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The significance of intramedullary cancellous bone formation in the repair of bone marrow tissue.

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The Significance of Intramedullary Cancellous
Bone Formation in the Repair of
Bone Marrow Tissue '
S. AMSEL? A. MANIATIS, M. TAVASSOLI AND W. H. CROSBY
Blood Research Laboratory, New England Medical Center Hospitals and the
Department of Medicine, Tufts University School of Medicine,
Boston, Massachusetts 02111
ABSTRACT
The repair of hemopoietic bone marrow following evacuation of the
tibia1 or femoral cavity of the rat was sequentially studied with the light microscope.
A stereotyped train of histologic events occurred. These were capillary invasion of the
cavity, appearance of primitive mesenchymal cells, osteoblastic proliferation, cancellous bone formation, development of sinusoids, reappearance of hemopoietic tissue and
resorption of cancellous bone. The studies suggest that restoration of marrow sinusoids
takes place only in the interstices of cancellous bone. Mechanical disruption of the
sinusoidal system is one method of triggering cancellous bone formation. The cancellous bone which appeared after injury was thought to be produced by endosteal
osteoblasts and osteoblasts derived from cells residing in normal hemopoietic tissue.
Localized radiation of the tibia followed by mechanical disruption of hemopoietic
tissue demonstrated that cancellous bone production and the repair process were
unimpaired by 1,000 r but were completely blocked by 4,000 r. This would imply
that the cell which can differentiate into a n osteoblast is resistant to 1,000 r.
Cancellous bone is an integral part of
the true callus formed in the repair of bone
fractures. Its origin is chiefly the osteoblasts of the endosteum and periosteum
and its function is to strengthen and bridge
the severed bone while permanent lamellar
bone is being laid down (Ham and Harris,
'56; Enneking, '57). The cancellous bone
is then resorbed. Following mechanical
removal of bone marrow from long bones
cancellous bone generates in the intramedullary cavity in positions distant from
the aperture created to remove marrow
(Steinber and Hufford, '47; Mital and Cohen, '66). It also appears deep in the marrow space following freezing of the bone
when the cortex has not been fractured
(Gage et al., '66). After transplantation
of autologous bone marrow, cancellous
bone is consistently present during the restoration of the new hematopoietic organ
even though no bone was present in the
transplanted tissue (Urist and McClean,
'52; Lacroix, '57; Tavassoli and Crosby,
'68). The same is true when the formation
of ectopic bone marrow is artificially induced (Huggins, '31; Heinen et al., '49).
In all these instances the cancellous bone
ANAT.REC.,164: 101-112.
is temporary and its significance, excluding the repair of fractures, is unknown.
The purpose of this report is to provide
evidence that reconstruction of the sinusoidal vasculature of the bone marrow following injury is intimately related to the
presence of cancellous bone and that cancellous bone formation can be triggered by
disruption of the marrow sinusoids as well
as by endosteal and periosteal injury.
MATERIALS AND METHODS
Five hundred gram, randomly bred,
white, male Wistar rats were used throughout. Nembutal (25 mg/kg intraperitoneally) served as anesthesia. Aseptic surgical
technique was used. Bone tissue was fixed
in 10% buffered fonnalin, decalcified in
Decal (Scientific Products, Inc.), embedded in paraffin, sectioned and stained with
hematoxylin and eosin or reticulum stain
(Gordon and Sweets, '36).
Received Nov. 7, '68. Accepted Jan. 9, '69.
1These studies were aided with funds from the
Atomic Energy Commission contract AT (30-1) 3808
and U. S. Public Heal!h Service q a n t AM 12444-01
from the National Institute of Arthrltls and Metabolic
Diseases.
2 S. Amsel is a Trainee in Hematology under U. S.
Public Health Service Graudate Training grant AM
5210-09 from the Naional Institute of Arthritis and
MeGbolic Diseases.
101
102
S.
AMSEL, A.
MANIATIS, M. TAVASSOLI AND W. H. CROSBY
Chemical injuy. The femur was preStudies of bone marrow repair. For sequential studies of bone marrow repair, the pared as described above, and a 90 polyright tibial plateau was surgically exposed, ethylene catheter was inserted to the proxand holes were drilled into the cortex with imal epiphysis. A 21-gauge needle was ata low speed dental drill (Emesco 90) and tached to the catheter and 20 ml of saline
dental burr 557 (S. S. White). A flap of were flushed through the intramedullary
bone was turned, the marrow lifted out on cavity removing most of the endogenous
a spatula and the flap and wound closed marrow. Then 0.5 ml of 2% phenol was
with 4-0 silk. Removal of marrow by this instilled as the catheter was withdrawn.
technique was incomplete with marrow The hole in the distal femur was closed
cells remaining along the diaphysis and with bone wax and the wound sutured. The
among the bony spicules of the epiphysis. opposite femur was flushed only with saAnimals were killed daily until the twenty- line. Animals were killed at 30, 60 and 120
first day, then weekly until eight weeks. days.
Terminology. The concepts of bone
A final group was killed at six months. The
marrow anatomy and terminology are
left tibia was used as a control.
Freezing injury. Freezing injury of the those proposed by Weiss ('65, '67). The
marrow was produced by holding dry ice sinusoidal system in the long bones runs
to the exposed tibial plateau for three min- radially from the arteries at the periphery
utes. Animals were killed at 1, 3, 5, 10, 15, to a central longitudinal vein. The sinusoidal wall is composed of one or two struc20, 30 and 80 days.
Irradiation plus mechanical disruption turally similar layers of fixed reticular cells
of the tibial marrow.
Using a 250 KV separated by an incomplete basement
x-ray machine, both tibias were exposed membrane. The outer or adventitial reticto either 4,000 or 1,000 r at 55 r/second ular cells, when present, may be connected
in a single dose. The body and feet were by spurs with other sinuses. These spurs
shielded with lead. To disrupt the mar- serve to compartmentalize the marrow. The
row a hole was drilled transcutaneously reticular cells of this interconnecting systhrough the proximal tibial epiphysis with tem are also responsible for reticuloendothe knee flexed, and a 21-gauge needle thelial function (Ito, '65; Weiss, '65; Adwas inserted into the intramedullary cav- achi, '66). The primitive mesenchymal
ity and moved up and down twice. Disrup- cell is a pluripotential cell capable of diftion was performed at 2, 21, 60 and 120 ferentiating into a fibroblast, osteoblast,
days following the 4,000 r dose and at 2 reticular cell or hemopoietic stem cell. The
and 21 days following the 1,000 r dose. latter cells may themselves further differAfter disruption, animals were killed at entiate. The morphology of the primitive
3, 6, 9, 12 and 45 days. Control animals mesenchymal cell has been described by
given 4,000 r were killed at 1, 2, 3, 6, 8, Bloom and Fawcett ('62). Recognition of
12, 20, 30, 50, 90, 120 and 240 days the cells in transition from the primitive
while controls given 1,000 r were killed at mesenchymal cell to its differentiated progeny cannot be done with the light mi3, 6, 9, 12 and 45 days.
Mechanical injuy without ablation The croscope.
marrow cavity of the femur was exposed
RESULTS
by incising the flexed knee joint and drillBone marrow repair following ablation
ing a 2 mm-diameter hole through the distal epiphysis into the diaphysis. A thin 50 (fig. 1). The cells present in the first five
polyethylene catheter was inserted to the days following ablation of the marrow have
proximal epiphysis and withdrawn. No previously been described (Steinberg and
marrow was removed. Animals were killed Hufford, '47), and the response is similar
at ten days. For complete disruption of the to that occurring in autologously transmarrow without removing hemopoietic tis- planted marrow (Tavassoli and Crosby,
sue, a 21-gauge needle was inserted and '68). Repair begins promptly after injury
withdrawn from the femoral cavity 20 and in the first 24 hours capillaries pierce
times. Animals were killed at 10, 20, 30 the clot that has filled the space where the
marrow was removed. A loose connective
and 60 days.
SIGNIFICANCE OF INTRAMEDULLARY CANCELLOUS BONE
tissue forms containing fibroblast-like cells.
Arteries, veins and capillaries are present,
but sinusoids are conspicuously absent.
Large primitive mesenchymal cells can be
seen in the proximal tibia. By the third
day, osteoblasts proliferate in the loose connective tissue and osteoid is forming in the
proximal tibia, both adjacent to the endosteum and in the center of the marrow
cavity. The primitive mesenchymal cells
diminish in numbers as the osteoblasts
proliferate. The osteoid develops into cancellous bone by the sixth day. After proliferation of endosteal osteoblasts, cancellous bone also extends contiguously from
the proximal endosteum into the intramedullary space. Eight days following ablation
the proximal marrow cavity is completely
filled with cancellous bone while osteoblasts, still actively producing osteoid, can
be seen in the loose connective tissue of
the middiaphysis. The area of cancellous
bone was enlarged by the osteoblasts forming osteoid in the distal loose connective
tissue adjacent to the bone rather than
osteoblasts adding new bone directly to the
outer edge of the older cancellous bone.
A similar phenomenon has been described
by Williams ('57). The interstices of this
newly formed cancellous bone contain
loose connective tissue, primitive mesenchymal cells (fig. 2), several capillaries,
and a net of argyrophilic fibers stemming
from the bone (fig. 5). Now the sinusoids
develop. As the cancellous bone matures,
vacuoles and breaks appear in the loose
connective tissue. The primitive mesenchymal cells circle these enlarging vacuoles and flatten out (fig. 2). The flattened
cells in the walls of the new vessels have
argyrophilic fibers and resemble reticular
cells. Occasionally spindle-shaped cells line
the vessels and whether these cells originated from primitive mesenchymal cells or
from capillary endothelial cells could not
be determined. The smaller vessels enlarge
and coalesce, except for tufts of connective
tissue, until one vessel only fills the interstice forming a sinusoid (figs. 3, 4). By
the twelfth day, hemopoietic marrow cells
appear in the tuft of tissue. Resorption of
proximal cancellous bone continues, and at
approximately 35 days, only normal sinusoidal marrow remains. Cancellous bone
undergoes a continual process of forma-
103
tion distally and resorption proximally
(fig. 1). Sinusoids can be seen in all stages
of development in any section containing
cancellous bone.
Response to physical and chemical injuries. Complete disruption of the bone
marrow using a 21-gauge needle without
removing marrow led to the same sequence
of histologic events described above. Inserting and withdrawing a 50 polyethylene
catheter through a drill hole made in the
distal epiphysis of the femur produced
cancellous bone only in the path of the
catheter (fig. 6). The cancellous bone was
near no endosteal surface, and the endosteal osteoblasts did not proliferate. When
removal of the marrow was accompanied
by instillation of 2% phenol, a patchy fibrosis was produced in which the osteoblasts did not proliferate. The stable histologic picture at four months was of
scattered areas of sinusoidal marrow adjacent to endosteum under a dome of lamellar bone. Freezing bone and marrow by
holding dry ice to the tibial plateau for
three minutes produced necrosis of bone
and marrow tissue in the area beneath the
cortex. Cancellous bone extended from the
endosteum and the normal marrow parenchyma, and it gradually replaced the necrotic tissue. By 80 days the histologic
picture of the tibia was normal. This is
similar to the experience of Gage et al.
('66).
Irradiation plus mechanical disruption.
Irradiation of the rat's tibia with 1,000 r
followed by disruption of the marrow 2 or
21 days following irradiation led to formation of cancellous bone in both instances.
The stereotyped sequence of histologic
events described above occured in the r e p
arative process. By day 45, the marrow had
sinusoidal architecture, minimal hypocellularity and the cancellous bone had disappeared. Following disruption of the bone
marrow 2, 21, 60 or 120 days after irradiation by 4,000 r, there was no cancellous
bone formation. The stable histologic picture was of a dense connective tissue.
Control animals receiving 1,000 or 4,000 r
to the tibia developed no cancellous bone
at any time. A detailed account of bone
marrow changes following localized irradiation in the rat has been previously published (Knospe et al., '66).
104
S.
AMSEL,
A. MANIATIS, M. TAVASSOLI AND W. H. CROSBY
DISCUSSION
The reparative process following the removal of hemopoietic tissue from a long
bone consists of capillary invasion of the
cavity, appearance of primitive mesenchyma1 cells, osteoblastic proliferation, cancellous bone formation, development of
sinusoids, reappearance of hemopoietic
tissue and resorption of cancellous bone.
Many of the sequential events occur simultaneously at different levels giving the
cancellous bone the appearance of moving
down the shaft of the tibia (fig. 1 ) . Our
studies suggest that when hemopoietic tissue is reconstructed a temporary phase of
cancellous bone is an essential preliminary
for sinusoidal formation. When phenol
produces a patchy but dense fibrous tissue
limiting osteoblast proliferation, sinusoidal
marrow only develops in areas where osteoblasts and cancellous bone had existed.
Reconstruction of marrow sinusoids apparently takes place only in the interstices of
cancellous bone. An exception to this may
be the sinusoidal hemopoietic tissue which
is found in unusual locations unassociated
with bone (Collin, ’33), but one may postulate that the bone necessary for its development has been already completely
resorbed. When heterotopic marrow is associated with hemolytic disease (Hartfall
et al., ’ 3 3 ) the expanded hemopoietic tissue could possibly have extruded through
its bony confines to form a mass attached
to the intraosseous marrow only by a
pedicle.
A striking parallel exists between phases
of the reparative response to injury described here and the embryological development of the bone marrow of long bones
(Langman, ’63). In both, the same pattern
occurs : capillary invasion, osteoblastic proliferation, cancellous bone formation, development of sinusoids and hemopoietic
elements and the final resorption of intramedullary cancellous bone. It is difficult
to determine whether the sinusoids in the
embryo are derived from capillaries which
enlarge in the ambience of cancellous bone
or whether the cell which lines the sinusoid is a mesenchymal cell which then differentiates into a reticular cell. The ability
of the reticular cells lining the sinusoids to
phagocytize particulate matter as compared
with the inability of endothelial cells of
the capillary suggests that the latter hypothesis is correct. In discussing the embryogenesis of the reticulo-endothelial
system, Jaff6 (’38) quotes Maximow’s observation that the reticular cells lining the
sinusoidal channels of the bone marrow
develop directly from the primitive mesenchymal cells. Thiel and Downey (’21), in
their review of the development of the
sinusoids in the mammalian spleen, described as the earliest sinusoids, mesenchymal cells at the margins of “slits” which
appear in mesenchymal tissue. Our observations also favor the primitive mesenchymal cells as being the precursor of the reticular cells which line the sinusoids.
Disruption of the marrow and sinusoids
without removing any tissue triggers a
response identical to that described after
marrow ablation. Inserting a catheter with
a diameter only 15% of that of the intramedullary cavity and withdrawing it without removing marrow. induced cancellous
bone formation along its path, despite the
endosteum’s not being injured (fig. 6). Local necrosis of hemopoietic tissue produced
by freezing with dry ice caused sinusoidal
disruption without entering the marrow
cavity, and again, cancellous bone formation preceded complete marrow repair.
These experiments indicate disruption
rather than removal of tissue is the event
which induces osteogenesis.
The osteoblasts which construct the new
bone evidently are not necessarily derived
from endosteal osteoblasts (fig. 6). When
marrow without bone is autotransplanted
the osteoblasts, after about three days, appear evenly distributed through the transplant, and preliminary to this there is an
active proliferation of primitive mesenchyma1 cells which rapidly fill the graft (Tavassoli and Crosby, ’68). The osteoblasts
evidently differentiate from these cells. The
formation of ectopic bone following injury
(for example, the intramuscular injection
of alcohol) indicates that cells which produce osteoblasts are not confined entirely
to the bone marrow. Injury or disruption
may be one of the requirements for the
differentiation and proliferation of these
cells.
We attempted to establish the radiosensitivity of this precursor cell by exposing
the marrow in situ to graded doses of Y
SIGNIFICANCE OF I N T R A M E D U L L A R Y C A N C E L L O U S BONE
irradiation followed by mechanical disruption to provoke formation of cancellous
bone. In these experiments delivery of a
single dose of 4,000 r to the tibial marrow
followed by mechanical disruption produced neither cancellous bone nor tissue
repair. However, disruption after 1,000 r
was followed by cancellous bone formation
and repair of the marrow. This indicates
that the cells which differentiate into osteoblasts are more radioresistant than
hemopoietic stem cells, which are destroyed
by 1,000 r. Evidence exists that the marrow sinusoids (Knospe et al., '66) and the
reticuloendothelial system (Nelp et al.,
'68) function after 1,000 r, but that heavier radiation causes progressive loss of
function. Since the two systems are probably identical and composed of reticular
cells (Weiss, '65; Ito, '65), these observations suggest that the reticular cell is also
resistant to 1,000 r but that higher doses
are lethal. One can hypothesize that the
reticular cell of the sinusoidal system and
the cell which differentiates into an osteoblast are the same cell; given a proper
stimulus which, in this instance, is mechanical disruption, the reticular cell differentiates into an osteoblast and thereby
initiates the repair process. Radiation with
greater than 1,000 r is lethal to the reticular cell and prevents the differentiating
process.
LITERATURE CITED
Adachi, Y. 1966 Structural and cytological studies on injury and regeneration of the rat bone
marrow following total body irradiation. Bull.
Tokyo Med. Dent. Univ., 13: 35-57.
Bloom, W., and D. W. Fawcett 1962 Textbook
of Histology. W. B. Saunders, Philadelphia.
Collins, D. C. 1932 Formation of bone marrow
i n the suprarenal gland. Am. J. Path., 8: 97105.
Enneking, W. J. 1957 Histologic investigation
of bone transplants in immunologically prepared animals. J. Bone Joint Surg., 39A: 597615.
Gage, A. A., G. W. Greene, Jr., M. E. Neiders and
F. G. Emmings 1966 Freezing bone without
excision. J. A. M. A., 196: 770-774.
105
Gordon, H., and H. H. Sweets, Jr. 1936 A simple method for the silver impregnation of reticulum. Am. J. Path., 12: 545-551.
Ham, A. W., and W. R. Harris 1956 The Biochemistry and Physiology of Bone. Academic
Press, New York, pp. 475-505.
Hartfall, S. J., and M. J. Stewart 1933 Massive paravertebral heterotopia of bone marrow
i n a case of acholuric jaundice. J. Path. and
Bact., 37: 455-459.
Heinen, J. H.,Jr., G. H. Dobbs and H. A. Mason
1949 The experimental production of ectopic
cartilage and bone i n muscles of rabbits. J.
Bone Joint Surg., 31 A: 765-775.
Huggins, C. 1931 The formation of bone under
the influence of epithelium of the urinary tract.
Arch. Surg., 22: 377408.
Ito, V. 1965 Electron microscopic studies on
benzene intoxicated rat bone marrow with special reference to its reticuloendothelial structures. Bull. Tokyo Med. Dent. Univer., 22: 1-29.
JaffC, R. H. 1938 The Reticuloendothelial System. In: Handbook of Hematology 11. H. Downey, ed. Hoeber, New York, p. 991.
Knospe, W. H., J. Bloom and W. H. Crosby 1966
Regeneration of locally irradiated bone marrow. Blood, 28: 398-415.
Lacroix, P. 1957 Le choix du site dans l'ktude
des greffes de tissus squelettiques. Colloques
Intern. du Centre National de la Recherche
Scientifique, 78: 41-48.
Langman, J. 1963 Medical Embryology. Williams and Wilkins, Baltimore.
Mital, M., and J. Cohen 1966 Repair of experimental bony intramedullary injuries varying in degree. Surg. Forum, 17:451-452.
Nelp, W. B., S. M. Larson, M. N. Gahil and L. D.
Grouse 1968 RES and erythron activity in
the marrow following irradiation. Clin. Res.,
16: 122.
Steinberg, B., and V. Hufford 1947 Development of bone marrow in adult animals. Arch.
Path., 43: 117-126.
Tavassoli, M.,and W. H. Crosby 1968 Transalantation of marrow to extramedullary sites.
Science, 161: 54-56.
Thiel, G. A.. and H. Downey 1921 The development of the circulation in the spleen of a
rabbit. Am, J. Anat., 28: 279-295.
Urist, M. R., and F. C. McClean 1952 Osteogenic potency and new bone formation by induction in transplants to the anterior chamber
of the eye. J. Bone Joint Surg., 34A: 443-476.
Weiss, L. 1965 The structure of bone marrow.
J. Morph., 117: 467-538.
1967 The histophysiology of bone marrow. Clin. Orthop., 52: 12-23.
Williams, R. G. 1957 A study of bone growing
from autografts of marrow in rabbits. Anat.
Rec., 129: 187-209.
PLATE 1
EXPLANATION OF FIGURE
1
106
Longitudinal section of the diaphysis of a rat’s tibia 13 days following
marrow ablation. The tissue active in the reparative process can be
divided into three zones. The zones run diagonally and zone A is
distal. A, loose connective tissue nourished by arteries, capillaries and
veins. B, osteoblasts, osteoid and cancellous bone. Sinusoids develop
in the bony lattice of the cancellous bone. C, normal sinusoidal
marrow.
Osteoblasts from mne B will soon proliferate in the loose connective tissue of zone A. By day 20, zone A will contain cancellous bone
and early sinusoids. At that time the cancellous bone of zone B will
have resorbed leaving hemopoietic marrow. The dynamic process of
bone formation distally and resorption proximally gives the reparative process the appearance of moving down the shaft of the tibia
leaving hemopoietic tissue beyond it. Hematoxylin and eosin. x 150.
SIGNIFICANCE OF INTRAMEDULLARY CANCELLOUS BONE
S. Amsel, A. Maniatis, M. Tavassoli and W. H. Crosby
PLATE 1
107
PLATE 2
EXPLANATION OF FIGURES
108
2
Cancellous bone of the rat’s tibia nine days following ablation. The
loose connective tissue of this interstice has begun to vacuolate. In
the center, large, pale, primitive mesenchymal cells are partially flattened (arrow) and lining a rudimentary sinusoid. Hematoxylin and
eosin. x 1,800.
3
Cancellous bone and sinusoids of the rat’s tibia nine days following
marrow ablation. The sinusoids are in different stages of development. Coalescence of two sinusoids to form a larger vessel causes
irregularity and fraying of the sinusoid wall (wide arrow). Hematoxylin and eosin. x 450.
SIGNIFICANCE OF INTRAMEDULLARY CANCELLOUS BONE
S. Amsel, A. Maniatis, M. Tavassoli and W. H. Crosby
PLATE 2
109
PLATE 3
EXPLANATION O F FIGURES
4
Cancellous bone and sinusoids of a rat’s tibia ten days following
marrow ablation. The upper interstice contains loculated sinusoids.
In the lower, coalescence has produced one large sinusoid. Hematoxylin and eosin. x 320.
5 Cancellous bone of a rat’s tibia nine days after marrow ablation.
Argyrophilic fibers form a meshwork in the bony lattice. Sinusoids
of different sizes can be seen (arrows). Reticulum stain. X 320.
6
110
Transverse section of a rat’s femur ten days after insertion and
withdrawal of a small catheter. No marrow was removed. Cancellous
bone can be seen in the path of the catheter. Endosteal osteoblasts
are inactive. Hematoxylin and eosin. X 200.
SIGNIFICANCE OF INTRAMEDULLARY CANCELLOUS BONE
S. Amsel, A. Maniatis, M. Tavassoli and W. H. Crosby
PLATE 3
111
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