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Differentiation kinetics of osteoclasts in the periosteum of embryonic bones in vivo and in vitro.

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THE ANATOMICAL RECORD 214:418-423 (1986)
Differentiation Kinetics of Osteoclasts in the
Periosteum of Embryonic Bones In Vivo and In
Vitro
BEN A.A. SCHEVEN, ELISABETH W.M. KAWILARANG-DE HAAS, ANNE-MARIE
WASSENAAR, AND PETER J. NIJWEIDE
Laboratory of Cell Biology and Histology, University of Leiden, 2333 AA Leiden, The
Netherlands
ABSTRACT
Osteoclast progenitors are seeded via the blood stream in the mesenchyme surrounding embryonic long bone models long before the appearance of
multinucleated osteoclasts. The proliferation and differentiation of these progenitors
in embryonic mouse metatarsal bones was studied with acid phosphatase (AcP)
histochemistry and 3H-thymidine autoradiography.
In vivo, tartrate-resistant, acid phosphatase-positive, mononuclear cells appear in
the periosteum (AcPP-P cells) at the age of 17 days (after conception). On day 18,
AcP-positive, multinucleated osteoclasts invade the bone rudiment and start resorbing the calcified cartilage matrix, resulting in the formation of the marrow cavity.
The kinetics of osteoclast formation in vitro was studied in metatarsal bones of
embryonic mice of different ages cultured in the continuous presence of 3H-thymidine. In young bones (15 days), mainly proliferating, 3H-thymidine-incorporating
progenitors gave rise to AcPP-P cell and osteoclast formation. In older bones (16 and
17 days) osteoclasts were progressively more derived from postmitotic, unlabeled
precursors. Irradiation of the metatarsal bones with a radiation dose of 5.0 Gy prior
to culture resulted in a selective elimination of the proliferating progenitors, whereas
the contribution of postmitotic precursors in AcPP-P cell and osteoclast formation
remained unchanged. The results demonstrate that in the periosteum of embryonic
metatarsal bones a shift occurs from a population composed of proliferating osteoclast progenitors (15 days) to a population composed of postmitotic precursors (17
days) before multinucleated osteoclasts are formed (18 days). Obviously, postmitotic
AcP-negative precursors, already present in 16-day-old bones, differentiate into
precursors characterized by tartrate-resistant AcP activity, the preosteoclasts (17
days), which in their turn fuse into osteoclasts.
In long bone development the primitive marrow cavity
is formed by resorption of the calcified cartilage matrix
in the center of the long bone model. The multinucleated
giant cell responsible for cartilage and bone resorption,
the osteoclast, belongs to a n extraskeletal, hematogenous cell lineage (Marks, 1983). Its progenitors are
transported via the blood stream and seeded in the periosteum of long bones at a n early stage of embryonic
development, long before the actual formation of the
marrow cavity (Thesingh and Burger, 1983; Burger et
al., 1984). Only little information is available about the
processes of proliferation and differentiation, i.e., osteoclast formation kinetics, which take place between the
homing of progenitors and the appearance of multinucleated osteoclasts. In a n in vitro study of embryonic
mouse long bones we recently found that the radiosensitivity of osteoclast formation is age-dependent (Scheven
et al., 1985). Our results suggested that in the periosteum of long bones radiosensitive osteoclast progenitors
proliferate and differentiate into postmitotic preosteoclasts before the first multinucleated osteoclasts are
0 1986 ALAN R. LISS. INC
formed by fusion. In order to obtain more insight into
the proliferation and differentiation kinetics of the osteoclast and its progenitor population, we studied the
appearance, in vivo, of tartrate-resistant acid phosphatase-positive (AcPP) mononuclear and multinucleated
cells in the periosteum and marrow cavity of metatarsal
bones of different ages. Subsequently we investigated
the 3H-thymidine (3H-TdR)incorporation into periosteal
osteoclast progenitors of embryonic long bones by assessing the number of labeled nuclei in histochemically (acid
phosphatase) recognizable osteoclasts and preosteoclasts. Finally we determined the effects of ionizing irradiation on the incorporation of 3H-TdFt in these cells.
MATERIALS AND METHODS
Tissue Preparation
The middle three metatarsal bones of the hind limbs
of Swiss albino mouse embryos in the age range of 1519 days (day of vaginal plug discovery was equated with
Received July 24, 1985; accepted November 18, 1985.
DIFFERENTIATION OF OSTEOCLAST PROGENITORS
419
day 0, day 19 is day of birth) were dissected and either
immediately processed for the demonstration of acid
phosphatase (AcP) activity (see below) or cultured
(Fig. 1).
Culture Procedure
After dissection, the bone rudiments were transferred
to a culture dish containing 1ml semisolid medium that
consisted of 60% Hanks’ balanced salt solution, 10%
embryonic extract of 10-day-oldchick embryos, 10% rat
serum, and 20% cock plasma. The bones of one of the
hind limbs were irradiated with a dose of 5.0 Gy by a
Philips RT 250 X-ray generator at 200 kV (HVL: 1.5
mm Cu, dose rate of 1.18 Gy/min). The corresponding
bones of the other hind limb were used as nonirradiated
controls. Subsequently, the explants were placed on fresh
medium containing 0.025 pCi 3H-thymidine (3H-TdR,
specific activity: 50 mCi/mmol) in 60% Eagle’s minimal
essential medium with the same supplements as described above. Culturin was performed in a 5% COa/air
atmosphere a t 37°C. ‘H-TW medium was refreshed
every 2 or 3 days. At different times the bones were
fixed and prepared for the AcP staining procedure and
autoradiography (Fig. 1).
Acid Phosphatase Histochemistry
The en bloc AcP staining a s described here proved to
be specific for the demonstration of osteoclastic, tartrateresistant AcP activity (Hammerstrom et al., 1971;
Minkin, 1982) in embryonic long bones.
Metatarsal bones were washed in cold Hanks’ balanced salt solution and fixed in 10% neutral buffered
formalin for 16 h r a t 4°C. Then the bones were decalcified in 5% formic acid and 5% formalin for 3 hr at 4°C.
After a quick wash in distilled water the specimens were
incubated for AcP staining with naphthol As-B1 phosphate as substrate and hexazonium pararosanilin as
coupler in Michaelis veronal acetate buffer for 1 hr a t
37 “C (Barka and Anderson, 1962). L( +)-tartaric acid
W S E
15
METATARSL
16
BONES
17
18
AGE
19
(days after cmccplion)
+AcP
HISTOCHEMISTRY
immediately after dissection
Fig. 2. Parts of noncounterstained histologic sections of embryonic
metatarsal bones stained for tartrate-resistant AcP activity. In the
periosteum (P) of 17-day-oldbones (a) AcP-positive cells (double arrows)
are visible. No osteoclasts have as yet developed. In 18-day-oldmetatarsal bones (b) AcP-positive osteoclasts (arrows) have invaded the
center of the bones. Note the absence of AcP-positive cells in the
periosteum. The bone collar is indicated by asterisks. The hypertrophic
cartilage is marked with HC. x 200.
dissolved in 0.1 M acetate buffer (pH 5.0) was added to
the substrate solution to a final concentration of 10 mM.
Omission of naphthol As-B1 phosphate or hexazonium
pararosanilin from the incubation medium resulted in
total absence of the azo dye staining. Then the bones
were washed in distilled water, kept overnight in 70%
ethanol, and embedded in paraffin. Sections of 5 pm
were counterstained with 0.1% methyl green and 0.01%
thionin in 0.02 M citrate buffer (pH 5.8) or with
hematoxy lin.
Light Microscope Autoradiography
‘i I i
I
CULTURE
in the presence of 3H-TdR
vL
3,i7,10 3.i7.K)
3.i.6 days
Paraffin sections were dipped in Ilford L4 emulsion
with a dipping apparatus and exposed for 4 weeks at
4°C. After development, the autoradiographs were examined by light microscope.
Quantification and Statistical Evaluation
In the metatarsal bones of different ages all nuclei of
the osteoclasts in the primitive marrow cavity (if present) and of the acid phosphatase-positive cells in the
LM AUTCUADIOGMWY
periosteum (AcPP-P cells) were enumerated from serial
sections. The AcP-positive cells (osteoclasts and AcPP-P
Fig. 1. Schematic representation of the experimental set-up. The cells) were easily discriminated from the AcP-negative
mesenchyme surrounding the bone models is indicated by dots. Resorption area (primitive marrow cavity) is illustrated as a light area in the cells by their specific, intense reddish azo dye staining
(Fig. 2).
calcified cartilage zone (black painted).
AcP
H15TCXHEMISTRY
B.A.A. SCHEVEN, E.W.M. KAWILARANG-DE H AAS, A,-M.WASSENAAR, AND P.J. NIJWEIDE
420
In the autoradiographs all labeled and unlabeled nuclei of osteoclasts and AcPP-P cells per cultured bone
were counted. Nuclei with more than 10 silver grains
were regarded as positively labeled (background labeling was minimal). Paired (control-irradiated) observations were statistically compared by the Student t test.
RESULTS
A schematic outline of the experiments performed is
given in Figure 1. The in vivo metatarsal development
with respect to the appearance and kinetics of tartrateresistant AcP-positive cells is described in the first part
of the results section. Subsequently, the results of the in
vitro 3H-TdR labeling experiments are presented.
Kinetics of Osteoclast Formation in vivo
On day 17 the first AcPP cells were observed in the
periosteum of embryonic mouse metatarsal bones (Fig.
2a). These AcPP-P cells were localized adjacent to the
periosteal osteoblasts or the newly formed bone collar.
Most cells were mononuclear but some multinucleated
AcPP-P cells, apparently about to invade the bone, were
also observed. In younger explants positive cells could
not be demonstrated, except for a n occasional weakly
colored cell in the periosteum of 16-day-oldbones. The
first osteoclasts resorbing the calcified cartilage matrix
appeared in metatarsal bones of 18-day-oldmouse embryos (Fig. 2b). The invasion process was accompanied
with a sharp decrease in the number of AcPP-P cells
(Fig. 3). On the 19th day of development the number of
osteoclast nuclei had further increased, coinciding with
a further decrease in the number of AcPP-P cells (Fig.
3).
Kinetics of Osteoclast Formation In Vitro
Metatarsal bones in which no multinucleated osteoclasts had been formed at the time of explantation (i.e.,
postconception age of 15, 16, and 17 days) were used for
continuous in vitro 3H-TdR labeling experiments (Fig.
1).Pilot culture experiments showed that the presence
of labeled thymidine did not significantly affect osteoclast formation (data not shown). Osteoclasts developed
during culture possessed either labeled or unlabeled nu.-
-
I
w
Z
c
40
L
0,
D
E
3
z
20
0
I
16
17
ia
19
AGE
days after conception
Fig. 3. Kinetics of AcPP-P cell ( 0 )and osteoclast (0)
formation
during metatarsal bone development in vivo. Bones of different ages
were stained for tartrate-resistant AcP activity, and the number of
nuclei of AcP-positive cells in the periosteum (AcPP-P cells), as well as
in the marrow cavity (osteoclasts), were counted. Values are plotted as
the mean & SD of 9-18 bones.
Fig. 4. Light-microscope autoradiographs of osteoclasts developed in
a control (a) and irradiated (b) 16-day-old bone labeled continuously
with 3H-TdR in vitro. Note the presence of labeled (arrows) and nonlabeled (asterisks) nuclei in the same osteoclast in the control and the
absence of labeled nuclei in the osteoclast formed following irradiation.
X 980.
clei or both. Especially in cultured 16-day-old bones,
both labeled and unlabeled nuclei were found together
in the same osteoclast (Fig. 4a). This was also the case
in the multinucleated AcPP cells, which were occasionally observed in the periosteum.
The results of a quantitative autoradiographic analysis of the sections are shown in Figures 5-7. Metatarsal
bones of 15-day-oldmouse embryos demonstrate a gradual accumulation of newly formed, 3H-TG-labeled
AcPP-P cells during a culture period of 7 days (Fig. 5 ,
hatched bars). Osteoclast formation started between days
5 and 7. Subsequently, the number of osteoclast nuclei
strongly increased, whereas the number of AcPP-P cells
slightly decreased. Almost all AcPP-P cells and osteoclasts were derived from proliferating progenitors. Nonlabeled AcPP-P cells or osteoclast nuclei (open bars) were
not present or only in very small numbers. Correspondingly, labeling indices ranged between 86% and 100%.
The kinetics of osteoclast formation in cultured 16day-old bones corresponded well with that of the AcPPP cell kinetics (Fig. 6). During the first days of culture
DIFFERENTIATION OF OSTEOCLAST PROGENITORS
15-day- old bones
-3
-
C I C I
L
C
I
C
l
I
C l C l
5
3
C
I
C
C
I
0
I
C
3
I
C l C l
5
C
l
C
7
l
I
I
10
7
0
C
16-day- o Id bo nes
I
T
421
l
C
l
C
C i C l
3
I
C
I
C I C I
5
7
I0
5
7
10
C l C l
10
Culture lime (days)
Fig. 5. AcPP-P cell and osteoclast kinetics in 15-day-oldmetatarsal
bones in vitro. The number o f labeled and nonlabeled nuclei were
enumerated in cultures labeled continuously with 'H-TdR. Open columns represent unlabeled nuclei, shaded columns represent labeled
nuclei. I, irradiated bones (5.0 Gy); C, paired nonirradiated controls.
Values are plotted as means (+SD) of 3-20 bones of two independent
experiments. (*): P < 0.01, compared to paired control bones.
3
Culture hme (days)
Fig. 6. AcPP-P cell and osteoclast kinetics in 16-day-oldmetatarsal
bones labeled continuously with 'H-TdR in vitro. See also legend to
Figure 3. Values are plotted as means k SD o f 6-8 bones of three
independent experiments. (*) P < 0.01, compared to paired control
bones.
17-day-old
the AcPP-P cells and osteoclasts mainly originated from
nonproliferating precursors (labeling index, day 3: 2025%). In time, the proportion of labeled nuclei increased
(Fig. 6, hatched bars). The mean labeling indices on days
5, 7, and 10 of culture were respectively 42%, 55%, and
68%for the AcPP-P cells and 42%, 57%, and 58%for the
osteoclast nuclei.
In metatarsal bones of 17-day-oldembryos the number
of osteoclasts and AcPP-P cells remained constant in the
culture period from 3 to 6 days (Fig. 7). The contribution
of proliferating, 3H-TdR-incorporating cells to either cell
type was minimal (labeling indices: 4-7%). During the
first 3 days of culture a massive decline in AcPP-P cell
numbers, already present at the time of explantation,
coincided with a rapid increase in osteoclast numbers
(Fig. 8).
I
C
I
bones
C I C I
C
:
c
I
6
4
3
Radiation Effects on Kinetics of Osteoclast Formation
Irradiation of metatarsal bones with 5.0 Gy prior to
culture specifically inhibited the appearance of labeled
AcPP-P cells and osteoclast nuclei, leaving the kinetics
of unlabeled nuclei in both cell types unchanged (Figs.
4b, 5-7). The radiation-induced inhibition in 15-day-old
bones was almost absolute, resulting in complete elimination of osteoclast formation (Fig. 5). In cultured 16day-old bones differentiation of H-TdR-incorporating
osteoclast progenitors was partially but significantly inhibited, causing severe interference with AcPP-P cell
and osteoclast kinetics (Fig. 6).
In older bones the radiation-induced reduction of the
number of labeled nuclei did not markedly affect osteoclast kinetics, since the pool of nonproliferating precursors principally determined the total osteoclast nuclear
population (Fig. 7).
1
C
I
C I
3
c i
C I
4
c
I
C
I
6
Culture time (days)
Fig. 7. AcPP-P cell and osteoclast kinetics in 17-day-oldmetatarsal
bones labeled continuously with 3H-TW in vitro. See also legend to
Figure 3. Values are plotted as means (f SD) of 6-8 bones of two
independent experiments. (*) P < 0.05, compared to paired control
bones.
422
B.A.A. SCHEVEN, E.W.M. KAWILARANG-DEHAAS, A.-M. WASSENAAR, AND P.J. NIJWEIDE
the findings of others (Burger et al., 1982; Seifert, 1984;
Horton et al., 1984).
-U
17-day-old
bones
The autoradiography experiments using continuous
3
3H-TdR
labeling in vitro enabled us to investigate the
Z
proliferation of osteoclast progenitors in the periosteum
of embryonic metatarsal bones. The results indicate that
% 40
in embryonic long bones of different ages osteoclast proL
W
genitor populations, which give rise to osteoclast formaf 20
tion in vitro, are in different kinetic phases. In cultured
3
15-day-oldbone rudiments (prelosteoclasts derived from
z
proliferating,
3H-TdR,-incorporatingprogenitors. Radia0
tion caused complete inhibition of (pre)osteoclast forma3
0
tion and consequently of marrow cavity development.
Bones of 16-day-old embryos mainly yielded unlabeled
Culture time (days)
(pre)osteoclast nuclei during the first days of culture,
but a n increasing proportion of proliferating progenitors
Fig. 8. Kinetics of AcPP-P cells ( 0 )and formation of osteoclasts (0)
during the first days of culture of 17-day-oldmetatarsal bone. Bones contributed to osteoclast formation a t longer culture
explanted from embryos of one mother mouse were either immediately times. This age group did not contain preexisting AcPPfixed for AcP staining (day 0)or cultured for 3 days. Means f SD of 9P cells, suggesting that the unlabeled (pre)osteoclast
12 bones.
nuclei were derived from postmitotic AcP-negative precursors. Hence, tartrate-resistant enzyme activity is obtained and/or becomes operative during the postmitotic
phase of the osteoclast differentiation. In older bones, a
DISCUSSION
pool of postmitotic precursors, mainly consisting of AcPPThe present study was designed to investigate the P cells, was involved in osteoclast formation. The contriproliferation and differentiation of periosteal osteoclast bution of proliferating progenitors to (pre)osteoclast deprogenitors during embryonic long bone development. velopment during culture of 17-day-oldbones is obviously
The osteoclast progenitors originate from a hemopoietic a minor one. In this age group the number of bloodstem cell (Marks, 1983) and are deposited in the perios- borne progenitors may be lowered owing to the declining
teum via the blood circulation at an early stage of em- number of circulating progenitor cells, as a result of the
bryonic development (Kahn et al., 1981; Thesingh and diminishing hemopoietic activity of the liver during the
Burger, 1983). The blood-borne progenitors then differ- late embryonic and neonatal development (Moore and
entiate and fuse into multinucleated osteoclasts which Williams, 1973; Burger et al., 1984). Furthermore, a
invade the calcified cartilage zone, resulting in the for- limitation of the proliferation ability of the present immation of a primitive marrow cavity. It should be em- mature osteoclast progenitors due to the advanced dephasized, however, that the osteoclast cell lineage, in velopmental stage of the 17-day-oldbones may in part
particular a s far as the early progenitors are concerned, account for the small participation of these cells in osteohas not yet been elucidated (Marks, 1983; Chambers, clast kinetics. In any case, the role of the periosteum as
1985). Several studies have described a direct mononu- secondary source of osteoclast precursors appears to declear precursor, the so-called preosteoclast, showing cline with the formation of the marrow cavity (Fig. 3).
morphological and cytochemical features that are simi- Nevertheless, the periosteum still remains a homing
lar to those of the multinucleated osteoclast (Scott, 1967; place of bone marrow-derived (pre)osteoclasts involving
Rifkin et al., 1980; Ejiri, 1983). In this study the appli- periosteal bone remodeling.
cation of tartrate-resistant AcP histochemistry resulted
In the 16- and 17-day-oldbones irradiation also inhibin specific staining of osteoclasts in the marrow cavity ited the appearance of labeled (prelosteoclast nuclei.
as well as of mononuclear cell types in the periosteum. However, unlike in younger bones, radiation did not
These periosteal AcPP cells are localized adjacent to the lead to total elimination of proliferating progenitors.
bone collar and appear just before multinucleated osteo- This may be explained by the assumption that osteoclast
clasts develop in 18-day-old bones. In vitro, this accu- progenitors in the explanted 15-day-old bones need to
mulation process prior to osteoclast formation could also undergo more cell divisions to reach the mature, postbe demonstrated (Fig. 5). The kinetic pattern of the mitotic stage.
AcPP-P cells appeared to be reciprocal to osteoclast forThis study also showed a discrepancy between the in
mation during both in vivo and in vitro development vivo vs. in vitro differentiation kinetics of the osteoclast.
(Figs. 3 and 8). Taken together, it is plausible that the In vitro, osteoclast formation appeared to be considerAcPP-P cell represents the mature, mononuclear preos- ably retarded compared to in vivo development (compare
teoclast, ready to fuse into multinucleated osteoclasts Figs. 3 and 5). However, once invasion of osteoclasts into
(Ejiri, 1983; Scheven et al., 1985).The corresponding 3H- the bone rudiment had started, the number of osteoTdR labeling pattern of the AcPP-P population and the clasts in the developing marrow cavity rapidly inosteoclast nuclear population is in support of this as- creased. Nonetheless, relatively high numbers of
sumption. Since both mono-/and multinucleated macro- preosteoclasts and some binucleated osteoclasts remain
phages do not express tartrate-resistant AcP activity in the periosteum of all age groups under the in vitro
(Seifert, 1984; own observations), our results make it conditions while disappearing in vivo. Possibly, the reunlikely that osteoclasts are derived from mature mono- tardation and ultimate stunting of the bone developnuclear phagocytes. This supposition is compatible with ment in vitro reduced the stimuli necessary for
.u
I
DIFFERENTIATION OF OSTEOCLAST PROGENITORS
activation and mobilization of the resident periosteal
(pre)osteoclast population.
In conclusion, the results provide conclusive evidence
for the earlier postulated differentiation pathway of periosteal osteoclast progenitors during metatarsal bone
development (Scheven et al., 1985).Obviously, osteoclast
progenitor differentiation is closely related to the development of the bone model, especially to initiation of
hypertrophic cartilage matrix calcification (16 days) and
the formation of the bone collar (17 days). In the periosteum mitotic active, blood-borne progenitors (15-16 days)
differentiate into postmitotic osteoclast precursors (16
days). Subsequently, the postmitotic precursors obtain
tartrate-resistant AcP activity (17 days). The latter identifiable cell may be called the preosteoclast, which forms
multinucleated osteoclasts by fusion (18 days).
It is evident that the kinetics and therefore the radiosensitivity (Scheven et al., 1985) of osteoclast differentiation in vitro is dependent upon the age of the bones.
In the in vitro system using metatarsal bones of different ages, it appears to be possible to separate the mitotic
phase (15 days) from the postmitotic phase (17 days) of
the osteoclast differentiation pathway. The experimental set-up may therefore provide a suitable model for the
study of factors influencing proliferation of osteoclast
progenitors on the one hand (i.e., long-term effects) and
for the study of factors regulating fusion, mobility, and
activity of postmitotic preosteoclasts (i.e., short-term effects), on the other hand.
ACKNOWLEDGMENTS
We are greatly indebted to Mr. A. Boon for performing
the irradiation procedures and to Prof. Dr. J. P. Scherft
and Dr. C.W. Thesingh for critical reading of the manuscript. This study was supported by the J.A. Cohen Institute for Radiopathology and Radiation Protection,
Leiden.
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