File Name: SLS1_04.DOC

EXPERIMENT SLS1 BSP4

OSTEOGENESIS - TISSUE FACTORS OF REGULATION

[PIs]{.underline}

[Russia:]{.underline}

V. S. Oganov, Institute of Biomedical Problems, Moscow

[US:]{.underline}

E. Morey-Holton, NASA Ames Research Center, Moffett Field, California

[Introduction]{.underline}

Bone physiological restructuring at a local level is regulated by
short-distant factors which are synthesized by bone cells and released
as a result of destruction of extracellular bone organic matrix \[5,
4\]. Environmental effects may modify substantially the status of bone
by disrupting the equilibrium of osteogenic and resorptive processes or
by changing the level of bone self-maintenance \[3, 1\].

Among bone remodeling regulators special attention should be given to
bone morphogenetic protein (BMP) that has been described in detail by
Urist \[6\] and its inhibitor which has been detected by Sumarokov
\[2\].

The amount of these factors in bone is measured in terms of their
biological activity. The concentration of BMP in bone under study is
evaluated with respect to the degree of ectopic osteogenesis induced by
its demineralized matrix, and the concentration of BMP inhibitor is
determined in relation to the inhibition or degree of inhibition of
ectopic osteogenesis produced by a standard matrix. The latter is done
by reconstructing an inhibitory substance on the matrix from acid
extracts of bone samples under examination \[7, 2\].

[Materials and Methods]{.underline}

Fragments of demineralized diaphyses of femoral bones of rats flown for
9 days on SLS-1 as well as HCl extracts of these bones dissected from
the following groups of animals were investigated: basal control (5
rats - BC), space flight (5 rats - R+0), vivarium control (5 rats -
C+0), flight rats allowed to get readapted for 9 days after recovery (5
rats - R+9), vivarium control to the readaptation rats (5 rats - C+9).
Altogether 2 bone samples and 1 HCl extract were obtained from each
animal.

Another category of bone samples under investigation comprised
demineralized bone matrix of femoral diaphyses of untreated noninbred
male rats weighing 160 - 180 g. Twelve samples were used to compare
osteoinductive activity while the remaining were subdivided into 5
groups (5 samples each) and used to reconstruct inhibitory substance
from HCl extracts: C - osteoinduction control, I-BC - inhibitor from BC
rats, I-C+0 - inhibitor from C+0 rats, I-R+0 - inhibitor from R+0 rats,
I-C+9 - inhibitor from C+9 rats, I-R+9 - inhibitor from R+9 rats.

The experimental protocol used for studying ectopic osteogenesis is
shown in Figure 1.

[Method for preparing demineralized matrix.]{.underline} Fragments of
diaphyses of rat femurs were cleaned from soft tissue and blood
(mechanically cleaned and washed by saline solution) and then placed in
0.5 N HCl (0.5 ml HCl per 1 mg bone) for 24 hours. The initial weight of
the fragment was recorded and designated for subsequent calculations as

Wi (weight initial). Demineralized matrix was washed in running
distilled water to remove excessive acid, and the extracts were
collected and stored. Frozen matrix fragments can be stored for as long
as a month.

[Method for preparing inhibitory substance.]{.underline} The acid
extract was measured, transferred into a measuring beaker and
supplemented with concentrated ammonium hydroxide that made 1/4 of the
extract volume. The precipitate was allowed to stand for an hour and
then was centrifuged. The supernatant was collected and checked: if the
precipitation was complete the supernatant was discarded. The
precipitate was measured and 5 acetone volumes were added. The mixture
was allowed to stand for 24 hours at ambient temperature. Then the
precipitate was separated and supplemented with 2 volumes of fresh
acetone, suspended and centrifuged. The precipitate was separated and
the whole procedure was repeated again. Then the precipitate was placed
on filter paper and washed with acetone (1 volume) through a funnel. The
precipitate was dried at 37

oC in a thermostat to produce granules. The precipitate was weighed and
put in a tightly stoppered vial where it was kept until further
analysis.

[Method for reconstructing inhibitory substance on demineralized
matrix.]{.underline} Powdered inhibitory substance was dissolved in
water acidified with concentrated HCl

to adjust to pH = 2 (100 mg powder in 1 ml of solvent). Then it was
dialyzed against distilled water for 24 hours at ambient temperature.
Dialysis was completed when Cl tests in the dialyzation liquid proved
negative. The inhibitor was water insoluble, and during dialysis was
reconstructed on the matrix.

[Method for measuring osteoinductive activity of bone
samples.]{.underline} The method is based on the ability of
demineralized bone matrix to induce ectopic osteogenesis. When matrix is
placed beyond the skeleton (in a muscle pocket, anterior chamber of the
eye, kidney capsule), it causes new bone formation at this site at the
expense of recipient cells \[4\]. It is recognized that recipient
pericytes can differentiate to yield osteogenic cells. This implies that
there is a direct relationship between the capability of transplantable
matrix to be vascularized and its ability to be replaced with *de novo*
generated bone. The entire procedure consists of 4 stages: 1)
implantation of the material under study; 2) incubation period during
which osteoinduction develops (6 weeks); 3) explantation of the
conglomerate made of the tissue implanted and newly generated bone, and
4) evaluation of the amount of new bone formed.

The first stage (implantation) is performed as follows. The
rat-recipient is under ether anesthesia; the inner side of the hips and
the chest are notched, muscles are bluntly drawn apart, and a graft is
inserted into the resultant pocket. Each recipient had four grafts
implanted in this manner. With the purpose of having objective results,
each recipient was implanted with 4 grafts from different rat groups,
and grafts from the same group were implanted at various sites (zones)
of implantation to different recipients . Skin wounds were sutured with
sterile silk (samples, surgical tools and silk were sterilized in 70%
ethanol).

The second stage: grafts were allowed to remain in the recipient\'s body
for 6 weeks.

The third stage: explantation procedure. The tissue conglomerate
consisting

of demineralized matrix of the graft and newly generated tissues was
isolated, stripped from soft tissue and evaluated. Each explanted sample
was consecutively shown to three experts who evaluated it with respect
to the following three parameters: density, vascularization and
roughness (the score was from 0 to 3). The experts were not aware

of the group of origin of the sample.

The score 000 was given to an explanted fragment which did not differ
from the graft: soft, light-yellow, smooth.

The score 111 was given to an explanted fragment that could not be
easily bent, had vessels inside, and rough layers on the surface.

The score 222 was given to an explanted fragment that could not be bent,
had at least half of the surface colored pink, and rough layers that
covered at least half of the surface.

The score 333 was given to an explanted fragment which was prickly,
stiff, pink and rough all over.

The final score was given on the basis of scores agreed upon by two
experts in the form of a three-digit number, e.g., 232.

[Evaluation of the amount of new bone.]{.underline} The amount of *de
novo* bone was measured on the basis of the tissue removed relative to
Wi (initial weight) and expressed as a percentage (W%). Previous
investigations demonstrated that this parameter at first declined to
approximately 20-30%, then began to grow and 6 weeks later reached
approximately 50%. If the parameter was less than 50%, it was considered
as a low rate of replacement and a low osteoinductive activity.

It is known that 70% of bone weight are made of mineral component, 40%
of which being Ca and P. It is also known that these compounds occur in
bone in a certain proportion termed the calcium/phosphorus molar
coefficient (Ca/P molar coefficient = 1.33 - 2.1). While measuring these
components, it is possible to identify bone tissue and indirectly assess
its quantity (the more Ca and P, the more bone we have). An additional
parameter indicating that the composition of explanted tissue is close
to that of bone is AEC, or acid extractable component which is the
difference between the weight of extracted tissue and the weight of its
demineralized matrix dried to reach constant weight as related to the
weight of the explantate and expressed as a percentage. For all
practical purposes, this is the sum total of bone mineral components,
water, and acid soluble proteins. For non-bone tissues this value varies
by about 10% and for young (hydrated) bone tissue it can be as high as
85%.

In bone about 50% of AEC are made of Ca and P. Due to this, the ratio
between the total amount of Ca and P and AEC indicates the level of bone
maturation (tends from 0 - no bone at all toward 0.5 - mature bone) and
is termed coefficient of maturity (CM).

All these indicators can be supplemented with the previously described
visual criterion of osteogenesis (VC).

The resultant data were treated using the traditional Student\'s t-test.
The visual criterion was considered as a single three-digit number,
added, and a mean value was calculated.

[Results and Discussion]{.underline}

Table 1 shows the results of measuring osteoinductive activity (OIA) of
demineralized matrix of rats from the flight and control groups.

Analysis of W% shows that it tended to decline in all groups when
compared to BC; however, the difference was significant only in the R+9
group. Comparison of the parameter in the R+9 and R+0 rats gives
evidence that readaptation failed to normalize it (to make it closer to
the control value) but stimulated its further decrease. The parameter
AEC remained unchanged in different groups.

The concentration of Ca tended to grow in all groups but increased
significantly only in R+0 and R+9 rats. The same holds true for the
concentration of P. Variations in these parameters are at variance with
W%.

As compared to BC, the Ca/P-molar coefficient was also slightly
increased.

The parameter CM showed that bone did not yet reach the stage of
maturity but it was higher than in the controls in all groups, except
C+9 rats.

The VC level was the highest in the R+0 rats; in other words, *de novo*
generated bone in those rats could be discerned with naked eye. The VC
value approached the maximally possible level. As a result of
readaptation, this parameter diminished markedly and significantly \--
at R+9 it was below BC. In the C+0 and C+9 groups the pattern of changes
was the opposite: in the C+0 rats it tended to diminish relative to BC
and in the C+9 rats it increased significantly and, consequently, the
intensity of osteogenesis grew.

Table 2 shows that the quantity of the powder containing inhibitory
substance is different in various groups: it is the largest in the C+0
rats and the lowest in the R+9 rats.

From Table 3 it follows that in all groups, except I-R+9 rats, M% tends
to decrease, the decrease being highly significant in the I-R+0 rats.
The parameter AEC is slightly lowered in the I-BC rats and in other
groups it is the same as in the controls.

The concentration of Ca and P in the I-BC and I-R+0 rats is not lower
but is even higher than in the controls; in other words, in terms of
this parameter osteogenesis is not inhibited and, hence, inhibitory
activity is at a minimum in these groups.

The Ca/P-molar coefficient is minimal in the I-C+0 and I-C+9 groups; in
the latter it is below the lower limit of theoretically plausible Ca-P
compounds. It can be

postulated that part of detectable P is bound with other than Ca
compounds and, therefore, is not involved in the formation of new bone.

The CM value is at a minimum in these groups (I-C+0 and I-C+9) as well;
in the latter group it is less than the theoretically possible limit for
bone. The VC in these groups is 0.

Thus it can be asserted that in the I-C+0 and I-C+9 groups the quantity
of inhibitory substance is sufficient to completely inhibit
osteoinduction in the demineralized matrix of control animals.

In other groups inhibitory activity was also observed. It was the lowest
in the

I-BC and I-R+0 rats. In the I-R+9 group it was higher than in the
corresponding control and lower than in the I-C+0 rats.

Summarizing the results and comparing the tabulated data, the following
patterns can be identified:

1\. During space flight osteoinductive activity of bone matrix varies
qualitatively. The rate of ectopic osteogenesis increases, the quantity
of *de novo* generated bone is less than in the controls and it is
strongly mineralized. Acid extracts maintain their inhibitory activity
but its level remains unclear because the observations are
controversial.

2\. In the flight rats exposure to the Earth\'s gravity reduces
osteoinductive potentials of the matrix and enhances the inhibitory
activity of acid extracts, i.e., diminishes bone regenerative capability
and, hence, stimulates osteoporosis.

3\. The C+0 rats (ground-based hypokinetic rats) occupy an intermediate
position between basal controls and flight rats. Exposure to the
Earth\'s gravity levels off the hypokinesia effect on osteoinductive
activity. In relation to inhibitory activity the situation is just the
opposite. Readaptation at 1 g stimulates the inhibitory activity of acid
extracts of rat bones from this group.

Comparison of these findings with observations made in the previous
study when the flight duration was 2 weeks shows that the results are
very similar; in both studies the trend of changes is the same; however,
the difference between groups can be regarded only as tendencies in a
short-term flight while it becomes significant in a 14-day flight.

Earlier Sumarokov \[2\] demonstrated that calcitriol can change the
pattern of osteo-inductive activity in qualitative terms. As was the
case in space flight, calcitriol reduced the amount of ectopic
osteogenesis and increased its rate (enhanced formation of mature
mineralized structures). Changes in inhibitory activity seen in the
flight animals were very close to those detected in calcitriol-treated
animals. This allows a hypothesis to be advanced that changes in
osteoinductive potentials of bone in space flight are induced by
increased production of calcitriol.

[Table 1]{.underline}

Variations in Parameters of Osteoinductive Activity of Bone Matrix

  ------- ------- -------------------------------------------------------
                  Parameters

  ------- ------- -------------------------------------------------------

  ------- -------- ------- ------- ------- ------- ------- ------- -------
  Rat     Number   W%      AEC     Ca      P       Ca/P    MC      VC
  group   of rats                                                  

  ------- -------- ------- ------- ------- ------- ------- ------- -------

  ------- ------- ------- ------- ------- ------- ------- ------- -------
  BC      10      22.8    77.6    47.1    24.1    1.53    0.08    197

  ------- ------- ------- ------- ------- ------- ------- ------- -------

  ------- ------- ------- ------- ------- ------- ------- -------- -------
                  ±2.5    ±1.5    ±4.0    ±2.3    ±0.08   ±±0.01   ±25

  ------- ------- ------- ------- ------- ------- ------- -------- -------

  ------- ------- ------- ------- ------- ------- ------- ------- -------
  C+0     13      18.2    74.4    57.4    29.6    1.61    0.12    177

  ------- ------- ------- ------- ------- ------- ------- ------- -------

  ------- ------- ------- ------- ------- ------- ------- ------- -------
                  ±2.1    ±3.3    ±4.5    ±1.9    ±0.07   ±0.01   ±19

  ------- ------- ------- ------- ------- ------- ------- ------- -------

  ------- ------- ------- ------- -------- -------- ------- ------- --------
  R+0     13      20.8    76.6    64.5\*   32.1\*   1.56    0.12    271\*#

  ------- ------- ------- ------- -------- -------- ------- ------- --------

  ------- ------- ------- ------- ------- ------- ------- ------- -------
                  ±1.6    ±2.1    ±6.0    ±2.3    ±0.06   ±0.01   ±18

  ------- ------- ------- ------- ------- ------- ------- ------- -------

  ------- ------- ------- ------- ------- ------- ------- ------- -------
  C+9     15      19.5    77.2    52.3    23.3    1.74    0.10    243

  ------- ------- ------- ------- ------- ------- ------- ------- -------

  ------- ------- ------- ------- ------- ------- ------- ------- -------
                  ±2.8    ±2.7    ±4.1    ±1.4    ±0.06   ±0.01   ±16

  ------- ------- ------- ------- ------- ------- ------- ------- -------

  ------- ------- --------- ------- -------- -------- ------- ------- --------
  R+9     17      14.7\*@   75.5    63.5\*   31.4\*   1.61    0.12    170\*#

  ------- ------- --------- ------- -------- -------- ------- ------- --------

  ------- ------- ------- ------- ------- ------- ------- ------- -------
                  ±1.4    ±2.4    ±4.4    ±1.2    ±0.04   ±0.01   ±15

  ------- ------- ------- ------- ------- ------- ------- ------- -------

\* Significantly different from BC

\# Significantly different from C+0

@ Significantly different from R+0

[Table 2]{.underline}

Isolation of Substance Containing Osteogenesis Inhibitor from Acid
Extracts

  -------------- -------------- -------------- -------------- -----------------
  Rat group      Volume of acid Amount of      Amount of mg   M [+]{.underline}
                 extract, ml    powder         powder in ml   m
                                prepared       extract        

  -------------- -------------- -------------- -------------- -----------------

  -------------- -------------- -------------- -------------- --------------
  BC             38             113            2.9            

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
                 37             113            3.0            

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
                 31             106            3.4            2.9±0.3

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
                 40             90             2.2            

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
                 35             100            2.8            

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
  C+0            40             139            3.5            

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
                 42             149            3.5            

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
                 44             240            5.4            3.9±0.5

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
                 35             124            3.5            

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
                 35             121            3.4            

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
  R+0            39             93             2.4            

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
                 37             105            2.7            

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
                 43             121            2.8            2.6±0.1

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
                 35             94             2.7            

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
                 44             115            2.6            

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
  C+9            40             112            2.8            

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
                 41             70             1.7            

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
                 40             116            2.9            2.6±0.4

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
                 41             155            3.8            

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
                 42             74             1.8            

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
  R+9            35             61             1.7            

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
                 43             46             1.0            

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
                 42             93             2.2            2.0±0.4

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
                 43             123            3.0            

  -------------- -------------- -------------- -------------- --------------

  -------------- -------------- -------------- -------------- --------------
                 38             75             2.0            

  -------------- -------------- -------------- -------------- --------------

[Table 3]{.underline}

Variations in Parameters of Osteoinductive Activity of Bone Matrix After

Reconstruction of Inhibitory Substance

  ------- ------- -------------------------------------------------------
                  Parameters

  ------- ------- -------------------------------------------------------

  ------- -------- ------- ------- ------- ------- ------- -------- -------
  Rat     Number   W%      AEC     Ca      P       Ca/P    MC       VC
  group   of rats                                                   

  ------- -------- ------- ------- ------- ------- ------- -------- -------

  ------- ------- ------- ------- ------- ------- ------- -------- -------
  C       8       61.3    62.4    45.0    21.3    1.63    0.11     160

  ------- ------- ------- ------- ------- ------- ------- -------- -------

  ------- ------- ------- ------- ------- ------- ------- -------- -------
                  ±8.1    ±4.6    ±3.9    ±1.3    ±0.14   ±0.1     ±27

  ------- ------- ------- ------- ------- ------- ------- -------- -------

  ------- ------- ------- ------- ------- ------- ------- -------- -------
  I-BC    3       52.2    49.8    52.0    22.1    1.83    0.15\^   40\^

  ------- ------- ------- ------- ------- ------- ------- -------- -------

  ------- ------- ------- ------- ------- ------- ------- -------- -------
                  ±4.0    ±5.0    ±8.0    ±1.7    ±0.27   ±0.01    ±20

  ------- ------- ------- ------- ------- ------- ------- -------- -------

  ------- ------- ------- ------- ----------- ----------- ----------- ----------- ----------
  I-C+0   3       48.0    63.0    14.3\^\*@   15.7\^\*@   0.71\^\*@   0.05\^\*@   0.0\^\*@

  ------- ------- ------- ------- ----------- ----------- ----------- ----------- ----------

  ------- ------- ------- ------- ------- ------- ------- -------- -------
                  ±5.0    ±4.0    ±3.0    ±2.1    ±0.09   ±0.01    ±20

  ------- ------- ------- ------- ------- ------- ------- -------- -------

  ------- ------- -------- ------- ------- ------- ------- -------- -------
  I-R+0   5       34.8\^   75.2    59.2    30.9    1.43    0.12     66

  ------- ------- -------- ------- ------- ------- ------- -------- -------

  ------- ------- ------- ------- ------- ------- ------- -------- -------
                  ±6.0    ±6.0    ±6.0    ±3.0    ±2.4    ±0.01    ±30

  ------- ------- ------- ------- ------- ------- ------- -------- -------

  ------- ------- ------- ------- ----------- ------- ----------- ------------ ----------
  I-C+9   5       54.4    70.8    10.6\^\*@   20.8@   0.45\^\*@   0.01\^\*@#   0.0\^\*@

  ------- ------- ------- ------- ----------- ------- ----------- ------------ ----------

  ------- ------- ------- ------- ------- ------- ------- -------- -------
                  ±5.1    ±2.5    ±1.5    ±1.7    ±0.21   ±0.01    ±0.0

  ------- ------- ------- ------- ------- ------- ------- -------- -------

  ------- ------- ------- ------- -------- --------- ------- -------- -------
  I-R+9   5       64.8    59.1    24.8\*   15.2\*@   1.3     0.08     22

  ------- ------- ------- ------- -------- --------- ------- -------- -------

  ------- ------- ------- ------- ------- ------- ------- -------- -------
                  ±7.1    ±5.0    ±8.0    ±3.1    ±0.25   ±0.04    ±25

  ------- ------- ------- ------- ------- ------- ------- -------- -------

\^ Significantly different from C

\* Significantly different from BC

\# Significantly different from C+0

@ Significantly different from R+0

![](media/image1.pct){width="6.958333333333333in"
height="4.263888888888889in"}

[References]{.underline}

1\. Knyazeva, M.V. Specific Features of Carbohydrate Metabolism in
Spinal Column

Upon Hypokinesia and Emotion-Pain Stress. Abstr. Cand. Dissert. Kharkov,
1988 (in Russian).

2\. Sumarokov, D.D. Experimental Study of Regulatory Factors in Bone
Reparative Regeneration Using Mandible Fracture as a Model. Doct.
Dissert., Moscow, 1987. (in Russian).

3\. Stupakov, G.P., Volozhin, A.I. Bone System and Microgravity.
Problems of Space Biology. Moscow, Nauka, 1989, vol. 63 (in Russian).

4\. Friedenstein, A.Ya., Lalykina, K.S. Bone Induction and Osteogenic
Cells-Precursors. Moscow, Nauka, 1973, 223 pp. (in Russian).

5\. Ham, A. Kormack, G. Histology. Moscow, Nauka, 1983, vol. 3, pp.
19-131 (in Russian).

6\. Urist, M.R. New Bone Formation Induced in Postfetal Life by Bone
Morphogenetic Protein. Mechanisms of Growth Control. Springfield, 1981,
pp. 406-434.

7\. Urist, M.R. Bone Morphogenetic Protein. US Paten \# 4455256, Appl.
May 5, 1981. \# 260726 Publ. June 19, 1984. MKI C 07 G 7/00 NKI 260/112
R.

[Summary]{.underline}

The effect of a 9-day space flight and a 9-day recovery period on bone
osteogenesis was investigated using induction of ectopic osteogenesis by
a demineralized matrix of a femur of flight rats (donors). Preliminary
analysis of the results has shown that in space osteoinductive activity
of bone matrix increased but remained unaltered in qualitative terms.
The amount of *de novo* generated bone was not large in recipient rats
(less than in the controls) but the level of its mineralization was
significant. During 9 days after flight osteoinductive potentials of the
matrix decreased and inhibitory activity increased; in other words, bone
regenerative potentials declined, thus stimulating osteoporosis.