Strategies of hemopoietic stress adaptation within the medullary cavity.

THE ANATOMICAL RECORD 216:528-533 (1986)
Strategies of Hemopoietic Stress Adaptation Within
the Medullary Cavity
DENNIS C. GIULIANI, JAMES C. HALL, AND BERNARD S. MORSE
Department of Medicine-Hematology Division, Monte-Scaglione Memorial Research
Laboratory, New Jersey Medical School, University of Medicine and Dentistry at New Jersey,
Newark, N J 07103 (D.C.G.,B.S. M.); Department of Zoology and Physiology, Rutgers
University, Newark, N J 07102 (J.C.H.)
ABSTRACT
Gravimetric determination of total bone water space was used as
a n index of available bone marrow space in mice following various specific stressors,
i.e., splenectomy, hypoxia, bone fracture, and estrone-induced osteosclerosis. Data
was corrected for bone weight and was reported as specific bone marrow volume
(total bone water spacetmg dry bone x 100).
A direct relationship was observed between specific bone marrow volume and
medullary hemopoietic activity induced by stress. Absolute andior relative marrow
space increased following splenectomy, hypoxia, and fracture. Osteosclerotic animals
shift most hemopoietic activity from marrow to spleen, and splenectomized osteosclerotic animals become anemic. Both intact and splenectomized hypoxic animals
develop increased specific bone marrow volume and successfully compensate for
hypoxia with enhanced erythropoiesis. Animals sustaining a fracture callus increase
both specific bone marrow volume and hemopoietic activity a t the callus without an
increase in hemopoietic demand.
Increased specific bone marrow volume extends the marrow bone interface, where
primitive stem cells accumulate, while expanding marrow stromal space, where
stem cells lodge, proliferate and differentiate. Therefore, it would appear that availability of competent marrow space may play a n integral part in passively permitting
hematopoiesis and in determining hemopoietic reserve capacity.
Stem cell migration increases during intensified hemopoietic demand, which also
may be related to available marrow space. Mice have a low medullary hemopoietic
reserve capacity; subsequently, when available medullary hematopoietic stroma
becomes occupied, stem cells are more likely to migrate from the marrow to extramedullary sites where they mature before entering the circulating pool.
Adaptation of hemopoietic tissue to stress has received
considerable attention in the literature. Yoffey (1966)
suggested strategies by which the hematopoietic tissue
may adapt in order to support the erythroid hyperplasia
following hypoxic hypoxia. He proposed: 1) accelerated
stem cell maturation, 2) increased numbers of mitoses
during maturation (amplification), 3) increased numbers
of stem cells (proliferation), and 4)expansion of hematopoietic reserve space. Current thought on these adaptive
strategies Worse et al., 1969) indicates that progenitor
cells may shorten their cell cycle, primarily by reducing
the time in G1 phase. Questions regarding the number
of mitoses preceding final maturation remain unresolved, and although stem cells reportedly proliferate in
order to support increased demand, the mechanism for
maintaining the differentiated pool (Bessis and Brecher,
1975)remains uncertain. Finally, hematopoietic reserve
space may be gained at the expense of scattered fat cells
within the predominantly active (red) marrow in those
species with greater hemopoietic reserve, and available
marrow space may be expanded through bone resorption, as reported by Yoffey (1966).
0 1986 ALAN R. LISS, INC.
Independent studies in dogs by Drinker et al. (19221,
Johnstone (1922), and Gong (1965) present data that
support a direct relationship between hemopoietic demand and available hematopoietic tissue space within
the medullary cavity. In contrast, Hudson (1958a,b) reported no change in either total or red marrow space of
guinea pigs exposed to hypoxia. Increased spleen weight
in the hypoxic guinea pigs indicates extramedullary hemopoietic adaptation in this species, which has a low
hemopoietic marrow reserve capacity according to Yoffey (1966).
In this study, hemopoietic adaptation was studied in
mice, a species with low medullary hemopoietic reserve,
as they responded to various specific stresses. Both normal and splenectomized animals were exposed to the
following: hypoxic hypoxia, estrone-induced osteosclerosis, and fracture repair. Hematologic parameters as
well as dry bone weight, bone mineral volume, and total
Received February 3, 1986; accepted July 15, 1986.
HEMOPOIETIC STRESS AND BONE MARROW VOLUME
529
bone water space were measured. Strategies of adapta- bone water space were made by projecting and tracing
tion to these stressors are discussed.
cross- and longitudinal sections of fully calcified bone
(Carl Ziess projection microscope). For this study, bones
MATERIALS AND METHODS
previously digested in pronase were embedded in clear
Animals
casting resin and allowed to harden under vacuum. MiFemale 8-week-old, 20-g CF1 outbred albino mice croscopic sections were made with the aid of Isomet saw
(Charles River Farms) were used exclusively in these and Ecomet I1 grinder-polisher. Each cross-sectional cut
experiments.
was 0.10 inch thick. Area and length measurement were
Animals were randomly divided into four groups: con- made from the tracings by means of a computer aided
trol, experimental, splenectomized control, and splenec- planimeter. Absolute medullary cavity volume and bone
tomized experimental. Spleens were surgically removed volume were calculated from the tracings by projecting
from the animals so designated, and individual experi- a stage micrometer along with the bone sections as
ments were started 2 weeks after splenectomy.
modified from reports by Green et al. (1981).Differences
between medullary cavity volume and total water space
Peripheral Blood Parameters
in the whole bone were taken to represent bone vascular
Mice were exsanguinated via cardiac puncture under and lacunae space as well as water of hydration.
light ether anesthesia. Whole blood, anticoagulated with
Bone Mineral Density
disodium ethylenediamine tetraacetic acid (EDTA), was
used to measure hematocrit and hemoglobin concentraBone mineral density was obtained by employing Artion; peripheral blood smears (Wright’s-Giemsa stain) chimedes’ Principle. Mineral volume was measured in
were prepared according to standard methodology re- pronase-digested, defatted bone. Bones were first satuported by Wintrobe (1972).
rated with water in vacuo then weighed in a 1-ml pycnometer in order to determine the weight of water
Bone Marrow Volume Measurement
displaced by the bone mineral. Weight of displaced water
Animals were weighed to the nearest gram, exsan- was converted to volume (dividing by the density of
guinated, then killed by cervical dislocation while under water at laboratory temperature). Density (dry bone
ether anesthesia. Intact bones (tibias and femurs) were weightlunit volume of bone) reflects contributions of both
dissected free from the soft tissue. Bones were then the bone mineral and organic matrix.
incubated in vacuo at 37°C in 0.01 M Tris buffer (pH
Fracture and Fracture Repair
7.2) containing 5 mglml pronase in order to digest the
marrow. Incubation was continued for 2 weeks, during
Midshaft fracture of the left tibia was performed by
which the buffered pronase solution was changed every digital manipulation under light ether anesthesia. Aniother day. Following pronase digestion, fat was ex- mals were sacrificed 23 days later. Both fractured and
tracted from the bone by refluxing in a mixture of 4:l contralateral unfractured tibias were processed a s preethyl etherabsolute alcohol for 1 hour at room temper- viously described.
ature. The remaining soluble substance was removed by
three changes of deionized water in vacuo, with one
Estrone
change per day.
Experimental
osteosclerosis
was induced by weekly
Microscopic examination of representative bones after
subcutaneous
injections
of
a
n
aqueous
estrone suspendigestion showed them to be completely devoid of all
marrow tissue. These bones were then dried under vac- sion (0.5 mg) for 6 weeks (Simmons, 1966).
uum a t 80°C for 6 hours, allowed to reach room temperHypoxic Hypoxia
ature in a dessicated chamber, and weighed to the
Hypoxia
was
induced
by housing mice in cages lined
nearest 0.0001 g. Intact bones were then placed in deionized, distilled water under vacuum for 24 hours. Upon with a silicone membrane enclosure, which selectively
removal from the water, bone surfaces were wiped dry, reduces oxygen permeability. Maintaining seven mice
and the bone was immediately weighed to the nearest per cage has been found (Lang et al., 1966) to lower the
0.0001 g. Both dry and water-saturated weights were oxygen concentration to 9% which is equivalent to a n
altitude of 21,000 feet. Cages were changed twice weekly
repeated until constant values were achieved.
Marrow volume (total bone water space) was deter- during the 23-day course of the experiment, and animals
mined by subtracting the dried bone weight from the unable to sustain hematocrits of 60% or greater were
water-saturated weight. Differences in these weights eliminated from the experiment.
represented the total weight of water within the bone.
Data Analysis
This value was converted to total volume within the
bone by dividing by the density of water at laboratory
Data was analyzed by means of standard statistical
temperature. These volume measurements include bone methods. Sample size with mean values are reported
lacunae space, vascular space, water of hydration, and k standard error. One-way analysis of variance was used
available marrow space. Bone marrow volume measure- to analyze for differences between means. If means were
ments were repeated using selected bones that had been significantly different by analysis of variance, (P < .05),
cleaved longitudinally in order to remove medullary Duncan’s multiple range test was used to determine
cavity water. This made it possible to determine the significant differences between all possible pairs of group
contribution of nonmedullary cavity water space to the means. Animals that sustained a fractured tibia were
total bone water space.
statistically analyzed using the two-tailed paired t-test.
Morphometric studies designed to confirm the contri- Comparison was made between the fractured tibia and
bution of nonmedullary cavity water space to the total the contralateral unfractured tibia.
D.C. GIULIANI, J.C. HALL, AND B.S. MORSE
530
TABLE 1. Cumulative data'
Body weight
(gm)
Mean
No.
f SE
Hematocrit
9'0
Mean
No.
f SE
Hemoglobin
conc.
(gram %I
Mean
No.
f SE
No.
Control
22
22
Hypoxic
14
Hypoxic
splenectomy
15
13.81
f 0.41
14.4
f 0.73
14.3
f 0.38
27.11"
f 0.88
25.35*
f 0.67
22
13
45.7
.-t 0.54
.
45.5
-t
.
- 0.76
47.1
-t 0.67
78.0"
.- 1.32
-t
76.5*
-t
.
- 0.69
22
Control
splenectomy
Shamoperated
28.6'
f 0.71
27.8
f 1.34
28.7
1.21
16.8"
f 0.38
15.9*
f 0.32
29.3
f 0.44
28.6
f 0.55
9
42.8"
.- 1.09
-t
33.1"
.- 1.44
-t
8
12.0"
f 0.07
9.77"
f 0.61
8
Estrone
Estrone
splenectomy
9
9
14
*
Body weight
Mean
No.
f SE
11
9
9
15
14
Hematocrit
_____
%
No.
Mean
k SE
11
9
9
15
14
Hemoglobin
conc.
(gram %)
Mean
No.
f SE
12
7
13
13
14
No.
Dry bone weight
(mg)
Tibia
No. Femur
30.3
f 0.93
27.8
f 1.36
30.2
f 1.07
22.3"
f 0.52
21.0"
f 0.67
22
43.8"
f 1.37
43.2"
f 0.86
8
13
9
14
14
14
Bone marrow volume
(total bone water space)
(mm3)
No.
Tibia
No. Femur
37.0
f 1.10
33.3
2.00
33.9
f 1.71
22.8*
k 0.51
21.9"
It 0.60
21
54.7"
f 1.53
51.1"
1.23
8
*
Dry bone weight
(mg)
Tibia
No. Tibia2
11
7
11
12
8
11.8
f 0.27
11.8
f 0.38
11.5
k 0.53
11.3
f 0.35
10.3"
f 0.51
22
8.5"
f 0.2
8.1"
k 0.17
8
13
9
14
13
14
16.6
f 0.46
16.7
f 0.51
16.1
f 0.64
14.6"
f 0.39
14.7"
f 0.62
11.8*
k 0.16
12.2*
f 0.25
Bone marrow volume
(total bone water space)
(mm3)
No.
Tibia
No. Tibia2
Fracture
(tibia only)
10
28.4'
k 0.72
10
4-6.1
._
-t
0.80
9
13.42
f 0.64
9
45.8"
f 1.17
9
30.7
f 0.45
9
26.6"
f 1.51
9
12.0
+ 0.39
-
Fracture
(tibia only)
splenectomy
10
27.7
f 0.58
10
4.4.8
._
-t
0.55
10
12.83
f 0.23
10
42.7"
f 1.76
10
30.1
f 0.84
8
25.6"
k 1.37
8
12.25
f 0.36
(continued)
RESULTS
Hematologic and bone and marrow volume parameters are listed in Table 1.
Splenectomized animals respond with a small decrease in bone mineral volume (tibia and femur) and
increased specific bone marrow volume (femur only).
Other parameters did not vary significantly from those
of control animals. Sham-operated splenectomized animals showed no variation in any parameter when compared to controls.
Mice exposed to atmospheric hypoxia showed significant variation from control animal values. Body weight,
dry bone weight, bone mineral volume, and bone marrow volume (total bone water space) decreased. Hematocrit, hemoglobin concentration, and specific bone
marrow volume (total bone water space/mg bone x 100)
all increased versus control values. Only tibia1 bone
marrow volume remained unaltered in nonsplenectomized animals exposed to hypoxia.
Animals subjected to bone fracture and fracture repair
were studied in order to assess their influence on the
bone and marrow. Peripheral blood parameters remained unchanged. Bone and bone marrow parameters
in the contralateral unfractured tibia also did not vary
from that of control. All bone and marrow parameters
in the fractured tibia, both measured and derived, increased-with the exception of bone density, which remained unchanged. Splenectomized animals responded
similarly to fracture.
Estrone-treated animals show a slight but significant
reduction in hematocrit and hemoglobin concentration.
Bone and marrow parameters, on the other hand, all
change in response to estrone. Dry bone weight increases, total bone water space decreases, and bone mineral volume increases. Derived parameters were also
altered; bone density increases, and specific marrow volume decreases. Splenectomized animals treated with
estrone respond with a pronounced reduction in hemoglobin concentration and hematocrit. Both normal and
splenectomized estrone-treated animals had similar
changes in their bone and marrow parameters.
Conformational studies of the displacement technique
are shown in Table 2. Both morphometric measurement
(projection microscopy)and displacement technique (longitudinally cleaved bones) are in close agreement and
confirm the contribution of medullary cavity water space
to total bone water space. Increased specific marrow
volume in hypoxic animals indicates a redistribution of
bone water space, with a larger portion of this water
distributing in the marrow cavity.
DISCUSSION
Enigmas associated with the stem cell concept of hematopoiesis reported by Cronkite (1964) remain unresolved. For example, whether individual stem cells
multiply sequentially as they mature into a specific
functional pool or have continuous access among the
various stem cell compartments is not clear (Mackey
531
HEMOPOIETIC STRESS AND BONE MARROW VOLUME
TABLE 1. (Continued)
Bone mineral volume
(mm3)
No.
Tibia
No.
Femur
No.
Tibia
No.
Femur
Control
21
21
22
Control
splenectomy
Shamoperated
12
Hypoxic
13
Hypoxic
splenectomy
Estrone
12
Estrone
splenectomy
14
2.30
f 0.026
2.33
f 0.031
2.28
0.063
2.43
0.069
2.43
k 0.069
2.51*
& 0.028
2.55"
f 0.032
2.32
f 0.027
2.38
f 0.044
2.28
f 0.054
2.43
k 0.068
2.47
k 0.063
2.53*
f 0.023
2.48*
f 0.017
7
8
13.4
f 0.29
12.0"
f 0.45
13.4
f 0.51
9.2"
f 0.40
8.9"
f 0.45
17.5"
f 0.62
17.1*
ir 0.36
22
13
9
14
14
8
14
16.0
f 0.49
14.0*
& 0.83
14.5
0.91
9.4"
f 0.43
9.0*
f 0.30
21.5"
& 0.71
20.6*
k 0.46
*
Bone density
12
7
13
12
8
14
Bone mineral volume
(mm3)
Fracture
(tibia only)
Fracture
(tibia only)
splenectomy
Tibia
No.
Tibia2
No.
Tibia
9
19.2
f 0.54
19.6*
f 0.76
9
13.1
f 0.39
13.0
f 0.45
8
2.34
0.017
2.30
f 0.024
8
9
13
14
8
14
Bone density
No.
8
13
9
No.
8
9
Tibia'
2.33
f 0.05
2.40
f 0.039
Specific bone marrow
volume
No.
Tibia
No.
Femur
21
11
7
11
12
8
14
38.9
f 1.48
42.5
& 1.58
37.5
f 1.01
49.1*
f 1.58
48.8*
f 1.96
19.5"
f 0.39
18.7*
f 0.33
22
13
9
14
13
8
14
45.1
f 1.59
51.6*
f 1.35
47.6
3.13
64.1"
f 2.06
65.8*
f 2.01
21.7"
f 0.58
24.1*
& 0.49
Specific bone marrow
volume
No.
Tibia
No.
Tibia2
8
10
56.0*
+ 1.87
55.6*
f 2.51
9
10
39.1
f 1.15
39.8
f 0.81
'Values are mean k standard error. Data analyzed by means of Duncan multiple comparison procedure. No., No. of animals.
'Contralateral unfractured tibia-two-tailed paired t-test with fractured tibia. All other data analyzed with Duncan multiple comparison
procedure.
*P < .05.
and Dormer, 1982; Dormer and Ucci, 1984). Furthermore, the mechanism of commitment to a functional
pool, studied by Patt and Maloney (1972) and Till (1982),
also remains in question. Stem cells may enter the committed stage of development randomly, as suggested by
the stocastic theory, or may be induced into final commitment by the hemopoietic microenvironment, according to the deterministic theory.
Further insight into the mechanism of stem-cell-mediated hematopoiesis in mice might be gained by studying hemopoietic stress adaptation. Hemopoietic adaptation to specific stresses includes changes in available
tissue space. Present studies evaluate such changes that
occur in the medullary cavity and relate these changes
to hemopoietic activity.
Strategies linking medullary cavity space and hemopoietic activity remain uncertain. However, expanded
specific marrow volume, as seen in mice following hypoxia, suggests a direct relationship between increased
marrow volume and the adaptive strategy, which compensates for increased erythroid demand.
Similarly, the medullary cavity expands at the site of
a fracture callus. Both 59Fedistribution and histological
inspection of the callus during repair was studied by
Van Dyke and Harris (1969) and Giuliani et al. (1977).
These reports indicate localized increased hemopoietic
activity. Data support localized expansion of active he-
matopoietic tissue into space made available through
resorption of the callus, as reported by Raisz (1981).
Although the influence of trauma during fracture cannot be fully assessed, it would appear that increased
marrow space alone may serve to support active hematopoietic stroma in the callus.
Splenectomized control animals show significant loss
of bone mineral volume. This, however, contributes litTABLE 2. Conformation-morphometric measurement and
cleaved bone studies'
Medullary cavity space
as percentage of
bone marrow volume
(total bone water spacetibias only)
Morphometric measurement
projection microscopy
Cleaved bone method
Control
Hypoxic
48.6 f 4.0 control animals
45.6 f 3.2
57.8 f 0.8*
'Data indicate that medullary space is less than half the total
bone water space. Hypoxic animals with increased specific
marrow volume contribute a greater portion of their bone
water space to the marrow. Values are mean SE.
* P = < ,051vs. control.
532
D.C. GIULIANI, J.C. HALL, AND B.S. MORSE
tle to available marrow space, since specific marrow
volume increases only in the femur (12.2%). Present
data tend to support reported increases in marrow space
(Drinker et al., 1922; Johnstone, 192%)associated with
the loss of normal splenic hemopoietic activity following
splenectomy.
Animals treated with estrone show a slight decrease
in hematocrit and hemoglobin concentration, which reflects a combination of mild hemolysis and decreased
production, as previously reported by Morse et al. (1974).
Bone and marrow parameters are consistent with endosteal deposition of a denser mineral complex. Histological
examination further confirms the loss of marrow space
to bone. Splenectomized, estrone-treated animals respond similarly, but they show a more marked decrease
in hematocrit and hemoglobin concentration. These data
suggest a greater dependence upon splenic hemopoietic
capacity following osteosclerosis.
There appears to be a direct relationship between the
availability of hematopoietic tissue space and hemopoietic activity in mice. During hypoxia, relative marrow
space expands at the expense of bone, as the animals
respond to increased erythroid demand. Furthermore,
active hematopoietic tissue infiltrates space made available through resorption of a fracture callus in the absence of altered hemopoietic demand, according to earlier
studies by Van Dyke and Harris (1969). Increased splenic
hematopoiesis accompanies the loss of medullary space
to estrone-induced endosteal bone deposition, while splenectomized estrone-treated animals demonstrate frank
anemia.
Quantitative study has shown that active hematopoietic tissue expands in response to increased demands
made upon the functional blood cell pool. This increased
space may be achieved 1)at the expense of medullary
fat in species with high medullary hemopoietic reserve
capacity, 2) through extramedullary hematopoiesis (primarily in the spleen) (Kozlov et al., 1980a, 1980b; Aggio
et al., 1973),and 3) through bone resorption. Though the
data suggest that increased medullary space may serve
as a direct incentive for increased hematopoietic activity, the mechanism for this response remains obscure.
Mass action phenomenon, however, might serve to explain foci of intensified hemopoietic activity in this space
even in the absence of increased hemopoietic demand.
In the mouse, intensified hemopoietic demand is compensated, in part, by selective migration of stem cells
from the marrow to the spleen (Kozlov et al., 1980a,b;
Aggio et al., 1973). Selective migration might be explained by direct competition between stem cells, i.e.,
preferential differentiation into one stem cell compartment a t the expense of another. Rather, competition for
a limited medullary niche remains indistinguishable
from direct competition.
If marrow constitutes a preferred site for stem cell selfperpetuation, while the spleen provides a preferred site
for their maturation, stem cell migration becomes more
understandable. Competition for a limited medullary
niche preferred by the self-perpetuating pool of stem
cells might initiate the expulsion of daughter cells from
the marrow. Multiplication of mature circulating cells
from the differentiated pool might proceed in the marrow, if space were available, andor a t extramedullary
(migratory) sites, such as the spleen.
Evidence in support of this proposal is indirect, though
compelling. Clinically, marrow volume increases during
conditions of chronic persistant hemopoietic demand, as
seen in patients with sickle cell disease and thalassemia
major (Raisz 1981). In these diseases, a s in the present
studies, active marrow fills the space made available by
bone resorption. Proliferating primitive stem cells, (CFUS),tend to distribute in greatest numbers at the marrowbone interface Cord et al., 1975); pronounced increases
in specific marrow volume will extend this site while
providing increased potential stromal space required for
lodging, differentiation, and maturation. Therefore, expanded (endosteal) surface area a t the site of the fracture
callus may permit the localized increase in hemopoietic
activity, which also potentially increases hemopoietic
reserve capacity.
Results from estrone-treated animals provide further
support for this proposal. Although a lead-shielded limb
from a lethally irradiated estrone-treated mouse may
provide increased endogenous (migrating) stem cells,
splenectomized estrone-treated animals become anemic
much more rapidly than normal estrone-treated animals
(Morse et al., 1974). Therefore, without adequate sites
for proliferating marrow stem cells to lodge and multiply into the functional pool, normal erythroid homeostasis cannot be maintained. In contrast, normal splenectomized animals, exposed to hypoxia, but with increased specific marrow volume, may effectively
compensate for the increased erythroid demand.
These data strongly support a direct relationship between hemopoietic activity and the availability of hematopoietic tissue space. Furthermore they support a
functional distinction between splenic and medullary
hematopoiesis, proposing the marrow a s a preferential
site for stem cell proliferation. Maturation of stem cells
into the function pool may also proceed in the marrow;
however, this depends, a t least in part, upon availability
of hematopoietic tissue space. During conditions of hemopoietic stress or when available medullary space is
otherwise occupied, mouse stem cells preferentially
lodge, proliferate, and differentiate at extramedullary
(migratory) sites, primarily in the spleen.
ACKNOWLEDGMENTS
This report was taken in part from a thesis submitted
by Dennis Giuliani to the faculty of the Graduate School
of Zoology-Physiology in partial fulfillment of the requirements for the Degree of Doctor of Philosophy a t
Rutgers University, Newark, New Jersey.
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