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Ultrastructural and histochemical studies of the epiphyseal plate in normal chicks.

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THE ANATOMICAL RECORD 242:29-39 (1995)
Ultrastructural and Histochemical Studies of the Epiphyseal Plate in
Normal Chicks
MASATO TAKECHI AND CHITOSHI ITAKURA
Mitsubishi Kasei Institute of Toxicological and Environmental Sciences, Hasaki, Kashima,
Department of Comparative Pathology, Faculty of
Ibaraki 314-02, Japan (M.T.);
Veterinary Medicine, Hokkaido University, Sapporo 060, Japan (C.I.)
ABSTRACT
Background: Chondrocytes in the epiphyseal plate undergo a series of well-defined stages, each stage containing a morphologically homogeneous cell population. However, biochemical studies show
that there are some functionally heterogeneous cell types in the calcifying
zone of the chick epiphyseal plate.
Methods: We studied the sequence of chondrocytic maturation in the normal chick epiphyseal plate ultrastructurally and histochemically. Chondrocytes in the calcifying zone were of three distinct types, the appearance of
each cell type being closely related to the stage of matrix calcification.
Results: Clear cells were observed in the upper calcifying region, stellate
cells appeared in the middle calcifying region, and hypertrophic clear cells
appeared in the lower calcifying region. Rough endoplasmic reticulum
(RER) and lysosome-rich cells were found, these being limited to the outermost layers of the calcifying zone and containing ACPase-positive products. Osteoclasts were attached to the matrix near the RER and lysosomerich cells in the poorly calcified regions.
Conclusion: We hypothesized that each cell type played a different role in
the initiation, progression, and maintenance of cartilage calcification. RER
and lysosome-rich cells may be responsible for the resorption of uncalcified
cartilage matrix, this resulting in induction of the osteoclastic resorption of
the calcified matrix. In addition, the fate of the chondrocytes was twofold
hypertrophic clear cells died, while the RER and lysosome-rich cells survived, suggesting that these cells were transformed into osteogenic cells.
0 1995 Wiley-Liss, Inc.
Key words: Chondrocytes, Calcifying zone, Chondrocytic maturation,
chicks
The ultrastructure of the epiphyseal plate has been
studied in mammals (Anderson, 1964; Thyberg and
Friberg, 1971; Holtrop, 1972a,b) and in chicks (Lutfi,
1974; Howlett, 1979). In the calcifying region of the
mammalian epiphyseal plate (Hwang, 1978; Wilsman
et al., 1981;Carlson et al., 1985),it has been shown that,
although chondrocytes are generally a morphologically
homogeneous cell population a t each stage of maturation, there are some morphologically discrete populations.
Because of their entrapment in a nutrient-poor and
oxygen-poor calcified matrix, the chondrocytes of the
calcifying zone have been regarded as degenerating or
dying cells (Brighton et al., 1973; Stambaugh and
Brighton, 1980). However, more recent studies have
indicated that chondrocytes in the calcifying zone are
viable and play a major role in regulating cartilage
calcification (Hunziker et al., 1984; Boyde and Shapiro,
1987;Farnum et al., 1990).It is generally believed that
the initial calcification of cartilage matrix is caused by
the release of intracellular calcium in chondrocytes,
this being induced by depletion of the energy source, due
to low oxygen tension or the consumption of glycogen
0 1995 WILEY-LISS, INC.
granules (Brighton and Heppenstall, 1971; Brighton,
1978). Scanning electron microscopic and biochemical
studies of the chick epiphyseal plate have, however,
demonstrated that the initial calcification is due to a
change in phenotypic expression related to chondrocyte
maturation and not to environmental factors (Shapiro
and Boyde, 1987). The progress of further calcification
may be controlled by the chondrocytes, in terms of their
production of matrix components (Ali, 1992).The study
of collagen mRNA in the chick epiphyseal plate has
identified two functionally discrete populations of chondrocytes in the calcifying region (Leboy et al., 1988).
These observations suggest the presence of functionally
heterogeneous cell types in the calcifying region of the
chick epiphyseal plate, each of the types playing an
important role in the initiation and progression of cartilage calcification. In this paper, we describe the ultrastructure and histochemical activity of the normal chick
epiphyseal plate, classifying the chondrocytes in the
calcifying zone and referring to the fate of these cells.
Received July 27, 1994; accepted December 12, 1994.
30
M. TAKECHI AND C. ITAKURA
MATERIALS AND METHODS
The birds used in this study were 1-day-old male
commercial broiler chicks. They were housed in floor
pens heated with a n electric bulb and were fed a commercial diet and given water ad libitum. Two chicks
each were selected a t random a t 4, 7, 14, 18, and 21
days old and sacrificed for necropsy. They were anesthetized with ether, and 2% glutaraldehyde in cacodylate sucrose buffer (pH 7.4) was perfused into the right
ventricle for approximately 15 min. For light microscopy, the bone tissues from the left femur and tibia
were fixed in 10% formalin solution, subjected to decalcification in 5% formic acid, and embedded in paraffin wax. Sections were stained with hematoxylin and
eosin. For electron microscopy, thin vertical sections
were sliced from the cartilaginous epiphysis of the
proximal ends of the right femur and tibia. Some samples were immersed in 2% glutaraldehyde in cacodylate sucrose buffer and kept at 4°C for 2 h. Others were
decalcified in 5% ethylenediaminetetraacetic acid (pH
7.4) at 4°C for 3-5 days. The tissues were washed several times in buffer, postfixed in 1%0,04in buffer for
1h at 4"C, dehydrated in a series of graded concentrations of cold ethanol, and embedded in Epon 812 after
alcohol was replaced with propylene oxide. Thick sections, cut with glass knives on a LKB ultramicrotome,
were stained with toluidine blue. Thin sections were
cut with diamond knives and stained with uranyl acetate and lead citrate. The sections were examined with
a Hitachi HT-7000 electron microscope.
For histochemical studies, decalcified sections from
21-day-old chicks were immersed in cold cacodylate
buffer containing 20% dimethylsulfoxide and subsequently quenched in isopentane chilled with liquid nitrogen. Specimens were sectioned a t about 40 pm
thickness in a cryostat at -30°C. For the examination
of ALPase activity, the frozen sections were pretreated
with a 10 mM MgC1, solution to reactivate enzyme
activity. The reactivated sections were then rinsed in
cold tris-maleate sucrose buffer, followed by incubation
for 20 min at room temperature in reactive medium,
performed by the metal-salt technique, using p-glycerophosphate as a substrate and lead citrate as a coupler. For the examination of ACPase activity, thick
sections were incubated for 20 min a t room temperature in the reactive medium, according to Gomori's
metal-salt technique, using p-glycerophosphate as a
substrate and lead nitrate a s a coupler. Following incubation, the sections were rinsed, postfixed with 1%
0,04,dehydrated, and embedded in Epon 812 for electron microscopic examination.
For the quantitative analysis of cellular profiles in
the chondrocytic lacunae a t the outermost layer of the
calcifying zone, about 50 lacunae, including cellular
elements from the upper, middle, and lower calcifying
regions of each animal, were examined ultrastructurally. Chondrocytic lacunae were classified as unopened
and opened. To calculate the percentage of osteoclasts
coexistent with the RER and lysosome-rich cells in the
calcifying zone, about 10 osteoclasts attached to the
surface of the cartilage columns from each of the upper,
middle, and lower calcifying regions of each animal
were examined ultrastructurally. We defined osteoclasts as being coexistent with RER and lysosome-rich
cells when the osteoclasts were attached to the matrix
Fig. 1. Longitudinal section through the proximal epiphyseal plate
of the tibia of a 14-day-old chick. The epiphyseal plate is divided into
four zones: resting (R), proliferating (P),maturating (M), and calcifying (upper, CU; middle, CM; lower, CL). Decalcified. H-E stain. x 40.
near these cells and their cytoplasmic branches invaded the lacunae of the RER and lysosome-rich cells.
RESULTS
The epiphyseal plate consisted of four cellular zones:
resting, proliferating, maturating, and calcifying (Fig.
1). The morphologic features were essentially the
same, regardless of the age of the chicks.
Chondrocytes in the resting zone were oval and had
many short cell processes (Fig. 2). The RER showed
parallel arrays of several flattened cisternae filled
with moderately electron-dense materials. The Golgi
apparatus consisted of flattened cisternae and small
vesicles. Some secretory vesicles containing light floc-
STUDIES OF CHICK EPIPHYSEAL PLATE
31
Fig. 2. A chondrocyte in the resting zone of the tibial epiphyseal
plate of a 14-day-old chick. The cell has well-developed RER and a
moderately developed Golgi apparatus. A secretory vesicle is releasing its contents (arrow).Decalcified. X 9,600.
Fig. 3. Chondrocytes in the proliferating zone of the tibial epiphyseal plate of a 14-day-oldchick. These cells have well-developed RER.
Their nuclei are located at the center, are elongated, and have large
nucleoli. Decalcified. x 6,400.
culent and granular materials were observed in the
Golgi area and the periphery of the cytoplasm. These
vesicles were close to the plasma membrane and were
releasing their contents.
Chondrocytes in the proliferating zone were spindleshaped, with short cell processes (Fig. 3). The nuclei,
located at the center, were elongated and had large
nucleoli and dispersed chromatin granules. Mitotic figures were often observed in this zone. The RER was
well developed, and ribosomes were arranged randomly or formed rosettes. The Golgi apparatus was
small, and some secretory vesicles were scattered in
the cytoplasm.
Chondrocytes in the maturating zone were rich in
cytoplasm and large (Fig. 4). An enlarged Golgi apparatus, associated with numerous secretory vesicles,
was present in the center of the cytoplasm. The peripheral area of the cytoplasm was rich in enlarged RER,
some of which were dilated and contained moderately
electron-dense materials. The pericellular matrix area
was apparent; this was pale, free from collagen fibrils,
and rich in granular materials. Long cell processes extended into the matrix and showed matrix vesicle production by the budding and detachment of their bulbous tips (Fig. 5). Numerous vesicles were scattered in
the matrix in this zone, but these vesicles contained no
apatite crystals.
The calcifying zone was divided into three regions,
the upper, middle, and lower, in terms of the stage of
matrix calcification and chondrocyte morphology. In
the upper region of the calcifying zone, matrix calcification was initiated with the deposition of apatite crystals in the matrix vesicles (Fig. 6). The change from
maturating to calcifying features in chondrocytes occurred abruptly, so that, in a pair of adjacent cells, one
exhibited the features of a maturating zone cell and the
other the characteristics of an upper calcifying region
cell. Chondrocytes in the upper region were scallop-like
in shape, with long cell processes; the cells were rich in
electron-lucent cytoplasm and had reduced cell organelles; these cells we termed clear cells (Fig. 6 ) .The
RER was irregularly arranged and widely separated or
segmentary. The Golgi apparatus was small and had
few secretory vesicles. These cells were surrounded by
moderately widened pericellular matrix. With the proliferation and growth of crystals within the matrix vesicles, the vesicle membrane was disrupted and the crystals formed feather-like clusters (Fig. 7). These crystal
clusters spread through the longitudinal septum.
In the middle region of the calcifying zone, small
crystal clusters were also present and had coalesced,
with the adjacent deposition of apatite crystals on collagen fibrils (Fig. 8).Thus, the clusters formed calcified
bands in the longitudinal septum. However, the peri-
32
M. TAKECHI AND C. ITAKURA
Fig. 4. Chondrocyte in the maturating zone of the tibial epiphyseal
plate of a 14-day-old chick. The cell has many long cell processes,
well-developed RER, and a n enlarged Golgi apparatus, associated
with numerous secretory vesicles, in the center. Pericellular matrix
(PM) is apparent and contains granular materials. Decalcified.
x 9.600.
Fig. 5. Matrix vesicles in the maturating zone of the tibial epiphyseal plate of a 14-day-old chick. The cell processes of the maturating
cell extend into the matrix, in which matrix vesicles seem to be
formed by detachment of the bulbous tips of the cell processes (arrow).
Numerous vesicles are present in the matrix. Decalcified. x 11,000.
cellular matrix, territorial matrix, and transverse septum were not calcified. The chondrocytes in this zone
were electron-dense and stellate, with long cell processes; we termed these stellate cells (Fig. 8). The pericellular matrix area was markedly wide and filled with
granular and flocculent materials. The nuclei of the
stellate cells contained diffusely dispersed chromatin
granules and large nucleoli. The cytoplasm of these
cells was rich in RER and ribosomes. Some RER were
dilated and filled with moderately electron-dense materials. Ribosomes were arranged randomly o r formed
rosettes. The Golgi apparatus was small to medium,
and there were no secretory vesicles in the cytoplasm.
In the lower region of the calcifying zone, the longitudinal septum was completely calcified, and endochondral ossification was in progress. The chondrocytes
in this region were extremely pale and ballooned, and
had short cell processes; we termed these cells “hypertrophic clear cells” (Fig. 9). The pericellular matrix was
obscure, so that the cells were surrounded by the territorial matrix. The hypertrophic clear cells were poor
in cell organelles; they had segmented RER, small
Golgi apparatus, some mitochondria and variable sized
vacuoles. There were no secretory vesicles.
The lowest transverse septa of the cartilage columns
(i.e., the uncalcified portions) were sometimes destroyed by monocyte-like mononuclear cells (Fig. 9).
Some mononuclear cells had invaded the chondrocytic
lacunae, through the opened transverse septa, and had
phagocytized degenerative hypertrophic clear cells
(Fig. 10).
In addition to the three major cell types described
above, cells of another type were observed in the calcifying zone. These cells were limited in location to the
outermost layer and the surface of the cartilage columns. They were characterized by well-developed RER
and dense lysosomal bodies (Fig. 11).RER in the cytoplasm consisted of flattened cisternae filled with moderately electron-dense materials. Both primary and
secondary dense lysosomal bodies were present, varying considerably in both number and size. The Golgi
apparatus was small but without secretory vesicles.
These cells were found in both opened and unopened
lacunae. In the unopened lacunae of these cells, the
pericellular matrices were wide but contained few matrix components. The territorial matrix surrounding
the RER and lysosome-rich cells varied in width; the
zone facing the marrow cavity was narrow and occasionally disappeared (Figs. 11,12). The cell processes of
these cells extended into the remaining territorial ma-
STUDIES OF CHICK EPIPHYSEAL PLATE
Fig. 6. A Upper calcifying region of the tibial epiphyseal plate of a
14-day-old chick. One chondrocyte (MC) exhibits features of a maturating zone cell, and other cells (CC) are characteristic of calcifying
zone cells (clear cells). Clear cells have electron-lucent cytoplasm with
reduced cell organelles, including segmented RER, small Golgi apparatus, and a few secretory vesicles. Many matrix vesicles are scattered
in the matrix (arrowheads). Not decalcified. x 3,200. B: Matrix vesicles
contain needle-like apatite crystals (arrows). Not decalcified. x 71,000.
33
Fig. 7. A Upper calcifying region of the tibial epiphyseal plate of a
14-day-old chick. Crystal clusters are diffusely distributed in the longitudinal septum. Clear cells are observed. Not decalcified. X 4,400. B:
Apatite crystals grow to form crystal clusters. Not decalcified.
x 40,000.
34
M. TAKECHI AND C. ITAKURA
Fig. 8. A Middle calcifying region of the tibia1 epiphyseal plate of a 14-day-old chick. The calcified band
spreads through the longitudinal septum, but the pericellular matrix (PM), territorial matrix (arrowheads), and transverse septum (TS)are not. Stellate cells (SC) are electron-dense, with long cell processes; their cytoplasm is packed with RER and ribosomes. Not decalcified. x 3,200. B Apatite crystals
deposited on collagen fibrils around crystal clusters. Not decalcified. x 88,000.
trix but not into the side where the territorial matrix
had disappeared.
Quantitative analysis of the cellular profiles within
the chondrocytic lacunae in the outermost layer of the
calcifying zone is shown in Table 1.The morphology of
the cells in the lacunae was assessed as “chondrocyte”
or “RER and lysosome-rich cell” according to the characteristics described above. In the upper and middle
calcifying regions, 93% and 94%, respectively, of the
unopened lacunae of each region were occupied by
chondrocytes, and 7% and 6%, respectively, were occupied by RER and lysosome-rich cells. In these regions,
78% and 73%, respectively, of the opened lacunae were
occupied by RER and lysosome-rich cells, and 22% and
27%, respectively, were occupied by chondrocytes. In
the lower calcifying region, most unopened lacunae
were occupied by chondrocytes, and 78% of the opened
lacunae were occupied by chondrocytes and 22% were
occupied by RER and lysosome-rich cells. The total
number of RER and lysosome-rich cells combined in
the unopened and opened lacunae of all regions was
one-fifth of the total number of chondrocytes.
Many osteoclasts in the upper and middle calcifying
regions were attached to the matrix near the RER and
lysosome-rich cells. They began to resorb the calcified
matrix forming a ruffled border (Fig. 11).Their cyto-
plasmic branches invaded the chondrocytic lacunae of
the RER and lysosome-rich cells (Fig. 12). The frequency of osteoclasts coexisting with the RER and lysosome-rich cells in the calcifying zone is shown in Table 2. In the upper and middle calcifying regions, 43%
and 34%, respectively, of the osteoclasts of each region
attached to the cartilage surface coexisted with RER
and lysosome-rich cells. In the lower calcifying region,
only 5% of the osteoclasts coexisted with RER and lysosome-rich cells. In the middle and lower calcifying
regions, collagen fibrils and osteoblasts covered the
cartilage surface, and they invaded the opened lacunae
of the RER and lysosome-rich cells (Fig. 13). The RER
and lysosome-rich cells were liberated from the lacunae to the marrow cavity or osteoid tissue (Fig. 14).
These liberated cells had normal cell architecture, with
well-developed RER and ribosomes. Pinocytic vesicles
were often observed in the periphery of the cytoplasm.
In the histochemical studies, ALPase activity reaction was observed in the chondrocytes of the proliferating, maturating, and calcifying zones but not in
the resting zone cells. Reaction products were seen on
the plasma membrane of chondrocytes and in association with matrix vesicles (Fig. 15). Intensive reaction
products were also seen in the osteoblasts. RER and
lysosome-rich cells showed no ALPase activity. The
STUDIES OF CHICK EPIPHYSEAL PLATE
35
Fig. 9.Lower calcifying region of the tibial epiphyseal plate of a
14-day-old chick. Hypertrophic clear cells (HC) are poor in cell organelles, are extremelypale, and have short cell processes. The lowest
transverse septum (TS)is resorbed by a monocyte-like mononuclear
cell (arrow). OC, osteoclasts. Decalcified. x 3,200.
Fig. 10. Lowest calcifying region of the tibial epiphyseal plate of a
7-day-old chick. A monocyte-like mononuclear cell (M) has invaded a
chondrocytic lacuna through the opened transverse septum and is
phagocytizing a degenerative chondrocyte (C). Decalcified. X 6,300.
ACPase activity reaction, on the other hand, was not
seen in the chondrocytes of any zones. The RER and
lysosome-rich cells, however, showed ACPase activity.
The ACPase reaction products of these cells were seen
in the Golgi apparatus and lysosomes (Fig. 16). Similar
ACPase reaction products in the Golgi apparatus were
also observed in the osteoblasts.
appear that the chondrocytes in the maturating zone
may prepare for matrix calcification.
Glycogen granules have often been observed in chondrocytes in the upper zones of the mammalian epiphyseal plate (Anderson, 1964; Holtrop, 1972a,b; Carlson
et al., 1985); however, we did not find these granules,
or glycogen lakes, in any zone of the chick epiphyseal
plate. Glycogen granules have not been reported in previous studies in chicks (Lutfi, 1974; Howlett, 1979). In
the mammalian epiphyseal plate, stored glycogen
granules are used as an energy source for anaerobic
metabolism in chondrocytes in the hypertrophic zone,
an avascular zone associated with low oxygen tension
(Brighton et al., 1973; Brighton, 1978).However, in the
epiphyseal plate in birds, tissue vascularity is high in
comparison with mammals (Wise and Jennings, 1973;
Howlett et al., 1984), and the chondrocytes of the chick
epiphyseal plate, even those in the late hypertrophic
and calcifying zones, are not far from blood vessels
(Boyde and Shapiro, 1987). These findings indicate
that chondrocytes in the lower region of the chick
epiphyseal plate are adequately supplied with blood.
Thus, it is presumed that, in chicks, there is no requirement to store glycogen granules as an energy source for
anaerobic metabolism.
DISCUSSION
The morphology of the chondrocytes in the resting,
proliferating, and maturating zones was essentially
similar to that reported previously (Lutfi, 1974;
Howlett, 1979). The resting zone cells suggested active
protein synthesis, and the proliferating zone cells had
the characteristics of duplicate cells, showing mitotic
figures. The maturating zone cells seemed to synthesize maximum amounts of proteins such as proteoglycans and collagen. It is probable that type X collagen,
a calcium-tolerating collagen, was produced, since immunoperoxidase localization studies with chick tibiotarsus have demonstrated strong anti-type X collagen
reactivity in the cartilage matrix just prior to calcification (Kwan et al., 1986). Maturating zone cells also
produced many matrix vesicles, resulting in a sharp
increase of matrix vesicles in this zone. Thus, it would
36
M. TAKECHI AND C. ITAKURA
Figs. 11-14.
STUDIES OF CHICK EPIPHYSEAL PLATE
37
TABLE 1. Morphological profile of cells within the chondrocytic lacunae in the outermost
layer of the calcifying zone'
Chondrocytes
RER and lysosome
-rich cells
Upper region
UL
OL
932
22
(368)3
(23)
7
78
(28)
(83)
Middle region
UL
OL
94
27
(372)
(29)
6
73
(24)
(80)
Lower region
UL
OL
99
78
(348)
(114)
1
22
(3)
(32)
Total4
UL
OL
95
46
(1,088)
(166)
5
54
(55)
(195)
'UL, unopened lacuna; OL, opened lacuna.
'Percentage for all animals combined.
3Number of cells for all animals combined.
4Combined for all regions.
We found the chondrocytes in the calcifying zone to
be of three distinct major cell types. Similar cell types
have been described in the swine growth plate (Wilsman et al., 1981; Carlson et al., 1985);it was suggested
that the stellate cells produce a major matrix component, while the other two cell types, clear and hypertrophic clear cells, produce lesser amounts of this component or a different component. Our findings indicate
that each cell type in the calcifying zone of the chick
epiphyseal plate plays a different role and function in
cartilage calcification, since the appearance of each cell
type was closely related to the stage of matrix calcification.
TABLE 2. Frequency of osteoclasts coexistent with
RER and lvsosome-rich cells in the Calcifying zone
Coexistent' cells
Upper
region
432
Middle
region
34
Lower
region
5
'Osteoclasts were defined as coexistent if they were attached to the
matrix near the RER and lysosome-rich cells with invasion of the
lacunae.
'Percentage for all animals combined.
Fig. 11. Outermost layer of the upper calcifying region of the tibial
epiphyseal plate of a 14-day-old chick. RER and lysosome-rich cells
(RL) have well-developed RER and dense lysosomal bodies. The chondrocytic lacuna of the left RER and lysosome-rich cell is open, but that
of the right cell is not. The pericellular matrix (PM) is wide and
contains few matrix components. The territorial matrix area facing
the marrow cavity (arrowheads) is narrow in comparison with that on
the opposite side. An osteoclast (arrow) is adjacent to a RER and
lysosome-rich cells. Decalcified. x 4,500.
Fig. 12. RER and lysosome-rich cell (RL) in the upper calcifying
region of the tibial epiphyseal plate of a 14-day-old chick. The territorial matrix facing the marrow cavity has almost disappeared. Cell
processes of a RER and lysosome-rich cell extend into the remaining
territorial matrix (arrowheads).An osteoclast (OC) is attached to the
matrix, forming a ruff led border; its cytoplasmic branches invade the
chondrocyte lacuna. Decalcified. x 3,500.
Fig. 13. Lower calcifying region of the tibial epiphyseal plate of a
14-day-old chick. Osteoblasts (OB) and collagen fibrils invade the lacunae of a RER and lysosome-rich cell (RL). Decalcified. x 4,100.
Fig. 14. A liberated RER and lysosome-rich cell (RL) in the bone
tissue of the lower calcifying region of the tibial epiphyseal plate of a
14-day-old chick. This cell has normal cell architecture with welldeveloped RER and Golgi apparatus. OB, Osteoblast. Decalcified.
x 5,400.
In the upper calcifying region, initial calcification,
characterized by the deposition of apatite crystals
within the matrix vesicles and the formation of crystal
clusters, occurred in the matrix. The clear cells in this
region appear to be well differentiated and to have calcium-releasing capacity, and they play an important
role in this initial calcification. We presumed that
these cells were involved only in the early stage of
calcification, since they were not observed in the middle and lower calcifying regions. Stellate cells were observed in the middle calcifying region, where further
calcification proceeded with the deposition of apatite
crystals on collagen fibrils. The stellate cells had welldeveloped RER and free ribosomes, suggesting active
protein synthesis. However, no secretory vesicles containing proteoglycan (Howlett, 1979) were found in
these cells. It is conceivable that these stellate cells
may produce mainly type X collagen, the major collagen protein of the calcifying zone matrix in the chick
epiphyseal plate (Schmid and Linsenmayer, 1985;
Kwan et al., 19861,and it appears that they control the
progress of further calcification. Hypertrophic clear
cells appeared in the lower calcifying region, where
endochondral ossification was in progress. Examination of collagen mRNA in the chick epiphyseal plate
has indicated that chondrocytes in the endochondral
bone produce type X as the major or sole collagen and
that these chondrocytes differ from those found in the
hypertrophiclcalcifying cartilage region (Leboy et al.,
1988). Thus, it is possible that the hypertrophic clear
cells participate in the process of cartilage calcification
by maintaining levels of matrix components, including
that of type X collagen, during endochondral bone formation.
A number of our findings here suggest that the RER
and lysosome-rich cells that we observed were involved
with the resorption of uncalcified cartilage matrix: 1)
the cells had well-developedRER and many lysosomes;
2) the territorial matrix facing the marrow cavity was
narrow and occasionally disappeared; 3) the cell processes extended into the remaining territorial matrix
but not into the side where the territorial matrix had
disappeared; and 4) the pericellular matrix contained
few matrical substances, despite displaying the characteristics of protein-synthesizing cells. In addition to
the morphological evidence, ACPase-positive products
were seen in the Golgi apparatus and lysosomes of
these cells. This histochemical finding indicated that
RER and lysosome-rich cells could possibly synthesize
38
M. TAKECHI AND C. ITAKURA
Fig, 15. ALPase activity in the maturating zone cells of a 21-day-old
chick. Intense reaction product is seen on the plasma membrane of
chondrocytes and in association with matrix vesicles (arrows).
x 3,500.
Fig. 16. ACPase activity in the RER and lysosome-rich cell of a
21-day-old chick. Reaction product is seen in the Golgi apparatus
(arrows) and lysosomes (arrowheads). x 9,000.
hydrolytic enzymes such as collagenase. Similar
ACPase-positive products were also observed in the osteoblasts. Immunohistochemical studies have demonstrated the synthesis and secretion of collagenase by
osteoblasts (Heath et al., 1984; Otsuka et al., 1984;
Sakamoto and Sakamoto, 1984). It has been suggested
that osteoblasts induce osteoclastic resorption through
the secretion of collagenase, which, by digesting the
surface osteoid, exposes bone minerals to osteoclastic
contact (Chambers et al., 1985; Chambers and Fuller,
1985).The same process would seem to be operating in
the relation between osteoclasts and the RER and lysosome-rich cells, since approximately 40% of the osteoclasts attached to the cartilage matrix in the upper
and middle calcifying regions, where matrix calcification was immature, were coexistent with the RER and
lysosome-rich cells, whereas in the lower calcifying region, where matrix calcification was mature, only 5%
of the osteoclasts were coexistent with the RER and
lysosome-rich cells. We hypothesize that, in the chick
epiphyseal plate, the RER and lysosome-rich cells induce osteoclastic resorption by digesting the uncalcified cartilage matrix in the scantily calcified regions.
Further, we suggest that cartilage resorption induced
by these cells may promote the replacement of cartilage by bone tissue, as indicated by the invasion of
osteoblasts and collagen fibrils into the lacunae of the
RER and lysosome-rich cells in the middle and lower
calcifying regions.
Cells similar t o RER and lysosome-rich cells have
been described in the mouse and the embryonic chick
epiphysis (Sorrel1and Weiss, 1980; Cole and Wezeman,
1985); it was suggested that these cells were responsible for cartilage resorption and that they may have
been macrophages. In the present study, these RER
and lysosome-rich cells were more often found within
opened lacunae and on the cartilage surface, and they
differed from the chondrocytes with respect to histochemical activity-findings that indicate that the RER
and lysosome-rich cells may have invaded from the
marrow cavity. However, a small number of these cells
was also found within unopened lacunae without chondrocytes and celullar debris in the lacunae. These findings, although not conclusive, suggest that these cells
are a specialized chondrocyte, different from other cell
types. Indeed, the freeing of chondrocytes from the matrix surrounding the canal has already been described
(Lutfi, 1971).
Two hypotheses have been advanced regarding the
ultimate fate of chondrocytes: death (Brighton et al.,
1973; Hanaoka, 1976; Stambaugh and Brighton, 1980;
Farnum and Wilsman, 1987) and modulation of phenotype to some type of bone precursor cell (Crelin and
Koch, 1967; Lutfi, 1971; Holtrop, 1972b; Kalayjian and
STUDIES OF CHICK EPIPHYSEAL PLATE
Cooper, 1972; Shapiro and Boyde, 1987). In the present
study, the hypertrophic clear cells in the lowest calcifying region were phagocytized by monocyte-like
mononuclear cells invading the lacunae. It has been
suggested that monocytes invading the last transverse
septum recognize membrane antigens of the terminal
chondrocytes as foreign and actively kill terminal hypertrophic chondrocytes (Hunziker et al., 1984). Our
findings in this study support this hypothesis. However, we found t hat the RER and lysosome-rich cells
liberated from the chondrocytic lacunae to the marrow
cavity or osteoid tissue had normal cell architecture,
with relatively well-developed RER and pinocytic vesicles. That is, these liberated cells were viable. The
morphological similarity of the small clear cells to osteogenic cells such as osteoblasts and osteoid osteocytes
indicated that these RER and lysosome-rich cells could
possibly develop into osteogenic cells. It has been reported that cultured chondrocytes derived from the
epiphyseal plate have remarkable osteogenic potential
(Shimomura et al., 1973; Shimomura and Suzuki,
1984) and that they switch their synthesis of collagen
from type I1 or type X (cartilage) to type I (bone) collagen (Mayne et al., 1984; Gibson and Flint, 1985).
These observations also support the idea that the RER
and lysosome-rich cells we observed would develop into
osteogenic cells.
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