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Does static precede dynamic osteogenesis in endochondral ossification as occurs in intramembranous ossification.

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THE ANATOMICAL RECORD PART A 288A:1158–1162 (2006)
Does Static Precede Dynamic
Osteogenesis in Endochondral
Ossification as Occurs in
Intramembranous Ossification?
MARZIA FERRETTI, CARLA PALUMBO, LAURA BERTONI,
FRANCESCO CAVANI, AND GASTONE MAROTTI*
Dipartimento di Anatomia e Istologia, Sezione di Anatomia Umana,
Università di Modena e Reggio Emilia, Modena, Italy
ABSTRACT
Endochondral ossification takes place with calcified cartilage cores
providing a rigid scaffold for new bone formation. Intramembranous ossification begins in connective tissue and new bone formed by a process of
static ossification (SO) followed by dynamic ossification (DO) as previously described. The aim of the present study was to determine if the process of endochondral ossification is similar to that of intramembranous
ossification with both a static and a dynamic phase of osteogenesis. Endochondral ossification centers of the tibiae and humeri of newborn and
young growing rabbits were studied by light and transmission electron
microscopy. The observations clearly showed that in endochondral ossification, the calcified trabeculae appeared to be lined first by osteoclasts.
The osteoclasts were then replaced by flattened cells (likely cells of the reversal phase) and finally by irregularly arranged osteoblastic laminae,
typical of DO. This cellular sequence did not include osteoblasts seen in
the phase of SO. These findings clearly support our working hypothesis
that SO only forms in soft tissues to provide a rigid framework for DO,
and that DO requires a rigid mineralized surface. The presence of osteocytes in contact with the calcified cartilage also suggests the existence of
stationary osteoblasts in endochondral ossification. Stationary osteoblasts
did not appear to be a unique feature of SO. The presence of stationary
osteoblasts may appear to provide the initial osteocytes during osteogenesis
that may function as mechanosensors throughout the bone tissue. If this is
the case, then bone would be capable of sensing mechanical strains from its
inception. Anat Rec Part A, 288A:1158–1162, 2006. Ó 2006 Wiley-Liss, Inc.
Key words: endochondral ossification; intramembranous ossification; static osteogenesis; dynamic osteogenesis
In a series of investigations carried out in recent years
on intramembranous ossification centers (Ferretti et al.,
2002; Palumbo et al., 2004), we pointed out the existence
of two types of bone deposition we respectively named
static osteogenesis (SO) and dynamic osteogenesis (DO).
SO always occurs first; it is characterized by stationary
osteoblasts, arranged in cords, which form at a rather
constant distance from the network of blood capillaries
and, without moving, they transform into osteocytes in
the same site where they differentiated. DO is the more
familiar mechanism of bone deposition. DO is characterÓ 2006 WILEY-LISS, INC.
Grant sponsor: 2004 MURST Cofinancing; Grant number:
2004067411_001.
*Correspondence to: Gastone Marotti, Dipartimento di Anatomia e Istologia, Sezione di Anatomia Umana, Via del Pozzo 71,
41100 Modena, Italy. Fax: 39-059-4224861.
E-mail: marotti.gastone@unimore.it
Received 6 March 2006; Accepted 26 July 2006
DOI 10.1002/ar.a.20386
Published online 9 October 2006 in Wiley InterScience
(www.interscience.wiley.com).
1159
EARLY OSTEOGENESIS IN OSSIFICATION CENTERS
Fig. 2. LM micrograph of an undecalcified cross-section of the
epiphysial plate in the humerus of an 8-day-old rabbit. The arrow
points to spindle-shaped cells of the reversal phase lining the cartilage
surface previously eroded by osteoclasts. CC, calcified cartilage; B, bone.
Scale bar ¼ 15 mm.
Fig. 1. LM micrograph of an undecalcified cross-section of the epiphysial plate in the tibia of a newborn rabbit. Note an osteoclast
(OCL) resorbing the calcified cartilage (CC) in an osteocartilaginous
trabecula. B, bone. Scale bar ¼ 15 mm.
ized by movable osteoblasts arranged in pseudoepithelial
laminae, which move away from the mineralizing surface during preosseous matrix secretion and osteocyte
transformation. In the case of intramembranous ossification, we observed that DO takes place only after SO on
the surface of the bony trabeculae laid down by SO. The
main speculation we drew from these investigations was
that SO is needed in soft mesenchymal tissue to provide
a more rigid framework for DO and DO can only occur
on a preexisting rigid mineralized surface.
To verify the validity of our working hypothesis, the
process of endochondral ossification was studied to determine the sequence of ossification. Specifically, to compare
the SO and DO previously described in intramembranous
ossification. Contrary to intramembranous ossification,
endochondral ossification begins with a rigid scaffold of
calcified cartilage.
MATERIALS AND METHODS
Metaphyseal plates from the tibiae and humeri of
three newborn rabbits and four growing rabbits aged
8–10 days were used for light and electron microscopy.
The care and use of animals were in accordance with
the National Institutes of Health guidelines. All specimens were fixed for 2 hr with 4% paraformaldehyde in
0.13 M phosphate buffer, pH 7.4, postfixed for 1 hr with
1% osmium tetroxide in 0.13 M phosphate buffer at pH
7.4, dehydrated in graded ethanol, and embedded in epoxy resin (Durcupan ACM). Decalcified specimens were
prepared by placing the tissue in 2.5% EDTA (0.13 M
phosphate buffer, pH 7.2) until soft and then postfixing
in osmium tetroxide prior to embedding in epoxy resin.
The epoxy blocks were sectioned with a diamond knife
using an Ultracut-Reichert Microtome. The methaphyseal plates were longitudinally and transversely sectioned with respect to the main axis of the shaft. Semithin sections (1 mm) were stained with toluidine blue
and examined with a Axiophot-Zeiss light microscope
(LM). Ultrathin sections (70–80 nm) were mounted on
Formvar- and carbon-coated copper grids, stained with
1% uranyl acetate and lead citrate, and examined with a
Zeiss EM109 transmission electron microscope (TEM).
RESULTS
In all metaphyseal plates, the following sequence of
events of endochondral ossification were observed: columns of proliferating chondrocytes undergoing gradual
hypertrophy towards the midshaft level; cartilage mineralization; chondrolysis and partial calcified cartilage disruption by osteoclasts; and the formation of osteocartilaginous trabeculae.
Our LM and TEM analyses were particularly focused
on the surface of the trabeculae of calcified cartilage
reabsorbed by osteoclasts (Fig. 1) and the early stages of
bone matrix deposition. Prior to the appearance of osteoblasts, a consistent observation was the presence of flattened or spindle-shaped cells, similar to those of a reversal phase, lining the cartilage lacunae eroded by osteoclasts (Figs. 2 and 3). After the disappearance of the
spindle-shaped cells, they were replaced by osteoblasts
arranged in laminae from their inception (Fig. 4). Osteoblasts were sometimes irregularly grouped in such laminae, particularly where the eroded surface of calcified
cartilage was irregularly indented by small and narrow
lacunae. Most osteoblasts appeared to be very active,
being rounded in shape and displayed a well-developed
endoplasmic reticulum and Golgi apparatus. All osteoblasts were functionally polarized toward the cartilaginous or the osteoid seam surface, as shown by the posi-
1160
FERRETTI ET AL.
Fig. 3. TEM micrograph of a decalcified specimen showing a flattened cell of the reversal phase (asterisk) lining the remnant of an
eroded cartilaginous trabecula. Scale bar ¼ 2.5 mm.
Fig. 5. TEM micrograph of a decalcified specimen showing a lamina of osteoblasts (OB) polarized toward the cartilaginous surface. CH,
chondrocytes. Scale bar ¼ 2.5 mm.
Fig. 4. LM micrograph of an undecalcified cross-section of the epiphysial plate in the tibia of a newborn rabbit showing laminae of plump
osteoblasts lining lacunae of calcified cartilage. Scale bar ¼ 15 mm.
tion of their organelles with respect to the nucleus (Fig. 5).
Gap junctions were sometimes observed between adjacent osteoblasts (Fig. 6).
The bone covering the remnants of calcified cartilage
contained osteocytes, whose cell body displayed a globous or ovoid shape, enclosed in single lacunae. Confluent lacunae were never observed (Fig. 2). An interesting
and fairly frequent finding was the presence of osteocyte
cell bodies very close to the calcified cartilage (Figs. 7
and 8; see also Figs. 1 and 2). Osteocyte dendrites
showed an asymmetrical arborization with short mineral
dendrites radiating toward the calcified cartilage and
longer vascular dendrites directed toward the osteoblastic laminae and the vessels (Fig. 8). Gap junctions or
simple contacts were observed between the cytoplasmic
Fig. 6. TEM micrograph showing a gap junction (arrows) between
movable osteoblasts. Scale bar ¼ 0.25 mm.
processes of adjacent osteocytes and between osteocyte
dendrites and osteoblasts (Fig. 9).
DISCUSSION
In intramembranous ossification centers that surround the developing shafts of long bones, transverse
periosteal bone growth occurs by progressive extension
of osteoblastic cords inside the surrounding mesenchyme
at about midway between blood capillaries (mean distance of cords from vessels, 28 6 0.4 mm). The cords are
EARLY OSTEOGENESIS IN OSSIFICATION CENTERS
Fig. 7. TEM micrograph showing two osteocytes, derived from stationary osteoblasts, close to calcified cartilage (asterisks). The three
arrows point to osteocyte mineral dendrites radiating toward the calcified cartilage. Decalcified specimen. Scale bar ¼ 5 mm.
1161
Fig. 8. TEM micrograph of an osteocyte (OC), derived from stationary osteoblasts, with a short mineral dendrite directed toward the calcified cartilage (top) and longer vascular dendrites extending toward
an osteoblast (OB). Undecalcified specimen. Scale bar ¼ 2.5 mm.
Fig. 9. A: Gap junction between osteocyte dendrites in a cross-sectioned canaliculus. B: Gap junction
between a vascular dendrite of an osteocyte (OC) and a short cytoplasmic processes of an osteoblast
(OB). Undecalcified specimens. TEM micrographs. Scale bars ¼ 0.2 mm (A); 0.3 mm (B).
made up of 2–3 layers of active plump osteoblasts, all
functionally polarized, but in a different direction with
respect to the adjacent ones. Additionally, these osteoblasts are stationary since they transform into osteocytes without moving from the site where they appeared
to differentiate. It is from these observations that we
proposed the term ‘‘static osteogenesis’’ to describe this
process (Ferretti et al., 2002). As a consequence, the
bony trabeculae, laid down in this manner, contain several globous-shaped osteocytes, often irregularly grouped
inside confluent lacunae and displaying short dendrites
having about the same length all around the cell body
(Palumbo et al., 2004). Afterward, typical monostratified
laminae of movable osteoblasts differentiate along the
surface of these trabeculae laid down by static osteogenesis. These osteoblasts form new bone by dynamic osteogenesis that results in thicker trabeculae that cause the
narrowing of the enclosed primitive Haversian spaces
(primary osteons). Osteocytes derived from movable
osteoblasts generally are ovoid and display an asymmetrical dendrite arborization. It should be noted that the
network of dendrites extending throughout all osteocytes, derived by both static and dynamic osteogeneses,
forms a functional syncytium since they were found to
be connected by gap junctions (Ferretti et al., 2002; Palumbo et al., 2004).
1162
FERRETTI ET AL.
In endochondral ossification, static osteogenesis never
seems to take place. In fact, the osteoblasts in contact
with the remnants of calcified cartilage are directly
arranged in movable laminae and all appear to be functionally polarized in the same directions, i.e., toward the
calcified cartilage. Additionally, the osteocytes inside the
bone surrounding the calcified cartilage are never grouped
inside confluent lacunae and all display an asymmetrical
arborization of dendrites. Thus, in endochondral ossification, dynamic osteogenesis is not preceded by static osteogenesis. We wish to stress, however, that one must not
confuse static osteogenesis with stationary osteoblasts. In
endochondral ossification, the presence of osteocytes close
to the calcified cartilage clearly indicates that such osteocytes are derived from stationary osteoblasts that remain
in the same site where they differentiated. The thin layer
of bone matrix separating the osteocyte cell bodies from
the calcified cartilage generally contained osteocyte mineral dendrites. Therefore, this layer does not indicate that
the parental osteoblasts moved; it instead reflects the diminution in size of osteoblast protoplasms and the radiation
of cytoplasmic processes that occurred during the differentiation of the osteoblast into an osteocyte. Osteocytes
derived from stationary osteoblasts can frequently be
observed close to the reversal lines of secondary Haversian systems; hence, stationary osteoblasts are not a distinctive feature of static osteogenesis. At the onset of bone
formation, it is necessary for some stationary osteoblasts
to be present, independently of whether static or dynamic
osteogenesis is taking place, to ensure the presence of
osteocytes, thus providing a network of strain-sensitive
cells throughout the developing bone tissue. Without these
stationary osteoblasts, the first layers of bone tissue would
not contain osteocytes. We wish to stress that a continuous osteocyte functional syncytium was observed throughout the bone laid down in both intramembranous and
endochondral ossification and that such syncytium comes
into contact with the osteoblasts covering the bone growing surfaces. Such a cellular organization suggests that
the bone is capable of sensing and answering mechanical
signals since from its inception.
Osteoblast differentiation seems to depend on a variety of inductive factors; in intramembranous ossification,
it probably depends on endothelial cell-derived cytokines, such as endothelin-1 (Sasaki and Hong, 1993;
Kasperk et al., 1997; Inoue et al., 2000) and growth factors (EDGF) (Guentheret al., 1986; Canalis et al., 1989;
Streeten and Brandi, 1990; Villanueva and Nimni,
1990). Since static and dynamic osteogeneses were
observed to occur in the same manner and sequence during bone repair (Marotti, 2004), osteoblast differentiation
could also depend on platelet-derived growth factor (PDGF)
(Zheng et al., 1992; Lind, 1998; Chaudhary et al., 2004)
in bone healing. In endochondral ossification, osteoblast
differentiation can depend on endothelial-cell derived cytokines as in intramembranous ossification; however, it
seems likely that it might also depend on coupling factors
released by the cells of the reversal phase, as has been
suggested in the bone remodeling cycle (Baron, 1976,
1989). Though the origin of these cells is still unknown,
they may be stromal cells in a preosteoblast phase. The
main difference between intramembranous and endochondral ossification is that in the latter, osteoblasts differentiate only after osteoclastic resorption of calcified cartilage
followed by a transient reversal phase.
In conclusion, this study of endochondral ossification
supports the hypothesis that dynamic osteogenesis needs
a rigid mineralized surface to occur and static osteogenesis only occurs in soft tissues where a rigid framework is
lacking. This is not only true in the mesenchyme during
intramembranous bone histogenesis but also in soft callus during bone repair (Marotti, 2004).
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