growing rat tibia. FASEB j 4: 16-23;. 1990. Key Words: spaceftight bone morphology bone vascularity. EXPERIMENTS FLOWN. ON SOVIET Cosmos biosatellites.
Cosmos 1887: morphology, histochemistry, vasculature of the growing rat tibia STEPHEN *Columbia 94035,
B. DOTY,1 University,
USA,
and
EMILY
New lnstitute
R. MOREY-HOLTON,
York, New of Biomedical
York 10032, Problems,
ABSTRACT
Light microscopy, electron microscopy, and enzyme histochemistry were used to study the effects of spaceflight on metaphyseal and cortical bone of the rat tibia. Cortical cross-sectional area and perimeter were not altered by a 12.5-day spaceflight in 3-month-old male rats. The endosteal osteoblast population and the vasculature near the periosteal surface in flight rats compared with ground controls showed more pronounced changes in cortical bone than in metaphyseal bone. The osteoblasts demonstrated greater numbers of transitional Golgi vesides, possibly caused by a decreased cellular metabolic energy source, but no difference in the large Golgi saccules or the cell membrane-associated alkaline phosphatase activity. The periosteal vasculature in the diaphysis of flight rats often showed lipid accumulations within the lumen
of the vessels,
occasional
degeneration
of the
vascular wall, and degeneration of osteocytes adjacent to vessels containing intraluminal deposits. These changes were not found in the metaphyseal region of flight animals. The focal vascular mia of bone or a developing
changes fragility
a result of spaceflight.-DoTY, E. R., DURNOVA, G. N., AND 1887: morphology, histochemistry, growing rat tibia. FASEB j Key Words:
spaceftight
may be due to ischeof the vessel walls as
S. B., KAPLANSKY,
4:
MOREY-HOLTON,
A. S. Cosmos
and
vasculature
16-23;
1990.
bone morphology
of the
bone vascularity
EXPERIMENTS FLOWN ON SOVIET Cosmos biosatellites and Spacelab 3 (SL3) suggest that growth in tibial diameter is suppressed in the long bones of growing rats during spaceflight (1, 2); also, osteoblast numbers appear to decrease in the tibial metaphysis (3-5). Bone resorption kinetics (6) and osteoclast numbers in the secondary spongiosa (3, 5) are not increased during spaceflight, but osteoclast numbers in the primary spongiosa of the tibia do increase in growing male rats during flight (4). Since bone formation is consistently suppressed during spaceflight while changes in bone resorption appear to be site specific (4), this study concentrated on the bone-forming cells, the osteoblasts, and the vascular supply within bone
USA, Moscow,
G. N. DURNOVA, TNASA-Ames 123007
and
AND A. S. KAPLANSKY1
Research Center, Moffett
Field,
Ca4fornia
USSR
matrix. The vascular supply provides all the nutrients for bone matrix formation, including oxygen for the hydroxylation of proline to form hydroxyproline (7, 8), an important step for mature collagen fibril formation. In the younger SL3 flight animals, randomly selected osteoblasts in the primary spongiosa of the proximal tibial metaphysis appeared to be slightly, but not significantly, smaller after 7 days of flight (2). Recently, we found that osteoblasts along the endosteal surface of diaphyseal bone tend to have a more uniform size than osteoblasts along trabeculae in the metaphyseal region of the long bones. Therefore, this study concentrated on cells and sampling sites within the tibial diaphysis after flight in Cosmos 1887. MATERIALS
AND
METHODS
Five, specific pathogen-free, growing male Wistar rats from the Institute for Experimental Endocrinology of the Slovakian Academy of Sciences were exposed to spaceflight for 12.5 days. Ground control groups included five baseline rats (samples collected at the beginning of the flight period), five vivarium rats, and five simulated flight (synchronous) animals. Unanticipated postilight complications delayed killing of the animals for about 2 days after the spacecraft returned to Earth. Details of the experimental protocol may be found in the paper by Grindeland et al. (9). The proximal tibia including some diaphysis and the tibial shaft was dissected free of muscle tissue; placed in a vial of 2% paraformaldehyde in 0.1 M cacodylate buffer, plus 0.5% glutaraldehyde, pH 7.4, at 4#{176}C for 48 h; rinsed three times with 0.1 M cacodylate buffer, pH 7.4; and shipped immersed in the buffer. All samples arrived at the .laboratory in excellent condition. The tibial shafts were dehydrated in ethyl ether and embedded undecalcified in polyester casting resin (Chemco,
San Leandro,
Calif.). The portion
of the tibial shaft im-
mediately proximal to the tibiofibular junction (TFJ)2 was sawed into 50-tm-thick cross sections with a GillingsHamco thin-sectioning machine. Bone area and perimeter were measured with an interactive computer system (2).
‘Current address: Hospital for Special Surgery, New York, NY 10021, USA. 2Abbreviations: MP, macrophages; PC, pericytic cells; VS, vascular space; OC, osteocyte; BM, demineralized bone matrix; TFJ,
tibiofibular junction; EM, electron active Data Analysis System.
microscopy;
ZIDAS,
0892-6638/89/0004-0016/$01
Zeiss Inter-
.50. © FASEB
TABLE
1. Cosmos tibial bone parameter? Basal
Bone area, Marrow
area,
Periosteal Marrow
mm3 mm2
perimeter, perimeter,
aValues
are
mm mm
means
± SD.
Flight
Synchronous
3.43
±
0.23
3.60
±
0.27
3.94
± 0.37k
0.79
±
0.06
0.77
± 0.16
0.84
±
7.93
±
0.20
7.97
± 0.29
8.38
±
3.37
±
0.13
3.32
±
3.46
±
0.34
bSignificandy
different
from
basal (P
< 0.05)
0.31 by two-tailed
test.
Sampling
Vivarium
3.75
±
0.34
0.17
0.79
±
0.17
0.29k
8.11
± 0.41
3.36
±
site
was imm ediately
proximal
0.37 to the
TFJ.
The techniques for electron microscopy (EM) and histochemistry have been extensively reviewed (10, 11). For histochemical analysis, the proximal tibia was decalcified in Tris-buffered 10% EDTA, pH 7.4. Decalcified sections 50 tm thick were obtained with a vibrotome. The sections were incubated in the appropriate media for alkaline phosphatase or NADPase (12). Lipid staining was done on frozen sections or on vibrotome sections using Nile Red (13) or Oil Red 0 (14). Sections were embedded in LR White resin for light microscopy or Spurr resin for EM. The vibrotome sections consisted of complete cross
sections through the mid-diaphyseal region of the tibia and contained 50-100 cross sections of vascular channels within bone. Several vibrotome sections per animal were used to measure vascular channel size and to compare relative histochemical activities. Comparisons were made between animals of the flight group and the synchronous control group. Morphometry was carried out on light or electron micrographs using the Zeiss Interactive Data Analysis System (ZIDAS). This system uses a dedicated computer and a hand-held light pen to quantitate structures, determine areas, etc. The EM used was a JEOL
Figure
1. Electron micrograph of a blood vessel within diaphyseal bone from a synchronous control animal. Thin-walled endothelial cells (arrows) surround an open lumen that contains two macrophages (MP). Perivascular or pericytic cells (PC) are situated between the endothelium and the bone matrix. An osteocyte (OC) is located in bone matrix adjacent to the vascular space. The bone matrix has been demineralized. x 5600.
SPACEFLIGHT
EFFECTS ON RAT TIBIA
17
because of the extreme variation in cell size even within the control groups. Blood vessels within normal diaphyseal bone had typical endothelial cells enclosing a large open lumen. The endothelium was surrounded by pericytes and some undifferentiated cells (Fig. 1). In large vascular spaces, the undifferentiated cells were often replaced by osteoblasts and new bone formation was evident. In diaphyseal bone from flight animals, blood vessels near the periosteal surface often showed very dense intraluminal deposits (Figs. 2, 3). The control tissues occasionally showed a similar pattern, but this pattern could be attributed to packing of intact red blood cells within the vascular lumen. The deposits within blood vessels from flight animals showed no recognizable cell structure and were not red blood cells. In addition, near these vessels, darkly staining osteocytes were found. This unusual arrangement of intraluminal deposits and adjacent degenerating osteocytes was seen only along the periosteal portion of cortical bone. Electron
Figure 2. Light micrograph of a vascular space in bone filled with a large lipid (L) inclusion that stains with Oil Red 0. Osteocytes are present in nearby matrix (arrows). This sample is from the diaphysis
of a flight
animal.
microscopy
Osteoblasts from randomly selected sites throughout the metaphysis were variable in size within each experimental group but otherwise were normal morphologically. In the diaphysis at the bone-marrow interface, the osteoblasts showed considerable variation in size but no specific changes in morphology.
x 1200.
100C microscope. Statistical treatment was accomplished with the two-tailed t test with n = number of animals per group and level of significance set at P < 0.05.
RESULTS Animals
The body weight of the basal group was 316 ± 18 g (SD). The initial weights of the other groups were: vivarium, 317 ± 12 g; synchronous controls, 313 ± 12 g; and flight rats, 294 ± 7 g. Light
microscopy
Visual observations of TFJ cross sections under brightfield or polarized light did not show any obvious differences between groups. Area and perimeter measurements (Table 1) showed no significant changes between flight and control groups. The only differences appeared in the periosteal perimeter and cross-sectional bone area at the TFJ in the basal rats compared with the synchronous group (P < 0.05). Light microscopy of trabecular and cortical bone and the included osteoblast populations showed no obvious morphological differences as a result of spaceflight. Attempts to quantitate osteoblast size were abandoned
18
Vol. 4
Jan. 1990
Figure 3. Light micrograph of a normal vascular space from the tibia of a vivarium control rat. The lumen (L) of the vessel and the endothelial x 1200.
The FASEB Journal
cells
(arrows)
can
be seen;
no lipid
deposits
are
present.
DOTY ET AL.
Figure
4. Electron
animal.
Both
micrograph
structures
are
showing a portion darkly
stained
and
of the vascular structurally
space (VS) and an osteocyte
degenerate,
suggesting
either
(OC) from the periosteal
a lipid
accumulation
region of a flight
or cell death.
x 9200.
In the periosteal region, adjacent to vessels that were filled with an osmiophiic material, empty osteocytic lacunae or lacunae filled with osmiophiic material were found (Fig. 4). The blood vessels in this region showed endothelial cell disruption and lipid accumulations within the lumen of the vessels (Fig. 5). In some cases, cell debris or debris mixed with lipid deposits was found within the lumen. The degeneration of the vascular wall was often evident. It was also apparent by electron microscopy that these osteocytic and vascular changes were confined to the periosteal portion of the the diaphyseal bone. These results were not found in the metaphyseal region of the long bones.
Histochemistry The histochemistry of the NADPase activity indicated that the enzyme was localized to the intermediate Golgi saccules of the osteoblast and to the small vesicles and
granules
within
the Golgi complex
(Fig.
6). All osteo-
blasts contained such activity within their Golgi, whether they were from flight or control animals. A quantitative count of the saccules and small vesicles associated with the Golgi showed that the number of saccules containing reaction product was similar for flight and control osteoblasts; flight animals averaged 11.3 ± 6.1 saccules per cell, and the vivarium controls averaged 14.4 ± 3.4 SPACEFLIGHT
EFFECTS ON RAT TIBIA
Figure 5. Electron micrograph showing a large osmiophilic lipidlike (L) inclusion that was typical of those seen in the vascular space of the diaphyseal vessels from the flight animals. Some structures resembled the remains of degenerated endothelial cells (arrows). x 18,000.
19
saccules per cell. However, when we counted the small vesicles (the transitory or intermediate vesicles) that bud off the saccules and that also contained NADPase activity, the flight animals averaged 17.0 ± 6.2 vesicles compared with the control average of 10.5 ± 2.7. These reactive vesicles were found only in the Golgi region and not in any other area of the cytoplasm. The histochemistry of alkaline phosphatase activity showed that the osteoblasts had a very high enzyme content along the external surface of the cell membrane (Fig. 7). Very rarely, activity was also found within the large Golgi saccules before formation of a secretory granule. Alkaline phosphatase activity was very apparent in the osteoblasts of the metaphyseal and diaphyseal regions; no differences in staining between flight and control animals could be observed.
( Figure blast
6. Electron that reacted
micrograph for NADPase
des (arrows) were more numerous x 16,000.
Figure
7. Electron
Enzyme
20
reaction
Vol. 4
micrograph
of the Golgi region of a single activity. The small transitory
in osteoblasts
showing
from flight animals.
the alkaline
can also be seen in the underlying
Jan. 1990
osteovesi-
phosphatase
A strong alkaline phosphatase reaction occurred within vascular channels of diaphyseal bone. This reaction was not limited to the osteoblasts adjacent to blood vessels but included the endothelial cells of these vessels (Fig. 8). Therefore, at the light microscope level of observation, the alkaline phosphatase activity associated with the vascular space is a combination of reactions from endothelial cells and perivascular cells. This phosphatase activity was decreased in flight animals compared with control rats. The EM observations showed that the alkaline phosphatase activity in the blood vessels of bone was located along the basement membrane surrounding the endothelial cells and along adjacent cells (Fig. 8). The pericytes and
reaction
bone matrix
(BM).
(arrows) along x 16,000.
The FASEB Journal
the external
cell membrane
of a single
osteoblast.
DOTY ET AL.
Figure blood
o. ri.... vessel
...i
in bone.
micrograph L, Lumen
of the a.aline phosphatase activity of blood vessel; BM, demineralized
nonosteoblastic cells associated with the vessel were surrounded with alkaline phosphatase activity, as were the osteoblasts adjacent to the vessels but associated with the bone surface. In the flight animals, the alkaline phosphatase activity was significantly lessened around the vascular endothelium and often appeared as an incomplete line of reaction compared with the complete reaction found in the controls (Figs. 9, 10). To determine whether the vascular channels per se had been affected by flight, the area of the vascular spaces in diaphyseal bone was measured. The data show that for flight animals the average area of each vascular space was 762 ± 157 tm2, whereas the same measurement in the synchronous control group was 921 ± 267 m2. The difference was not significant (P < 0.2) because of the large standard deviation but does suggest a trend to smaller vascular spaces within the diaphyseal bone of the flight animals. The numbers of vessels per area of bone showed a signficant difference (P < 0.001): flight, 72.3 ± 6.8 vessels/mm2 of bone area; synchronous controls, 49.0 ± 8.8 vessels/mm2 of bone area. These results indicate more vascular space per area of bone in flight animals than in the synchronous controls.
(arrows) bone
distributed matrix.
among
the endothelial
cells and pericytes
of a
x 10,000.
cant differences are in bone area and periosteal perimeter between the synchronous and basal groups; these differences may reflect the more rapid growth of the synchronous group. The periosteal perimeter of flight animals
DISCUSSION Data from Cosmos 1887 are more difficult to interpret than data from previous missions because of the unanticipated postflight problems and the signficant difference in mean body weight between the flight and control groups (9). Whether the weight difference reflects a postflight or an in-flight difference between the groups is not known.
Bone parameters including area and perimeter at the TFJ are similar between groups (Table 1). The only signifiSPACEFLIGHT
EFFECTS ON RAT TIBIA
Figure
9. Light micrograph showing the alkaline tion (arrows) among the blood vessels of normal matrix. x 700.
phosphatase bone. BM,
reacBone
21
cumulate whenever the energy sources of the cell have been depleted (17). Also, in osteogenesis imperfecta, a collagen deficiency disease of bone, these vesicles were present in unusually large numbers (10). Thus the data suggest that spaceflight creates an energy loss within the osteoblast that could alter collagen synthesis. However, postflight readaptation complicates the interpretation of these data since the increased number of vesicles could also reflect postflight bone formation. The vasculature within bone matrix also appears to be altered by spaceflight. The normal strong alkaline phosphatase reactivity, located between the endothelial cells and the pericytes and including the basement membrane between cells, may be decreased by spaceflight. These histochemical results indicate that the vessels within bone matrix contain enzyme activities that could be important in regulating ion transport between serum and bone matrix. Many of the vessels near the periosteal surface contained lipid inclusions and/or morphological signs of
endothelial
Figure 10. Preparation is from a flight animal.
identical to that in Fig. 9 except that the bone The intensity of the reaction is less than in
the control and some areas (arrows) of enzyme
activity.
show a definite reduction
or loss
x 700.
is very similar to that of basal group, suggesting that little bone was formed at this site during flight; this value does not quite reach significance (P < 0.1) when compared with synchronous rats. Similar data from the older rats in SL3 also show no significant changes in bone area between flight and synchronous controls; however, use of a bone marker in the SL3 experiment revealed a significant decrease in bone mineralization during flight (2), which demonstrates the importance of bone markers for detecting changes in bone during short-duration spaceflights. The decrease in new bone formation due to the spaceflight has been documented (1, 4, 5, 15) and most results suggest that the primary defect is in the bone-forming cells, the osteoblasts. Bone resorption in weight-bearing bones appears to function essentially normally during hypogravity exposure (1, 3-6, 15). Therefore, the osteoblast has been the bone cell of interest for determining the effect of gravity on the growing skeleton. The Golgi saccules and transitional vesicles of matrixsecreting cells are specifically stained by the NADPase reaction (12) and are also directly involved in collagen synthesis and secretion (7, 8). The appearance of increased numbers of transitional vesicles in cells from flight animals compared with controls was unexpected since collagen synthesis must be altered if new bone formation is reduced during spaceflight (1, 16). However, in other nonbone cell types, these vesicles have been shown to ac22
Vol. 4
Jan. 1990
degeneration.
Intravascular
deposits
of lipid
can result in necrosis of bone (18), and ischemia may cause cell death and degeneration in bone (19). Whether ischemia and the appearance of lipid are related events is not known. The centrifugal microcirculation of bone (20) may make the vessels nearest the periosteal surface more sensitive to reduced blood flow, decreased oxygenation, or intraluminal lipid deposits than vessels closer to bone marrow. Also, the smaller cross-sectional area per vessel in compact bone from the flight animals might be a factor in large droplets being trapped within these vessels. The factor or factors responsible for decreased bone formation in growing animals as a result of hypogravity is unknown. However, vascular changes may occur during flight and influence the bone-forming ability of the osteoblasts in bone. It is also possible that the vascular damage could occur by injury of these fragile vessels during the impact and hypergravity forces of reentry from space or during recovery after spaceflight. The time course and extent of bone changes during spaceflight must be understood before long-duration spaceflight without compensating forces is considered. A major concern is that adaptation to spaceflight might impair the ability to return to earth or other planets. Thus, we must understand what is happening to the mammalian skeleton during spaceflight. The Cosmos missions provide data for understanding skeletal adaptation in the growing rat to spaceflights lasting longer than I week. The authors thank the many Soviet scientists who assisted with the experiment by dissecting tissues, preparing samples, and expediting the shipment of biological specimens to the United States. We also thank the NASA personnel who made this experiment possible. We are grateful for the technical assistance provided by Christopher Maese.
REFERENCES T. J., and Morey, E. R. (1983) Effect of spaceflight on bone formation in rats. Am.]. Physiol. 244, R305-R309 T. J., Morey-Holton, E. R., Doty, S. B., Maese, A. C., and Walsh, C. C. (1987) Histomorphometric analysis of rat skele-
1. Wronski, periosteal 2. Wronski,
The FASEB Journal
DOTY ET AL.
3.
ton following space flight. Am. j PhyioL 252, R252-R255 Jee, W. S. S., Wronski, T. J., Morey, E. R., and Kimmel, D. B. (1983) Effects of spaceflight on trabecular bone in rats. Am. j PhysioL 244, R3l0-R314
A. S., Durnova, G. N., Sakharova, Z. F, and ilyinaY. I. (1987) Histomorphometric analysis of rat bones after spaceflight aboard Cosmos-1667 biosatatellite. Space Biol. Med. 21, (5), 33-40 5. Vico, L., Chappard, D., Palle, S., Bakulin, A. V., Novikov, V. E., and Alexandre, C. (1988) Trabecular bone remodeling after seven 4. Kaplansky,
Kakuyeva,
days
of weightlessness
exposure
(BIOCOSMOS
1667).
Am. j
PhysioL 255, R243-R247 6. Cann, mineral
C. E., and Adachi, R. excretion in rats during
R. (1983) spaceflight.
R327-R331 7. Bornstein, P. (1974) The biosynthesis chem. 43, 567-603 8.
Kruse,
N.
J.,
and
Bornstein,
for transcellular movement 250, 4841-4847
P. (1975) and secretion
Bone
resorption
Am.].
of collagen.
and
Physiol. 244, Annu. Rev. Bio-
Metabolic requirements of collagen.j BioL Chem.
9. Grindeland, R. E., Popova, I. A., and Vasques, M. F. (1990) Cosmos 1887 mission overview; effects of microgravity on rat body and adrenal weights and plasma constituents. FASEBJ 4, 105-109 10. Doty, S. B., and Mathews, R. (1971) Electron microscopy and histochemical studies of osteogenesis imperfecta. Gun. Ortlzop.
12. Smith,
Ultrastructural phosphatase of the Golgi
localization of nicotinamide (NADPase) activity to the inapparatus in rat incisor amelo-
135-137,
Little,
Brown,
Boston
D., Alexandre, C., Palle, S., Minaire, P., Riffat, G., Novikov, V. E., and Bakulin, A. B. (1987) Effects of weightlessness on bone mass and osteoclast number in pregnant rats after a five day spaceflight (Cosmos 1514). Bone 8, 95-103 16. Patterson-Buckendahl, P., Arnaud, S. B., Mechanic, G. L., Martin, R. B., Grindeland, R. E., and Cann, C. E. (1987) Fra15. Vico,
17.
18. 19.
20.
L, Chappard,
gility and composition of growing rat bone after one week in spaceflight. Am. j PhysioL 252, R240-R246 Saraste, J., Palade, G. E., and Farquhar, M. G. (1986) Temperature-sensitive steps in the transport of secretory proteins through the Golgi complex in exocrine pancreatic cells. Proc. NaIl. Acad. Sci. USA 83, 6425-6429 Jones, J. P. (1985) Rat embolism and osteonecrosis. Orthmp. Clin. North Am. 16, 515-633 Chryssanthou, C. P. (1978) Dysbaric osteonecrosis. Cue. Orthop. Relat. Res. 130, 94-106 Lopez-Curto, J. A., Bassingthwaighte, J. B., and Kelly, P. J. (1980) Anatomy of the microvasculature of the tibial diaphysis of the adult dog. j Bone ]t. Surg. 62A, 1362-1369 .Feceived for publication March 22, 1989. Accepted for publication
Paris
SPACEFLIGHT
dinucleotide
termediate saccules blasts.]. Histoc/zem. Cytochem. 28, 16-26 13. Fowler, S. D., and Greenspan, P. (1985) Application of Nile Red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with Oil Red 0. j Histochem. Cytochem. 33, 833-836 14. Troyer, H. (1980) Principles and 72chniques of Histochemistry, pp.
Relat. Res. 80, 191-200 11. Doty, S. B. (1980) Histochemical investigations of secretory activity in the osteoblast. In Third International Workshop on Bone Histomorphometry (Jee, W. S. S., and Parfitt, A. M., eds), pp. 201-206, Soci#{233}t#{233} Nouvelle de Publications M#{233}dicales et Dentaires,
C. E. (1980)
adenine
EFFECTS ON RAT TIBIA
July
21, 1989.
23
