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Multinucleated cells formed on calcified dentine from mouse bone marrow cells treated with 1╬▒ 25-dihydroxyvitamin D3 have ruffled borders and resorb dentine.

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THE ANATOMICAL RECORD 224379-391 (1989)
Multinucleated Cells Formed on Calcified Dentine
From Mouse Bone Marrow Cells Treated With
1a,25Dihydroxyvitamin D3 Have Ruffled Borders
and Resorb Dentine
TAKAHISA SASAKI, NAOYUKI TAKAHASHI, SHOHEI HIGASHI,
AND TATSUO SUDA
Department of Oral Anatomy (T.S., S.H.) and Biochemistry (N.T., T.S.), School
Dentistry, Showa University, Tokyo 142, Japan
of
ABSTRACT
Osteoclast-like multinucleated cells were formed from mouse
bone marrow mononuclear cells, and their morphology on coverslips and on calcified dentine slices was compared by means of transmission electron microscopy.
Addition of la,25-dihydroxyvitamin D3 [ la,25(OH),D3] to bone marrow cells cultured on coverslips greatly stimulated the formation of multinucleated cells within
8 days. These multinucleated cells had the cytological features of osteoclasts
(abundant pleomorphic mitochondria, a large number of vacuoles and lysosomes,
many stacks of Golgi membranes, and a n extensive canalicular system), but they
developed neither ruffled borders nor clear zones. The multinucleated cells appeared to result from direct fusion of mononuclear progenitor cells, whose structural features were similar to those of multinucleated cells. Like isolated osteoclasts, both multinucleated cells and their precursors exhibited a n intense reaction
for tartrate-resistant acid phosphatase (TRACP) in the cisterns of endoplasmic
reticulum and lysosomes. Multinucleated cells formed from alveolar macrophages
in the presence of k ~ ,2 5 (OH)~ D
were
3 totally negative for TRACP reaction. When
marrow cells were cultured on dentine slices in the presence of la,25(OH),D3, some
of the multinucleated cells were located in the shallow resorption lacunae of dentine surfaces, and they developed the characteristic ruff led borders and clear
zones. The narrow extracellular spaces of the ruffled borders, the adjacent pale
endocytotic vacuoles, and the dark lysosomes located in the perinuclear cytoplasm
of the multinucleated cells contained numerous apatite crystals delete in resorption lacunae. These results indicate that 1) the multinucleated cells formed on
coverslips from mouse marrow cells treated with 1a,.%(OH)2D3 exhibit non-functional basic features of osteoclast morphology, and 2) differentiation of the multinucleated cells into functional osteoclasts requires some components of calcified
dentine.
It is known that osteoclasts, the principal cells responsible for bone resorption, are multinucleated giant
cells formed by fusion of mononuclear precursors derived from hematopoietic progenitor cells (Chambers,
1980; Bonucci, 1981; Roodman et al., 1985; Mundy and
Roodman, 1987). The precise mechanism by which the
progenitors in the hematopoietic cell population differentiate into osteoclasts, however, has not been established (Ibbotson et al., 1984; Roodman et al., 1985;
MacDonald et al., 1987; Takahashi et al., 1988a).
Recently, we developed a mouse marrow culture system in which the progenitor cells fuse into multinucleated cells and have the following characteristics in
common with authentic osteoclasts (Takahashi et al.,
1988a). 1) The multinucleated cells formed exhibited
tartrate-resistant acid phosphatase (TRACP) activity;
TRACP is known as a marker enzyme of osteoclasts. 2)
The formation of TRACP-positive multinucleated cells
0
1989 ALAN R. LISS, INC
was markedly stimulated by osteotropic hormones such
as la,25-dihydroxyvitamin D3 [ la,25(OH),D3] and parathyroid hormone. Calcitonin strikingly inhibited the
formation of TRACP-positive multinucleated cells induced by both osteotropic hormones. 3) As in the case of
authentic osteoclasts, TRACP-positive multinucleated
cells were formed by fusion of mononuclear precursors.
4) Perhaps most important, when marrow mononuclear
cells were cultured on calcified dentine slices in the
Received June 7, 1988; accepted January 25, 1988.
Address reprint requests to Dr. T. Sasaki, Department of Oral Anatomy, School of Dentistry, Showa University. 1-5-8Hatanodai, Shinagawa-ku, Tokyo 142, Japan.
Abbreviations used: la,25(OH)zD3, la,25-dihydroxyvitamin D,;
TRACP, tartrate-resistant acid phosphatase; FCS, fetal calf serum;
PBS, phosphate-buffered saline; a-MEM, alpha-minimal essential
medium; rER, rough endoplasmic reticulum.
380
T. SASAKI ET AL.
presence of l c ~ , 2 5 ( O H ) ~ the
D ~ ,TRACP-positive multinucleated cells resorbed dentine by creating resorption
lacunae.
In the present study, we demonstrate further ultrastructural and cytochemical evidence that the multinucleated cells formed on dentine slices from mouse
bone marrow mononuclear cells have ruff led borders
and clear zones, the characteristic cytological features
of functional osteoclasts, and they resorb calcified dentine in resorption lacunae exactly as do the authentic
osteoclasts.
MATERIALS AND METHODS
Animals and Hormone
Seven- to 9-week-old male mice and 3-day-old delete
mice, both ddy strain, were obtained from Shizuoka
Laboratories Animal Center (Shizuoka, Japan).
l ~ i , 2 5 ( O H ) ~was
D ~ the generous gift of Dr. I. Matsunaga, Chugai Pharmaceutical Co. (Tokyo, Japan).
Bone Marrow Culture
The mice were killed by cervical dislocation under
light ether anesthesia, and their tibiae were aseptically removed and dissected free of adhering tissues.
The bone ends were cut off with scissors, and the marrow cavity was flushed out with l ml of a-minimal
essential medium (a-MEM) by slowly injecting a-MEM
at one end of the bone using a sterile 25 gauge needle.
The marrow cells were collected into tubes and washed
twice with a-MEM. The marrow cells were then suspended in a-MEM containing 10% heat-inactivated fetal calf serum (FCS, Gibco, Grand Island, NY) at 1.5 x
lo6 celldm1 and cultured for 8 days on either Lux coverslips (15 mm, Miles Scientific, Naperville, IL) or
whale calcified dentine slices in 24-well plates (Sumitom0 Bakelite Co., Tokyo) (0.5 ml/well). The blocks of
whale dentine were provided by Dr. A. Boyde, London
University, London, UK. In brief, the dentine slices,
0.5-1 mm thick, were cut using a water-cooled diamond saw from blocks of sperm whale dentine, washed
by ultrasonication for 10 min and further sterilized by
ultraviolet light irradiation for 4 hr before use. Cultures were fed every 3 days by replacing 0.4 ml of old
medium with fresh medium. 1a,25(OH)2D3(lop8 M)
was added at the beginning of the culture and each
time the medium was changed. All cultures were maintained at 37°C in a n atmosphere of 5% COz in air.
Isolation of Osteoclasts and Alveolar Macrophages
Osteoclasts were obtained from long bones of 3-dayold mice a s described previously (Takahashi et al.,
1988b). Femora and tibiae of the mice were removed
and curetted into a-MEM with 10% fetal bovine serum
(eight bones/ml). The cell suspension was pipetted onto
the center of Lux coverslips in 24-well plates (0.5 mll
well) and incubated for 2 h r at 37°C. The coverslips
were then washed with PBS and/or a-MEM and processed for electron microscopy.
Alveolar macrophages were obtained by the tracheobronchial lavage method (Abe et al., 1983). Lavaged
macrophages were washed with a-MEM, suspended in
the same medium containing 5% heat-inactivated human serum a t a concentration of 5 x lo6 cells/ml, and
plated in the center of the coverslips in 24-well plates
(30 pUwe11). After incubation for 30 min at 37"C, nonadherent cells were removed, and only adherent cells
were cultured in a-MEM with 5% human serum.
M to
la,25(OH)2D3was added to the cultures at
induce fusion of alveolar macrophages. After culture
for 4 days, the cells were rinsed with a phosphate-buffered saline (PBS, pH 7.4) and processed for electron
microscopy.
Electron Microscopy
For conventional thin sectioning, adherent cells on
either coverslips or dentine slices were rinsed with PBS
and fixed with a mixture of 2% acrolein, 2.5% glutaraldehyde, and 3% formaldehyde in 0.06 M sodium cacodylate buffer (pH 7.3) containing 0.05% CaC12. They
were then rinsed in the same buffer overnight and
postfixed with 1.5% potassium-ferrocyanide-reduced
1% osmium tetroxide for 2 h r at 4°C. Then, they were
block-stained with 1% uranyl acetate in 10% ethanol
for 30 min at room temperature, dehydrated through a
graded ethanol series, and embedded as a monolayer in
Poly Bed 812. For ultracytochemical detection of
TRACP activity, we modified the medium of acid pnitrophenyl phosphatase (Miyayama et al., 1975). This
medium consisted of 3 mM p-nitrophenyl phosphate
(Sigma Chemical Company, St. Louis, MO) as a substrate, 3 mM lead nitrate as a capture reagent, 80 mM
Tris maleate buffer (pH 6.7), 8% sucrose, and 10 mM
sodium tartrate. Final pH was adjusted to 5.5-5.8 by
adding 0.1 M nitric acid. Adherent cells on the coverslips were rinsed once with PBS and fixed in culture
flasks containing a mixture of 1% glutaraldehyde and
1%formaldehyde in 0.1 M sodium cacodylate buffer
(pH 7.3) for 2 h r at 4"C, rinsed in the same buffer overnight, and incubated for 30 min at room temperature
(25°C) in the medium described above for TRACP
staining. Control tissues were incubated in the same
medium lacking substrate. After incubation, all specimens were postfixed with 1.5% potassium-ferrocyanide-reduced 1%osmium tetroxide for 10 min at 4°C
and processed for embedding as described above.
Sections perpendicular to the coverslips or the dentine slices were cut using a diamond knife on a Reichert-Jung Ultracut OmU-4 and counter-stained with
aqueous uranyl acetate and lead citrate before being
examined in a Hitachi HU-12A electron microscope at
a n operating voltage of 75 kV.
To study the inorganic elements in the above specimens, unstained ultrathin sections mounted on formvar-coated copper grids were analyzed with a Hitachi
H-600 electron microscope fitted with a scanning device and Kevex 7000A energy-dispersive X-ray microanalyzer. Analysis points were selected using the
scanning images of ultrathin sections. The microprobe
conditions were 75 kV accelerating voltage, 2 x lop9A
specimen current, 15 nm spot size, and 200 sec counting time.
RESULTS
Tartrate-ResistantAcid Phosphatase (TRACP) in lsolated
Osteoclasts and Multinucleated Cells Derived From Either
Alveolar Macrophages or Bone Marrow Cells
TRACP activity was detected as electron-dense precipitations of lead phosphates in membrane-bounded
OSTEOCLAST FORMATION I N MOUSE MARROW CULTURE
bodies resembling lysosomes and cisterns of rough endoplasmic reticulum (rER) in isolated osteoclasts (Fig.
la), but it was never seen in any subcellular structures
of multinucleated cells derived from alveolar macrophages (Fig. lb).
When marrow cells were cultured on coverslips in
the presence of ~ c L , ~ ~ - ( Ofor
H 8) days,
~ D ~a number of
mature mononuclear cells and their fused multinucleated cells appeared (Fig. lc, d). The cytoplasm of the
multinucleated cells and their mononuclear progenitors was characterized by the presence of many mitochondria, lysosomal membrane-bounded bodies, a moderate number of rER cisterns and stacks of Golgi
membranes, numerous free polyribosomes, and several
small membrane-bounded azurophilic granules. The
cell surface formed numerous microvillous projections
and complicated cytoplasmic membrane foldings associated with a number of pale vacuoles (Fig. lc-f).
Unlike the multinucleated cells derived from alveolar macrophages, a n intense TRACP reaction was seen
in the multinucleated cells and their precursors derived from bone marrow cells. In Figure lc, five mononuclear precursor cells undergoing fusion were recognized, and all of them exhibited TRACP activity. These
structural and cytochemical features of mononuclear
precursors were similar to those of multinucleated cells
described below. In the multinucleated cells, TRACP
activity was demonstrated in the rER cisterns (Fig. le)
and membrane-bounded bodies resembling lysosomes
(Fig. If) but was never found in the Golgi saccules and
the plasma membranes.
In the control experiments, omission of the substrate
p-nitrophenyl phosphate from the incubation medium
led to a complete disappearance of TRACP activity in
all cell types, but omission of sodium tartrate did not
alter the intensity and localization of TRACP in both
isolated osteoclasts and marrow cell-derived multinucleated cells (data not shown).
38 1
with mononuclear progenitor cells (Fig. 2a, b). The
mononuclear progenitor cells showed no membrane specialization for cell-to-coverslip contact.
Multinucleated cells formed on coverslips also
showed smooth cell surfaces devoid of any membrane
specializations for cell-to-coverslip contact. The free
cell surface facing the culture medium extended numerous long microvilli associated with a number of
membrane infoldings and pale vacuoles of various sizes
(Fig. 2c). Although these multinucleated cells contained many cell organelles, they appeared to lack the
morphological polarity of cytoplasmic organization: the
central part of cytoplasm of the multinucleated cells
was occupied by many stacks of Golgi saccules (Fig. 2c,
inset), mitochondria, and cisterns of rER, whereas nuclei having both euchromatin and heterochromatin
were arranged along the peripheral cytoplasm. Nucleoli were clearly distinguished in these nuclei. Nuclear
mitosis never appeared during the formation of multinucleated cells. Mitochondria, rER, free polyribosomes,
and lysosomes were scattered throughout the cytoplasm of the multinucleated cells (Fig. 2c). The basal
cell surface of the multinucleated cells facing the coverslip and its subsurface cytoplasm showed no membrane specializations, except for shallow plasmalemma1 infoldings (Fig. 2d, inset). Thus, it is concluded
that the multinucleated cells formed on coverslips
lacked ruffled borders. The cell periphery of the multinucleated cells showed a clear zone-like region devoid
of most cell organelles, except for numerous free ribosomes and cytoplasmic filaments (Fig. 2d).
Multinucleated Cells Formed on Dentine Slices
When marrow mononuclear cells were cultured on
dentine slices in the presence of l ~ i , 2 5 ( 0 H ) ~multiD~,
nucleated cells with somewhat different morphological
features were formed. These multinucleated cells were
located on the shallow resorption lacunae of the dentine surface (Fig. 3a). In contrast with the multinucleMultinucleated Cells Formed on Coverslips
ated cells formed on coverslips, those formed on dentine
When bone marrow cells were cultured for 8 days with slices in vitro appeared to satisfy almost all the morlop8 M 1a,25(OH)2D3on coverslips, typical multinu- phological criteria of fully differentiated osteoclasts.
The multinucleated cells had several nuclei with eucleated cells and their mononuclear progenitors were
formed. On the coverslips, a number of mature mono- chromatin and heterochromatin and prominent nuclenuclear cells thought to be progenitors of multinucle- oli located at the cell periphery facing the culture meated cells were observed (Fig. 2a). The nucleus of such dium (Fig. 3a). Many stacks of the Golgi membranes,
cells possessed both euchromatin and heterochromatin each stack consisting of about five Golgi saccules and
and sometimes had a prominent nucleolus. The cyto- related vesicles, were localized in the perinuclear cytoplasm was characterized by the presence of many mi- plasm (Fig. 3b). The multinucleated cells exhibited
tochondria, lysosomal membrane-bounded bodies, a well-developed ruff led borders consisting of deeply inmoderate number of rER cisterns, the stacks of Golgi folded plasma membranes adjacent to the dentine sursaccules, numerous free polyribosomes, and several face. The depth of these membrane infoldings was
small membrane-bounded azurophilic granules. Facing 1.5-4 Km; the adjacent cytoplasm contained many pale
the culture medium, the cells extended numerous long endocytotic vacuoles of various sizes and configuramicrovilli (pseudopodia). Another cell surface special- tions, some of which were apparently continuous with
ization feature was the complicated deep membrane the narrow extracellular spaces of the ruffled border
infoldings (Fig. 2a). These structural features of mono- (10-40 nm in width) (Fig. 3b, c).
The extracellular space of the ruff led border was parnuclear progenitors were similar to those of multinucleated cells described below. The mononuclear cells tially filled with numerous small apatite crystals of
appeared to fuse with each other (indicated by arrow dentine matrix. Many pale endocytotic vacuoles adjaheads in Fig. 2b). When these mononuclear cells came cent to or continuous with the ruffled border also coninto partial contact with the coverslips, osteoblast-like tained apatite crystals (Fig. 3b, c). Characteristically,
cells appeared to intervene between the mononuclear the surface dentine layer facing the ruffled border excells and the coverslips and were in very close contact hibited lower crystal density compared with the deeper
382
T. SASAKI ET AL.
Fig. 1. Ultracytochemical localization of tartrate-resistant acid
phosphatase (TRACP). a: An isolated osteoclast exhibits intense
TRACP activity in lysosomes and tubulovesicular structures.
x 10,000. b A multinucleated cell formed from mouse alveolar macrophages in the presence of 1 1 ~ , 2 5 ( 0 H ) ~ItDis~totally
.
negative for the
enzymatic reaction. x 6,250. c: Mononuclear bone marrow cells cultured with 1 1 ~ , 2 5 ( 0 H ) ~These
D ~ . cells undergoing multinucleation
show intense TRACP activity. Two mononuclear monocyte-like cells
(arrowheads) are negative for the enzymatic reaction. x 3,750.
OSTEOCLAST FORMATION IN MOUSE MARROW CULTURE
Fig. 1, d A multinucleated cell formed from bone marrow cells in
the presence of l c ~ , 2 5 ( O H ) ~exhibits
D~
TRACP activity. Note the presence of TRACP-positive mononuclear cells (arrowheads) around the
383
multi-nucleated cell. cs: coverslip. x 5,000. e: TRACP activity in cisterns of endoplasmic reticulum of a multinucleated cell. x 33,750. fi
TRACP activity in lysosomes. x 27,000.
384
T. SASAKI ET AL.
layer (Figs. 3b, 4a). The peritubular dentine had higher
crystal density than the intertubular dentine. At the
border between the electron-lucent surface dentine
layer and the electron-opaque deeper layer, an electron-dense line of crystals appeared (Fig. 4a, arrows).
Toward the peripheral region of the multinucleated
cells facing the dentine surface, the so-called clear zone
appeared adjacent to the ruffled border (Fig. 4b). The
plasma membrane of the clear zone showed small undulations but never formed deep membrane infoldings.
The cytoplasm of the clear zone was 0.5-1.2 pm in
depth and totally devoid of cellular organelles, except
for numerous cytoplasmic filaments and a small number of free ribosomes (Fig. 4b). The dentine surface fac-
ing the peripheral clear zone contained more mineralized matrix similar to the deeper zone of dentine than
that facing the ruffled border (Fig. 4a and b).
X-ray microanalysis of dentine in various regions
was also performed on the same but unstained sections.
Several major elemental peaks of calcium (Ca: Ka =
3.691 KeV; KP = 4.012 KeV), phosphorus (P: Ka =
2.013 KeV), and sulfur (S: Ka = 2.307 KeV), as well as
some minor peaks derived from plastic embedding medium (Cl), fixative (K and Os), and block-staining solution (Ur) were recognized. The lowest spectrum
peaks of Ca, P, and S were obtained from the spaces of
the ruffled border containing apatite crystals (Fig. 5a).
The surface dentine layer showed moderate peaks of
OSTEOCLAST FORMATION IN MOUSE MARROW CULTURE
385
Fig. 2. a: A mononuclear progenitor cell has come in contact with
a coverslip (CS) (marked by arrow). An osteoblast-like cell (OBC)
intervenes between a mononuclear cell and a coverslip and has close
contact with the mononuclear cell (arrowheads). The inset figure indicates the perinuclear cytoplasm of the mononuclear cell including
the Golgi saccules (Go), mitochondria, azurophilic granules, and pale
vacuoles. x 7,500; inset, X 25,000. b: Fusion of two mononuclear progenitor cells at marked areas (arrowheads). These cells have partial
contract (arrows) with a coverslip (CS). An osteoblast-like cell (OBC)
is present between the progenitors cell and a coverslip. x 10,000. c: A
low-magnification view of a multinucleated cell formed on a coverslip
(CS). The cell contains several nuclei and abundant cell organelles
such as mitochondria and cisterns of rER. Many vacuoles are scattered throughout the cytoplasm. The cell surface facing the culture
medium has a number of microvilli, while that facing a coverslip is
quite smooth. The inset figure indicates the perinuclear cytoplasm of
the multinucleated cell, including the stacks of Golgi saccules (Go)
and pale vacuoles. ~3,750;
inset, ~ 8 , 0 0 0 .d The peripheral cytoplasm of multinucleated cell facing a coverslip (CS) is devoid of cell
organelles except for numerous free ribosomes and microfilaments.
The cell surface at the central part of a multinucleated cell facing a
coverslip (CS) shows shallow membrane infoldings (MI) but not ruffled border (inset figure). x 12,000.
Ca, P, and S (Fig. 5b). The highest spectrum peaks of
Ca, P, and S were detected from regions deeper in the
dentine layer (Fig. 5c).
The multinucleated cells contained various types of
phagosomes and lysosomes. As has been described,
many pale endocytotic vacuoles in the ruffled border
zone contained apatite crystals (Figs. 3c, 6a). These
vacuoles are regarded as phagosomes. Round dark lysosomes in the perinuclear cytoplasm also contained a
smaller number of apatite crystals (Fig. 6b). Multivesicular bodies and coated vesicles were always free of
apatite crystals. X-ray microanalysis of the pale en-
386
T.SASAKI ET AL.
OSTEOCLAST FORMATION IN MOUSE MARROW CULTURE
docytotic vacuoles and dark lysosomes containing crystals detected energy spectrum peaks of Ca, P, and S to
some degree, but the peak heights were apparently
much lower than those in the surface and deeper dentine layers (inset figures in Fig. 6 a, b). The sulfur peak
was sometimes not distinct in the dark lysosomes,
while it was evident in the pale endocytotic vacuoles
adjacent to the ruffled border (inset figures in Fig. 6a,
b).
387
The bone resorption-stimulating hormone, 101,25
(OHhD3, is directly or indirectly involved in the differentiation and fusion of mononuclear precursors to form
multinucleated cells (Abe et al., 1983; Bar-Shavit et al.,
1983; Ibbotson et al., 1984; Roodman et al., 1985; MacDonald et al., 1987). Recently, we developed a mouse
marrow culture system, in which multinucleated cells
with several characteristics of osteoclasts, including
tartrate-resistant acid phosphatase (TRACP), were
formed (Takahashi et al., 1988a). The formation of
multinucleated cells by 101,25(OH)~D~
was greatly inhibited by simultaneously adding salmon calcitonin.
When marrow cells were plated on coverslip to start a
culture, no multinucleated TRACP-positive osteoclastlike cells were found (Takahashi et al., 1988a).
The multinucleated cells formed on coverslips in the
presence of 101,25 (OH)ZD3 exhibited several ultrastructural features of osteoclasts such as abundant
pleomorphic mitochondria, many stacks of Golgi saccules, an extensive system of microvilli and related
membrane infoldings, and a peripheral cytoplasm lacking organelles (a clear zone-like structure) (Kallio et
al., 1971; Gothlin and Ericsson, 1976; Holtrop and
King, 1977; Ibbotson et al., 1984; MacDonald et al.,
1987). As seen in isolated authentic osteoclasts,
TRACP, a reliable marker enzyme of osteoclasts
(Burger et al., 1982; Minkin, 1982; van de Wijngaert
and Burger, 1986), was detected in the bone marrowderived multinucleated cells and their mononuclear
precursors a t an ultrastructural level, using p-nitrophenyl phosphate as a substrate. Multinucleated cells
formed from mouse alveolar macrophages in the presence of 101,25(OH)~D~
showed no TRACP activity. Acid
trimetaphosphatase and f3-glycerophosphatase activities, reliable marker enzymes of lysosomes, were observed both in the alveolar macrophage-derived and in
the bone marrow-derived multinucleated cells (data
not shown). Previous cytochemical investigations of lysosomal acid phosphatases in osteoclasts have demonstrated the intense enzymatic activities of P-glycerophosphatase, arylsulfatase, and trimetaphosphatase in
the Golgi-GERL-lysosomesystem of the osteoclasts and
their precursors (Lucht, 1971; Doty and Schofield,
1972; Baron et al., 19861, but none of these acid phosphatase isoenzymes are specific for osteoclasts. The ultracytochemical demonstration of TRACP shown in
this study is therefore thought to be a useful biological
tool for determining whether they are osteoclasts and
their lineage cells.
It should be noted that the ruffled border, the most
important morphological feature of functional osteoclasts, was not observed in the multinucleated cells
formed on coverslips. When mononuclear marrow cells
were cultured on dentine slices, the ruffled border was
observed in the multinucleated cells that formed. We
used dentine slices instead of bone slices, since dentine
slices do not have any lacunae for osteocytes in the
matrices, and there is no risk of misjudging them to be
newly formed resorption lacunae (Boyde et al., 1984).
Since ruffled borders were detected on the cell surface of the multinucleated cells facing dentine slices, it
is suggested that some unidentified components of calcified dentine are responsible for inducing the formation of ruffled borders. The appearance of ruffled borders on multinucleated cells affects the cytoplasmic
polarization. From the surface toward the inside of the
cells, the functional arrangement, in order, is the ruffled border, an adjacent endocytotic vacuole region, a
perinuclear Golgi region, and dark lysosomes in the
deeper cytoplasm. This is the characteristic organization of the Golgi-lysosome system in osteoclasts (Kallio
et al., 1971; Gothlin and Ericsson, 1976; Holtrop and
King, 1977). We call these multinucleated cells fully
differentiated functional osteoclasts.
It is therefore likely that there are two morphological states of osteoclasts: 1)the common, non-functional
osteoclast morphology and 2) the acquired specialized
features characterizing functional osteoclasts. To induce the latter, calcified tissues such as bone and dentine are thought to be required. It is quite understandable that previous investigators have failed to
demonstrate multinucleated cells with ruff led borders,
since they cultured mononuclear marrow cells on plastic dishes or coverslips (Ibbotson et al., 1984; Roodman
et al., 1985; MacDonald et al., 1987). Our recent autoradiographic study using ['2511-labeled calcitonin has
revealed that mouse marrow cell-derived TRACP-positive multinucleated cells and mononuclear cells grown
on coverslips have calcitonin receptors as do authentic
osteoclasts (Warshawsky et al., 1980; Takahashi et al.,
198813). Therefore all morphological characteristics of
osteoclast-like multinucleated cells, except the ruff led
border, should be regarded as being non-functional basic features of osteoclasts. In the absence of calcified
tissues in the culture, the marrow cell-derived multi-
Fig. 3. a: A low-magnification view of multinucleated cell on a
calcified dentine slice (CDe). This multinucleated cell has numerous
mitochondria, cisterns of rough endoplasmic reticulum, many pale
endocytotic vacuoles, and dark lysosomes. Facing the calcified dentine, the cell shows a characteristic ruffled border. x 3,750. b The
perinuclear cytoplasm and the ruffled border zone (RB) of the same
multinucleated cell as in a in a resorption lacuna on a calcified dentine slice (CDe). The cytoplasm contains several stacks of Golgi suc-
cules (Go),several mitochondria (Mt), multivesicular bodies (MVB),
and pale endocytotic vacuoles (V). The ruffled border is composed of a
deeply infolded plasma membrane. The calcified dentine facing the
ruffled border is reduced in crystal density. ~ 1 2 , 5 0 0 c:
. A higher
magnification of the ruffled border. Note that numerous small apatite
crystals are present in the narrow extracellular space of the ruffled
border and in adjacent newly formed pale endocytotic vacuoles (arrowheads). x 27,500.
DISCUSSION
388
T. SASAKI ET AL.
OSTEOCLAST FORMATION IN MOUSE MARROW CULTURE
389
nucleated cells exhibited neither ruffled borders nor
polarized organization of cytoplasmic organelles (Ibbotson et al., 1984; Roodman et al., 1985; MacDonald et
al., 1987). The components in calcified tissues that are
responsible for inducing the ruffled border need to be
determined.
The present study also points out the resorption
process of calcified tissues by multinucleated cells: liberation of crystals from the collagen fibers by decalcification of dentine and cellular absorption and intracellular degradation of liberated crystals. First, the
surface dentine layer facing the ruffled border of the
multinucleated cells is partially decalcified, resulting
in numerous liberated apatite crystals. The surface
dentine facing the clear zone is not decalcified and remains intact. X-ray microanalysis of the dentine overlaid by the ruffled border revealed that the peaks of Ca
and P in the surface layer were lower than those in the
deeper layer. The identical analytical results were obtained by in vivo dentine resorption mediated by human odontoclasts (Sasaki et al., 1988a,b). In addition,
the structural porosity of the bone matrix facing the
osteoclast ruff led border has been indicated by the penetration of intravenously injected horseradish peroxidase into the matrix zone from 1 to 2 pm below the
surface (Sasaki et al., 1985). A partial decalcification of
the bone surface under the osteoclast ruffled border
has been recognized by the presence of loose apatite
crystals and disrupted collagen fibers at that matrix
zone; this decalcification is considered to be the first
extracellular phase in bone resorption (Kallio et al.,
1971; Bonucci, 1974). Such a partial decalcification of
the bone matrix may result in liberation and release of
apatite crystals from the matrix for subsequent resorption by osteoclasts.
These findings agree well with a recent hypothesis
that osteoclasts produce protons and hydrolytic enzymes, including acid protease, and secrete them into
the local milieu between the ruffled borders and resorbing bone surface, to decalcify and degrade bone matrix (Fallon, 1984; Baron et al., 1986; Blair et al., 1986;
Chambers et al., 1987). In fact, Fallon (1984) reported
that the local pH in resorption lacunae on bone surfaces covered with ruffled borders of osteoclasts is below 5. Baron et al. (1985) and Silver (1988) have recently achieved similar results.
The liberated apatite crystals of dentine are distributed in the extracellular canals of the ruffled borders,
in the newly formed pale endocytotic vacuoles, and in
the dark lysosomes of the multinucleated cells. The
ruffled border of the multinucleated cells therefore appears to be the site of formation of endocytotic vacuoles
containing apatite crystals. Absorption of apatite crystals by multinucleated cells is thought to be due to a
fluid-phase endocytosis that has been demonstrated in
the osteoclast ruffled border (Sasaki et al., 1985). Similar absorption of apatite crystals into the extracellular
channels of the ruffled border has been observed in
osteoclastic bone resorption (Bonucci, 1974; Gothlin
and Ericsson, 1976).
An X-ray microanalysis revealed that the apatite
crystals showed lower peak heights of Ca, P, and S in
the dark lysosomes than in the newly formed pale endocytotic vacuoles and the ruff led borders of multinucleated cells. In particular, the S peak was scarcely
detected in the dark lysosomes, suggesting that digestion of the sulfur-containing material had taken place
a t this phase. Therefore, the multinucleated cells are
thought to resorb liberated apatite crystals with sulfated organic materials via the ruff led borders and endocytotic vacuoles and subsequently to digest them in
lysosomes. It has also been well documented that the
osteoclasts contain various lysosomal enzymes in membrane-bounded bodies around the Golgi complexes such
as the dark lysosomes shown in this study (Lucht,
1971; Doty and Schofield, 1972, 1976; Walker, 1972;
Rath et al., 1981; Baron et al., 1985, 1986).
Takagi et al. (1982) have demonstrated that the
complex carbohydrates of bone matrix are incorporated by osteoclasts into pale endocytotic vacuoles by
way of the ruffled borders. These vacuoles then fuse
with primary lysosomes derived from the Golgi
vesicles and consequently become the phagolysosomes
where intracellular digestion of the incorporated
materials takes place. It is therefore likely that the
pale endocytotic vacuoles containing apatite crystals
in the multinucleated cells have obtained hydrolytic
enzymes from the Golgi apparatus, resulting in the
dark lysosomes, where incorporated materials are
digested. Taken together with these results, osteoclasts are thought to resorb free inorganic crystals and
dissolve them and to digest the organic matrix of bone
in an acidic microenvironment mediated by the ruff led
border.
In conclusion, we developed a culture system to make
functionally fully differentiated osteoclasts. This culture system provides a useful experimental model for
further examining the mechanisms of osteoclast formation and bone resorption in normal and pathological
states.
Fig. 4. a: The surface dentine layer (marked by a bracket) facing
the ruff led border (RB) consists of relatively electron-lucent intertubular dentine (ID) showing low crystal density and electron-dense
peritubular dentine (I'D) surrounding the dentinal tubules (DT). Beyond the electron-dense border (arrows), both the intertubular and
peritubular dentine matrices in the deeper layer show a much higher
packing density of apatite crystals, x 15,000. b The surface dentine
layer facing the clear zone (CZ) of multinucleated cell. Toward the
peripheral region of the multinucleated cell (at the top right-hand
corner), the surface dentine consists of densely packed crystals,
whereas the crystal density becomes reduced in the dentine adjacent
to the ruffled border region (RB, at the bottom left-hand corner). DT:
dentinal tubule, x 12,500.
Fig. 5. Energy spectrum peaks from an X-ray microanalysis of a:
an area in the ruffled border, b the surface dentine layer facing the
ruffled border, and c: the deeper dentine layer beyond the dense border in Figure 4a. The peaks of calcium (Ca) and inorganic phosphorus
(PI derived presumably from apatite crystals were lowest in a: the
ruffled border, highest in c: the deeper dentine layer, and intermediate in b: the surface dentine layer. The phosphorus peak is combined
with the osmium peak ( 0 s ) .
390
T. SASAKI ET AL.
Fig. 6. a: Four pale endocytotic vacuoles (arrowheads) adjacent to
the ruffled border of a multinucleated cell containing apatite crystals.
The inset figure indicates the high peaks of Ca and P and a low peak
of sulfur (S) in these vacuoles. x 37,500. b Five dark lysosomes (arrowheads) containing crystal-like dense materials in the deeper cyto-
plasm of a multinucleated cell. An X-ray microanalysis of this dense
material revealed lower but significant peaks of Ca and P. The peak
of S was almost undetectable. x 37,500. Os, osmium; Ur, uranyl acetate.
OSTEOCLAST FORMATION IN MOUSE MARROW CULTURE
ACKNOWLEDGMENTS
This study was supported by Grants-in-Aid
(60440086, 61570894, and 61870047) from the Ministry of Science, Education, and Culture of Japan.
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dihydroxyvitamin, dentin, mouse, former, bones, cells, multinucleate, marrow, border, calcified, resort, ruffles, treated
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