JOURNAL OF BONE AND MINERAL RESEARCH
Volume 12, Number 5, 1997
Blackwell Science, Inc.
q 1997 American Society for Bone and Mineral Research
New Aspects of Endochondral Ossification in the Chick:
Chondrocyte Apoptosis, Bone Formation by Former
Chondrocytes, and Acid Phosphatase Activity in
the Endochondral Bone Matrix
HELMTRUD I. ROACH
ABSTRACT
A detailed histological study of the growth plates from 9- to 20-day-old embryonic chick long bones was carried out
with the aim of clarifying the long-debated question of the fate of the hypertrophic chondrocytes. Since resorption
in chick bones does not occur synchronously across the plate as it does in mammals, specialized regions develop
and the fate of the chondrocyte depends on its location within the growth plate. Where resorption took place, as
at the sites of primary vascular invasion or at the main cartilage/marrow interface, chondrocytes underwent
apoptosis before the lacunae were opened. In addition, spontaneous apoptosis of chondrocytes occurred at
apparently random sites throughout all stages of chondrocyte differentiation. In older chick bones, a thick layer
of endochondral bone matrix covered the cartilage edge. This consisted of type I collagen and the typical
noncollagenous bone proteins but, in addition, contained tartrate-resistant acid phosphatase in the mineralized
matrix. Where such matrix temporarily protected the subjacent cartilage from resorption, chondrocytes differentiated to bone-forming cells and deposited bone matrix inside their lacunae. At sites of first endochondral bone
formation, some chondrocytes underwent an asymmetric cell division resulting in one daughter cell which
underwent apoptosis, while the other cell remained viable and re-entered the cell cycle. This provided
further support for the notion that chondrocytes as well as marrow stromal cells give rise to endochondral osteoblasts. (J Bone Miner Res 1997;12:795–805)
INTRODUCTION
takes place whenever preformed cartilage is replaced by bony tissue, for example, at the growth plate, the secondary center of ossification,
and during fracture repair. The overall cellular strategies
for the cartilage-to-bone transition are well known: parts of
the cartilage matrix are resorbed and replaced by vascular/
marrow elements(1–3) and new bone is then laid down onto
the scaffold of unresorbed cartilage spicules. One important
unresolved question is the precise origin of the bone-forming cells. Although it is generally assumed that they differentiate from marrow-derived osteogenic stem cells (reviewed by Marks and Popoff(4) and Beresford(5)), the
evidence rests on the osteogenic potential of marrow stro-
E
NDOCHONDRAL OSSIFICATION
mal cells rather than direct proof that this actually occurs
during endochondral ossification in vivo. An alternative
view is that chondrocytes themselves become bone-forming
cells. Observations over many years have been consistent
with a chondrocyte-to-osteoblast transition,(6 –18) but unambiguous proof that this occurs in vivo has not yet been
obtained, and the notion has remained controversial.
The currently accepted view is that the terminal chondrocytes die and that marrow-derived osteoprogenitor cells
migrate to the cartilage edge, differentiate into osteoblasts,
and lay down the new bone matrix. Since developmental
cell death usually takes the form of apoptosis, the expectation has been that terminal chondrocytes are eliminated by
this process. However, verification of this and in situ quantification of apoptosis in chondrocytes has proven difficult.
Academic Orthopaedic Unit, University of Southampton, General Hospital, Southampton, United Kingdom.
795
796
ROACH
The morphological characteristics, such as apoptotic bodies, are very small and thus easy to miss. In addition, the
number of cells with apoptotic morphology is low at any
given time point— even if many cells undergo programmed
cell death with time.(19) Using end-labeling of DNA strand
breaks (TUNEL method of Gavrieli et al.(20)), a relatively
large percentage of hypertrophic chondrocytes were labeled: 20 –30% in chick sterna(21) and 30 – 40% in adult
chick tibia.(22) However, using flow cytometry, the latter
authors estimated that only 8% of chondrocytes were apoptotic at any one time, which suggested that the TUNEL
method sometimes overestimates the number of apoptotic
chondrocytes. Bronckers et al.(23) found far fewer chondrocytes with positive TUNEL staining in the growth plate of
rodents.
The problem in ascertaining whether chondrocytes undergo apoptosis or become bone-forming cells in vivo is
that, once released from their lacunae, any cartilage-derived cells would become interspersed among the marrow
cells and hence impossible to trace. Previous work had,
therefore, concentrated on in vitro situations where resorption was reduced so that lacunae that would have opened in
vivo remained intact in vitro. In organ cultures of cut embryonic chick bones, the chondrocytes became bone-forming cells which produced mineralized osteoid matrix within
intact lacunae after 9 –12 days.(14) More recently, we established that one of the crucial early events in the chondrocyte-osteoblast transition was an asymmetric cell division,
where the fate of the daughter cells diverged: one cell
remained viable, while its sister was programmed to die.(16)
These in vitro experiments provided convincing data on
changes in phenotypic expression, but they did not directly
address the question of the fate of the terminal hypertrophic chondrocyte in vivo. In the present study, many growth
plates from 9- to 19-day-old chick embryos were examined
in great detail. Particular attention was paid to the characteristics of the cells at, or just behind, the cartilage/marrow
edge.
MATERIALS AND METHODS
Tissue processing and light microscopy
Femurs and tibiae were dissected from 9- to 20-day-old
chick embryos and either fixed immediately in 4% phosphate buffered paraformaldehyde (pH 7.4) or the whole
bones were cultured for 18 h as previously described.(24) It
is known that hypertrophic chondrocytes are extremely
fragile cells which shrink with these conventional fixation
methods. However, fixation with gluteraldehyde and ruthenium hexammine trichloride, which preserves cartilage
morphology,(25,26) led to impaired enzyme- and immunoreactivity. The shrinkage of the late hypertrophic chondrocytes was therefore accepted as inevitable. The bones were
processed without decalcification for 9- to 17-day femurs,
whereas the bones from 17- to 20-day-old chick embryos
were decalcified in 5% EDTA (in 0.1 M Tris/HCl buffer,
pH 7.4) for 2–3 days. The bones were processed through
graded ethanols and chloroform into paraffin wax.
Twenty to eighty sequential longitudinal sections were
cut off each bone at 5– 6 mm, with the number depending on
the age and hence thickness of each bone. The sections
were stained by one of the following methods: Weigert’s
hematoxylin/alcian blue/sirius red (after Lison(27)) was used
as a nuclear stain and to distinguish bone matrix (red) from
cartilage matrix (blue). Acid phosphatase activity was demonstrated with an azo dye coupling method (modified from
Bancroft and Stevens,(28) described in detail in Roach and
Shearer(29)). To test for tartrate or fluoride inhibition, 50
mM Na tartrate or 0.1–10 mM NaF was present in the
reaction mixture. Sections were counterstained with 0.2%
light green (1 minute) and 1% alcian blue (1 minute).
Immunocytochemistry
The following polyclonal antibodies were used. The antibody for type I collagen (LF-67 from Prof. L. Fisher) had
been raised against the human C-terminal propeptide of the
a-1 chain. This antibody detects pro-a-1, but also the fully
processed a-1 chain.(30) The antibodies to the noncollagenous proteins were all specific for chick. The osteopontin
(details in McKee et al.(31)), osteocalcin (details in Neugebauer et al.(32)) and bone sialoprotein (details in Yang et
al.(33)) antisera were gifts from Prof. L. Gerstenfeld (Boston, MA, U.S.A.). The osteonectin (LF-8; details in Pacifici
et al.(34)) antiserum was donated by Prof. Larry W. Fisher
(Bethesda, MD, U.S.A.). The primary antibodies were visualized using the avidin/biotin method with peroxidase and
3-amino-9-ethylcarbazole (AEC). The sections were counterstained with 0.2% light green and 1% alcian blue. Control sections were incubated with rabbit serum (negative
control, Sigma Chemical Co., Poole, Dorset, U.K.), then
treated as above. No staining was found in controls.
Autoradiography
Proliferating cells were identified by incubating the whole
femurs with 3H-thymidine (2 mCi/ml) for 18 h followed by
autoradiography of the paraffin sections. Sections were
prestained with light green/alcian blue, dipped in photographic emulsion (K-5, 1:1 dilution, 458C, Ilford, Mobberly,
Cheshire, U.K.), exposed in a dark box at 48C for 3 weeks,
developed with Kodak Dektol (2 minutes), and fixed with
30% Na-thiosulphate (5 minutes).
Confocal microscopy
Forty-micrometer-thick paraffin sections were cut. Type I
collagen was visualized with antirabbit IgG-FITC as the
second antibody, and nuclei were stained with propidium
iodide. Dual series of 36 confocal optical slices were obtained by two-channel scanning, using a Leica TCS confocal
microscope with a krypton/argon laser light source (Leica
UK Ltd., Milton Keynes, U.K.).
In situ detection of apoptotic cells
Localizing DNA breaks by end-labeling (TUNEL): The
end-labeling method of Gavrieli et al.(20) was used, except
that and no predigestion with proteinase K or Triton X-100
ENDOCHONDRAL OSSIFICATION IN THE CHICK
was carried out after preliminary experiments had shown
that it led to overstaining. The terminal transferase and
dUTP-DIG labeling mix were purchased from Boehringer
Mannheim (Mannheim, Germany). The incorporation of
digoxigenin-labeled dUTP was detected using monoclonal
antidigoxigenin Fab-fragments, linked directly to either
horseradish peroxidase or alkaline phosphatase (Boeringer
Mannheim; 1:1000 dilution; 1–2 h incubation), followed by
color development with AEC and H2O2 (15 minutes) or 4-Nitroblue tetrazolium chloride/5-bromo-4-cloro-3-indolylphosphate (NBT/BICP) (40 minutes). For some sections,
the TUNEL method was followed by the reaction for acid
phosphatase activity.
Negative controls were processed without any enzyme or
without the anti-DIG antibody. Some negative controls
were pretreated with DNAse as above. No reaction was
observed in negative controls, even when DNA breaks had
been created with DNAse.
Morphological assessment: Condensed nuclei and apoptotic
bodies: Since the TUNEL method is selective rather than
specific for apoptosis and also detects DNA strand breaks
in other physiological and pathological states,(35) and
since nuclear morphology is still the most reliable
method for quantifying apoptosis,(36) those sections routinely stained with hematoxylin/sirius red/alcian blue
were examined for the presence of pycnotic nuclei and
apoptotic bodies.
RESULTS
The growth plates of embryonic chick femurs
Although the cellular events of endochondral ossification
are similar in birds and mammals, there are several crucial
differences in the strategies used to achieve longitudinal
growth. In the mammalian growth plate, vascular invasion
and resorption occurs in synchrony at a similar horizontal
“plane” throughout the disc-like growth plate. Expansion by
proliferation is more or less matched by resorption so that
the thickness of the growth plate stays reasonably constant.
This is in marked contrast to the avian growth region where
the overall thickness of the growth plate increases considerably. For example, the thickness of the hypertrophic zone
increases from around 0.5 mm (9 days in ovo, Fig. 1A) to 1.8
mm (14 days, Fig. 1B) and nearly 3 mm (19 days, Fig. 1C),
whereas the thickness of the corresponding zone in a fastgrowing rat tibia is just 0.37 mm.(37) In addition, there is no
secondary center of ossification until after hatching, although some vascular canals (vc, Fig. 1B) are present in the
epiphyses of embryonic bones.
Primary resorption takes place at the tip marrow “tunnels” (mt), followed by secondary resorption at the “walls”
of the tunnels. Since these tunnels push up to the region of
flattened cells, it is not only hypertrophic chondrocytes that
are in proximity to the vascular/marrow elements. At the
lateral walls of the marrow tunnels, resorbing surfaces alternate with bone-forming surfaces where new bone matrix
is deposited onto the tunnel walls analogous to the deposition of bone onto cartilage spicules below the mammalian
growth plate. Specialized regions, not present in the mam-
797
malian growth plate, can be distinguished within the hypertrophic zone of the avian growth plate. Where endochondral bone covers the cartilage, the subjacent region is
temporarily protected from further resorption (protected
regions). Marrow tunnels tend to be positioned around the
periphery near the bone shaft (Fig. 1D), and regions between two marrow tunnels appear to “anchor” the bulk of
the epiphysis to the bone shaft (anchor regions, p in
Fig. 1C). Resorption at the main cartilage/marrow interface is carried out by both mono- and multinuclear osteoclasts, depending on whether the cartilage matrix has
calcified. Protected anchor regions frequently survive as
cartilage remnants in the main marrow space for some
time.
Identification of apoptotic cells with the TUNEL
method and morphological criteria
Preliminary experiments had shown that when the tissues
were predigested with proteinase K or treated with Triton,
staining was extremely variable and inconsistent. Even without such treatments, the number of cells containing
TUNEL-labeled nuclei was often higher than the number
of cells with “apoptotic morphology” (pycnotic nuclei or
apoptotic bodies). When sections were stained with hematoxylin/alcian blue/sirius red, normal chondrocytes appeared as pale cells with light, scarcely visible nuclei
(Fig. 2A). The weak nuclear staining with hematoxylin was
due to partial removal of the stain by the subsequent alcian
blue/sirius red counterstain, but reflected the diffuse chromatin of interphase chondrocytes. Other cells, such as chick
erythrocytes or osteoblasts, retained a more intensely
stained nucleus. Interspersed among these pale chondrocytes were cells with an intensely stained, pycnotic nucleus
(arrows). At low magnification, such cells were easy to miss,
but at high magnification their intense nuclear staining
clearly distinguished them from their pale neighbors. Very
occasionally, smaller fragmented bodies were found that
contained DNA strand breaks, as indicated by the TUNEL
reaction (p in Fig. 2B).
Incidence and localization of apoptotic chondrocytes
In the proliferative region, 0 –2% of chondrocytes contained TUNEL-labeled nuclei, and a similar number had an
apoptotic morphology, as assessed by hematoxylin staining.
In the hypertrophic region away from marrow tunnels,
0 –5% of cells were labeled with the TUNEL method,
roughly corresponding to the number of hypertrophic chondrocytes with pycnotic nuclei. These apoptotic chondrocytes
were found at apparently random locations throughout the
epiphyses and were present in femurs from 9-day-old embryos
as frequently as in 20-day-old embryos. It is noteworthy that in
some sections no apoptotic cells were present, whereas in
others several apoptotic cells were found in clusters. In addition to single apoptotic cells, cell doublets were occasionally
present where only one nucleus labeled with the TUNEL
reaction (arrow in Fig. 2B).
The incidence of apoptosis was increased near sites of
resorption. At the “top” of marrow tunnels, 5–15% of
798
ROACH
FIG. 1. Overall structure of the growth plates in femurs from 9- to
19-day-old chick embryos. All four sections are of the same magnification (315, bars 5 100 mm). (A, B, and C) Longitudinal sections of chick
femurs, stained with sirius red and alcian blue. (A) Nine-day embryo;
(B) 14-day embryo; (C) 19-day embryo; (D) transverse section through
the distal epiphysis of a femur from a 17-day-old embryo, Von Kossa
stain. The areas marked by squares show the approximate locations of
regions shown in higher magnification in later figures, the numbers
corresponding to the figure numbers. “Anchor” regions between two
marrow tunnels are indicated by stars in (C). bs, bone shaft; pz, proliferative zone; fz, zone of flattened cells; mz, maturing zone; hz, hypertrophic zone; mt, marrow tunnel; vc, vascular canals.
chondrocytes contained condensed nuclei (Fig. 2A). Such
cells were found up to 10 cell diameters around the distal
tip of the marrow tunnel. At the “resorbing” lateral walls of
marrow tunnels, apoptotic bodies were occasionally found
in opened lacunae (not shown). At the main cartilage/
marrow interface, up to 5% of cells contained a condensed
nucleus (not shown) or DNA breaks (Fig. 2C). In the chick,
the interface between the marrow space (M) and the cartilage (C) sometimes contained intermediate regions (I) in
which the “ground substance” of the cartilage matrix had
disappeared, but some structure still remained. Very condensed TUNEL-labeled cells were frequently found within
this structure, as if temporarily “trapped.” It is likely that
these cells were apoptotic chondrocytes that would otherwise have dispersed within the marrow space. The staining
in the cells in the area marked by the open circle is an
artifact.
Endochondral bone formation
To obtain clues as to the origin of the bone-forming cells,
the lateral “walls” of marrow tunnels were examined in
great detail. In the bones from younger chick embryos, the
majority of edges appeared to be “quiescent,” i.e., there was
no evidence for either bone formation or resorption. “Late”
bone-forming regions, i.e., regions where the walls of marrow tunnels were already covered with bone matrix, were
readily identified, particularly in bones from 18- to 20-dayold embryos. It was, however, extremely difficult to find
“early” bone-forming regions, i.e., where a resorbing sur-
ENDOCHONDRAL OSSIFICATION IN THE CHICK
face was just changing to a bone-forming surface. Such
regions were identified by the presence of a thin layer of
type I collagen (not shown). If chondrocytes became boneforming cells and if this process involved an asymmetric cell
division,(16) then one would expect to find “asymmetric”
cell doublets, i.e., where only one cell showed evidence of
apoptosis, or only one cell entered the cell cycle. However,
if chondrocytes underwent apoptosis, one would expect
single apoptotic chondrocytes in lacunae near the edge. The
presence of one apoptotic and one osteogenic cell inside
opened lacunae near the edge would be consistent with
either possibility.
At bone-forming surfaces, cell doublets were frequently
present, whereas away from the edge chondrocytic lacunae
only contained one cell. Incubation was with 3H-thymidine–
labeled cells that had entered the S-phase, for example,
most cells in the proliferative zone and in the osteogenic
layer of the periosteum (not shown). Some cells in the
marrow tunnels were also labeled (small arrows in Fig. 2D)
and an “asymmetric” lacuna was present, still separated
from the marrow space by a thin layer of matrix, where only
one cell had taken up the label (large arrow). The cells in
the marrow tunnel could either have been released from the
cartilage or migrated to the edge. The “asymmetric” doublet, however, provides evidence that an asymmetric division had occured and that one daughter cell had re-entered
the cell cycle. Cell doublets were not present in the hypertrophic region away from marrow tunnels.
At a slightly later stage, identified by a thicker layer of
type I collagen along the edge (Fig. 2E), osteoblasts (ob)
now lined the edge and had covered the surface with bone
matrix. Some intact lacunae “behind” this edge also contained type I collagen and usually two or more cells (arrows). Since this matrix had been formed inside lacunae, it
will be termed intralacunar bone matrix. As the endochondral bone matrix thickened, the edge no longer had a
scalloped appearance but became smooth and contained
osteocytes (Figs. 2F–2K). This temporarily protected the
subjacent cartilage from further resorption. In those areas,
cell doublets were found within intact lacunae, in which one
cell contained DNA strand breaks while the other one did
not (lacuna 4, large arrow in Fig. 2K).
TRAP activity in the new endochondral bone matrix
The endochondral bone matrix contained the typical
noncollagenous proteins of bone, such as osteopontin, osteonectin, bone sialoprotein, and osteocalcin (not shown).
In addition, the matrix contained TRAP activity (Figs. 2G
and 2I), which colocalized with type I collagen in lacunae 1,
2, and 3. The activity was inhibited by 2 mM NaF, whereas
osteoclast-associated TRAP activity required 10 mM NaF
for inhibition. TRAP activity was absent from a thin line of
unmineralized osteoid immediately beneath the cuboidal
osteoblasts (small triangles in Figs. 2G and 2I). In the same
sections, no TRAP activity was found in the mineralized
matrix of the intramembraneous bone shaft (bs in Figs. 2F
and 2G), except sometimes at “reversal lines” (arrows in
Fig. 3A) where it indicated previous osteoclast activity.(38,39)
799
Intralacunar bone formation in anchor regions
In the bones from 17- to 20-day chick embryos, small
pieces of former anchor regions were sometimes found
within the main marrow cavity. Since the a type I collagen
antibody had been raised against the extension peptide of
the a1(I) chain,(30) it always stained the recently synthesized collagen more strongly than the mature, processed
collagen. In Fig. 2F, the bone matrix of the bone shaft (bs,
on right) and the mineralized part of the endochondral
bone matrix stained weakly, indicating that this was mature
collagen. However, the intralacunar bone matrix and the
osteoid of the endochondral bone matrix showed very
strong immunoreactivity. This indicated that the latter two
were formed more recently than the first types of bone
matrix.
In anchor regions, the occasional cell doublet was found
in which only one cell contained DNA strand breaks
(Figs. 3A and 3B). More frequently, lacunae were filled
with type I collagen matrix and contained several cells. Such
lacunae were usually close to the marrow tunnel, but not
always connected to it, as shown by a series of confocal
images (Figs. 3C–3F), confirming that the type I collagen
had been formed inside intact lacunae.
Figure 3G shows the last remnant of an anchor region,
where the endochondral bone matrix had already been
resorbed by giant osteoclasts (oc) which will proceed to
resorb the whole anchor region. Here the intralacunar bone
is positive for acid phosphatase, whereas the bone of the
shaft (bs, lower right) does not contain acid phosphatase
activity. It is likely that this intralacunar bone had acquired
tartrate-resistant acid phosphatase (TRAP) activity with
mineralization, just as the endochondral bone had.
DISCUSSION
Since the requirement for rapid longitudinal expansion in
birds is much greater than in mammals, they have adopted
different strategies to achieve longitudinal growth. In mammals, the majority of longitudinal growth results from advancing the whole “plate,” i.e., cell proliferation and matrix
synthesis at the top of the plate is more or less matched by
resorption at the bottom of the plate. In birds, the thickness
of the growth plate increases in addition to distal expansion.
Resorption does not occur synchronously across the plate,
rather primary vascular invasion takes place at the tip of
“marrow tunnels,” which are regularly spaced around the
periphery, leaving the bulk of the cartilage initially intact.
Resorption of the remaining cartilage takes place at a later
time, at the lateral walls of the marrow tunnels and, even
later, at the main interface of cartilage with the marrow
cavity. There are some consequences of this different strategy: (1) most chondrocytes have a longer life span than in
mammals, particularly in the hypertrophic stage; (2) the
stages of resorption and endochondral bone formation are
temporally and spatially separated to a much greater extent
than in mammals, which facilitates study of the sequence of
events.
800
ROACH
ENDOCHONDRAL OSSIFICATION IN THE CHICK
801
FIG. 2. Apoptosis in chondrocytes and endochondral bone formation. (A) LS of the hypertrophic region around a
marrow tunnel in a 19-day femur, alcian blue/sirius red/hematoxylin. Many chondrocytes with morphological signs of
apoptosis (arrows) are present around the “tip” of the marrow tunnels. (B) TUNEL of the proliferative region of a 14-day
femur (see B for location). Apoptotic bodies are seen in one lacuna (p), while another lacuna (open arrow) contains an
“asymmetric doublet” where only one cell shows positive TUNEL labeling. (C) TUNEL-labeled cells and TRAP-positive
mononuclear resorbing cells (red) at the main cartilage (C)/marrow (M) interface (19-day femur; see C for location). Note
that some cells with TUNEL-labeled nuclei seemed to be “trapped” in an intermediate region (I), which is sometimes
found in the chick, where some “structure” remains, although the “ground substance” of the cartilage matrix has been
resorbed. Some artifactual staining is present in the cells marked by the open circle, possibly due to endogenous alkaline
phosphatase activity. (D) Autoradiography of an early bone-forming region in a femur which had been incubated with
3
H-thymidine for 18 h. Isotope label overlies several cells in the marrow tunnel (small arrows) and also one cell of an
“asymmetric doublet” whose lacuna is still separated from the marrow by a thin cartilage bridge (large arrow). (E) Type
I collagen immunocytochemistry showing a slightly later stage in endochondral bone formation. A continuous layer of type
I collagen (brown) has been deposited onto the wall and is also present intracellularly in the osteoblasts (ob) that have
accumulated along the edge. “Behind” the edge, type I collagen is present on the inner perimeter of two lacunae (arrows)
which contain two smaller cells each. (F and G) Serial sections of a former anchor region, (F) showing Type I collagen and
(G) and TRAP activity. TRAP activity co-localizes with the more mature collagen, but is absent from the newly-synthesized
endochondral and the intra-lacunar bone. (H, I, and K) Serial sections of a “protected” edge showing the localization of
(H) Type I collagen; (I) tartrate-resistant acid phosphatase activity and (K) TUNEL-labeled nuclei. Lacunae that are
recognizable in more than one section have been numbered. Resorption has taken place at the lower part of the picture,
and lacuna 1 has been opened. TRAP activity colocalizes with type I collagen in lacunae 2 and 3 and in the endochondral
bone matrix, except for a thin line which represents nonmineralized osteoid (small triangles). Lacuna 4 contains an
“asymmetric” doublet where only one nucleus contains DNA strand breaks. Lacuna 1 contains three nuclei, of which one
is labeled by the TUNEL method. The nonspecific blue reaction product in the osteoblasts lining the edge is due to
endogenous alkaline phosphatase activity. Bars, 10 mm.
4
Programmed cell death in chondrocytes
At the bottom of the growth plate, cartilage tissue and
cells need to be eliminated to make way for vascular canals
or bone marrow. In mammals, hypertrophy precedes resorption, and the accepted view is that chondrocytes undergo apoptosis at the terminal stage of hypertrophy. The
corresponding areas in the chick would be the main cartilage/marrow interface or resorbing regions at the lateral
walls of marrow tunnels. The present study confirmed that
hypertrophic chondrocytes underwent apoptosis at these
sites. In addition, primary resorption at the “tip” of marrow
tunnels occurred in the regions of resting or maturing chondrocytes. The chondrocytes just ahead of the advancing
“tip” underwent apoptosis prior to vascular invasion, suggesting that hypertrophy was not a prerequisite for apoptosis but that chondrocytes at any stage of differentiation may
be programmed to die.
This notion was strengthened by the findings that a small
number of apoptotic chondrocytes were present at apparently random sites throughout the epiphyseal cartilage. This
suggested that sporadic apoptosis occurred continuously
throughout chondrocyte differentiation. Although at any
one time the apoptotic index was low (0 –2%), with time a
significant proportion of chondrocytes might be affected,
since the characteristic morphological structures of apoptosis remain visible histologically for only a few hours. In a
variety of experimental tumors, the apoptotic index was
0.3–2.2%, which nevertheless accounted for an overall cell
loss of up to 90%.(40) Even in thymocytes, the “classic” cells
in which apoptosis has been extensively studied, only a
small number of cells show an apoptotic morphology at any
one time. Nevertheless, this seems to be sufficient for the
elimination of autoreactive T-cell clones during the devel-
opment of cellular immune self-tolerance.(41) Apoptosis of
chondrocytes during differentiation suggests that, like thymocytes, chondrocytes are generated in excess of eventual
requirement. It is possible that apoptosis serves to correct
“mistakes” during differentiation by eliminating faulty or
inappropriate cell types.(17)
Origin of the osteoblasts in endochondral
bone formation
This has been the subject of considerable debate and
controversy for at least 30 years. On the one hand, the
evidence for differentiation from marrow-stromal cells rests
on the osteogenic potential of the marrow stroma, as indicated by marrow transplantation or diffusion chamber experiments.(4,5) On the other hand, autoradiographic studies,(6,7) chick-quail chimaeric studies,(8) organ culture
studies of mouse metatarsalia,(13,14) or cut embryonic chick
femurs(14,16) have provided convincing evidence of the osteogenic potential of chondrocytes. Since it is not possible
to follow the fate of individual marrow stromal cells or
chondrocytes in situ, the crucial question of whether these
osteogenic potentials are realized in vivo has remained
unanswered, although it is generally assumed that marrow
stromal cells give rise to the endochondral osteoblasts.
Our previous study(16) identified asymmetric cell divisions as early events during the chondrocyte-to-osteoblast
transition in vitro. This concept provided a new approach
for in situ investigations of the fates of chondrocytes. In
vitro, the osteogenic differentiation of chondrocytes was
induced by a cut through the hypertrophic region,(14,16)
which resulted in (as yet unknown) changes in the extracellular microenvironment of the chondrocytes. Since the dif-
802
ROACH
FIG. 3. Intralacunar bone formation
in an anchor region. (A) Overview of
an anchor region bordered by marrow
tunnels and the bone shaft. TUNEL
method combined with TRAP histochemistry. In the bone shaft (left),
TRAP activity is only present along reversal lines (small arrows). The strong
activity in the bone matrix in the upper
part (star) suggests endochondral origin of that matrix. (B) An enlargement
of the central area shows one lacuna
containing an “asymmetric doublet”
with one TUNEL-labeled cell. (C, D,
E, and F) Four confocal images of an
anchor region where the bone shaft is
on the left, the marrow tunnel at the
bottom. Five lacunae near the edge
contain type I collagen (green, FITC)
inside the lacunae and up to four nuclei
each (orange, stained with propidium
iodide). The lacunae more distal to the
tunnel are intact. (G) TRAP histochemistry of a late remnant of an anchor region undergoing resorption by
giant osteoclasts (oc). The activity
within the chondrocyte lacunae is
present in intralacunar bone matrix, as
seen in a parallel section stained for
Type I collagen. Note that many lacunae contain more than one cell. Bars,
10 mm.
ENDOCHONDRAL OSSIFICATION IN THE CHICK
ferentiation state of chondrocytes is partly regulated by
their extracellular environment,(42) it is likely that cartilage
resorption in vivo also caused changes in the environment
of those chondrocyte near the resorptive front. These
changes might alter the differentiation state of the chondrocytes and provide the stimulus for a change in cell type.
The present study showed that “asymmetric doublets” were
located near regions where the cartilage matrix had been
resorbed, supporting the above concept. However, commitment to change phenotype was confined to chondrocytes
near bone-forming surfaces, i.e., at any one time the vast
majority of hypertrophic chondrocytes were not destined to
change phenotype. This would explain why isolated chondrocytes rarely become bone-forming cells unless additional
conditions are fulfilled, e.g., intrarenal transplantation,(11)
coculture with brain tissue,(13,43) or the addition of retinoic
acid,(15) although mouse Meckel’s cartilage chondrocytes
seem to have the capacity to transform to osteocyte-like
cells in vitro without any additional factors.(17)
Even at bone-forming surfaces, the incidence of “asymmetric doublets” was low, suggesting either that only a few
chondrocytes transdifferentiated to bone-forming cells at
any one time and/or that an asymmetric cell division was a
very short-lived event. The occurrence of an asymmetric cell
division in itself did not prove that the viable cell of that
division would become osteogenic. This required further
evidence, such as the expression of typical bone proteins or,
at a later stage, the deposition of bone matrix. This evidence
was readily obtained where the surface of the cartilage had
been covered with endochondral bone matrix, as in the
“protected” and “anchor” regions of the chick growth plate.
Here the lacunae remained intact, and unambiguous proof
that chondrocytes had became bone-forming cells was obtained. Similarly, a direct chondrocyte-osteoblast transition
had been demonstrated in the cartilaginous center of the
so-called “mixed” spicules during fracture repair.(44) In
view of this, the possibility that the viable cell of an asymmetric cell division became osteogenic after release into the
marrow space cannot be discounted.
TRAP activity in the endochondral bone matrix
TRAP is generally recognized as a histochemical marker
for skeletal resorbing cells, such as osteoclasts(45,46) and
their precursor cells. Hence, it was surprising to find TRAP
activity in the mineralized matrix of endochondral bone,
although in all other aspects the endochondral bone matrix
was identical to intramembraneous bone matrix. The presence of TRAP activity thus provided a way to distinguished
the two types of bone matrix. Similar results were obtained
for the medullary bone which is formed in egg-laying
hens.(47– 49) No enzyme activity was detected in osteoblasts
or the osteoid of endochondral bone, just as the osteoblasts
or osteoid of medullary bone did not have any TRAP
activity.(49) Either an inactive form of the enzyme was
secreted by osteoblasts, which became activated with calcification of the matrix,(49) or acid phosphatase was present in
the osteoblasts, but activity was below the level of detection.
There have been reports that chick(50) and rat(51) osteoblasts contained fluoride-sensitive TRAP activity, albeit
803
much less than osteoclasts. Hence, the osteoblasts that lay
down the endochondral bone matrix are in at least one
respect different from the periosteal osteoblasts which lay
down intramembraneous bone matrix.
The presence of TRAP also raises the question as to the
function of the enzyme in endochondral and medullary
bone. Both are temporary bone structures, destined for
resorption once they have fulfilled their function. Nevertheless, during an interim time period, the presence of
endochondral bone is crucial in protecting selected regions
of cartilage from resorption. TRAP dephosphorylates osteopontin and bone sialoprotein and thereby detaches osteoclasts,(52) so the presence of TRAP activity throughout
the matrix of endochondral bone might temporarily prevent
remodeling.
The fate of the hypertrophic chondrocytes at the avian
growth plate
Apart from the sporadic apoptosis that occurred in a
small number of chondrocytes at all stages of differentiation, the fate of avian chondrocytes depended on their
location within the growth plate. Where resorption was the
prime requirement, as at the top of marrow tunnels or at
the main cartilage/marrow interface, the available evidence
suggested that chondrocytes underwent apoptosis. However, where selected areas of cartilage required “strengthening,” as in anchor regions, bone formation took place
within lacunae by former chondrocytes. In bone-forming
regions at the walls of marrow tunnels, chondrocytes underwent asymmetric cell divisions, consistent with a chondrocyte-to-osteoblast transition. This strengthens the evidence that chondrocytes as well as marrow stromal cells
give rise to endochondral osteoblasts. The novel finding
that the avian endochondral bone matrix contains TRAP
activity indicates that endochondral osteoblasts may be different from periosteal osteoblasts.
Do chondrocytes become bone-forming cells at the
mammalian growth plate?
There have been many excellent and detailed histological
studies of the terminal chondrocytes in the mammalian
growth plate,(53–55) but evidence of an osteogenic differentiation was never found in that region. This is not surprising,
since the site of vascular invasion in mammals would correspond to the top of marrow tunnels in birds, where no
evidence of osteogenic differentiation was found either. In
mammals, most of the cartilage spicules that provide the
scaffolds for endochondral bone formation do not contain
cells, and hence the conditions for a chondrocyte-osteoblast
transition are not present. However, sometimes chondrocytes do persist within these such spicules and become
trapped by the deposition of endochondral bone, analogous
to the “mixed spicules” of the fracture callus. It is in those
areas, which are found at the side or below the actual
growth plate, where one might expect bone-forming cells
within intact chondrocytic lacunae.
804
ROACH
ACKNOWLEDGMENTS
The author gratefully acknowledges the technical assistance of Mr. Adrian Wilkins, the provision of antisera by
Prof. Larry Fisher (Bethesda) and Prof. Louis Gerstenfeld
(Boston), and the financial support of the Wishbone Trust.
REFERENCES
1. Pechak DG, Ilujawa MJ, Caplan AL 1986 Morphology of bone
development and bone remodeling in embryonic chick limbs.
Bone 7:459 – 472.
2. Lewinson D, Silbermann M 1992 Chondroclasts and endothelial cells collaborate in the process of cartilage resorption. Anat
Rec 233:504 –514.
3. Hunziker EB 1994 Mechanism of longitudinal bone growth
and its regulation by growth plate chondrocytes. Microscop
Res Tech 28:505–519.
4. Marks SC, Popoff SN 1988 Bone cell biology: The regulation of
development, structure and function in the skeleton. Am J
Anat 183:1– 44.
5. Beresford JN 1989 Osteogenic stem cells and the stomal system
of bone and marrow. Clin Orthop Rel Res 240:270 –280.
6. Holtrop ME 1966 The origin of bone cells in endochondral
ossification. In: Fleisch H, Blackwood HJJ, Owen M (eds.)
Third European Symposium on Calcified Tissues. Springer,
New York, NY, U.S.A., pp. 32–36.
7. Crelin ES, Koch WE 1967 An autoradiographic study of chondrocyte transformation into chondroclasts and osteocytes during bone formation in vitro. Anat Rec 158:473– 484.
8. Kahn AJ, Simmons DJ 1977 Chondrocyte-to-osteocyte transformation in grafts of perichondrium free epiphyseal cartilage.
Clin Orthop Rel Res 129:299 –304.
9. Weiss A, Von der Mark K, Silbermann M 1987 Fully differentiated chondrocytes alter their phenotype expression in vitro
and transform into bone cells. In: Hurwitz S, Sela J (eds.)
Current advances in Skeletogenesis/3. Heiliger Publishing,
Jerusalem, Israel, pp. 40 – 43.
10. Yoshioka C, Yagi T 1988 Electron microscopic observations on
the fate of hypertrophic chondrocytes in chondylar cartilage of
rat mandible. J Craniofacial Genet Dev Biol 8:253–264.
11. Moskalewski S, Malejczyk J 1989 Bone formation following intrarenal transplantation of isolated murine chondrocytes: Chondrocytebone cell transdifferentiation. Development 107:473–480.
12. Strauss PG, Closs EI, Schmidt J, Erfle V 1990 Gene expression
during osteogenic differentiation in maudibular condyles in
vitro. J Cell Biol 110:1369 –1378.
13. Thesingh CW, Groot CG, Wassenaar AM 1991 Transdifferentiation of hypertrophic chondrocytes in murine metatarsal bones,
induced by co-cultured cerebrum. Bone Miner 12:25–40.
14. Roach HI 1992 Trans-differentiation of hypertrophic chondrocytes into cells capable of producing a mineralized bone matrix.
Bone Miner 19:1–20.
15. Descalzi-Cancedda F, Gentili C, Manduca P, Cancedda R 1992
Hypertrophic chondrocytes undergo further differentiation in
culture. J Cell Biol 117:427– 435.
16. Roach HI, Erenpreisa J, Aigner T 1995 Osteogenic differentiation of hypertrophic chondrocytes involves asymmetric cell
divisions and apoptosis. J Cell Biol 131:483– 493.
17. Erenpreisa J, Roach HI 1996 Epigenetic selection as a possible
component of trans-differentiation. Mech Aging Dev 487:165–182.
18. Ishizeki K, Takigawa M, Nawa T, Suzuki F 1996 Mouse Meckel’s
cartilage chondrocytes evoke bone-like matrix and further transform into osteocyte-like cells in culture. Anat Rec 245:25–35.
19. Wyllie AH 1992 Apoptosis and the regulation of cell numbers
in normal and neoplastic tissues: an overview. Cancer Metast
Rev 11:95–103.
20. Gavrieli Y, Sherman Y, Ben-Sasson SA 1992 Identification of
programmed cell death in situ via specific labeling of nuclear
DNA fragmentation. J Cell Biol 119:493–501.
21. Gibson GJ, Kohler WJ, Schaffler MB 1995 Chondrocyte apo
ptosis in endochondral ossification of chick sterna. Dev Dynam
203:466 – 476.
22. Hatori M, Klatte KJ, Teixeira CC, Shapiro IM 1995 End
labelling studies of fragmented DNA in the avian growth plate:
Evidence of apoptosis in terminally differentiated chondrocytes. J Bone Miner Res 10:1960 –1968.
23. Bronckers ALJJ, Goei W, Luo G, Karsenty G, D’Souza RN,
Lyaruu DM, Burger EH 1996 DNA fragmentation during bone
formation in neonatal rodents assessed by transferase-mediated end labelling. J Bone Miner Res 11:1281–1291.
24. Roach HI 1990 Long-term organ culture of embryonic chick
femurs: A system for investigating bone and cartilage formation at an intermediate level of organization. J Bone Miner Res
5:85–100.
25. Hunziker EB, Herrmann W, Schenk RK 1982 Improved cartilage fixation by ruthenium hexammine bichloride is a prerequiste for morphometry in growth cartilage. J Ultrastruct Res
81:1–12.
26. Nuehring LP, Steffens WL, Rowland GN 1991 Comparison of
the ruthenium hexaminine bichloride method to other methods
of chemical fixation for preservation of avian physeal cartilage.
Histochem J 23:201–214.
27. Lison L 1954 Alcian blue 8G with Chlorantine fast red 5B: A
technique for selective staining of mucopolysaccharides. Stains
Tech 29:131–138.
28. Bancroft DJ, Stevens A (eds.) 1982 Theory and Practice of
Histological Techniques, 2nd Ed. Churchill Livingstone, Edinburgh, U.K., pp. 384 –387.
29. Roach HI, Shearer JR 1989 Cartilage resorption and endochondral bone formation during the development of long
bones in chick embryos. Bone Miner 6:289 –309.
30. Fleischmajer R, McDonald ED, Perlish JS, Burgeson RE,
Fisher LW 1990 Dermal collagen fibrils are hybrids of type I
and type III collagen molecules. J Struct Biol 105:162–169.
31. McKee MD, Nanci A, Laudis WJ, Gotoh Y, Gerstenfeld LC,
Glimcher MJ 1990 Developmental appearance and ultra-structural immunolocalization of a major 66 KDa phosphoprotein
in embryonic and postnatal chicken bone. Anat Rec 228:77–92.
32. Neugebauer BM, Moore MA, Broess M, Gerstenfeld LC,
Hauschka PV 1995 Characterization of structural sequences in
the chicken osteocalcin gene: Expression of osteocalcin by
maturing osteoblasts and by hypertrophic chondrocytes in
vitro. J Bone Miner Res 10:157–163.
33. Yang R, Gotoh Y, Moore MA, Rafidi K, Gerstenfeld LC 1995
Characterization of an avian bone sialoprotein (BSP) cDNA:
Comparison to mammalian BSP and identification of conserved structural domains. J Bone Miner Res 10:632– 640.
34. Pacifici M, Oshima O, Fisher LW, Young MF, Shapiro IM,
Leboy PS 1990 Changes in osteonectin distribution and levels
are associated with mineralization of the chicken tibial growth
cartilage. Calcif Tissue Int 47:51– 61.
35. Ansari B, Coates PJ, Greenstein BD, Hall PA 1993 In situ
end-labelling detects DNA strand breaks in apoptosis and
other physiological and pathological states. J Path 170:1– 8.
36. Martin SJ, Green DR, Cotter TG 1994 Dicing with death:
Dissecting the components of the apoptosis machinery. Trends
Biochem Sci 19:26 –30.
37. Hunziker EB, Schenk RK 1989 Physiological mechanisms
ENDOCHONDRAL OSSIFICATION IN THE CHICK
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
adopted by chondrocytes in regulating longitudinal bone
growth in rats. J Physiol 414:55–71.
Reinholt FP, Widholm SM, Ex-Rylander B, Andersson G 1990
Ultrastructural localization of a tartrate-resistant acid ATPase
in bone. J Bone Miner Res 5:1055–1060.
Roach HI 1994 Why does the bone matrix contain non-collagenous proteins? The possible roles of osteocalcin, osteonectin,
osteopontin and bone sialoprotein in bone mineralisation and
resorption. Cell Biol Int 18:617– 627.
Sarraf CE, Bowen ID 1988 Proportions of mitotic and apoptotic cells in a range of untreated experimental tumors. Cell
Tissue Kinet 21:45– 49.
Surth CD, Sprent J 1994 T-cell apoptosis detected in situ
during positive and negative selection in the thymus. Nature
372:100 –103.
Cancedda R, Cancedda FD, Castagnola P 1995 Chondrocyte
differentiation. Int Rev Cytol 159:265–358.
Groot CG, Thesingh W, Wassenaar AM, Scherft JP 1994
Osteoblasts develop from isolated fetal mouse chondrocytes
when co-cultured in high density with brain tissue. In Vitro Cell
Dev Biol 30A:547–554.
Scammell BE, Roach HI 1996 A new role for the chondrocyte
in fracture repair: Endochondral ossification includes direct
bone formation by former chondrocytes. J Bone Miner Res
11:737–745.
Minkin C 1982 Bone acid phosphatase: Tartrate-resistant acid
phosphatase as marker of osteoclast function. Calcif Tissue Int
34:285–290.
Hermanns W 1987 Identification of osteoclasts and their differentiation from mononuclear phagocytes by enzyme histochemistry. Histochemistry 86:225–227.
Yamamoto T, Nagai H 1992 A histochemical study of acid
phosphotase in medullary bone matrix and osteoclasts in laying
japanese quail. J Bone Miner Res 7:1267–1273.
Yamamoto T, Nagai N 1994 Tartrate-resistant acid phosphatase accumulated in the matrix of developing medullary bone
805
49.
50.
51.
52.
53.
54.
55.
induced by estrogen treatment of male japanese quail. J Bone
Miner Res 9:1153–1157.
Yamamoto T, Yamagata A, Nagai H 1995 Histochemical reactivity of tartrate-resistant acid phosphatase in the matrix of developing medullary bone. Acta Histochem Cytochem 28:319 –322.
Lundy MW, Lau K-HW, Blair HC, Baylink DJ 1988 Chick
osteoclasts contain fluoride-sensitive acid phosphatase activity.
J Histochem Cytochem 36:1175–1180.
Bianco P, Ballant P, Bonnucci E 1988 Tartrate-resistant acid
phosphatase activity in rat osteoblasts and osteocytes. Calcif
Tissue Int 43:167–171.
Ek-Rylander B, Flores M, Wendel M, Heinegard D, Andersson G 1994 Dephosphorylation of osteopontin and bone sialoprotein by osteoclastic tartrate-resistant acid phosphotase.
J Biol Chem 269:14853–14856.
Brighton CT, Sugioka Y, Hunt RM 1973 Cytoplasmic structures
of epiphyseal plate chondrocytes. J Bone Joint Surg 55A:771–784.
Hanaoka H 1976 The fate of hypertrophic chondrocytes of the
epiphyseal plate. J Bone Joint Surg 58A:226 –229.
Farnum CE, Wilsman NJ 1989 Cellular turnover at the chondro-osseous junction of growth plate cartilage: Analysis by
serial sections at the light microscpical level. J Orthop Res
7:645– 666.
Address reprint requests to:
Dr. H.I. Roach
Academic Orthopaedic Unit
CF 86, MP 817
Southampton General Hospital
Tremona Road
Southampton, SO16 6YD, U.K.
Received in original form September 9, 1996; in revised form
December 6, 1996; accepted January 8, 1997.