JOURNAL OF MORPHOLOGY 262:546 –565 (2004)
Structure of Long Bones in Mammals
Michael Locke*
Department of Biology, University of Western Ontario, London, Ontario N6A 5B7, Canada
ABSTRACT Techniques for staining (silver, osmium,
metal sulfides, ink) and microphotography (epiillumination) of polished bone surfaces have been developed to visualize the three-dimensional structure of the
shafts of mammalian long bones. Bone is a twocompartment system with capillaries and some kinds of
connective tissue in one compartment separated from fibers of bone collagen, often forming lamellae, in the other.
Laminar bone consists of stacks of lamellae separated by
vascular spaces containing capillary network sheets. It is
deposited at the periosteal and endosteal surfaces. Osteonic bone, well described in the literature, consists of
cylinders of lamellae with central vascular spaces. The
primary structure of the shafts of mammalian long bones
is laminar and laminae often remain as the main component. Secondary osteons are a replacement within laminae. As laminar bones mature, some of the irregular longitudinal capillary spaces in the network sheets enlarge
and become less crooked to form secondary osteons. Parts
of the random networks become ordered longitudinal ones,
resulting in collapse of those network spaces not converted
to osteons. The residual capillaries become bloodless,
making the surviving network spaces difficult to resolve.
This may account for them being overlooked in descriptions of bone structure. For example, laminar bone occurs
with osteonic bone in the human femur, although it is
rarely figured. Nearly mature bones switch the kind of
primary bone deposited at the peripheral (periosteal) surface from laminar to primary osteonic. J. Morphol. 262:
546 –565, 2004. © 2004 Wiley-Liss, Inc.
KEY WORDS: bone structure and staining; bone as a
two-compartment system; laminar bone; osteon
This study is concerned with the structure of the
shafts of mammalian long bones at the low microscopic level. It supplements the prevailing view that
bone is constructed from osteons by showing that
compact bone is formed primarily from laminae. In
the young growing animals examined (beef, sheep,
pigs), the shaft is completely made from circumferentially oriented laminae without osteons. In older
or even mature animals much of the shaft is still
laminar (human, buffalo, deer, horse, oxen, as well
as beef, sheep, and pigs). Enlow and Brown’s (1956,
1957, 1958) often-quoted but perhaps rarely examined classical comparative study of bones describes
what we now call laminar bone in many mammals.
In spite of this, descriptions of osteons with no mention of laminae were found in a survey of more than
40 textbooks of general biology, comparative mor© 2004 WILEY-LISS, INC.
phology, veterinary, medical, and functional histology. Longitudinal profiles of laminae have sometimes even been labeled osteons (Locke and Dean,
2003, in a note aimed at biology teachers). Most
research articles follow this tradition, which has
changed little since Todd and Bowman’s description
of Haversian bone in 1845 (Martin and Burr, 1989b),
ignoring the capillary network sheets that separate
layers of bone lamellae. Enlow and Brown (1958, p.
211) noted this almost 50 years ago, saying “Most
vertebrates do not possess the classic pattern of bone
tissue structure described in modern textbooks of
histology. The descriptions of bone tissue in current
texts are concerned primarily with human bone.”
Laminar and osteonic structure is well known to
those studying physical properties (Wainwright et
al., 1976; Weiner et al., 1999; Currey, 2002), but
many researchers are not aware of the complexity of
bone structure and its variation with species, kind,
orientation, and developmental stage that was described by Enlow and Brown. For example, Halstead
(1974, p. 68) follows Currey, by commenting that
laminar bone has “received scant attention” compared to “Haversian systems,” but still figures the
two kinds of bone incorrectly.
Some literature error has resulted from a confused use of terms: for example, circumferential lamellae rather than laminae have been described as
smoothing off the endosteal and periosteal surfaces.
Lamella is the diminutive of lamina. Laminar bone
is made from 4 –20 lamellae. This article is concerned with the shape of the bone compartment,
which is usually some variation of a sheet (laminar
bone) or a cylinder (osteonic bone). It does not discuss fiber orientation within these compartments,
which contributes another layer of definitions at a
finer order of microscopy. A brief glossary of the
terms used here clarifies terminology (see Appendix).
Contract grant sponsor: NSERC; Contract grant number: A6607.
*Correspondence to: Michael Locke, Department of Biology, University of Western Ontario, London, Ontario N6A 5B7, Canada.
E-mail: mlocke@uwo.ca
Published online 16 September 2004 in
Wiley InterScience (www.interscience.wiley.com)
DOI: 10.1002/jmor.10282
Fig. 1. The primary structure of compact bone is laminar. A: Vascular compartments cut transversely and separated from one
another by layers of bone in a transverse profile at 90° to the long axis. Vc, Volkmann’s canals. Silver-stained 3-year-old buffalo
humerus. B: The vascular compartments form an intricate reticular network in a tangential profile (face view parallel to the surface).
Ink-injected 2-year-old beef humerus. C: Three layers of reticular compartments in a slightly oblique tangential profile. Ink-injected
giraffe radius. D: Layers of bone alternate with obliquely or transversely cut vascular compartments connected by Volkmann’s canals
(Vc) in a radial profile (longitudinal view from center to periphery). Silver-stained 3-year-old buffalo humerus. Scale bars ⫽ 1 mm.
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M. LOCKE
Figure 2
STRUCTURE OF LONG BONES IN MAMMALS
549
ciation is that studies on bone have traditionally
been either anatomical or have relied on grinding
thin sections (Enlow and Brown, 1956). Grinding
sections is a tedious and difficult process appropriate for high-resolution light microscopy (Yuehue et
al., 2003), but at high resolution it is often difficult to
determine the plane of section and threedimensional arrangement. There has also been a
reluctance to observe profiles other than transverse
(as in Enlow and Brown, 1956, 1957, 1958). Modern
techniques of scanning electron microscopy of resin
casts of the vascular system are time-consuming
(Moller et al., 1997), although the results are exceptionally beautiful (Pannarale et al., 1997). Microangiography is restricted to those with specialized
X-ray units (Berry, 1979). This article uses simple
stereo observation at intermediate magnifications to
study the relationships between osteons and laminae in mammalian long bones at different stages of
development. It introduces ways to stain carefully
oriented samples for viewing by epi-illumination in
order to describe the most common compartment
patterns and microvasculature of the bone matrix in
three dimensions.
Fig. 2. Compartments forming laminae contain capillary network sheets. A: Unstained blood-filled capillaries form reticular
networks in the plane of the lamina. Natural unstained blood in a
tangential profile of cleared beef humerus close to the periosteal
surface. B: Staining of the capillary lumens confirms their reticular
pattern in capillary sheets. The black speckles are osteocytes. Olive
oil-injected and osmium-stained tangential profile of beef humerus.
3D observations in A and B show that some of the denser spots are
connections between adjacent sheets. Single connections are more
common than Volkmann’s canals uniting several laminae. C: General staining of the vascular compartment shows a coarser reticulum than the capillary network that it encloses. The white speckles
are unstained osteocytes. Iron sulfide staining of tangential profile of
beef humerus. D: The vascular compartment is probably the prime
mover in forming new laminae at the periosteal surface. The vascular compartment (V, blue) is at first much wider (top, younger bone
at the periosteal surface) than the compartment destined to contain
bone (B, brown to black). Below the forming layer the vascular
compartment is reduced to a thin darkly staining layer. Silverstained transverse profile of 2-month-old pig humerus. E: The vascular compartments (V) are at first flat sacs connecting across the
bone compartment (B) to the older and newer vascular compartments on each side. Tangential views near the bone surface show
profiles of the tubular connections as circles. Each circle contains a
brown-stained fiber (3), perhaps concerned in its genesis. These
connecting fibers have the topological position of Sharpey’s fibers
described in mature bone. Silver-stained tangential profile of a femur of a 6-month-old calf. F: The bone compartment (B) connections
enlarge, reducing the vascular compartment (V) to the reticulum
that comes to contain the capillary network. Silver-stained tangential profile of a lamb femur further from the periosteal surface than
E. Scale bars ⫽ 1 mm. G: Laminar bone is a connective tissue
system of two compartments, one containing mineral reinforced
collagen fibers (and perhaps Sharpey’s fibers), the other, capillaries
and a different class of fibrous components (see also fig. 5.14 in
Wainwright et al., 1976; fig. 10.10 in Fawcett, 1994; fig. 1.5 in
Currey, 2002).
Information on laminar bone is scattered though
the literature, but rarely with the clarity and detail
that it deserves. One reason for this lack of appre-
MATERIALS AND METHODS
Sample Preparation
Bone samples were prepared from many different sources—
new kills, with and without aldehyde fixation, slaughterhouses,
mortuary specimens, worn museum preparations, subfossils, and
fossils. The periosteum and endosteum were pulled from fresh
bone with pliers where necessary. Only the central shaft of long
bones, uncomplicated by the cancellous bone at the ends, was
studied. Bone blocks were cut on a bandsaw with radial, transverse, and tangential faces. Blanks 1–2 mm thick were then cut
from these faces using a fine bandsaw or a jeweler’s saw. Test
samples were prepared as billets measuring about 10 ⫻ 10 ⫻ 1
mm or less, depending on the type of bone. It is important to make
the main faces parallel to one another to obtain uniformly flat
fields for photography. The blanks were ground flat on a wet 220
grit diamond lap followed by a wet 600 grit lap to give a surface
finish fine enough for microscopy.
Impregnation
Infiltration methods show spaces formerly occupied by blood
vessels or the spaces around them even in subfossil specimens.
Ink. Ink has been used to study blood circulation in bone (De
Saint-Georges and Miller, 1992) and to show sutures or the
spaces in trabecular bone. It is an easy way to display the spaces
in bone occupied in life by blood vessels. Clean dry bone samples
were left overnight in a jar of black carbon Indian ink with a few
drops of dishwasher detergent. Impregnation was helped by allowing infiltration at reduced pressure in a vacuum oven for 1–24
h before slowly (5 min) returning to atmospheric pressure. Surface ink was then washed off. Either the lumina of blood vessels
or the spaces left in the bone formerly occupied by blood vessels
and osteocytes, depending on age and preservation, stand out in
clear black patterns against the white bone (Figs. 1B,C, 6B).
Other colored inks gave similar results.
Olive oil and osmium black. Clean dry bone samples were
allowed to take up olive oil for hours to days in the reduced
pressure of a vacuum oven before a slow (1 h) return to atmospheric pressure. The bones had then become translucent light
yellow. They were wiped clean and left for 2–24 h in 1% osmium
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M. LOCKE
tetroxide in phosphate buffer, pH 7.5. The osmium was reduced to
a black mass of suboxides (osmium black) by the double bonds of
the oleic acid in the olive oil, leaving the blood vessels and/or the
spaces they occupied in the bone standing out in sharp black relief
(Figs. 2B, 6A).
Direct Observation of Blood-Filled
Capillaries
Most fine blood vessels in bone samples fill with air (see Results), but some, particularly in osteons and in newly formed
laminar bone, may contain red blood. After infiltrating the sample with glycerol for a few days, the surface layer becomes translucent or even transparent, allowing blood-filled vessels to be
resolved in three dimensions (Figs. 2A, 11A,B).
salts is that they penetrate through and across the walls of the
capillaries, binding to most of the contents of the vascular
compartment but not mature bone itself (iron does stain the
bone surface, which may have to be polished away). Hydrogen
sulfide penetrates easily, so that all cells and tissues adsorbing
the metals become dense black.
Conventional staining. Several stains (Coomassie blue,
hematoxylin) gave satisfactory colored representations of
bone cell components, but not the sharp, dark contrasts most
useful to show vascular distribution by low-magnification microscopy.
Ground sections. Conventional ground sections viewed by
transmitted illumination were also prepared for comparison with
the procedures above.
Microscopy and Photography
Staining
Staining techniques require the survival of some organic material and are not suitable for very old bones. Bones were fixed in
formaldehyde both for preservation and safety from infectious
agents in bone dust.
Silver impregnation and reduction. Bone samples were
equilibrated in 3–5% silver nitrate on a rotator in the dark for
1–24 h before being washed in water and fixed in 5% sodium
thiosulfate. Aldehyde-fixed material showed black silver staining
of blood, blood vessels, osteocytes, and epithelia. This makes it
ideal for distinguishing different types of bone— osteonic or
laminar—at a glance. Although it does not stain mature bone, it
does react with preosseus tissue or osteoid, i.e., bone lamellae, as
they are being deposited, before calcification. There is a gradient
from black in the youngest, outermost, newly forming bone laminae, to brown and yellow in those inner laminae formed earlier.
Bone being absorbed during osteoporosis or bone reconstruction
stained brown to black, as did recently deposited bone. Silver
staining is useful for quickly mapping areas of bone genesis or
osteoporosis. The expanded matrix around blood vessels in forming bones stained light blue to orange. Exposure to diffuse light
developed more silver after weak impregnation. It is important
not to overstain in the silver nitrate. Too long an exposure stained
everything black, with no components resolved. In some overstained preparations the thin black surface crust was ground
away on a wet 600 or 1800 grit lap to reveal the impregnated
capillary systems beneath. The best results were obtained with
bone that had been stored in weak formaldehyde, allowing complete penetration and fixation. Unreduced silver was washed out
in 5% sodium thiosulfate to make permanent preparations (Figs.
1A,D, 2D,E,F, 3F–H, 4A,B, 5A–I, 6D, 7A,B, 10A). For the purposes of this article, dark silver staining is taken to be a marker
for osteoid bone as it is being formed or resorbed.
Osmium fixation and staining. Bone samples were left in 1%
osmium tetroxide in phosphate buffer at pH 7.2 for 2–24 h.
Osmium tetroxide fixes most tissues and prolonged fixation adds
more precipitated osmium black. The blackness was further accentuated in some preparations by reacting for 1 h in 1% ethyl
gallate. New bone, osteocytes, and cells of the capillary lining
were contrasted in black against the unstained mature white
bone matrix (Figs. 3I, 11C).
Impregnation With Metal Salts
Billets were exposed to saturated solutions of metal salts for
1– 4 h, with and without a vacuum. They were then washed and
the adsorbed metals converted to black sulfides by treatment
with dilute yellow ammonium sulfide. After cleaning, and in
some cases repolishing, they were cleared in glycerol. The
following salts all gave excellent results: ferrous sulfate (Figs.
2C, 6C, 9A), mercurous acetate (Figs. 7C–F, 8A,B, 9B, 11D,E),
bismuth subnitrate, lead acetate, cobalt acetate. The general
result resembles osmium black staining, but with a greater
tendency to stain the cement sheath. The advantage of metal
Stained test samples were observed after glycerol infiltration.
This had a dual purpose for photography. It allowed deeper visualization by increasing the transparency of the surface, and it
eliminated reflections from surface scratches. This method of
tissue preparation and observation is useful for immunofluorescence (tetracyclines and antibodies) in addition to the staining
procedures described here. Photographs were taken with a Zeiss
photomicroscope and a ⫻1, ⫻2.5, or ⫻10 objective lens with
epi-illumination provided by two side lamps. Infiltration and
epi-illumination also allowed for 3D observation with a stereomicroscope. The conclusions illustrated by the photographs are
based on these 3D observations of several hundred preparations,
including those of beef (Bos taurus), sheep (Ovis aries), giraffe
(Giraffa camelopardalis), pig (Sus domesticus), human (Homo
sapiens), buffalo (Bison bison), deer (Odocoileus virginianus),
horse (Equus caballus), and goat (Capra hircus), some involving
several different stains and stages of development. Most preparations were made from the central shaft of the humerus or
femur, occasionally from other long bones. Because of their simplicity, the techniques may be useful for anyone who works with
bone— biologists, gerontologists, archaeologists, physical anthropologists, or forensic scientists.
The page orientation has been standardized for all illustrations. Transverse profiles have the periosteal edge to the top.
Tangential and radial profiles orient the length of the bone with
the length of the page.
RESULTS
Primary Structure of the Shafts of
Mammalian Long Bones Is Laminar
The diaphyses or shafts of long bones are made of
compact bone, the primary structure of which is
laminar. In all young growing animals examined
(beef, sheep, pigs), the shaft is completely made from
circumferentially oriented laminae without osteons.
In older or even mature animals much of the shaft is
still laminar (human, buffalo, deer, horse, oxen).
Staining showed transverse profiles resembling tree
rings. Circumferentially arranged bone laminae,
about 200 ␮m wide, separate endothelium-lined vascular compartments, 10 –50 ␮m across (Fig. 1A).
This is the primary lamellar or mature plexiform
bone of Martin and Burr (1989b). Viewed tangentially, i.e., facing the surface, each vascular compartment sheet is resolved as a reticulum (Fig. 1B,C)
very similar to the scanning electron microscope
images of resin casts of cow laminae (Arsenault,
1990). Radial profiles (longitudinal cuts from center
to periphery) show the capillaries and their com-
STRUCTURE OF LONG BONES IN MAMMALS
551
partments cut with all orientations from longitudinal to transverse and oblique (Fig. 1D). The shape
and orientation of the osteocytes show that the bone
lamellae are layered parallel to the vascular compartment sheets, not in cylinders around capillaries,
as in osteons. Layers of bone alternate with obliquely or transversely cut compartments connected
by Volkmann’s canals, venous escape routes from
pooled blood in the marrow to the network at the
periosteal surface (Seliger, 1970; Oni and Gregg,
1990). Laminar bone, i.e., stacks of vascular sheets
separated by bone laminae, with each lamina being
a plywood-like composite of several lamellae, is the
common, basic structure of compact bone. It is the
primary structure of most bones.
Capillary Network Sheets in the Vascular
Compartment of Laminar Bone
To understand the images seen in profiles of developing laminar bone it is necessary to visualize
them in three dimensions. There are two compartments, one containing bone lamellae, and the other
capillaries (and also nerves and connective tissue).
The capillaries form a reticular sheet within the
reticular vascular compartment.
Capillaries form a very fine reticular network in
the plane of the lamina. They are easily visualized
in tangential profiles of cleared, unstained preparations of bone that has not been exsanguinated
and that contains blood-filled capillaries (Fig. 2A).
Injection of the capillary lumens (olive oil and
osmium) confirms the reticular nature of the capillary sheets (Fig. 2B). Both kinds of preparation
allow 3D views under the stereomicroscope.
Stereo-views show the arrangement of the short
connecting capillaries between adjacent sheets
that had been visualized in radial profiles (Fig.
1D). The pattern of capillaries resembles that in a
rabbit tibia observed intravitally (Winet, 1989) or
in rats by corrosion casting (Ohtani et al., 1982).
Staining of the vascular compartment shows a
coarser reticulum than the capillary network that
it encloses (Fig. 2C). The vascular compartment is
probably the prime mover in forming new laminae
at the osteal surface. In young animals there may
be 5– 8 enlarged vascular compartments forming
new laminae. These make up the plexiform bone
described by Martin and Burr (1989b), the laminar bone described in the beagle by Jee et al.
(1970), or the lamellar bone in fig. 5.13 of Wainwright et al. (1976). Even in older, mature animals
there are usually some regions with newly forming
laminae. The vascular compartment is at first
much wider than the compartment destined to
contain bone. It declines in width as the lamellae
are deposited in the bone layer, eventually shrinking to contain little more than the capillary network (Fig. 2D). The vascular compartment
changes its form as well as its thickness. It is at
Fig. 3. The distribution of laminar and osteonic bone in an
adult human femur. A: Transverse profiles were cut at seven
levels along the length of the femur, 11, 14 (B), 19 (C), 23 (D), 25
(E), 29, and 33 cm from the proximal end. Mainly laminar bone,
with its characteristic vasculature, survives into the adult in a
layer below the periosteum. Within this is a layer of mainly
osteonic bone. Bone on the endosteal surface is cancellous. The
core consists of marrow. Right femur 42 cm long from a 52-yearold woman. B–E: The appearance of laminar and osteonic bone
marked in A at positions 14 (B), 19 (C), 23 (D), and 25 (E) cm from
the proximal end. Silver stained. Scale bar ⫽ 1 cm. Transverse
(F:), radial (G), and peripheral H tangential profiles at 23 cm
from the proximal end illustrate the histology. The structure was
similar at other levels, with osteons predominating over laminae.
Silver stained. I: The osteonic layer (O) still contains some fine
network capillaries (nc) surviving from its primary laminar structure. Conversely, some capillaries (o) in subperiosteal laminae (L)
have become osteons (see Osteons Occur Within Laminae and
Osteons Form by Erosion…). Thus, laminar (L) and osteonic
layers (O) each contain both kinds of bone. Osmium-stained
transverse profile at 23 cm. Scale bars ⫽ 1 mm.
first a flat sac with tubular connections between
the two sides that appear as circles in face view.
Each circle is a window on the bone compartment
and contains a brown-stained fiber (Fig. 2E). The
fibers are inside the bone compartment and could
be concerned in its genesis, making connections
between adjacent sheets. They are in the same
topological position inside the bone compartment
as Sharpey’s fibers (Bloom and Fawcett, 1975).
They are noted here because of their topological
position in the bone compartment, but with an
552
M. LOCKE
Figure 3. (Continued)
orientation normal to that of the bone collagen.
The windows through the forming vascular compartment should not be confused with the connections across the bone (Volkmann’s canals) that
unite adjacent vascular compartments. These circular windows enlarge to leave the remainder of
the vascular compartment as the reticulum holding the capillaries (Fig. 2F).
The structure of laminar bone and its development is most easily understood if it is thought of
as a system of two compartments, one containing
bone lamellae and their associated osteocytes, and
the other capillaries, nerves, and connective tissue
(Fig. 2G). The vascular compartment of laminar
bone is a reticulum and its bone a sheet. The
vascular compartment of an osteon is a tube and
its bone a cylinder. Both kinds of bone compartment are organized in lamellae having various
orientations. As Weiner says: “…lamellar bone is
present in two forms, namely, as parallel arrays or
as cylinders…” (Weiner et al., 1999, p. 253). The
lamellae may be interwoven fibers alternating
with collagen-rich and collagen-poor regions
(Marotti, 1993). Currey (2002, figs. 1.4, 1.5) distinguishes woven from lamellar bone by the woven
orientation of some of its fibers in the laminae.
The collagen fiber orientation in lamellae suggests
a plywood-like structure (Giraud-Guille, 1988;
Wiener et al., 1997, 1999; Weiner and Wagner,
1998), as in insect cuticle lamellae.
STRUCTURE OF LONG BONES IN MAMMALS
553
Figure 3. (Continued)
Laminar and Osteonic Bone in the Human
Femur
Human histology textbooks, e.g., Bargmann
(1967), Elias and Pauly (1966), Han and Holmstedt
(1981), Cormack (1984), Krstic (1978, 1984, 1991),
figure transverse sections of long bones made only
from osteons, sometimes with peripheral layers labeled “circumferential lamellae.” The work of Enlow
and Brown (1958) showing laminae and osteons has
been forgotten. Published photographs of human
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M. LOCKE
Fig. 4. Osteons occur within laminae. A: Although there are relict laminae, the mature human femur is mainly composed of
osteons, as the textbooks propose for a typical diaphysis. Transverse profile of a silver-stained human femur. B: The formation of
osteons (o) in laminar bone. Some buffalo bone can be entirely laminar (Fig. 1A) but other regions contain numerous osteons.
Silver-stained transverse profile of a 3-year-old buffalo femur. Scale bars ⫽ 1 mm.
bone are not easy to interpret. Typically, as in di
Fiore (1981), they show osteons and circumferentially arranged “lamellae” averaging 5 ␮m in thickness. A Neanderthal bone (Schultz, 1999, 2001)
shows osteons and “basic lamellae” in a subperiosteal peripheral layer in transverse section. The following study of the shaft of a human femur shows
that these peripheral circumferential lamellae are
probably unrecognized laminar bone.
Transverse profiles were cut and prepared for microscopy at seven levels along the length of a human
femur (Fig. 3A). Four regions were easily distinguished. Laminar bone, with its characteristic vasculature, survived into the adult in a layer below the
periosteum. In some positions there were only one or
two laminae; in others, as many as 10 laminae constituted up to a quarter of the shaft thickness. The
laminar region was often stained a darker shade of
brown by silver or osmium, suggesting that it had a
different composition from the layer below it (Fig.
3B–D). The main thickness of the shaft was largely
osteonic. Many of the osteons had wide, darkly
stained lumens, as if their bone lamellae were being
resorbed, perhaps to be expected from osteoporosis
in a 52-year-old woman. The endosteal surface had a
variable ragged edge of cancellous bone, as though
much of it had been eroded, also suggesting osteoporosis. There was no endosteal laminar surface.
The core consisted of marrow.
Transverse, radial, and peripheral tangential profiles show the human femur has the typical features
of laminar and osteonic bone (Fig. 3F–H). The circumferential lamellae so often described are therefore laminae in which the vascular compartments
Fig. 5. Osteons form in the vascular compartments of laminar
bone. Transverse (A) and radial (B) profiles. Osteons (o) in laminar bone line up exactly with capillaries in the surviving network
sheets as though they have replaced them. Some capillaries and
their cavities in the bone laminae have enlarged. These are presumed to be forming osteons because their dense staining resembles that in laminar bone deposition and resorption in osteoporosis and endosteal erosion. Silver-stained 3-year-old buffalo femur.
Scale bar ⫽ 1 mm. C–I: Transverse profiles of osteons can be
arranged in series suggesting stages in their development from
capillary network sheets. C: There is no synchrony within a
region. All stages of development are found together. A capillary
beginning to enlarge is adjacent to an almost completely formed
osteon. D: A vascular compartment enlarges locally, resorbing
laminar bone as it does so. E,F: A cylinder of osteonic lamellae
begins to replace the space carved in the laminar bone. The newly
deposited bone stains intensely at first like that during the deposition of laminae. G,H: Osteonic bone continues to be deposited
until only a space large enough for a small blood vessel remains.
I: The end result is a cylinder of lamellae around a blood vessel
little larger than that in the original capillary network but with
few lateral connections. The bone is only distinguishable from the
original laminar bone by the concentric orientation of its osteocytes and lamellae and the cement sheath. Transverse profiles of
silver-stained 3-year-old buffalo femur. Scale bar ⫽ 0.1 mm.
STRUCTURE OF LONG BONES IN MAMMALS
Figure 5
555
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M. LOCKE
have been reduced or made difficult to resolve (see
Why Bones Are White…). The structure was similar
at other levels, with osteons predominating over
laminae. The details of structure may be idiosyncratic to this particular bone, but we may conclude
that human compact bone contains laminae as well
as osteons. Separation of the osteonic and laminar
layers is not absolute with respect to their constituents (Fig. 3I). Some large osteons occurred among
the capillaries of the laminar layer (see following
two sections). Conversely, some capillaries in the
osteonic layer had the small cross section characteristic of capillary networks and lacked concentric
bone lamellae. They are presumably relicts from the
primary laminar structure after its replacement by
osteons.
Osteons Occur Within Laminae
The presence of osteons among laminae is a general feature of mature bones. Some bones, as in the
human femur, are almost entirely composed of osteons (Fig. 4A). The shaft structure in a 3-year-old
buffalo femur shows the replacement of laminar by
osteonic bone even more clearly. Much of it is entirely laminar (Fig. 1A), but other regions contain
numerous osteons. (Fig. 4B). All the mature bones
studied contained osteons. Descriptions of mature
bones, as if they represent the typical situation, may
account for laminar bone being so often overlooked.
they replace parts of the capillary network sheets of
laminar bone rather than carving new pathways.
The most probable sequence is shown in Figure
5C–I. It begins with enlargement of the vascular
cavity around one of the more axially oriented capillaries. Bone eroded from the surrounding lamina
begins to stain brown. The space enlarges as much
as 10-fold before deposition takes over from resorption. Osteonic lamellae continue to be deposited until only a space large enough for a small blood vessel
remains. The new circumferential lamellae progressively lose their dense staining from the outside to
the inside, until the only difference from laminar
bone lies in the orientation of lamellae and the osteoblasts that have secreted them. The faintly
stained density separating the new tubular lamellae
from the old flat lamellae may be traces of a cement
sheath like that found in other secondary osteons.
The result is a less crooked space (see also Fig. 11D),
and a blood vessel little larger than that in the
original capillary network, but with fewer lateral
connections. The loss of lateral connections confirms
a micrograph of a resin cast of osteons that have
separated from the reticular network but retained
connections to Volkmann’s canals (Arsenault, 1990).
Straightening and loss of lateral connections is a
general property of all osteons observed. The change
from radially connected stacks of network sheets, to
longitudinal vessels connected to other longitudinal
vessels, could result in a significant change in blood
flow (see Why Bones Are White…).
Osteons Form by Erosion of Laminae and
Secretion of Cylindrical Lamellae
Folded and Irregular Laminae
Primary bone is entirely laminar, growing at the
periosteal surface (Fig. 2D,E). Osteons arise later
(Fig. 4A,B). By this time the volume of bone may be
fixed, so that new bone can only arise in the space
created by resorption of the old. New osteons stained
strongly with silver, as would be expected if bone is
being deposited and/or resorbed. These new osteons
in the laminar bone lined up exactly with capillaries
in the surviving network sheets, as though they had
replaced them (Fig. 5A,B). They did not have the
irregular distribution of the basic multicellular
units (BMUs) of Martin and Burr (1989a), believed
to be the basic mechanism for replacing older bone
with new osteons. The new osteons in Figure 5 were
at all stages of development, from barely discernable
dark brown swellings in the profiles of the reticular
spaces, to large concentric cylinders of unstained
lamellae (Fig. 5C–I). There was no synchrony within
a region. All stages were found together (Fig. 5C).
Longitudinal profiles of these forming osteons (Fig.
5B) were each at a particular stage of development,
suggesting that the transverse profiles (Fig. 5C–I)
represent stages rather than different levels in the
length of newly forming osteons. These new osteons
resemble the BMUs of Martin and Burr (1989c), or
the remodeling osteons of Parfitt (1994), except that
In some regions, the shape of the bone compartment is not in flat laminar sheets but may be folded
or distorted, creating an irregular 3D network. (The
fiber orientation within these compartments creates
a further subdivision of categories, such as woven
[Stover et al., 1992], fibrolamellar, parallel fibered,
or plexiform [Martin and Burr, 1989c]). Lateral connections between capillaries are then oriented in all
directions (Fig. 6A). The 3D arrangement is most
clearly seen in tangential profiles (Fig. 6B). This 3D
network is very different from the stacked network
sheets of standard laminar bone (Fig. 1B,C). The
switch from flat to distorted laminae is often abrupt,
without a smooth transition from flat 2D laminae
into folded 3D structures (Fig. 6C). Regular and
irregular laminae may alternate. The positioning of
irregular layers shows that they have been created
in this way by changes in deposition at the periosteal surface, not by distortion of previously deposited regular laminae. (They may also be deposited at
the endosteal surface; see Endosteal Laminae
Form…). Vascular chambers in these irregular laminae can also become osteons, as they do in flat
laminae (Figs. 4, 5). They enlarge and resorb laminar bone before redepositing lamellae in winding
osteonic cylinders (Fig. 6D).
STRUCTURE OF LONG BONES IN MAMMALS
557
Fig. 6. Laminae may be folded or distorted as well as flat. A: Sheets of laminar bone may be deposited as 3D networks with lateral
connections between capillaries oriented in all directions. Olive oil-injected and osmium-stained transverse profile of a deer cannon
bone. B: The 3D arrangement is most clearly seen in tangential profiles. This 3D network is very different from the stacked network
sheets of standard laminar bone (Fig. 1B,C). Tangential profile of ink-injected beef femur. C: The switch from flat to distorted laminae
is often abrupt, without a smooth transition from flat 2D laminae into folded 3D structures. The outer layers are regular laminae, while
the inner layer formed earlier is irregular. Iron sulfide-stained radial profile of young beef humerus. D: Vascular chambers in these
irregular laminae also become osteons in the same way as in flat laminae (Figs. 4, 5). They enlarge and resorb laminae before
redepositing lamellae in winding osteonic cylinders. Silver-stained transverse profile of the endosteal surface of a young beef humerus.
Scale bars ⫽ 1 mm.
Fig. 7. Some vascular compartments are radially rather than circumferentially oriented. A,B: In a common variant of laminar
structure, the vascular chambers and their capillaries extend radially (Rl). The regions are often bounded by circumferential laminae
(Cl) but may extend to either surface. Silver-stained transverse profile of a humerus from a young pig. C: Radially arranged
components are usually diagonal to the long axis. Infolds from the surface continue as irregular vascular chambers resembling
laminae. Mercury sulfide-stained radial profile of a young pig humerus. D: Radial chambers often begin at the surface as continuations
of tendon insertions, containing connective tissue as well as or instead of capillaries. They are often interspersed with radial laminae.
Mercury sulfide-stained transverse profile of a young pig humerus. E: Tendon insertions may also continue into distorted laminae.
Mercury sulfide-stained transverse profile of a young pig humerus. F: Volkmann’s canals (Vc) are often radial-diagonally oriented
especially near tendon insertions like A–D. Mercury sulfide-stained radial profile of a young pig humerus. Scale bars ⫽ 1 mm.
STRUCTURE OF LONG BONES IN MAMMALS
559
Fig. 8. At the surface of mature bones there may be a switch from deposition of laminae to longitudinal tubes. A: Forming vascular
compartments below the periosteum of some mature bones are exactly axially oriented, forming a regular array in transverse profile.
Below them each completed compartment has cylindrical lamellae like an osteon. The exact orientation extends for several rows below
the surface. Mercury sulfide-stained mature deer cannon bone. B: Tangential profile close to the surface shows longitudinally arrayed
vascular compartments with few lateral connections, unlike laminar bone. Some have cylindrical lamellae like osteons. Mercury
sulfide-stained mature deer cannon bone. Scale bar ⫽ 1 mm.
Some Laminae Are Radially Oriented
In a common variant of the folded laminar structure, some laminae are radially oriented. Their vascular chambers and capillaries not only extend radially but also diagonally in the axial direction (Fig.
7A–D). In transverse profile they are leaf-like, flattened radially (Fig. 7B). They may be sandwiched
between circumferential laminae (Fig. 7A), or extend to either surface. Radial laminae begin at the
surface as tendon insertions and contain connective
tissue fibers in addition to blood vessels (Fig. 7D).
They pass diagonally across from the periosteal surface, continuing as irregular vascular chambers resembling laminae. (Fig. 7C). Tendon insertions may
also continue into distorted laminae (Fig. 7E). Radial laminae overlap in structure with Volkmann’s
canals, which are often radial-diagonally oriented,
especially near tendon insertions (Fig. 7F). These
radial-diagonal orientations show the complexity of
bone structure and demonstrate the need for study
in all planes before 3D reconstruction.
Longitudinal Tubes in Peripheral Laminae
Not all bone formed at the periosteal surface is
laminar. Late in development, when the size of the
bone has been determined, the reticular form may
become predominantly axial (Fig. 8A,B). Transverse
profiles show that these axial vascular spaces have
cylindrical lamellae like osteons. Developmentally,
they are primary osteons in the sense that laminar
bone is primary, osteons developing within laminae
being secondary (Currey, 1982). They have been described as primary osteons in transverse profiles of
horse metacarpals (Stover et al., 1992) and are assumed to be the main source of osteons by Ham
(1974) in fig. 15.36.
Endosteal Erosion With Periosteal
Deposition Is a Mechanism for Size and
Shape Change
The primary consequence of the deposition of circumferential laminae at the surface is increase in
girth. If lamellae continue to be deposited around
distorted and radially oriented vascular compartments as they are covered over below the surface,
there is also the possibility of a lateral increase in
area. However, the main cause of growth in the
cylinder of bone forming the shaft is periosteal deposition. At the endosteal surface many osteons and
all kinds of lamina may be eroded to different de-
560
M. LOCKE
Fig. 9. Changes in bone size and shape come from periosteal deposition coupled with endosteal erosion. A: The endosteal surface
of growing bones is often irregular, as though erosion has followed a different pattern from deposition. Iron sulfide-stained transverse
profile of young beef humerus. B: In some mature bones the endosteal surface cuts diagonally across circumferentially oriented layers
of laminae. This suggests that the final shape of a bone depends on specific patterns of erosion as well as outer surface deposition.
Mercury sulfide-stained transverse profile of deer cannon bone. Scale bar ⫽ 1 mm. C: Increase in bone diameter with enlargement of
the marrow core comes from erosion of the endosteal face coupled with deposition at the periosteal surface. Transverse profiles traced
from the middle of the femur and humerus of pigs about 2 and 6 months old. Bone deposited early in development may be largely
replaced during growth. Scale bar ⫽ 1 cm.
grees at the same time, allowing for a change in
shape from that originally mandated by the periosteal deposition (see fig 2.22 in Martin and Burr,
1989c). Change in bone size and shape come from
periosteal deposition coupled with endosteal erosion, perhaps related to the lack of load on bone
closest to the core. The endosteal surface of growing
bones has an irregular surface, as though bone of all
kinds is constantly being eroded (Fig. 9A, see also
Fig. 4A). The endosteal face of mature bones often
cuts diagonally across circumferentially oriented
layers of laminae, showing that erosion follows a
different pattern from deposition (Fig. 9B). The final
shape of a bone therefore depends on specific patterns and extent of erosion as much as growth on the
outer surface. Most bone deposited early in develop-
ment may be replaced during growth. For example,
transverse profiles from the middle of the femur and
humerus of pigs about 2 months old can fit completely inside the bones at 6 months (Fig. 9C).
Endosteal Laminae Form in Mature Bones
Periosteal laminae form continually to make a
cylinder of increasing diameter. Endosteal laminae deposited on the inner face at the same time
would progressively occlude the lumen. However,
endosteal bone is not at first deposited where it is
found, but is derived from periosteal laminae.
Laminae are only deposited at the endosteal surface at the conclusion of growth (Fig. 10A). They
often appear to tidy up the irregular surfaces sur-
STRUCTURE OF LONG BONES IN MAMMALS
561
Fig. 10. A: Endosteal laminae of old bones form in situ, covering over secondary osteons and the
eroded surface of laminae formed earlier at the periosteal surface. O, osteonic layer; L, endosteal
laminae. Silver-stained transverse profile of a 3-year-old buffalo femur. Scale bar ⫽ 0.1 mm.
viving erosion. Thus, although endosteal and periosteal bone cylinders may be structurally similar,
their genesis can be different. Endosteal laminae
may be the remains of periosteal laminae that
have survived erosion, but in older bones they are
a late-stage deposition on the inner surface after
erosion and osteon insertion is complete. These
observations confirm the very beautiful photomicrograph of late-stage endosteal deposition in fluorescently labeled horse bones by Stover et al.
(1992).
Why Bones Are White Rather Than Red or
Pink
In spite of their relatively rich blood supply, compact bones are white rather than red or pink. There
are two obvious reasons for this. Although even very
fine capillaries can be clearly resolved as pink
threads (e.g., Fig. 2A), the proportion of the profile
displaying blood vessels is small. Second, many
bones have been partially exsanguinated, although
even such bones contain many vessels with blood.
Bones from animals chosen because they had not
been exsanguinated were still not very pink. Cleared
but unstained bone showed blood restricted to only a
few vessels. While blood was usually present in capillaries of peripheral laminae and in osteons, it was
often absent from more centrally located laminae
containing osteons. Blood loss and the small diameter of capillaries may therefore not be the whole
reason for white bones.
To test whether parts of the vascular compartment were intact but bloodless, unstained bones
known not to be exsanguinated and bones with
clearly observable blood were studied. Typically,
capillary networks in newly formed outer laminae
contained blood, as did osteons. Capillaries in central laminae lacked blood and shone white and
empty, especially around newly forming osteons
(Fig. 11A). Unstained preparations of a human femur showed blood in osteons, but little or none in
surviving laminae (Fig. 11B). This was not because
the vascular compartments of the laminae had disappeared. Subsequent staining of the same preparation showed laminar structure all around the osteons (Fig. 11C). The idea that not all vascular
compartments contain blood-filled capillaries agrees
with the observations that osteons lose their connections with the capillary sheets from which they have
been derived (Fig. 11D, see Osteons Form by Erosion…). Blood might be expected to be absent from
such capillaries. The flow of blood to all bones in the
rat, rabbit, dog, and man has been estimated to be
4 –10% of cardiac output (Shim, 1968). It would
therefore be an economy to reduce the flow through
mature laminar bone that had minimal metabolic
activity. Blood is readily observed in the capillaries
of new network sheets (Fig. 1A) and in the center of
osteons of older bone (Fig. 11B), but not in both
where they occur together (Fig. 11A). The question
is not whether capillary network sheets cease to be
functional, but when. In many mature bones, the
only relict of primary laminar structure lies in the
arrangement of osteons that follows that of the capillary network sheets into which they have been
inserted (Fig. 11E). All blood has been transferred to
the osteons. The absence of blood in old capillary
network sheets may correlate with local degeneration of the vascular compartment, explaining why
562
M. LOCKE
Figure 11
STRUCTURE OF LONG BONES IN MAMMALS
laminar bone has so often been overlooked, why
lamellae but not laminae have been described between osteons.
DISCUSSION AND CONCLUSION
The human bone most intensively examined in
this study was from an older woman. The expectation that she would have had osteoporosis (Thompson, 1980) was confirmed by the endosteal erosion.
There were also forming osteons, as well as a high
frequency of degenerate ones, i.e., osteons enlarged
by resorption. The formation of osteons in laminar
bone throws a new light on osteoporosis. Most growing bones have all stages of osteon development,
with resorption followed by cylindrical lamellar deposition. Osteoporotic bone has more in the resorptive
phase, as if the message to complete a normal sequence has been blocked. The problem for osteoporosis may not only be increased resorption but a
block to resecretion. Manzke et al. (1982) report
cases of increased bone formation coupled with normal resorption that might be explained by increased
conversion of laminar to osteonic bone.
The seemingly great variety of compact bone described here can be resolved as the patterns made by
a two-compartment system. Bone is topologically a
tubular connective tissue compartment extending
into a bone collagen compartment. The simplification should bring its study into the range of developmental biology and genetics, like the studies of
tube growth in tracheal systems (Locke, 2001), or
lung and kidney development (Metzger and Krasnow, 1999; Lubarsky and Krasnow, 2003).
The bone two-compartment system may also be
thought of as a functional response to the need for a
separate environment that is both massive and locally controlled. Within it, collagen fibers can be
oriented in lamellae under conditions that allow hydroxyapatite crystals to be deposited. The varied
Fig. 11. Blood is often missing from laminar capillaries in
some regions. A: Cleared but unstained bone shows blood (b) in
newly forming laminae (l) at the surface and in osteons, but
missing from older capillary networks around osteons. The empty
capillaries (ecn) shine silvery white. Unstained transverse profile
of pig humerus. B: Cleared, unstained human bone shows blood
(b) in osteons but not in nearby laminae (L). Transverse profile of
an unstained human femur. C: Osmium staining of the preparation in B showed that laminar vascular compartments continued
to be present, suggesting that these parts of the vascular system
survive but are bloodless. D: Osteons (o) forming from capillaries
in laminae lose connections with capillary network sheets (cn).
This could cause capillaries like those in A to be bloodless. Mercury sulfide-stained tangential profile of 3-year-old buffalo femur.
A–D is the same magnification. Scale bar ⫽ 1 mm. E: In many
mature bones the only relict of primary laminar structure lies in
the arrangement of osteons that follows that of the capillary
network sheets from which they have been derived. All blood has
been transferred to the osteons. Mercury sulfide-stained transverse profile of horse femur. Scale bar ⫽ 1 mm.
563
patterns of laminae show the versatility of the system. Sheets of bone can be deposited with any orientation, transverse radial and diagonal radial as
well as axial circumferential. The idea of bone as a
two-compartment system also has implications for
its electrical properties. Conduction could be different in each compartment, as well as along the membrane that separates them.
An important difference between laminar and osteonic organization lies in the size of their bone
compartments. Although only lamellae in the immediate vicinity of a laminar capillary are eroded when
an osteon forms, laminar bone is essentially continuous, with a reticular blood supply. This might
make it difficult to effect local morphogenesis. Secondary osteonic bone, on the other hand, forms small
local compartments bounded by cement sheaths that
lend themselves to resorption and redeposition. During osteoporosis, osteons can be resorbed one by one.
Capillary network sheets are ideally patterned for
the function of supplying precursors for rapid bone
deposition. However, functional capillary network
sheets would cause a purposeless resistance to blood
flow were they to be retained after the completion of
a lamina. It is therefore perhaps not surprising that
they cease to contain blood at maturity, when the
osteonic capillaries flowing in straight cylinders
would give much less resistance. It may be significant that the network sheet capillaries are easily
infiltrated with oil, suggesting that they have lipophilic linings. A lipophilic lining could have the
dual property of low surface drag, giving reduced
resistance to aqueous fluid flow while functional,
combined with easy filling of the lumen with air, as
seems to happen after nearby osteon formation.
Capillaries with hydrophilic linings would have difficulty emptying blood or lymph due to their capillarity.
The results suggest that there is still a need for
descriptive work on different kinds of bone, their
development, structure in relation to function and
bone type, and phylogeny. A detailed inventory of
bone microstructure of the kind begun by Enlow and
Brown (1956, 1957, 1958) might make it possible to
identify species, kind of bone, region, and stage of
development from fragments, information of interest to systematists, archeologists, and forensic scientists. We may conclude by agreeing with von Ebner (1875) that “the major difficulty of skeletal
analysis lies in the heterogeneity of bone, so that its
distinctive structure and function may be understood only when every component has been analyzed
in all its ontogenetic aspects…” (quoted in Ascenzi,
1983, p. 233).
ACKNOWLEDGMENTS
I thank Drs. B.A. Flumerfelt and P. Haase, Anatomy Department, University of Western Ontario, for
supplying the human bone; Professors J.D. Currey,
564
M. LOCKE
R. Dean, B. Hall, D. McMillan, and S. Weiner for
careful review and very helpful comments on the
manuscript; and R. Nichol and P.C. Gould for invaluable help with literature searches.
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APPENDIX
Glossary
Basic multicellular unit (BMU) The collection of
cells involved in creating secondary osteons in bone
remodeling (Martin and Burr, 1989a; Parfitt, 1994).
Bone compartments The flat stacks (laminar) or
cylinders (osteonic) of bone separated from one another by vascular compartments.
STRUCTURE OF LONG BONES IN MAMMALS
Capillary network sheets The vasculature between bone laminae.
Cement sheath The boundary of uncertain composition and permeability that separates secondary
osteons from their surroundings.
Circumferential lamellae Lamellae at the endosteal and periosteal surfaces surviving from
stacks of laminae after the loss of their capillary
network sheets.
Circumferential system Laminae and/or lamellae
at the endosteal and periosteal surfaces surviving
from stacks of laminae after varying degrees of loss
of their capillary network sheets.
Endosteum The layer of cells, connective tissue
and blood vessels lining the inner face of hollow
bones.
Haversian system The older word for groups of
osteons (American journals) or osteones (British
journals). Secondary osteon (Currey, 1982).
Interstitial lamellae Derived from laminar bone,
filling some of the space between osteons after the
loss of capillary network sheets.
Lamellae Bone layers in both laminar and osteonic
bone, each consisting of a 20 –50-␮m thick layer of
collagen fibers on which calcium salts may be deposited. The fiber orientation varies, often changing
from lamella to lamella like the layers in plywood or
interspersed with nonlamellar orientations. The orientation determines the different kinds of bone (woven, parallel fibered, lamellar, fibrolamellar) described at the ultrastructural level by Weiner et al.
(1999), Currey (2002), and others.
565
Laminae Primary bone made from 4 –20 lamellae
laid down between capillary network sheets and surviving as interstitial lamellae between osteons and
in endosteal and periosteal layers. Laminar bone is
always primary.
Osteoblast Cell concerned with bone deposition.
Osteoclast Cell concerned with bone resorption.
Osteocyte Bone cell formed by the incorporation of
an osteoblast into the bone matrix (Aarden et al.,
1994).
Osteoid Preosseous tissue, lamellae before ossification.
Osteon A cylinder of concentric lamellae around a
tubular space containing blood vessels. Primary
osteons form at the periosteal surface of bones
nearing maturity and lack a cement sheath. Secondary osteons are a replacement of laminar bone
forming around blood vessels derived from capillary networks (Currey, 1982), or the basic multicellular unit (BMU) in bone remodeling (Parfitt,
1994). They have a cement sheath.
Periosteum The layer of cells, connective tissue,
and blood vessels that protects the outer surface of
bones.
Vascular compartment The compartment containing capillary networks or osteonic blood vessels and connective tissue, separated by an epithelium from the bone compartment.
Volkmann’s canals The radially oriented spaces
with blood vessels connecting capillary sheets and
to a lesser extent, osteons. They are often diagonal
to the long axis.