an ultrastructural study of cartilage resorption at the site of initial

J. Anat. (1997) 191, pp. 65–76, with 16 figures Printed in Great Britain 65

An ultrastructural study of cartilage resorption at the site of

initial endochondral bone formation in the fetal mouse

mandibular condyle

SHUNICHI SHIBATA1, SHOICHI SUZUKI2 AND YASUO YAMASHITA1

" 1st Department of Oral Anatomy and # 2nd Department of Orthodontics, School of Dentistry, Tokyo Medical and Dental

University, Tokyo, Japan

(Accepted 11 February 1997)

An ultrastructural study was undertaken on cartilage resorption at the site of initial endochondral bone

formation in the mouse mandibular condyle on d 16 of pregnancy. After resorbing the bone collar, the

osteoclasts extended their cell processes into the cartilage matrix and made contact with hypertrophic

chondrocytes. By means of cell processes or vacuolar structures, these osteoclasts entrapped the calcified

cartilage matrices, cell debris, and the degraded uncalcified cartilage matrices. In particular, since the

calcified cartilage matrices were sometimes seen to be disrupted within the osteoclastic vacuolar structures,

they were probably disposed of by the osteoclasts. Invading endothelial cells giving rise to capillaries also

directly surrounded the degraded uncalcified cartilage matrices and small deposits of cell debris. In addition,

hypertrophic chondrocytes that had attached to or were in the process of attaching to the invading

osteoclasts often enclosed the small calcified cartilage matrices. Other cell types that have often been

reported in other regions of cartilage resorption were not seen at the site of initial endochondral bone

formation in this study. Our findings in relation to cartilage resorption may therefore represent unique

features of the site of initial endochondral bone formation site. We consider that the manner of cartilage

resorption is likely to vary by site, age, and species.

Key words : Osteoclasts ; chondrocytes ; endothelial cells.

In the mammalian growth plate, which is often used

as a model for endochondral bone formation (Jee,

1988), the longitudinal septa are usually calcified and

are mainly resorbed by multinucleated osteoclasts}chondroclasts (Anderson & Parker, 1966; Schenk et

al. 1967; Kuettner & Pauli, 1983), although some

mononuclear cells are also involved in the resorption

of calcified cartilage (Knese & Knoop, 1961; Anderson

& Parker, 1966; Schenk et al. 1967). In contrast, the

transverse septa are usually uncalcified and it is

speculated that the endothelial cells of invading

capillaries and perivascular cells involved in their

resorption (Anderson & Parker, 1966; Schenk et al.

1967, 1968; Lee et al. 1995).

Recently,Lee et al. (1995) clarified the characteristics

of these cathepsin B-rich perivascular cells and termed

Correspondence to Dr S. Shibata, 1st Department of Oral Anatomy, School of Dentistry, Tokyo Medical and Dental University, 1-5-45,

Yushima, Bunkyo-ku, Tokyo 113, Japan. Tel : 03-5803-5441; Fax: 03-5803-0185; e-mail : S.Shibata.oan1!dent.tmd.ac.jp.

them ‘septoclasts ’. However, other cells are thought

to be implicated in the resorption of uncalcified

cartilage in various tissues, such as the epiphyseal

cartilage canals (Cole & Wezeman, 1985, 1987) and

the proximal part of Meckel’s cartilage in the rat

(Mu$ hlenhauser, 1986). In both of these examples, the

respective cartilage is not covered with bone tissue,

and fibroblast-like and macrophage-like cells are

involved in the resorption process.

In other instances of cartilage resorption, such as in

the distal part of rat Meckel’s cartilage (Savostin-

Asling & Asling, 1973, 1975), perichondral bone

surrounds the cartilage, this being followed by

cartilage calcification, after which osteoclasts resorb

both the perichondral bone and the calcified cartilage.

In Meckel’s cartilage in fish (Sire et al. 1990;

Huysseune & Sire, 1992), after the osteoclasts have

removed the perichondral bone, fibroblast-like cells

66 S. Shibata, S. Suzuki and Y. Yamashita

invade the cartilage. In terms of the site of initial

endochondral bone formation in avial embryonic long

bone, cartilage is not calcified when the osteoclasts

begin to resorb the bone collar, after which cartilage

is invaded by fibroblast-like and macrophage-like cells

(Sorrell & Weiss, 1980, 1982; Aceitero et al. 1988).

Some authors have suggested that osteoclasts may

be involved in the resorption of uncalcified cartilage

(Aceitero et al. 1988; Sire et al. 1990; Huysseune &

Sire, 1992). Furthermore, in the condylar cartilage of

the growing rat mandible, Lewinson & Silbermann

(1992) have speculated that multinucleated chondro-

clasts and tube-forming endothelial cells may colla-

borate in cartilage resorption. Recent studies have

also reported that hypertrophic chondrocytes exhibit

matrix metalloproteinase activity and are involved in

the degradation of the cartilage matrix (Sakiyama et

al. 1994; Fuller & Chambers, 1995; Gack et al. 1995).

Thus these various findings appear to indicate that the

details of cartilage resorption vary by site, age and

species.

We have recently reported (Shibata et al. 1996) that

in fetal mouse condylar cartilage, initial endochondral

bone formation begins on d 16 of pregnancy with the

resorption of the bone collar by osteoclasts. On the

same day, calcification had already commenced in a

small area of the cartilage matrix. Uncalcified cartilage

is subsequently resorbed and calcified cartilage is

exposed to the primary spongiosa.

In the present investigation we have extended our

observations and have undertaken an ultrastructural

study on cartilage resorption at the site of initial

endochondral bone formation in the mouse man-

dibular condyle.

Pregnant female ICR mice on d 16 of pregnancy

(8 a.m. on the day of vaginal plug formation was

designated d 0 of pregnancy) were used in this study.

Under ether anaesthesia, the mice were killed by

cervical dislocation, after which the head of each fetal

mouse was taken and immediately immersed in a 5%

glutaraldehyde–4% paraformaldehyde solution (0.1

γ-collidin buffer, pH 7.4, at room temperature) for

24 h. Tissue samples were postfixed in 1% osmium

tetroxide (0.1 γ-collidin buffer, pH 7.4, 4 °C) for

3 h, and embedded in Epon 812 (TAAB). Some

samples were decalcified with 10% EDTA before

postfixation.

Sections (1 µm) were cut in the frontal plane and

stained with toluidine blue for light microscopy.

Ultrathin sections were also prepared and contrasted

with uranyl acetate and lead citrate for electron

microscopy; they were examined in a Hitachi HS-9

electron microscope.

Figure 1a and b shows light micrographs of the

anterior end of the condylar cartilage in a fetal mouse

mandible on d 16 of pregnancy. Matrix composed of

calcified tissue surrounding the condylar cartilage is

seen. Electron microscopy of decalcified sections (Fig.

2) indicates that this calcified tissue consisted mainly

of thick collagen fibrils with distinctive cross striations,

confirming it to be the bone collar. Endochondral

bone formation had commenced with resorption of

the bone collar by osteoclasts, as previously described

(Shibata et al. 1996). These osteoclasts further invaded

the cartilage and contacted hypertrophic chondrocytes

which were spherical in shape with large round nuclei

and pale cytoplasm. In addition, many hypertrophic

chondrocytes at this site did not show degenerative

features and some were seen to have migrated into the

primary spongiosa (Fig. 1b). Within the cartilage,

small spherical calcified tissue areas were formed

distant from the bone collar (Fig. 1b). Electron

microscopy of decalcified sections (Fig. 3) indicated

that these spherical calcified tissue areas consisted

mainly of fine granular material and were thus

confirmed to be calcified cartilage matrices. An intact

uncalcified cartilage matrix was seen around the

calcified cartilage matrix ; the former consisted of thin

collagen fibrils and fine granular material, as pre-

viously described (Sheldon, 1983). Currently, using

improved fixation methods, the cartilage matrix is

classified histologically into 3 zones: territorial,

interterritorial and pericellular (Hunziker et al. 1984;

Marchi et al. 1991). In our samples of fetal condylar

cartilage observed using conventional fixation

methods, the territorial and interterritorial matrices

can barely be discriminated and, as the territorial

matrix gradually extended towards the pericellular

matrix, the border between them became indistinct.

Our term ‘uncalcified cartilage matrix ’ therefore

includes all 3 matrices. Capillary sprouts had also

invaded the cartilage and their endothelial cells

showed tube-like profiles (Fig. 1b).

Figure 4 shows the site of initial endochondral bone

formation adjacent to that in Figure 1a ; Figures 5, 6

and 7 show higher magnification views of areas A, B

and C, respectively, from Figure 4. A capillary

containing numerous erythrocytes is seen in the lower

part and its endothelial cell which had invaded the

cartilage showed a tube-like profile at area C. A

Cartilage resorption 67

All photographs except Figs 2 and 3, show undecalcified sections.

Fig. 1. Light micrographs of the anterior end of the condylar cartilage (CC) in the fetal mouse mandible on d 16 of pregnancy; (b) is a higher

magnification (a). Matrices of calcified tissue surrounding the condylar cartilage (arrows in a and b) are seen. Osteoclasts (OC) make contact

with hypertrophic chondrocytes (HC). Many chondrocytes at this site show intact profiles and some (o in b) are seen to have migrated into

the primary spongiosa. Within the cartilage, small spherical areas of calcified tissue have formed (arrowhead in b). Capillary (Ca) sprouts

have invaded the cartilage and their endothelial cells (EC) show tube-like profiles. Bars, 10 µm.

Fig. 2. Electron micrograph of calcified tissue matrix surrounding the condylar cartilage. This consists mainly of thick collagen fibrils (CF)

with distinctive cross-striations. Decalcified section. Bar, 0.2 µm.

Fig. 3. Electron micrograph of small spherical calcified areas within the condylar cartilage. These areas consist mainly of fine granular

material (arrowheads). Around these areas, intact uncalcified cartilage consisting of thin collagen fibrils (CF) and fine granular material is

seen. Decalcified section. Bar, 0.2 µm.

region of intact uncalcified cartilage matrix is seen

around area A. Higher magnification of area A (Fig.

5) showed that the intact uncalcified cartilage matrix

consisted of thin collagen fibrils and fine granular

material. This matrix gradually became degraded as it

extended from area A to area B (Fig. 4). Higher

magnification of area B (Fig. 6) showed that the

cartilage matrix was separated into small regions that

had a mesh-like appearance and broken collagen

fibrils which were shorter than those in area A. This


Fig. 4. Electron micrograph of the site of initial endochondral bone formation adjacent to that in Figure 1a. A capillary (Ca) containing

numerous erythrocytes is seen in the lower part and its endothelial cell (EC) which has invaded the cartilage shows a tube-forming profile

at area C. Intact uncalcified cartilage matrix is seen at area A and this matrix is gradually degraded as it extends from area A to area B

(indicated by thick arrow). In area C, the tube-forming endothelial cell has surrounded some small areas of matrix. Many hypertrophic

chondrocytes (HC) surrounded by the uncalcified cartilage matrix show intact profiles. Some undifferentiated cells (UC) are seen near the

endothelial cells. BC, bone collar. Bar, 1 µm.


Fig. 5. Higher magnification of area A in Figure 4. Intact uncalcified cartilage matrix consists of thin collagen fibrils (CF) and fine granular

material. Bar, 0.5 µm.

Fig. 6. Higher magnification of area B in Figure 4. The cartilage matrix is separated into small clumps which show a mesh-like appearance

(arrowheads) and interrupted collagen fibrils (CF) are shorter than those shown in Figure 5. Bar, 0.5 µm.

Fig. 7. Higher magnification of area C in Figure 4. The portions of matrix (arrowheads) entrapped by the endothelial cell are smaller than

those in Figure 6 and mainly consist of fine granular material. Bar, 0.5 µm.

Fig. 8. Electron micrograph of the capillary lumen near the site of initial endochondral bone formation in the fetal mouse mandibular

condyle. Small deposits of cellular debris (CD) are seen. E, erythrocyte. Bar, 0.5 µm.

finding indicates that some degradation had occurred

in the uncalcified cartilage matrix. In area C, a tube-

forming endothelial cell has surrounded some small

matrix material. Higher magnification of area C (Fig.

7) showed that these matrices were smaller than those

shown in Figure 6, and mainly consisted of fine

granular material, while collagen fibrils were rarely

seen. Judging from the sequential degradation process


Fig. 9. Another tube-forming cell (o) and an osteoclast (OC) extending cellular process (CP) into the cartilage matrix. Intact uncalcified

cartilage matrix is seen on the upper right side (n) and this matrix is gradually degraded towards the lower part of the figure. Deposits of

cellular debris (CD) are seen in the uncalcified cartilage matrix and this tube-forming cell is seen to surround small deposits of cellular debris

(arrowhead). This osteoclast has entrapped some calcified matrix (arrows) with its cellular process and vacuolar structures (V). Bar, 1 µm.


Fig. 10. Higher magnification of rectangular area in Figure 9. Two small spherical portions of calcified matrix (arrows) are entrapped by

the osteoclastic cellular process (CP). Bar, 0.2 µm.

Fig. 11. Another osteoclast that has entrapped some portions of calcified matrix (arrows). Although no ruffled borders but only a nucleus

(N) is seen in this section, the presence of numerous mitochondria (M) and vacuolar structures (V) indicates that this cell is an osteoclast.

One of the entrapped portions of calcified matrix has become disrupted within the vacuolar structure (arrowhead). Bar, 0.5 µm.

of the uncalcified cartilage matrix described above,

these small areas of matrix probably represent

degraded uncalcified cartilage.

Some hypertrophic chondrocytes were surrounded

by this degrading uncalcified cartilage matrix (Fig. 4).

These cells were spherical in shape and had large

round pale nuclei and light cytoplasm. Moderately

extensive rough endoplasmic reticulum was arranged

around their nuclei. Some undifferentiated cells were

seen adjacent to the endothelial cells (Fig. 4). Since

these cells were oval in shape, and had few cytoplasmic

organelles, they were readily distinguished from the

hypertrophic chondrocytes. No other cell types, such

as ‘septoclasts ’ as described by Lee et al. (1995) or

fibroblast-like cells as described by Cole & Wezeman

(1985, 1987) in other cartilage resorption areas, were

detected at the site of initial endochondral bone

formation in this study.

Small deposits of cellular debris were often seen in

the capillary lumen near the site of initial endo-

chondral bone formation (Fig. 8).

Figure 9 shows another tube-forming cell and

another osteoclast extending its cellular process into

the cartilage at the site of initial endochondral bone

formation. Intact uncalcified cartilage matrix is seen

at the upper right side and this matrix gradually

becomes degraded towards the lower side. Deposits of

cellular debris are seen in the cartilage matrix and this

tube-forming cell is seen to surround small deposits of

cellular debris.

While the osteoclast in Figure 9 had enveloped

some calcified matrices with its processes, seen at

higher magnification in Figure 10 and by vacuolar

structures, according to Lucht (1972) these vacuolar

structures include both genuine vacuoles and invagina-

tions of the cell membrane. However, since we were

unable to distinguish these invaginations in any of the

sections observed, their possible presence is included

in the overall term ‘vacuolar structures ’ in this paper.

Figure 10 shows a higher magnification of the

rectangular area in Figure 9. Two small spherical

areas of calcified matrix are enclosed by the osteo-

clastic process. Since these calcified matrices were

small in size and spherical in shape, and located at

some distance from the bone collar near the un-

calcified cartilage matrices, they can be regarded as

calcified cartilage.

Figure 11 shows another osteoclast enclosing


Fig. 12. An osteoclast has extended a cell process (CP) into the cartilage matrix. Although no ruffled borders but only a nucleus (N) is seen

in this section, the presence of numerous mitochondria (M) and vacuolar structures (V) indicates that this cell is an osteoclast. Uncalcified

cartilage matrix (n) is seen on the right side which is gradually degraded towards the left side. Cellular debris (CD) is also seen in the

uncalcified cartilage matrix. This osteoclast has entrapped calcified matrices (arrow). Bar, 1 µm.

Fig. 13. Higher magnification of rectangular area a in Figure 12. The degraded uncalcified cartilage matrices (arrowheads) are seen in the

vacuolar structures (V). Bar, 0.5 µm.


calcified matrix. Although no ruffled borders but only

a nucleus is seen in this section, the presence of

numerous mitochondria and vacuolar structures

indicates that this cell is an osteoclast. Also, for the

same reason as stated in Figure 10, these calcified

matrices can be regarded as calcified cartilage, and

one of them was disrupted within the vacuolar

structures.

Figure 12 shows another osteoclast, and Figures 13

and 14 are higher magnifications of the rectangular

areas a and b, respectively, in Figure 12. Although no

ruffled borders but only a nucleus is seen in this

section, the presence of numerous mitochondria and

vacuolar structures again indicates that this cell is an

osteoclast. An area of uncalcified cartilage matrix is

also seen on the right which gradually becomes

degraded towards the left side. Cellular debris is also

seen in the uncalcified cartilage matrix. This osteoclast

had entrapped calcified matrix (Fig. 12) as well as

degraded uncalcified cartilage matrix within the

vacuolar structures (Fig. 13). In addition, its cell

process surrounded cell debris (Fig. 14).

In Figure 15, an osteoclast is seen on the left side,

and the clear zone probably originating from this

osteoclast had attached to the bone collar and a

hypertrophic chondrocyte which was surrounded by

an area of uncalcified cartilage matrix. This hy-

pertrophic chondrocyte had similar structural features

to those shown in Figure 4. The inset in this figure

shows a higher magnification of the rectangular area

in Figure 15. This hypertrophic chondrocyte had

entrapped small areas of calcified matrix which can be

regarded as calcified cartilage for the same reason as

stated for Figure 10 (see inset).

Figure 16 also shows a hypertrophic chondrocyte

which had nearly made contact with an osteoclast.

This hypertrophic chondrocyte had also entrapped

some calcified matrix, which was probably calcified

cartilage.

In long bones, endochondral bone formation begins

with the osteoclastic resorption of the bone collar

(Silvestrini et al. 1979; Pechak et al. 1986; Aceitero et

al. 1988) which is the same method of endochondral

bone formation as in the fetal mouse mandibular

condyle (Shibata et al. 1996). In this study, after the

osteoclasts resorbed the bone collar, tube-forming

Fig. 14. Higher magnification of rectangular area b in Figure 12. A cell process (CP) of an osteoclast is seen to have entrapped cell debris

(CD). Bar, 0.5 µm.

endothelial cells of invading capillary sprouts directly

surrounded the degraded cartilage matrices and small

deposits of cellular debris. Capillary participation is

vital for endochondral bone formation to progress

and has often been cited as a factor in the resorption

of uncalcified cartilage (Anderson & Parker, 1966;

Schenk et al. 1967, 1968; Kuettner & Pauli, 1983).

Furthermore, Lewinson & Silbermann (1992) have

indicated that tube-forming endothelial cells and

chondroclasts collaborate in the process of condylar

cartilage resorption of the growing rat mandible.

However, as no other study has mentioned the direct

encirclement of degraded cartilage matrices by the

endothelial cells as being part of the resorption

process, we feel that our findings may only apply to

the site of initial endochondral bone formation.

The ultimate fate of the entrapped cartilage matrix

and cellular debris is unclear, but as small deposits of

cellular debris were sometimes noted in the capillary

lumen near the site of initial endochondral bone

formation, we believe that they are probably disposed

of through the vascular system. In the next step of the

probable cartilage resorption process, after resorbing

the bone collar, the osteoclasts invaded the cartilage

and their cellular processes and vacuolar structures

often entrapped the calcified cartilage matrix. Further,

since the calcified cartilage matrix was sometimes

disrupted within the vacuolar structures, the osteo-

clasts probably degrade them.

The functions of osteoclasts additional to their role

in bone resorption have been studied in various

tissues. The resorption of calcified cartilage by

osteoclasts or by multinucleated chondroclasts that

have similar structural features has been reported in

the growth plate (Anderson & Parker, 1966; Schenk et

al. 1967; Kuettner & Pauli, 1983), the mandibular

condyle of the growing rat (Lewinson & Silbermann,

1992), embryonic chick long bone at later stages

(Silvestrini et al. 1979; Sorrell & Weiss, 1980), the

distal part of rat Meckel’s cartilage (Savostin-Asling

& Asling, 1973, 1975) and the deep epiphyseal

cartilage canals (Cole & Wezeman, 1985, 1987).

However, with regard to the resorption of calcified

cartilage, the above-mentioned studies maintained

that the osteoclasts or chondroclasts mainly utilise

the ruffledborder for this function, and fewcommented

on phagocytosis of calcified matrices by osteoclasts

(Anderson & Parker, 1966). The mechanism of

calcified cartilage resorption other than via the ruffled

border is unknown and additional cytochemical


Fig. 15. A hypertrophic chondrocyte (HC) that has become attached to the clear zone (CZ) of an osteoclast (OC) has entrapped small

calcified matrices (rectangular area). Inset shows higher magnification of this rectangular area. BC, bone collar. Bar, 1 µm; inset, ¬22000.

Fig. 16. A hypertrophic chondrocyte (HC) that has nearly made contact with an osteoclast (OC) has entrapped small portions of calcified

matrices (arrows). Bar, 1 µm.


studies are required. We consider that the site of initial

endochondral bone formation is appropriate to

investigate this mechanism.

Cellular debris and degraded uncalcified cartilage

matrix were also seen to be entrapped by osteoclasts

in the present study. The fate of these entrapped

substances is unclear, but we speculate that they are

also disposed of by osteoclasts. Involvement of

osteoclasts in the resorption of uncalcified cartilage

has been suggested in embryonic chick long bone

during its early stages (Aceitero et al. 1988) and in fish

Meckel’s cartilage (Sire et al. 1990; Huysseune & Sire,

1992). Phagocytosis of cellular debris by chondroclasts

has also been described (Lewinson & Silbermann,

1992). These findings and our results indicate that,

based on their environment, osteoclasts are able to

perform functions other than bone resorption.

It should be pointed out that when the uncalcified

cartilage matrix was being surrounded by endothelial

cells and osteoclasts, it had already become degraded.

We therefore speculate that proteolytic enzymes must

have been released from an unidentified source to

degrade this matrix. Although we do not know which

cells may be involved, some recent studies have

indicated that hypertrophic chondrocytes appear to

be implicated in the activity of various matrix

metalloproteinases (Sakiyama et al. 1994; Fuller &

Chambers, 1995; Gack et al. 1995). Because numerous

hypertrophic chondrocytes of the fetal mouse man-

dibular condyle had intact profiles, we believe that

they are strong candidates as a source for these

proteolytic enzymes. At the same time, however, we

cannot exclude the possible involvement of osteoclasts,

since they are known to extend cellular processes into

the cartilage matrix.

In the present study the hypertrophic chondrocytes

that had attached to or were in the process of

attaching to invading osteoclasts were often seen to

surround small areas of calcified cartilage matrix,

and others have noted that mononuclear phagocytic

cells that had entrapped calcified matrix are often

seen in the primary spongiosa of long bone (Knese &

Knoop, 1961; Anderson & Parker, 1966; Schenk et al.

1967), in bone remodelling sites (Tran Van et al.

1982), and in vitro studies (Rifkin et al. 1979;

Takahashi et al. 1986). Also, as we have previously

reported (Shibata et al. 1996), since numerous sur-

viving hypertrophic chondrocytes at the site of initial

endochondral bone formation in the fetal mouse

mandible are released into the primary spongiosa,

these chondrocytes may play the role of mononuclear

phagocytic cells. If this role is confirmed, it would be

another unique feature of this site.

Perivascular cells are thought to be involved in the

resorption of uncalcified transverse septa of the

growth plate (Anderson & Parker, 1966; Schenk et al.

1967; Lee et al. 1995). In this regard, Lee et al. (1995)

have recently clarified the nature of these cells in the

rat growth plate and, as mentioned above, have

named these cathepsin-B rich cells ‘septoclasts ’. It

also has been reported that fibroblast-like and

macrophage-like cells are involved in the resorption of

uncalcified cartilage in the epiphyseal cartilage canals

(Cole & Wezeman, 1985, 1987). We have also

investigated the mouse tibial growth plate and

epiphyseal cartilage canals, and noted similar cell

types (data not shown) at each site, but could not

detect them at the site of initial endochondral bone

formation in this study.

Thus the diverse findings of this study and previous

investigations support the conclusion that the manner

of cartilage resorption varies by site, age, and species.

We believe that many factors must be taken into

consideration when investigating the process of

cartilage resorption.

This work was supported by a Grant-in-Aid for

Scientific Research (No. 07671963) from the Ministry

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