab mission. Aviat
Space Eituiron Med 1976; 47(4): 391-396.
GP. Microgravity induced changes in human bone strength. The
Physiologist 1989; 32(l) Suppl: S41-S44.
ER, Baylink D,T. Inhibition of bone formation during spaceflight.
Science 1978; 201: 1138-1141.
CE, Adachi RR. Bone resorption and mineral exceretion in rats
during spaceflight. Am J Physiol, 1983; 244:
PJ, Morey ER, Robert WE. Osteoblast histogenesis in periodontal
liganient and tibial metaphysis during simulated weightlessness.
Aviat Space Environ Med 1986; 57: 1125-1130.
S. Modification of bone atrophy seen with hind limb suspension
by exercise and dobutamine. The Physiologist 1989; 31(1)Supl)l:
SS, Banerjee PK, Jaiii PK. Studies on skeletomuscular deconditioning
and hematological changes in rats following antiorthostatic
hypokinesis induced by tail suspension. AFMRC Project
RB. Periodic icceleratioti stimulation in a weightlessness
environment (PAS-WE): A new scheme. The Physiologist t989;
RR. A human use centrifuge for space stations : Proposed ground
based studies. Aviat Space Environ Med 1988;
PK, Banerjee PK, Baboo NS et al. Physiological properties of
rat hind limb muscles after 15 days of simulated weightless
environment. Indian J Physiol Pharmacol 1997; 41(l):
Robert D, Dillaman RM. Bone growth and calcium balance during
simulated weightlessness in the rat. J Appl Physiol 1990;
PK, lyer EM, Banerjee PK et al. Bone changes during simulated
weightlessness in rats. Indian J Physiol Pharmacol
2000; 44(3): 359-362.
PK, lyer EM, Baneijee PK et al. Modification of bone atrophy
by daily 2 hour weight support during simulated weightlessness
in rats. Ind J Aerospace Med Winter 97; 41(2):
RL, Nath RK. Practical biochemistry in clinical medicine. 2nd
edn, Calcutta: Academic Publisher, 1990; 385-386.
CA, E Neil, N Joels. Samson Wriglit's Applied Physiology. 13th
edn, New York Toronto: Oxford University Press, 1984; 546-555.
AC. Textbook of Medical Physiology. 8th edn, Philadelphia:
WB Saunders Company, 1991; 469 and 872-874.
VV, Savina EA, Kaplansky AS et al. Effect of space flight factors
on the mammal : Experimental-morphological study. Aviat
Space Environ Med 1976; 47(8): 813-816.
T. Turner. Physiology of a Microgravity Environment; Invited
Review: What do we know about the effects of spaceflight on
bone ? J Appl Physiol 2000; 89(2): 840-847.