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The effects of high dose of parathyroid hormone on fetal osteoclasts and their precursors in vivoAn ultrastructural-cytochemical study.

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THE ANATOMICAL RECORD 243:421-429 (1995)
The Effects of High Dose of Parathyroid Hormone on Fetal
Osteoclasts and Their Precursors In Vivo: An
Ultrastructural-Cytochemical Study
HISAYUKI ISAKI AND HIDEYA HANAOKA
Department of Orthopedic Surgery, Keio University School of Medicine, Tokyo, Japan
ABSTRACT
Background: It is not well known how the immediate precursors of osteoclast develop into osteoclasts in the fetus. This ultrastructural-cytochemical study was designed to clarify the formation process of
the osteoclasts and their increased activities in the fetal mouse limb buds
after administration of high dose parathyroid hormone (PTH).
Methods: Twenty-four or forty-eight hours after the high doses of PTH
were injected into amniotic fluid of the pregnant C,H mice, the femoral limb
buds of embryos were dissected out. Tartrate-resistant acid phosphatase
(TRAP) reactions were performed while preparing specimens for electron
microscopy.
Results: Both control and PTH-given preosteoclasts and osteoclasts exhibited TRAP-positivities in dense bodies and vesicles. As effects of PTH, a
binucleated preosteoclast of tandem fashion was observed. More osteoclastic hyperactivities were observed in the diaphyseal bone marrow. An osteoclast with a large cytoplasm exhibited two sets of clear zones and ruffled
borders. Some osteoclasts demonstrated prominent amoeboid figures,
while other osteoclasts developed large cytoplasmic vacuoles, which contained pieces of calcified chondroid bars.
Conclusions: Our results revealed the progression of maturation from
young preosteoclasts to osteoclasts. An existence of a peculiar binucleated
preosteoclasts suggested one of the processes for multinucleation of the
osteoclast. Quite remarkable osteoclastic hyperactivities were obviously
the effects of high dose PTH. Our results also indicated the endophagocytic
ability of the osteoclast. How PTH affected the osteoclasts and their precursors in the diaphyseal bone marrow can be speculated.
0 1995 Wiley-Liss, Inc.
Key words: C,H mouse, Enchondral ossification, Fetus, Limb bud, Osteoclast, Parathyroid hormone (PTH), Tartrate-resistant acid
phosphatase (TRAP),Ultrastructure
The hypothesis of osteoclast derivation from hematopoietic stem cell is predominant (Burger et al., 1987;
Mundy and Roodman, 1987; Helfrich et al., 1989;
Sasaki et al., 1989; Nijweide et al., 1990). However, it
is not well known how the immediate precursors of
osteoclasts develop into osteoclasts in periosteum of the
uninvaded fetal limb buds where the osteoclasts are
first formed (Hanaoka et al., 1989), although the process of their differentiation in the periosteum of fetal
rat parietal bone was examined by Ejiri (1983).Several
studies on the ultrastructural changes in osteoclasts
after administration of parathyroid hormone (PTH)
have revealed that PTH causes an increase in the number of osteoclasts and that it activates osteoclasts to
resulting in an increase in various cytoplasmic organelles, as well as in the extent of the clear zone and
ruffled border regions (Lucht and Maunbach, 1973;
0 1995 WILEY-LISS. INC
Holtrop et al., 1974; King et al., 1978; Miller et al.,
1984; Klaushofer et al., 1989; Barengolts et al., 1990).
The following ultrastructural-cytochemical study
was carried out on the formation process of the osteoclasts and their increased activities in the fetal limb
buds after administration of high dose parathyroid hormone.
MATERIALS AND METHODS
Under ether drop anesthesia, laparotomy was performed on eight C,H mice that were in their 14th day
Received May 19, 1995; accepted August 21, 1995.
Address reprint requests to Dr. Hideya Hanaoka, Department of
Orthopedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160, Japan.
422
H. ISAKI AND H. HANAOKA
Fig, 1, A multinucleated osteoclast of control without administration of PTH is invading into the limb bud through the bone collar (B).
This cell has standard osteoclastic features including a TRAP-positive
vesicle (arrow). No vascular buds have invaded, yet. Note the hypertrophic chondrocyte (C) adjacent to the calcified chondroid bar
(CB). Also, note the primitive cells (P) of oval shape, which have
invaded into the limb bud. It remains uncertain whether these cells
are the macrophages, the very young preosteoclasts, or any other cells
(~3,400).
of pregnancy. Ten units of PTH (Asahi Kasei Co., Tokyo) were injected through the wall of the uterus into
the amniotic fluid to increase the number and activity
of osteoclasts and preosteoclasts. Either 24 or 48 hours
later, 60 femoral limb buds of 15- or 16-day-old embryos were dissected out together with their periosteum. Similarly, ten control femoral limb buds of the
same age without administration of PTH were dissected out. Tartrate-resistant acid phosphatase (TRAP)
reactions were performed while preparing specimens
for electron microscopy. Femoral limb buds were fixed
in 0.5% glutaraldehyde and 4% paraformaldehyde in
0.1 M cacodylate buffer at 4°C for 90 minutes. They
were washed with 8% sucrose in 0.1 M cacodylate
buffer a t 4°C overnight. After they were embedded in
5% agarose (Sigma Chemical Co., St. Louis, MO), sections of 50 to 80 bm were cut on a vibratome (Sorvall,
TC 2 Type). To demonstrate TRAP activity, preincubation was performed with 50 mM potassium sodium tartrate in 10 ml of 0.2 M acetate buffer (pH 5.5) a t 37°C
for 2.5 hours, followed by incubation in a reaction
buffer of 50 ml of 0.05 M acetate buffer (pH 5.0), 4 g of
sucrose, 50 mg of lead nitrate, and 5 ml of 3% p glyc-
erophosphate at 37°C for 30 minutes. Specimens were
then postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer a t 4°C for 1 hour, dehydrated in a graded
series of ethanol, and embedded in EPON 812. Thin
sections were cut on a Porter-Blum 11-B ultratome and
stained with lead citrate to increase contrast. They
were examined on a Hitachi HU-12AS electron microscope.
RESULTS
Individual femoral limb buds appeared to differ
slightly in the developmental stage, despite having the
same fetal age. The bone collars of the limb buds were
usually ossified, but the amount of ossification varied
to some degree from limb bud to limb bud. In addition,
osteoclasts and preosteoclasts of various developmental
stages were observed even in serial sections from the
same limb bud. The femoral limb buds of 15-day-old
embryos were just beginning to be invaded by osteoclasts, but were not yet invaded by vascular buds, so
that no bone marrow had been formed. Bone marrow
had formed in the diaphysis of the femoral limb buds of
the 16-day-old embryos, and osteoclasts, endothelial
EFFECTS OF HIGH DOSE PTH ON OSTEOCLASTS
423
Fig. 2. An apparently mononuclear control osteoclast, identified by the ruffled border facing the bone collar, is of round shape, and abundant
of mitochondria ( x 5,200).
Fig. 3. A control preosteoclast in the cambium layer shows TRAP-positivedense bodies and vesicles (arrows), despite less osteoclastic features
( x 6,200).
cells, erythrocytes, monocytic cells, some unidentifiable primitive cells, and a few possible preosteoblasts,
but no mature osteoblasts were observed there a t this
stage.
Figure 1demonstrated a multinucleated osteoclast of
control without administration of PTH, which was just
invading into the limb bud. An apparently-mononuclear control osteoclast, identified by the ruffled border
424
H. ISAKI AND H. HANAOKA
Fig. 4. A binucleated osteoclast situated outside the bone collar. Note the abundant mitochondria, TRAP-positive (arrows) dense bodies, and
vesicles and RER with narrow canaliculi in the cytoplasm ( x 5,000).
facing the bone collar, was of round shape, and abundant of mitochondria (Fig. 2). A control preosteoclast in
the cambium layer showed TRAP-positive dense bodies
and vesicles, despite less osteoclastic features (Fig. 3).
The following were the observations in the specimens of PTH administration.
Number of preosteoclasts and osteoclasts increased
and various unusual types of these cells were observed.
There were binucleated osteoclasts which were
TRAP-positive in both dense bodies and vesicles (Fig.
4). In addition to standard osteoclastic features including mitochondria and dense bodies, some osteoclasts
demonstrated relatively abundant rough endoplasmic
reticulum (RER), whose cisternae showed narrower
canaliculi than usual seen in the postnatal osteoclasts
(Fig. 4). Similarly, preosteoclasts were identified by
TRAP-positive reactions in both dense bodies and vesicles, and by their round or spindle shape and a n abundance of mitochondria (Fig. 5). Some also showed RER
with narrower sternal canaliculi than usual, like those
of the osteoclasts (Fig. 5). We also observed a peculiar
binucleated preosteoclast, which appeared to have been
formed by the fusion of two preosteoclasts in a tandem
fashion (Fig. 6).
A series of maturation was observed among these
preosteoclasts. The younger they were, the lighter
their nuclei and cytoplasm, the greater the nucleus/
cytoplasm (N/C) ratio, and the fewer the organelle in
the cytoplasm. The youngest osteoclast precursor we
were able to identify was a preosteoclast with a high
NIC ratio, and a light nucleus and cytoplasm (Fig. 7).
Although it showed few organelles in its cytoplasm,
this cell showed TRAP-positive dense bodies (Fig. 7,
Inset). Such preosteoclasts (Figs. 5-7) were scattered
among the osteoblasts and preosteoblasts of the cambium layer, and some of them were adjacent to the bone
collar.
Osteoclasts in the diaphyseal bone marrow were
more hyperactive than those in the periosteum. One
apparently mononuclear osteoclast had developed a
large cytoplasm, so that it had a goldfish appearance
(Fig. 8). And this cell had a clear zone with polar distribution. Another osteoclast with large cytoplasm exhibited two sets of clear zones and ruffled borders
which were resorbing the calcified chondroid bars (Fig.
9). Some osteoclasts demonstrated prominent amoeboid
figures (Fig. 10). Other osteoclasts exhibited many
large cytoplasmic vacuoles, possibly d.erived from lysosomes, which contained pieces of calcified chondroid
bars and other piece of possible autophagozomes (Fig.
9). In many instances, the mitochondria were enlarged
and vacuolated. No mature osteoblasts were observed
in the diaphyseal bone marrow as described before.
TRAP-positivities were less occasionally observed in
the osteoclastic cells in the diaphyseal bone marrow
than in the periosteum.
DISCUSSION
In our experimental study, high dose PTH caused the
appearance of more abundant preosteoclasts, including
very young preosteoclasts and a n unusual type of binucleated preosteoclast as well a s apparently mononu-
EFFECTS OF HIGH DOSE PTH ON OSTEOCLASTS
425
Fig. 5. A mononuclear preosteoclast situated outside the bone collar. Note the mitochondria, TRAP-positive (arrow) dense bodies and vesicles,
and RER with narrow canaliculi ( x 4,800).
clear osteoclasts, compared with those in the controls.
These preosteoclasts and osteoclasts exhibited not only
changes in the cytoplasmic organelles, but also far
more remarkable morphologic changes than those in
our controls or in any other prior report (Lucht and
Maunbach, 1973; Holtrop et al., 1974; King et al., 1978;
Klaushofer et al., 1989; Barengolts et al., 1990).
Since tartrate-resistant acid phosphatase was found
to be more specific to osteoclasts than acid phosphatase
(Minkin, 1982), ultrastructural histochemistry using
TRAP reaction have been performed on osteoclasts by a
few researchers (Clark et al., 1989; Sasaki et al., 1989).
Using ultrastructural staining by the TRAP reaction
and other ultrastructural features of osteoclasts and
preosteoclasts, we sought osteoclast precursors in the
cambium layer of uninvaded limb buds where osteoclasts are first formed, and where no hematopoiesis
occurs. In such limb buds we observed a gradual preosteoclast maturation series, with the youngest osteoclast precursor cell showing a high N/C ratio, a light
nucleus and cytoplasm, and TRAP-positive dense bodies, most of which were lysosomes. However, we were
unable to identify any younger precursors of osteoclasts than this stage.
The appearance of a binucleated preosteoclast may
indicate the strong effect of PTH to cause cell fusion.
The preosteoclasts apparently fused without first developing into mature osteoclasts. Hanaoka (1979) reported that a n osteoclast fused with a preosteoblastic
cell and concluded that the osteoclasts increased the
number of their nuclei by the fusion of osteoblastic and
osteoclastic cells or by the fusion between osteoclastic
cells. Ejiri (1983) observed a flattened binucleated cell,
one half of which was rich in mitochondria, a n osteoclastic feature, and another half of which had welldeveloped RER, a n osteoblastic feature. He presumed
that this cell had been formed by this type of cell fusion. These findings, including ours, indicate the possible steps in the process of multinucleation of the preosteoclasts and osteoclasts, although the main process
of multinucleation should be the cell fusion between
osteoclastic cells, as reported by Fukushima et al.
(1991).
The osteoclasts in the diaphyseal bone marrow became more hyperactive. We observed that a n osteoclast
exhibited two sets of clear zones and ruffled borders in
the same cytoplasm. This phenomenon has been suggested by a scanning electron microscopic study of
Kanehisa and Heersche (19881,but has never been confirmed by transmission electron microscopy. It is, in
truth, necessary to prove by serial sections whether or
not i t might be a transverse section of a single set of
clear zone and ruffled border. However, as seen in Figure 11, pieces of calcified chondroid bars are loosely
scattered in the diaphyseal bone marrow. Therefore, it
is more likely that the osteoclast has developed two
sets of clear zone and ruffled borders to absorb the
separate pieces of calcified chondroid bars which are
situated far away from each other.
Quite bizzare amoeboid figures of osteoclasts were
reported in a cultured osteoclast (Zambonin-Zallone
and Teti, 19911, but have never been reported in vivo.
The existence of pieces of calcified chondroid bar in
large vacuoles indicates that they have been endocy-
426
H. ISAKI AND H. HANAOKA
Fig. 6. A peculiar binucleated preosteoclast appears to have been
formed by the fusion of two preosteoclasts in a tandem fashion. It has
TRAP-positive vesicles (arrow). The majority of the cells adjacent to
bone collar are either osteoblasts or preosteoblasts, having well-developed RER. Note the fibroblasts in the fibrous layer. A mononuclear
cell in the left-hand side, having features similar to those of the binucleated preosteoclast, might be a preosteoclast, however, there is no
definite evidence to prove it, because it shows no TRAP-positivities
( x 4,000).
Fig. 7. The youngest preosteoclast type, with a high NIC ratio and a light nucleus ( x 6,800). Inset: Higher view of a TRAP-positive dense body
(arrow) ( x 20,000).
EFFECTS OF HIGH DOSE PTH ON OSTEOCLASTS
427
Fig, 8.An apparently mononuclear osteoclast with a goldfish appearance. Note TRAP-positive dense bodies (arrow). This cell has a clear zone
with polar distribution. Monocytic cells are also seen ( X 4,000).
Fig. 9. An osteoclast with a large cytoplasm exhibiting two sets of clear zones and ruffled borders is resorbing the calcified chondroid bars.
No TRAP-positivities are observed in this cell ( X 4,000).
Fig. 10. An osteoclast demonstrating a prominent amoeboid shape. The cytoplasm is extended quite
remarkable, and a thick clear zone and ruffled border facing the calcified chondroid bar are observed at
the end of the cytoplasm ( X 2,600).
Fig. 11. An osteoclast demonstrating many large cytoplasmic vacuoles which contain a piece of calcified
chondroid bar (arrow) and other small piece of possible autophagozomes. Unidentifiable primitive cells
are also seen ( x 4,000).
EFFECTS OF HIGH DOSE PTH ON OSTEOCLASTS
tosed prior to phagocytosis, a s noted for osteocytes in
the literature (Soskolne, 1978; Elmardi et al., 1990).
In the diaphyseal bone marrow of the limb buds of
16-day-old fetal mice, calcified chondroid bars were being resorbed to form the bone marrow space and no
bone formation took place. This is the reason why no
mature osteoblasts were observed there in our study,
while remarkable hyperactivity of the osteoclasts affected by PTH were observed.
It is believed that PTH affects the osteoclasts indirectly through the osteoblasts, since the former have
been believed not to express the PTH receptor while
the latter have been shown to express i t (McSheehy
and Chambers, 1986). How PTH affected the osteoclasts in the diaphyseal bone marrow where no mature
osteoblasts were present in our study is not certain.
One possibility is that the PTH affected the osteoclasts
and their precursors through the osteoblasts while the
former had been in the periosteum before their migration into the bone marrow. Another is that PTH affected the osteoclasts and their precursors in the bone
marrow directly, a s the osteoclasts have been reported
to have the PTH receptor (Teti et al., 1991; Agarwala
and Gay, 1992). The third is that PTH affected the
osteoclasts and their precursors either through the possible preosteoblasts in the bone marrow or through the
parathyroid target (PT) cells termed by Rouleau et al.
(1990). Such PT cells could exist in the diaphyseal bone
marrow in our study as well as in the metaphyseal
bone marrow in their study, although we could not
identify if they were present in our study.
ACKNOWLEDGMENTS
This work was supported by a grant-in-aid from J a pan Orthopaedics and Traumatology Foundation, Inc.
(JOTF), grant no. 0039
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