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The micocirculation of bone and marrow in the diaphysis of the rat hemopoietic long bones.

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THE ANATOMICAL RECORD 233169-177 (1992)
The Microcirculation of Bone and Marrow in the Diaphysis of the
Rat Hemopoietic Long Bones
L. DE SAINT-CEORCES AND S. C. MILLER
Division of Radbbwlogy, School of Medicine, University of Utah,
Salt Lake City, Utah 84112
ABSTRACT
The nature of the mimirculation of the diaphyseal portion of
long bones and the adjacent bone marrow is poorly understood. The purpose of this
study was to describe the blood supply in the diaphyseal cortex and the relationship of the bone vascular circulation to that of the bone marrow in the growing rat.
India ink-gelatin was infused in the arterial system of 3-month-old rats and the
vascularization was determined from histological sections. In some studies the
periosteal circulation was blocked but the nutrient and metaphyseal arteriole systems were left intact. In the growing rat, most of the vascular flow appears to be
centripetally through the diaphyseal cortex and this appears to be the primary
blood supply for the adjacent bone marrow. The India ink traversed the cortex and
entered the marrow through osteal canals at the endocortical surface. At the marrow-endocortical bone surface interface, ink exiting from the osteal canals filled
the adjacent marrow sinusoids in what appeared as “bush-like” structures. From
the bone marrow the ink appeared to drain into the central vein. Some arterioles
from the nutrient system were found to penetrate the inner two thirds of the
cortical bone and then reenter the bone marrow. The centripetal flow of blood and
the importance of the cortical flow for perfusion of the hemopoietic tissue was
further documented when periosteal flow was obstructed. In this situation, the
cortical bone and adjacent bone marrow were not perfused while the nutrient
system and central vein were filled with ink. These results show that in the growing rat, the diaphyseal circulation is almost entirely centripetal and the adjacent
hemopoietic tissues are perfused mostly with blood that must first traverse the
cortical bone. 0 1992 Wiley-Lm, Inc.
The long bones have three basic blood supplies: 1) the
nutrient; 2) the metaphyseal and epiphyseal, which is
combined after the growth plates are closed, and 3) the
periosteal (Rhinelander, 1972). It is quite clear that the
metaphyseal region, especially during endochondral
growth, is supplied by the metaphyseal and nutrient
system. There is, however, considerable diversity of
opinion on the contribution of the afferent systems to
the irrigation of the diaphyseal cortical bone as well as
the adjacent diaphyseal hemopoietic tissues. In particular, it is not clear if the flow of blood in the diaphyseal
cortical bone is centrifugal or centripetal and the exact
territories supplied by the afferent nutrient and periosteal systems are not defined. While the microcirculation of most other organ systems is well defined, technical difficulties encountered with processing osseous
tissues have likely made the study of the local vascularization of bone somewhat unappealing.
The circulation of the middiaphyseal cortical bone
in rabbits and rats was described by Brookes (1957, and
1971 for review) as a centrifugal blood flow that could
be reversed in certain abnormal conditions, such as
blockade of the nutrient afferent system. Later studies
conducted by Rhinelander (1972) in dogs generally s u p
ported centrifugal flow but noted that a periosteal s u p
ply of blood for the outer third or quarter of the com0 1992 WILEY-LISS. INC.
pacts only in areas adjacent to fascia1 attachments. De
Bruyn et al. (1970) reached a different conclusion in
studies with rodents and reported that the marrow and
cortical blood flow are in series, that is, that the primary function of the nutrient arterial system was to
supply the bone while most of the blood flow in the
marrow sinuses was from vessels in the osteal canals.
It was suggested that the blood flow through the osteal
canals was from the periosteal capillaries and from osteal capillaries arising from the nutrient arterial system.
In rats, vascular canals that enter the compacta from
the periosteal and endocortical surfaces are usually orientated transverse to the long axis of the bone and the
surface plane. This is usually the case in bones from
animals, such as shorter-lived rodents, that lack Haversian systems (Ruth, 1953). In addition, the afferent
nutrient system arising from the nutrient artery is
very conspicuous in histological sections from rats. For
Received August 6.1991; accepted November 22,1991.
Dr. de Saint-Ceorgee is now at CEN/SCK, Biologie Department,
Boeretang 200,B-2400Mol. Belgium.
Address reprint ueeta to Dr.Scott C. Miller, Building 586,University of Utah, M%ke
City, UT 84112.
170
L. DE SAINT-GEORGES AND S. C. MILLER
Fig. 1. A plastic hose clamp (a) and a piece of latex tubing (b) ensure the compression of the periosteal
vessels around the diaphysis in the position shown on this isolated rat femur. x 3.
Fig. 2. India-ink-gelatin perfusion of the femoral diaphyseal shaft illustrating the cortical bone canals
(cc), the marrow vascular spaces (MI, and the surrounding muscle vessels (ms). x 12.
these reasons, the rat model should be a convenient
system to study the respective contributions of blood
supply from the periosteal vessels and nutrient arterial
blood supply for the irrigation of the cortex and the
bone marrow. This study describes the relative contribution of the nutrient and periosteal blood supplies to
the irrigation of the bone cortex and the marrow in the
diaphyseal regions of the long bone by using a n India
ink-gelatin perfusion method with a periosteal blockage technique.
MATERIALS AND METHODS
Three-month-old male Sprague-Dawley rats (Simonsen Labs, Gilroy, CAI were used in this study. The
animals were maintained and used according to the
principles in the NIH Guide for the Care and Use of
Laboratory Animals (1985) and guidelines established
by the Institutional Animal Care and Use Committee.
For anesthesia, the rats were given a n intraperitoneal
injection of 1.5 ml of chloral hydrate (0.7 g/10 ml). In
some rats, the periosteal circulation was blocked by
local compression of the periosteum prior to perfusion
with India ink-gelatin. For this, the muscles of the femoral shaft, just distal to the third trochanter, were carefully displaced. A piece of latex tubing was placed
around the shaft of the bone and clamped with a small
plastic hose clamp to ensure compression of the periosteal vessels (Fig. l). All rats were then perfused
through the abdominal aorta with a mixture of polyvinylpyrrolidone, heparin, and procaine hydrochloride in
saline to clear the blood from the vascular network
(Forssmann et al., 1977). The perfusate was heated to
BONE-BONE MARROW MICROCIRCULATION IN T H E RAT
40°C by transit through a plastic coil immersed in a
water bath. The perfusion pressure was determined by
gravity at 160 cm of H,O (14 cm Hg). When the blood
cleared from the perfusate, the animals were then perfused with a India ink-gelatin mixture (Jee and Arnold, 1960). This consisted of 28 g purified calfskin gelatin (Eastman Kodak, Rochester, NY), 200 ml distilled
water, and 200 ml India-ink. This was also kept at 40°C
but was administered by manual pressure to achieve a
flow rate of 0.5 mlhec. During the perfusion procedure,
the animal was kept warm with a medicinal heating
lamp. When the perfusion was completed, the abdominal aorta and vein were clamped and the limbs were
placed on ice to solidify the gelatin in the perfusate.
The femurs and tibias were removed and cut either at
the diaphyseal-metaphysealjunctions or at the center
of the diaphysis and placed in fixative. The bones were
immersion fixed for 3 days in 4% glutaraldehyde and
1%formaldehyde in 0.1 M phosphate buffer a t pH 7.2.
The bones were then decalcified, dehydrated, and embedded in epoxy resin. The bone pieces were sectioned
longitudinally using a low speed diamond saw (Isomet,
Buehler, Inc., U.S.A.), mounted on plastic slides, and
ground to about 30 pm in thickness. Serial sections
were collected and were necessary for the interpretation of some of the material.
Because we were using histological and perfusion
methods similar to that described by De Bruyn (1970),
we found it appropriate to use the same terminology for
the vessels as described in detail in his work.
RESULTS
The perfusion method employed in this study was
very effective in filling the vascular network in bone
and bone marrow (Fig. 2). While complete vascular perfusions were frequently obtained, most of our results
were obtained from series of slides with incomplete perfusion areas, permitting the sequence of filling of the
vascular network to be determined.
The extensive vascular constitution of the marrow
and bone cortex was clearly evident. The marrow vascular space was distinctly demarcated from the surrounding hemopoietic tissues as no leakage of the ink
from the vessels into the domains of hemopoietic cells
was observed. The metaphyseal region, which in these
animals is a region of rapid growth and remodeling,
generally appeared to be the first part of the bone to be
perfused (Fig. 3), followed by the large central sinusoid
in the diaphysis (Fig. 4).The central sinus was filled
with ink even when the adjacent bone cortex and hemopoietic tissues showed little evidence of perfusion.
These observations were frequently noted and indicated the principal role of the nutrient system in supplying the growing region of the bone. These observations also emphasized the importance of the central
sinus as the drainage of the metaphysis and nutrient
system. The blood supply from the nutrient system in
the metaphysis appeared to reach the metaphyseal side
of the epiphyseal growth plate and then loop back (Fig.
5) towards the metaphyseal sinusoids.
Typically, the perfusion of the marrow hemopoietic
tissue was observed only when the diaphyseal cortex
vascular channels were filled. Thus, the perfusion appeared to be in a centripetal direction. At the exits of
the transverse cortical canals on the endocortical sur-
171
face, the vessels formed “bush-like” patterns as they
entered the marrow (Fig. 6). In perfusions that were
more complete, the individual “bush-like” vascular
patterns in the hemopoietic tissue were progressively
obscured, suggesting lateral connections between these
vascular domains in the marrow (Fig. 7). The perfusion
of the marrow sinusoids and the disappearance of the
bush-like formations progressively delineated the collecting sinusoids (Fig. 7).
Very narrow dividing branches of the nutrient artery
(Figs. 8, 9) were seen in the marrow but were never
observed with a sinusoid perfusion without simultaneous perfusion of the cortex. In some preparations,
some cortical vessels appeared to arise from the nutrient artery and usually curved back towards the bone
marrow (Fig. 8). These vessels entered at the endocortical surface and appeared to penetrate into the inner
two thirds of the cortex before exiting at the same surface. It was not possible to determine if these vessels
also formed a distinct marrow supply or if they communicated with the transcortical vessels before reaching the marrow sinusoids.
The serial disposition of the blood supply to the cortical bone and the adjacent marrow was further investigated in the bones where periosteal blood flow was
blocked by a clamp. Not only did the periosteal blockade prevent the underlying cortical bone from being
perfused but also prevented the perfusion of the underlying bone marrow (Fig. 10). The nutrient arterial system appeared therefore to be incapable of filling the
marrow in the diaphyseal region where the periosteal
flow was obstructed (Fig. 11).The perfusion of the nonblocked regions of the diaphysis and the filling of the
central sinusoid appear to exclude a lack of ink filling
due to a n exit blockade.
DISCUSSION
In general, our data supports the results of De Bruyn
et al. (19701, who suggested that the flow of blood in
rat, guinea pig, and rabbit bone is centripetal in the
cortex. The histological images obtained in the perfused material indicates that the blood supply to the
bone marrow in the diaphyseal regions of the long
bones in rats is transosteal. The “bush-like’’ vascular
structures of the marrow that arose from the ink exiting the osteal canals at the endocortical surface appeared to be progressively filled and were often observed not yet connected to the central sinus. This
provides evidence for a centripetal flow of blood but
does not support the suggestion that in the rat a substantial centrifugal flow might occur, even in the outer
portion of the cortex (Rhinelander, 1972).
The centripetal flow of blood was further demonstrated in the animals where the periosteal blood flow
was blocked. In these animals, not only was the cortical
flow restricted but flow in the adjacent bone marrow
was also restricted. In these same sections, however,
the nutrient vessels were filled suggesting that the afferent nutrient system does not perfuse most of the
diaphyseal marrow or cortex. The lack of the hemopoietic tissue perfusion in the absence of cortical bone perfusion emphasizes the serial connection of the cortex
and marrow with the obliged transit of blood through
the bone prior to perfusing the marrow. While it might
be argued that the India-ink may not have perfused the
172
L. DE SAINT-GEORGES AND S. C. MILLER
Fig. 3. Distal region of a partially perfused femur. The highly
vascular and metabolically active metaphyseal (Met) region of the
long bone was readily perfused. Portions of the diaphyseal (Dial
marrow were generally less perfusable than the metaphyseal areas.
x 6.
Fig. 4. Diaphysis from an incompletely perfused femur. India-ink
filled the central sinus (CS) but incompletely filled the marrow sinusoids (MS). X 18.
Fig. 5. Distal metaphysis of the femur illustrating nutrient arterial
branches (ab) beneath the growth plate (gp). x 18.
BONE-BONE MARROW MICROCIRCULATION IN THE RAT
Fig. 6. Femoral diaphyseal shaft illustrating the “bush-like” marrow sinusoids formations (bf) in the marrow (M) where the vessels
(Vc) exit the bone cortex at the endocortical surface. The marrow is
perfused only where the cortical canals (cc) are also perfused. The
central sinus (CS) is perfused in the absence of complete perfusion of
the marrow sinusoids. x 12,
173
Fig. 7. Femoral diaphyseal shaft with more filling of the marrow
sinusoids than illustrated in Figure 6. Many of the bush-like formations become indistinguishable but the collecting sinusoids (c0.s) become more apparent. x 12.
174
L. DE SAINT-GEORGES AND S. C. MILLER
Fig. 8. Femur diaphysis. Occasionally some arterial branches (ab) of
the nutrient system enter the cortex and curve back to the marrow.
The thickness of the sections (30 pm) makes it difficult to determine
the connectivity between arterial branches and other cortical vessels.
x 12.
Fig. 9. Femur diaphysis. Some small nutrient arterial branches (ab)
can be found entering the inner two thirds of the cortex (C). The
marrow is perfused only where cortical canals (cc) are also perfused.
x 20.
BONE-BONE MARROW MICROCIRCULATION IN THE RAT
Fig. 10. A plastic clamp was placed on the periosteal surface of the
diaphyseal shaft, in the area indicated (A). The cortical bone under
this clamp was not perfused. The marrow (M) adjacent to these nonperfused cortical areas ( C ) were also not perfused, but the central
sinus was fully perfused. X 10.
175
Fig. 1 1, A plastic clamp placed on a portion of the periosteal surface
area (A) prevented the perfusion of the underlying cortex (C) and
marrow (MI. Branches of the nutrient system (arrows) were well perfused with ink but the marrow around these vessels was poorly perfused. x6.
176
L. DE SAINT-GEORGES AND S. C. MILLER
very small arterial branches, it did, however, perfuse
the cortical vessels which are much smaller than the
sinusoids that we propose they supply.
The role of the small branches of the nutrient artery
that were observed entering and leaving the inner cortex is not clear. Such vessels were mostly observed in or
near the metaphyseal region. Nutrient and metaphyseal arterial branches were often difficult to distinguish from each other in sections that were partially
perfused and thus they are referred to here collectively
as the nutrient system, except if stated otherwise. The
scarcity of these nutrient vessels entering the cortex
and inability to supply blood in the area of the periosteal circulation blockade argue for a non-essential
role of the nutrient system in the diaphysis. They could
subsist as a remnant of the vascular network present
during earlier growth (endochondral osteogenesis)
when this region was formihg in the metaphysis of the
growing bone. This vascular network in the cortical
bone and bone marrow may serve as a collateral circulation when blood flow is reduced at the periosteum
(i.e., bone fracture).
A parallel circulation pattern between marrow and
cortical bone was suggested from blood flow studies in
the dog by Kelly (1973) and Morris et al. (1980). There
are two main systems of afferent blood supply in the
diaphyseal shaft: the nutrient and periosteal, and only
one major exit, the comitant vein. Thus the afferent
systems may appear to be in a parallel configuration
and in blood flow studies it was possible to obtain regional flow values of cortical bone separate from that of
marrow. This would not be the case if the afferent supply to the cortex and marrow was serially arranged. It
is possible, however, that there are changes in the contributions of the afferent systems to different tissue
compartments a s bone develops and ages and thus differences between shorter- and longer-lived animals.
For example, Haversian systems (osteons) develop in
bones from longer-lived animals, whereas they are not
normally found in bone from shorter-lived animals, except in very old animals or under unusual experimental conditions (Ruth, 1953). Thus the circulatory pathways of bone from longer-lived species may differ and/
or change as these bones develop a n osteonal structure.
Many years ago surgeons noted that when the periosteum was stripped from a bone, blood would ooze
from the bone surface (Brookes, 1971). This was taken
as evidence for a centrifugal flow of blood from the
cortex. While it is possible that in humans and other
long-lived animals, the flow of blood in the diaphysis is
centrifugal (Rhinelander 19721, as discussed above, it
could also be explained by a simple leakage effect
caused by the surgical removal of the periosteal supply.
The backflow of blood, in this case, could be due to the
high pressures that appears to exist in the bone marrow (Petrakis, 1954).
The sinusoid network of the hemopoietic marrow of
the young rat appears to have a slow centripetal flow
toward the central sinus. In the marrow, a light centrifugal pressure gradient should exist between the
cortex and the central sinus. If we consider the corticoendosteal surface as a cylindrical isobaric higher
zone and the central sinus surface as a isobaric lower
zone, intermediate isobaric surfaces could be traced as
concentric cylinders of intermediate diameters. Along
such a n isobaric surface there should be no flow. When
some of the cortical canals were obstructed, such as by
a brace around the bone, perfusion of the underlying
marrow was prevented, emphasizing the centripetal
flow and the inability of blood from the neighboring
zone to spread in the sinusoids along the bone axis.
The endocortical surface could be considered as a n
isobaric distribution surface with each distribution
point corresponding to a n osteal canal aperture. The
flow issued from each canal will then generate the
bush-like appearance of the sinusoids that may be interpreted as a functional manifestation of a centripetal
blood flow rather than a n actual anatomic sinusoid
structure. The sinusoids in the marrow may be interconnected and networked, but because the point source
of the blood flow is the canal aperture, there would be
local sinusoidal regions, or domains. The possible physiological significance, if any, of such “territories” is not
known.
When in traumatic situations the normal pressure
gradient is perturbed, such as after a fracture, a completely different situation may develop which might
allow blood flow to change directions, depending on the
nature of change in the pressure gradient. For example, disruption of the periosteum, which is much different from a periosteal blockade, as used in the present
study, might result in a leakage effect that would divert some sinusoidal blood from the comitant vein exit
or increase a centrifugal flow from the collateral arterioles from the nutrient system that enter the cortex.
These alternative, or collateral circulatory patterns
could help prevent tissue necrosis and promote healing
following trauma to the skeleton.
Based on our observations of extensive vascularization of the cancellous bone in the metaphyseal region
from the nutrient arterioles and metaphyseal vessels,
it is evident that blood flow to the metaphyseal cancellous bone is greater than to the cortical bone (Morris et
al., 1980). The afferent nutrient system is likely essential for the perfusion of the growth plate and the highly
active metaphyseal bone in more rapidly growing
younger animals. This area of the bone would not be as
readily accessible to the periosteal circulation. Metaphyseal veins have been observed (Brookes, 1971; Trueta, 1968; Rhinelander, 1972; Danckwardt-Lilliestrom,
1969) and it is likely that this venous system drains the
cancellous bone circulation into the comitant vein.
From the observations made in this study of the rodent long bone diaphysis, it appears that the bone marrow is more dependent on the periosteal, trans-osseous
blood flow than on the nutrient system. This is in
agreement with the conclusion of Sorrel (1988), who
found in embryonic chick bone that arterial development does not appear as a prerequisite for marrow hemopoiesis. In the metaphyseal region, however, the hemopoietic tissue would be dependent on the nutrient
supply. Assuming that our perfusion model of the long
bone is correct, it is interesting that the distribution of
hemopoietic tissues appears to occur in the regions of
higher vascular supply while fatty marrow appears to
occur in downstream regions. For example, fatty marrow deposits are usually seen in the central marrow
areas while red marrow is observed adjacent to endocortical surfaces. In some long bones in older dogs,
red marrow is also seen in metaphyseal areas while the
BONE-BONE MARROW MICR(ICIRCULATION IN THE RAT
adjacent epiphyseal region may contain yellow marrow
(Miller et al., 1980). In dogs, the perfusable vascular
space in red marrow is much greater than in fatty marrow (Miller and Jee, 1980; Smith et al., 1984). Similar
conclusions have been made in studies of human bone
(Trueta and Harrison, 1953) and other animals including rats, monkeys, and an opossum (Van Dyke, 1967).
In addition, osseous tissues in red marrow sites have a
greater metabolic activity than those found in yellow
marrow sites (Smith et al., 1984).A positive correlation
between the occurrence of yellow marrow and osteoporosis has been noted in humans (Meunier et al.,
1971) and the relationship between bone marrow composition and bone mass might be mediated, a t least in
part, by changes in the vascularization and/or blood
flow in bone.
ACKNOWLEDGMENTS
This study was supported by U S . Public Health Service grant DE-07006 from the National Institutes of
Health and US. Department of Energy Contract
DEAC0276EV00119.
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Danckwardt-Lillestrom, G. 1969 Reaming of the medullary cavity
and its effect on diaphyseal bone: A fluorochromic, microradiographic and histological study on the rabbit tibia and dog femur.
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