Effects of Periodic Weight Support in a Simulated Weightless Environment in Preventing Bone Demineralisation  

P. K. JAIN*, E. M. IYER, P. K. BANERJEE AND N. S. BABOO
Department of Physiology,
Institute of Aerospace Medicine
Vimanapitra Post,
Bangalore - 560 017
(Received on October 4, 2001)

Key words : hind, limb, unweighing, weightlessness, osteoporosis , bone mineralisation

 

INTRODUCTION
METHODS
RESULTS
DISCUSSION
CONCLUSION
REFERENCES

INTRODUCTION

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 bone formation.

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.
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METHODS

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).
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RESULTS

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.
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DISCUSSION

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, 17).

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.

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Conclusion

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 by HU.
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REFERENCES  

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