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The structure of developing bone marrow sinuses in extramedullary autotransplant of the marrow in rats.

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The Structure of Developing Bone Marrow Sinuses in
Extramedullary Autotransplant of the
Marrow in Rats '
Department of Anatomy, The Johns Hopkins University School of
Medicine, Baltimore, Maryland and the Blood Research Laboratory,
New England Medical Center Hospitals and the Department of
Medicine, Tufts University School of Medicine,
Boston, Massachusetts
The structure of the developing sinuses of the bone marrow was
studied in extramedullary autotransplants of the marrow in rats just before the
onset of hemopoiesis. Sinuses are highly branching channels with walls consisting of a single layer of endothelium. The wall thickness varies Considerably but
larger sinuses generally have a thinner wall. Endothelial cells may overlap and
interdigitate displaying junctional densities. Abrupt thinning of endothelium was
observed in some areas but no apertures were noted. The endothelium is sometimes surrounded by collagen, but no basement membrane is present. Twentyfour percent of the endothelial surface is covered by pericytes and often rarSed
cytoplasmic spurs extend into perisinus areas. Microfilaments were seen under
the cell membrane of pericytes. Medium-sized lymphocytes are frequently seen
in the sinal lumen and intersinal tissue and they may constitute hemopoietic
stem cells.
This study deals with the structure of
developing marrow sinuses in autologous
extramedullary transplants of bone marrow in the rat. The following sequence occurs in such tissue after transplantation
(Tavassoli and Crosby, '68, '70; Crosby,
'70). Within 12 hours, blood vessels from
the supporting tissue penetrate the implant, establishing a rich capillary circulation. After 24 hours, the implant is almost
devoid of hemopoietic cells and only a reticulum is discernible. By days 3-4 the implant is filled with fibroblasts richly interspersed with collagen. Few other cells are
present at this stage. Collagen then concentrates in some areas of the implant
giving rise to osteoid tissue. By day 6, the
calcification of osteoid substance gives rise
to osteoid bone. A loose connective tissue
lies within the interstices of the trabecular
bone and contains fibroblasts, occasional
macrophages and some granulocytes, particularly eosinophils. This space is the primordial marrow cavity. By day 7, highly
branching, intercommunicating vascular
sinuses appear within the loose connective
ANAT.REC., 171: 477-494.
tissue. Hemopoiesis then begins outside
the sinuses and gradually extends. Intense
hemopoiesis is associated with resorption
of trabecular bone so that after five weeks,
the implant consists of a bone marrow
nodule surrounded by a shell of bone. A
similar process takes place w3thin the
medullary cavity during the repair of
marrow tissue (Amsel et al., '69).
The major objective of this study is to
describe the structure of the developing
marrow sinuses in the autotransplant.
Since the development of marrow sinuses
preceeds the onset of hemopoiesis, there is
a period when hemopoiesis either has not
yet begun or is minimal; during this period,
the structure of developing sinuses can be
studied to advantage.
Received Feb. 18, '71. Accepted June 7. '71.
1 Supported in part by U.S. Public Health Service
grant AM 12444 from the National Institute of Arthritis and Metabolic Diseases and Atomic Energy Commission contract AT (30-1) 3808 and U. S. Public
Health Service grant CA5375-05 from the National
Cancer Institute.
ZResearch Fellow of the Medical Foundation of
Marrow implantation. More than 30
implants were made in 12 white male rats
weighing 250-300 gm. All surgery was carried out under aseptic conditions. The knee
joint was opened and the distal end of
the femur exposed. A hole was drilled in
the articular surface of the femur until the
marrow cavity was reached. A polyethylene
tube (gauge: 160) was inserted into the
marrow cavity and was gently pushed into
the whole length of the femoral shaft. The
free end of the tube was then clamped and
the tube was slowly removed. The tube
was packed with marrow. The marrow was
pushed out by using a syringe attached to
a needle tightly inserted at one end of the
tube. A small pocket was incised in the subcutaneous tissue of the abdomen and the
marrow fragment was implanted (Tavassoli et al., '70). Regenerating marrow implants were removed on days 4, 5, 7, 8, 10,
11 and 12. At least two implants were
studied on each day.
Tissue preparation. Formaldehyde-glutaraldehyde fixative (Karnovsky's mixture )
containing trinitrophenol (picric acid) was
used as the primary fixative (It0 and Karnovsky, '68). This fixative was made by
diluting Karnovsky's solution in distilled
water 3: 1 and adding CaCL (5 mg/ml)
and picric acid (0.2 mg/ml). The implants
were removed from anesthetized animals
and fixed for 24 hours at room temperature. The tissue was then rinsed in 0.1 M
sodium cacodylate buffer (pH 7.4) containing 1% sucrose, sliced into small cubes and
postfixed in 2% osmium tetroxide in cacodylate buffer for two hours at 4°C.
Decalcification of the tissue was carried
out in 10% cold EDTA dissolved in 0.1 M
tris buffer (pH 7.0) (Fullmer and Link,
'64). The solution was agitated in a cold
room for two to three days using a magnetic stirrer. EDTA solution was changed
daily. The tissue was then dehydrated in
graded alcohol and transferred to araldite
using propylene oxide as a transitional solvent. Thick sections ( 1 cL)were cut in a
Porter-Blum I1 microtome using glass
knives. They were stained with 1% toluidine blue buffered with borate (pH 11.0)
for light microscopy (Bencosme et al.,
'59). Thin sections in the range of gray
to silver were cut with a diamond knife.
They were stained with uranyl acetate
(Watson, '58) and lead ciaate (Karnovsky,
'61) and studied in Siemens Elmiskop 1.
In light microscopic sections, sinuses appear as highly branching, intercommunicating vascular channels. The lumen may
be packed with erythrocytes and other
blood cells, may contain no cells at all, but
typically, moderate numbers of blood cells
are present (figs. 1, 2). The sinus wall is a
single layer of continuous endothelium
(fig. 1). The endothelium varies considerably in thickness and in some
areas becomes barely visible. The intersinal tissue contains a ground substance
with the density varying from place to
place. It is denser in the vicinity of the
vessels and where cells are present. The
predominant cells in the intersinal tissue
are fibroblasts. Their nuclei are clear, ovoid
with occasional indentations and their
abundant cytoplasm may contain large
vacuoles. The fibroblastic cytoplasm has
long, slender processes. These are more
conspicuous in the perisinus area where
they tend to run parallel to the endothelium (fig. 2). Macrophages are seen in intersinal cords and are distinguished by
somewhat dark cytoplasm and moderately
dark granules. Granulocytes, particularly
eosinophils, are present in both the cords
and the sinuses. In materials from days
10, 11 and 12, hemopoietic cells are seen
in intersinal tissue. They are in different
stages of maturation and some are in
By electron microscopy, sinuses vary
considerably in size from very small channels with an almost closed lumen to large,
widely open vessels (figs. 3, 4). The luminal size is unrelated to the cellular content
of the lumen. Large channels are often
seen filled with plasma but no cells,
whereas the smaller vessels may be packed
with red cells and leukocytes. The thickness of the wall also varies considerably
from one sinus to the other as well as in
a single sinus. Larger sinuses generally
have a thinner wall (fig. ,4). The wall consists of a single layer of endothelium (lining cell) usually with flat nuclei and basal
cytoplasmic extensions which encircle the
lumen. Cytoplasmic spurs may enter the
perisinal tissue and reach similar extensions of other cells (fig. 3). The cytoplasm
is often dark but occasionally it may be
considerably rarified. It contains many
pinocytic vesicles, glycogen and scattered
polyribosomes. Granular endoplasmic reticulum (GER) is rare and only a few
lysosomes are to be seen. Microfibrillar
structures are usually present. Contiguous
endothelial cells often overlap and may
even interdigitate (figs. 5 , 6 ) . In these sites
junctional densities are frequent. Abrupt
areas of thinning, few micra in length,
occur in the endothelium and sometimes a
few such areas are seen in a row (figs. 3,
8). In these areas, the wall may consist of
little more than apposing cell membranes
(fig. 8). No apertures were observed in the
The endothelium is closely surrounded
by collagen, the fibers usually in parallel
array (figs. 3, 4, 5, 7, 8). Collagen may be
scant and dispersed or densely packed.
The latter is sometimes associated with a
branching sinus (fig. 4). The larger
sinuses are generally surrounded by heavier
condensation of collagen (fig. 4). Where
the collagen is dispersed, patchy condensation of subendothelial ground substance
may be observed.
Pericytes or adventitial cells are infrequent. They have a rarified cytoplasm with
moderate amounts of GER and polyribosomes (figs. 4 through 8 ) , but the dominating cytoplasmic characteristic is the
presence of lysosomes in great number.
Glycogen is frequently seen in pericytes
(figs. 7, 9). Microfilaments are also frequently seen in these cells; (figs. 6,8). They
are often located under the cell membrane
in parallel arrays forming a band but a
network of microfilaments may be seen
anywhere. The extent of this coverage
measured by the method used by Weiss
('70) to 24% of the total sinus area. Their
cytoplasmic spurs penetrate deep into the
intersinal cord reaching the extensions of
other cells or a nearby sinus. A pericyte
sometimes appears to be shared by adjacent sinuses. Sometimes a few strands of
ccjllagen appear within a vacuole in a pericyte (fig. 7).
In the intersinal tissue, the dominant
cell type is the fibroblast. Many fibroblasts
contain large autophagosomal structures
and appear to be degenerating. Macrophages, granulocytes, particularly eosinophils and mast cells are also present in the
In our materials from day 7, mediumsize lymphocytes are frequently seen
within the sinuses, passing through sinus
wall or in the intersinal tissue (fig. 9).
Clusters of these cells may be seen in the
intersinal tissue.
The vascular channels described in this
study are recognized as marrow sinuses
because of their association with hemopoiesis which gradually develops on days
10, 11 and 12. Similar structures have
been described during intramedullary repair of the marrow tissue (Amsel et al.,
The structure of fully developed sinuses
of bone marrow has been previously
studied in the rabbit (Weiss, '61), rat
(Weiss, '65), chicken and pigeon (Campell,
'67), guinea pig (Pease, '56; Zamboni and
Pease, '61; deBruyn et al., '66) and frog
(Campbell, '70) under various conditions.
The present study was focused primarily
on the structure of the developing sinuses
in the rat during regeneration of extramedullary marrow implants.
The sinus wall in our material consists
of a single layer of flattened endothelium
which varies considerably in thickness but
it is always a complete layer. This is somewhat different from the sinuses of fully
developed marrow and those of the liver
(Carr, '70) where the endothelium shows
apertures. Developing marrow sinuses in
transplants are different from both definitive splenic sinuses (Weiss, '57) and marrow sinuses because they lack a basement
membrane. A patchy condensation of the
ground substance occurs on the outside
surface of the endothelium, but this is not
consistent enought to be recognized as a
basement membrane. The structure of vascular sinuses is, in general, remarkably
similar to that of lymphatic channels
(Weiss, '66). This parallel applies to the
developing marrow sinuses in the present
study; the single layer of flattened endothelium lacking a basement membrane is
particularly similar to the structure of
lymphatic vessels. Furthermore, develop
ing marrow sinuses are typically surrounded by collagen as is the case in the
lymphatic vessels. Since lymphatics are
absent wherever sinuses are present, it is
likely that the sinuses subsume lymphatic
vascular functions.
The luminal size of the sinuses in our
material correlates negatively with the
overall thickness of the wall, the thickest
wall belonging to almost collapsed sinuses
(fig. 4). Perhaps when a channel opens,
the bulk of the endothelial cytoplasm
stretches over a larger area and consequently the wall becomes thinner. The
luminal size may well change as a result of
endothelial contraction. The presence of
microfilaments in the endothelium suggests that this may occur (Wessels et al.,
'71 ).
Transmural cellular passage in marrow
sinuses have been the subject of a recent
investigation (Weiss, '70). A correlation
between the extent of pericyte coverage of
the sinus wall and the volume of transmural cellular passage was demonstrated.
In fully developed rat bone marrow, 65%
of sinus endothelium is covered by pericytes (Weiss, '70). In developing marrow,
this does not exceed 24%. Transmural exchange in bone marrow sinuses is a transcellular process. The cells can push against
the wall, opening an aperture in the cytoplasm through which they can pass. The
aperture may then close or not. In well
developed rat bone marrow, the number of
apertures per unit of sinus wall correlates
well with the volume of transmural cellular passage (Weiss, '70). In our material,
the marrow having little or no hemopoiesis
shows a lack of apertures in the wall which
may represent the lack of appreciable
transmural cellular exchange.
Degeneration of fibroblasts in the intersinal cords is likely to be a developmental
phenomenon (Saunders, '66). It is soon
follewed by hemopoietic proliferation
which gradually becomes extensive and
requiring space. These processes take place
within rigid confines of bone so that degeneration of fibroblasts may seem necessary for hemopoietic proliferation to proceed.
The presence of mature eosinophils in
relatively large numbers is of interest,
since transplants of marrow in our study
are true autografts. Tissue eosinophilia is
generally associated with tissue allografts
(Rogers et al., '53).
The nature of the medium-sized lymphocytes in our materials is not clear. One
may assume that these cells are brought
to the newly formed marrow by circulating blood. They are (aside from granulocytes and erythrocytes) the first cells entering the marrow. Certain lymphoid cells
have long been suspected to be the pluripotential stem cells (Maximow, '24;
Cudkowicz et al., '64; Yoffey et al., '65;
Tyler and Everett, '66). There is good experimental evidence indicating that the
stem cell is a circulating cell (Robinson
et al., '65; Epstein et al., '66; McCredie
et al., '71 ) , selectively lodging in the bone
marrow or other hemopoietic tissues
(Osogoe and Omura, '50; Weiss, '58). Recent autoradiographic studies (Fliedner et
al.. '70; Orlic, '70) have tentatively identified a small round cell, lymphocytic in
morphology, as the stem cell. The lymphoid
cells we have observed in our material may
well constitute stem cells. Study of more
advanced stages of bone marrow implants
may, indeed, show the transformation of
these cells into more differentiated hemopoietic cells.
We are indebted to Dr. William H.
Crosby, for valuable advice and encouragement.
Amsel, S., A. Maniatis, M. Tavassoli and W. H.
Crosby 1969 The significance of intramedullary cancellous bone formation in the repair
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1 This light micrograph shows the primordial marrow cavity. The darker
areas are trabecular. A large sinus extends diagonally in this field.
Note the thin endothelium with flattened nuclei. Three smaller
sinuses are also seen i n the right. Few macrophages are seen i n the
intersinal tissue but the dominant cells here, are fibroblasts. x 600.
The primordial marrow cavity in this figure shows four sinuses; the
largest appear to be branching. One of the sinuses contain no cells
but others contain moderate numbers of blood cells. Few macrophages
and an occasional lymphocyte is seen in the intersinal tissue. Several
5broblasts appear to be degenerating. x 600.
Mehdi Tavassoli and Leon Weiss
A sinus occupies most of this field; its wall consists of a single layer
of endothelium surrounded by collagen. Short endothelial spurs extend
into the lumen which is open. Endothelial spurs also extend into
perisinus area (curved arrows). Note the variation in the wall thickness with abrupt areas of thinning (straight arrows). x 7,500.
Mehdi Tavassoli and Leon Weiss
Two sinuses are seen in this field and are outlined in the tracing.
The larger sinus has a n open lumen and portions of four endothelial
cells (End) participate in forming the wall. The section has included
three of the endothelial cell nuclei. Note the variation in the wall
thickness. Portions of six adventitial cells or pericytes ( Adv) partially
cover the endotheliuin. Note the rarified cytoplasm of the pericytea.
A deep indentation is seen i n the upper part of this sinus and here
the endothelium is surrounded by thick bands of collagen. This i c
probably a site of branching. A second sinus is seen in the upper
left with a n almost closed lumen. Its wall consists of portions of
three endothelial cells. Note the relative thickness of the wall. The
endothelium is not surrounded by coilagen and no adventitial coverage is present. The cell in the right upper corner of this figore is a
phagocyte containing inany lysosomes and some large vacuoles.
x 5,000.
Mehdi Tavassoli and Leon Weiss
Mehdi Tavassoli and Leon Weiss
A sinus is seen in the upper part of this field. The endothelium ( E n d )
is relatively thin and as it extends toward the right, it branches and
interdipitates with the cytoplasmic extension of another endothelial
cell. Portion of an adventitial cell (Adv) with rarified cytoplasm is
covering the endothelium and a Iew strands of collagen ( C ) cover
the wall. Several adventitial spurs are also seen, one of which contains glycogen ( G ) . The cell in the lcwer part of this figure is either
a mast cell or a n early eosinophil. x 20,000.
Mehdi Tavassoli and Leon Weiss
6 The endothelium of this sinus consists of two cells which overlap for
a short distance and two junctional desities are seen in this ares
(arrows). Note the band of microfilaments ( M ) underneath the cell
membrane of the adventitial cell (Adv). x 25,000.
Portion of a sinus is seen i n the left lower corner of this field ( S )
with thin endothelium anti a few strands of collagen ( C ) . Portion of
a pericyte occupies the right upper corner and contains glycogen (G).
Note collagen containing vacuoles in the pericyte, several of thein
are identified by arrows. x 30,000.
The lumen of a sinus is seen in the upper part of this field. Thc
endothelium (End) varies in thickness and an abrupt area of thinning is seen where the endothelium appears to consist of only
apposed cell membranes (arrow). Strands of collagen are present anc!
portions of two adventitial cells (Adv) with relatively rarified cytoplasm cover the wall. Note bands of micrcfilaments ( M ) under the
cell membrane of both adventitial cells. Portion of another adventitial
cell is seen in the left lower corner. x 20,000,
Mehdi Tavassoli and Leon Weiss
49 1
This figure is dominated by a cell morphologically similar to the
lymphocyte. This cell may constitute hemopoietic stem cell. Spur of
a pericyte containing large amount of glycogen ( G ) is also seen.
The cell in the right lower corner is likely a fibroblast. X 25,000.
Mehdi Tavassoli and Leon Weiss
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autotransplants, structure, marrow, developing, extramedullary, sinuses, rats, bones
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