Experiments were conducted on young growing rats. Hypokinesia lasting from 20 to 200 days caused retarded gain in weight and volume of the femur and delayed development of the cortical layer of the diaphysis. In contrast, the density of the cortical layer of the femoral diaphysis increased due to elevation of the mineral saturation of the bone tissue microstructures. Incorporation of Ca into the bone tissue in hypokinesia had a tendency to reduce. Partial normalization of the bone tissue mineral component occurred during a 20 day recovery period following hypokinesia

Muradov, I. S.

Pavlova, M. N.

Stupakov, G. P.

Volozhin, A. I.

English

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NASA TECf1TTICAL MEMORANE M NASA TM-76059
STATE OF THE ,MINERAL COMPON-IT OF RAT BONE TISSUE
DURING HYPOKINESIA AND THE R=1 JERY PERIOD
. 1. Volortun, G. P. Stupakov, M. N. Pavlova and I. Sh. Muradov
Translation of "Sostoyaniye mineral'nogo komponerta kostnoy tkani krys pri
gipokinezii i v vosstanitel l nom periode," Patologicheskaya Fiziolo-
giya i Eksperimental'n.aya Terapiya, No. 2, 1979, pp 30-33.
(NASAA-TM-76059) STATE OF THE MINERAL	 N80-19753
COMPONENT OF RAT DONE TISSUE DURING
HYPOKINESIA AND THE RECOVEF,.' PERIOD
(National .Aeronautics and Space	 U.nclas
Administration) 6 p HC A02/MF A01 CSCL 06C G3/51 47450
"^fsA ^^yFO
rg/^^,^5 DAC^/
NATIONAL AERONAUTICS AND SPACE ADMINISTRATION
WASHINGTON, D. C. 20546	 MARal 1980
STATE OF THE MINERAL COMPONENT OF RAT BONE TISSUE
DURING IiYPOKINESIA AND THE RECOVERY PERIOD
A. I. Volozhin, G. P. Stupakov, M. N. Pavlova and I. Sh. Muradov
Chair of Pathological Physiology, Semashko MedLcal Stomatological Institute;
Biophysical Laboratory, Central Institute of Trawnatology
and Orthopedics, Moscow
Long term mobility restriction in animals induces changes in the metabolism of /30*
calcium, phosphorus and protein in mineralized tissues [1, 2, 5] . However until the
present time no comprehensive study had been made in respect to the condition of the
mineral cowponent of bone tissue under conditions of hypokinesia and during the re-
covery period. The present investigation is devoted to this problem.
Method. The study was done on 135 mongrel albino rats weighing initially 140-150 g.
Background data were obtained from 10 intact rats. Hypokinesia was produced by put-
ting the animals in narrow cages for 20, 40, 60 and 100 hours. At he end of each
hypokinesic period part of the animals were put on a free exercise program (recovery
period).
After rats had been killed their femurs were removed and subjected to rentgeno-
graphy in the absence of an intensification screen on a TUR-60 apparatus using Ivak-
rat-200 film, shooting conditions: 10 niA, 25 kv, 4 sec. Viewing the sagittal Fa-id
frontal projections on the rentgenograms through an MSS-2 microscope equipped. with
an ocular micrometer the central portion of the bone was measured for di;aphysis and
osteomedullar canal as well as overall thickness of the cortical layer from opposite
sides. In the degreased and dehydrated femoral bone (entire right and the left in
the distal epiphysis) the following indices were determined! bone density (g/cm3),
r unit volume (mineral saturation
	
3
ash content pe
	 g/car.) and in percent of dry
	 /31
weight (ash content .:.r: percent) . Bone volume was determined by weighing on torsion
scales in the air a°r:,i distilled water. The bonE.:> were ashed in a muffle furnace at
700°C for 7 hours and the ash weighed. The formula used in calculations was:
Numbers in the margin indicate pagination in the foreign text.
A
1
Pn ufrayse ^' Pu n to	 b1	 P;, nwO.IbHOCTb	 P,VXO Oe 100
PCSVOt1p g 11Hepa lbHas► 11 aCWL4e 1JH © Crb =: r° wCf 11,i0THOCTb
Key:	 V. Bone volume. b' Ash content.
P. bone weight. rr. Ash.
a. Tit air. e. Dry.
b. In water. f. Density.
d. Specific gravity g. Mineral saturation.
of water.
From the central portion of the diaphysis of the left femur ring-shapccl fragments
3 Hatt across were sawed and measured for the sacra indices as the whole bone. For great-
er accuracy in water measurements surface tension on the bone was dijiii.nis ►ie by the ad-
dition of a small amount of detergent. Contact quantitative mi.crorenckJc^tography was
used for differential quantitative determination of microstructural mineralization (3,
6]. This method was used to determine optical density of microrentgenograma for trans-
verse sections of the central portion of the femoral diaphyr,- 1 0 as compared with the
density of a picture of a standard graded tapered sample. Photometry of the mi.crorent-
genograms was done in the subperiosteal, intermediary and subendostcal zones of the
section and the results were expressed in grams of the mineral component per cubic
centimeter. A study was also made of the effect of hypokinesia on the intensity of
Ca45 incorporation in the proximal epiphysis and diaphysis of the femur. For this
purpose 24 hours before being sacrificed the animals received Ca 45 intraperitoneally
in the form of a chlorite diluted in an isotonic NaCl solution at a dosage level of
2 microcuries per animal. The accepted method (4] was used for treating the materi-
al and determining radiochem:i.cally the Ca 45 in the ash. Data were expressed in per-
cent of the isotope dose administered.
Results and Evaluation. Under conditions of hypokinesia the weight of the rats and
the weight and volume of the femur were lower than the corresponding figures for the
control (Fig. 1). Hypokinesia for 20, 40 and 60 days led to a reduction in the thick-
ness_of the cortical layer of the diaphysis in a front-back direction by 6-11% and
after 100 days by 25.9% (P<0.05) as compared with the control. Muc,:h less marked was
the reduction of the cortical layer looked at from the lateral surfaces of the femur
on day 20 and especially uzi days 40 and 60 of the experiment (18.4% and 22.6% respec-
tively compared with the control, which is about twice larger than the parameters of
2
caR1GINAL PAGE 15
of POOR QUAL"
a OQH 20 av CU Cu iw i WIJ 1'"!
measurement in the front-back direction; Fig.
2). Density, ash content and mineral satura-
tion of the overall femur and epiphysis sepa-
rately diminished in the experimental animals,
yet there was no evidence of a direct lint: be-
tween the manifestness of these changes and
the lont,th of the experiment. Density and rd-
neralization of the diaphysis bone tissue in-
creased with the length of hypokinesia. For
example, by the 100th day of the experiment
the density, ash content and mineral satura-
Fig. 1. Change in body mass of rats 	 tion of the cortical layer of the diaphysis had
(1, in g) and of the 3mass (2, in mg) increased by 5.9 , 5 and 15- respectively (P<
and volume (3, in mm ) of femoral
bone in hyl^okinesia and during re-	 0.05-0.01), i. e. mobility restriction was not
covery period. I3ere and in Fig. 2	 resulting in the development of osteoporosis.
and 3: solid line - control, broken
line - hypokinesia, dot-dash - re-	 Decrease in the mineral content of the entire
covery period, X axis - time (days), bone was linked with attenuation of the corti-
a -background
cal layer while the width of the ost^omedullar
channel was maintained. The drop in epiphysis mineralization seems to be connected /32
with attenuation of the osseous trabeculae of the porous material.
The figure for mineral saturation, obtained by •the volume-weight method, reflects
the content of phosphoro-calcium salts in a unit volume of a fragment of the cortical
layer and is a direct function of the degree of mineralization of the microstructures
of the osseous tissue and conversely of the total volume of the vascular channels
and resorption cavities. The effect of these "nonmineralized" areas on the amount of
mineral saturation was elimLnated by the microrentgenological study. As a result it
was established (Fig. 3), that minerals were unequally distributed in different lay-
ers of the femoral cortical layer: the intermediary was the most mineralized and
the subperiosteal the least.
Under hypokinetic conditions mineral saturation of osseous tissue increases 9n
all zones of the femoral diaphysis, the dynamics of this indicator being the same as
for control animals but occurring at a higher level. There was a more marked increase
of mineral saturation for the strata of the subperiosteal layer than the intermediary.
3
A
r	
^
,r	 t^
2,0
The data obtained may be interpreted as severe
inhibition of bone tissue formation in hypokine-
sia corry
 ponding to severe attezutation of the
osseous cortical layer.
r
2 2[
2
2.0
to
1.2
1,0—
a
2.,d	 /4	 z
2
I's
!.2
3
By day 20 of the recovery period following
20, 40 and 60 days of hylvkinesia the animals ra-
pidly tool: on weight and their weight became per-
fectly normal; following 100 days restricted
mobility the animals' weight did not reach the
control level (scc Fig. 1). Femoral volume dur-
ing the recovery period normalized only with 20
or 40 day hypokinesia. Bone mass was not re-
stored in a single period, of the study because
of the marked loss of density. In the racovery /33
period the rats' overall femoral density went
down particularly in rr7e epiphysis. Density pa-
rameters and those for mineral saturation of os-
0,8	 1	 1 - i	 L r	 Ag L 	 t.
^1 d'oM 20 40 00 90 1 0	 2'0 a0 G0 s0
Fig. 2. Changes in cross-sections
of rat femoral bone in hypokine-
sia and during recovery. A -
front-)Jack, B - side, x axis -
time (days), Y axis - width of
diaphysis (1), medullar channel
(2), cortical layer (3) in ma. 	 seous tissue in the cortical layer of the diaphy-
sis increased to a larger extent, than under hyp-
okinesia alone. In contrast to these findings results of the mi.crorentgenographic
study following 60 and 100 days of hypokinesia showed normalization of mineral satur-
ation for microstructures in all investigated zones of the diaphysis (see Fig. 3).
The thickness of the cortical layer tended to normalize when measured front-back but
the lateral, measurenx.nt remained low (see Fig. 2). The width of the osseomedullar
charnel of the rat femur during recovery following hypokinesia of varying lengths in-
creased 7-111 when compared with the control (P< 0.05) thus explaining its density.
The radioisotope study showed, that for control rats the intensity of Ca 45 i.ncoipora-
tion in the femoral diaphysis was in inverse ratio to growth. 20 days of restricted
mobility caused the level of such incorporation in the diaphysis and epiphysis to go
down (85.8 and 77.8% in respect to the control). On day 40 of hypokinesia the in-
corporation of the isotope in these tissues increased slightly but on day 60 went
down once more on an average of 10% compared to the control. During the recovery
period following 20 and 40 days of hypokinesia the Ca 45 content inthe epiphysis and
diaphysis of the femur tended to reduction; after 60 days of hypokinesia it rose in-
significantly in the diaphysis and substantially in the epiphysis (21.5% compared to
4
1
Y^
-
^	 -	 /	 .	 ^	 . 1s JYl	 ^^^/t. ♦ 	 ..s•	 ^a,c :..:,-wYiw....^Yf Ye:J^^.:u.s1YLY.—s^.r.n.
1.4
12
1.0
08
00
1,4-
1.2 -
20 40	 80 100 120 14o
Fig. 3. Changes in mineral
saturation of microstruc-
tures in cortical layer of
rat femoral bone in hypo-
kinesia and in recovery,
Zones: 1 - subperiosteal,
2 - intermediary, 3 -sub-
endosteal. X axis - time
(days), Y axis - mineral
saturation iii
control; P < 0.05).
Titus, long term restricted mobility for growing
rats slays down increase in mass and femoral volume,
reduces the density of porous matter in the epiphyses
and retards developme-nt of the diaphysis' cortical lay-
er. At the same- time there is an increase in the den-
sity of this same layer due to an increase in mineral
saturation of osseous tissue microstructures. Mineral
saturation increases most in the subperiosteal layer,
evidence of retardation in the oppositional, growth of
the bone and consequently of the inhibition of young
bone formation. Ca 45 incorporation into bone tissue
under conditions of hypokinesia tends to reduction,
probably a reflection of the repression of the osteo-
genetic process. Normalization of the bone's mineral
component during the recovery period following various
lengths of hypokinesia is partial in character.
M ,
5
r.
rXi	 ^ i i RUCTI, A7
1. Volozhin, A. I., Pat. fiziol,., 6, 65-69 (1971).
l
2. Volozlun, A. 1., P. V. Vasil lyev, N. N. Uglova et al., Kosmi.cheskaya biol., 3,
10-15 (1972) .
3. Polyakov, A. N., Ortoped. travmatol., 3, 41-44 (1970).
4. Prolchonchukov,. A. A. and Lyu Din-Sin', Med. radiol., 7, 70-73 (1961).
5. Prokhonchukov, A. A., Ye. A. ICovalenko, A. G. Nolesnik et al., Stomatologiya, 4,
1-6 (1970).
6. Engstr8m, A., Aorta radiol. (Stockholm), 1946, Suppl. 63.
C0PYAi(,ri7 4111-A•f-	
^^04 ^
6
I


State of the mineral component of rat bone tissue during hypokinesia and the recovery period

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