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Surface modifications at the periosseous region of chick osteoclast as revealed by freeze-substitution.

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THE ANATOMICAL RECORD 222:323-332 (1988)
Surface Modifications at the Periosseous Region of
Chick Osteoclast as Revealed by
Freeze-Substitution
TOSHITAKA AKISAKA, GUS PERMANA SUBITA, AND YOSHIO SHIGENAGA
Department of Anatomy, School of Dentistry, Hiroshima University, Kasumi 1-2-3,
Minami-ku, Hiroshima 734, Japan
ABSTRACT
Improved preservation of osteoclast fine structure can be achieved
by quick freezing, freeze-substitution, or detergent extraction. With such techniques
the ruffled border mainly contains a disorganized, interconnected meshwork of microfilaments (5-7 nm in diameter), whereas in the clear zone a few ordered arrays
of intermediate-type filaments (10-12 nm in diameter) are detectable among the
network of microfilaments. In well-frozen samples, well-preserved matrix may have
occluded the cytoskeleton; detergent extraction permits visualization of the cytoskeletal components. In fresh-frozen cells an extracellular fuzzy coat overlays the ruffled
border. At the site of attachment of the clear zone to the bone surface, extracellular
cementing material is detected only after quick freezing. The superiority of quick
freezing to preserve ultrastructure is shown in various cytoplasmic organelles. Most
vesicles and vacuoles found close to the ruffled border seemed not to make contact
with the extracellular matrix. Anhydrous procedures using quick freezing and freezesubstitution stabilize bone mineral in some vacuoles and in the channels of the ruffled
border.
Osteoclasts (bone-destroying cells) are highly motile
and show morphological changes in response to calcium-regulating hormones and various substrates
(Hancox and Boothroyd, 1961; Cameron et al., 1967;
Kallio et al., 1972; Lucht, 1973; Lucht and Maunsbach,
1973; Holtrop et al., 1974; Weisbrode et al., 1974;
Miller, 1977; Chambers et al., 1984). On the surface
next to the calcified matrix, two distinct areas become
identifiable: the attaching, organelle-free clear zone and
the complicated membrane infoldings of the ruffled border, both of which appear to be active features of osteoclasts involved in bone resorption (Gothlin and
Ericsson, 1976; Holtrop and King, 1977). These two
membrane modifications seem t o endow the osteoclast
with its functional and morphological characteristics.
It is apparent that the osteoclast cytoskeleton may play
an important role in regulating cell shape, motility, and
adherence to bone.
Despite the considerable attention paid t o the surface
modifications of osteoclasts, their cytoskeletal organization has yet to be confirmed. Most information about
the osteoclast cytoskeleton has come from in vitro experiments (King and Holtvop, 1975; ZamboninZallone et al., 1983; Marchisio et al., 1984). However,
it is questionable whether the in vitro state always reflects the in vivo state. Morphological differences between osteoclasts in vitro and in vivo have been
suggested. The inability to preserve the cytoplasmic
structure of in vivo osteoclasts may in part be ascribed
to the deleterious effects of conventional processing.
Chemical fixatives cannot penetrate rapidly and evenly
into the deeper portion of calcified tissues, since the
0 1988 ALAN R. LISS, INC.
calcified matrix prevents a smooth and even penetration of fixatives. Such a delay in fixation causes various
postmortem changes, leading to distortion, destruction,
or loss of cytoplasmic structures. Probably, the high
concentration of hydrolytic enzymes or organic acids
both inside and outside of osteoclasts can accelerate the
postmortem changes. Also, soluble components may be
extracted or precipitated during aqueous treatment,
again resulting in an artifactual situation.
The main purpose of the present study was to elucidate the cytoplasmic structure of in vivo osteoclasts,
especially focusing on their ruffled border and clear
zone. To minimize the various disadvantages of conventional methods, the present study has employed quick
freezing, freeze-substitution, or detergent extraction,
by which the organization of cytoplasmic structure has
been successfully and recently clarified on various types
of cells (Small, 1981; Schnapp and Reese, 1982; Bridgman and Reese, 1984; Bridgman et al., 1986). As expected, these methods have contributed toward the
improved preservation of the ultrastructure of in vivo
osteoclasts.
MATERIALS AND METHODS
Forty white leghorn chicks (1-2 weeks old) were used
for the present study. After cervical dislocation without
anesthesia, tibial metaphyses were dissected out quickly.
Received September 21, 1987; accepted February 15, 1988.
Gus Permana Subita’s permanent address is Department of Oral
Medicine, Faculty of Dentistry, Indonesia University, JL Salemba Raya
4, Jakarta, Indonesia.
324
T.AKISAKA ET AL.
Fig. 1 . A low-power electron micrograph of a quick-frozen (QF) and
freeze-substituted(FS) osteoclast attaching to the metaphyseal bone.
The d e d border (RB)and clear zone (CZ) are identifiable. Bar
pm. x 7,800.
=
1
325
FREEZE-SUBSTITUTEDOSTEOCLAST
Direct Quick Freezing Followed by Freeze-Substitution
Slabs of tissue were frozen by the metal contact method
introduced by Van Harreveld and Crowell (1964). Immediately after tissue dissection, within 30 sec, samples
without any cryoprotectant were firmly contacted by
hand against a polished copper block precooled by liquid
nitrogen. Frozen samples were stored in a vial containing 1%tannic acid (Merk, West Germany) in absolute
acetone for 1-2 days a t -78°C and then transferred
into another vial containing 2%osmium tetroxide (OsO,)
(Merk) in absolute acetone and left there 1-2 days at
- 78°C. The vials were gradually brought to room temperature. Subsequently, the samples were washed in
absolute acetone and embedded in Epon-Araldite.
Chemical Fixation Followed by Freeze-Substitution
Tissue blocks were immersed in a fixative containing
1% tannic acid, 1%acrolein (Merk), and 2% glutaraldehyde (GA) (Taab, U.K.) in 0.1 M cacodylate buffer
(pH7.2) at 4°C for 2 hr. After fixation, the samples were
frozen by the metal contact method as described above
or by submersion into Freon cooled by liquid nitrogen.
Followingfreezing,the samples were processed for freezesubstitution as mentioned above.
Detergent Extraction
To extract the soluble components from the cytoplasm, tissues were immersed in buffered fixative containing 2%paraformaldehyde (Taab), and 0.2% saponin
(Katayama Chemicals, Japan) for 1-2 hr. Some tissues
were soaked for 20-60 min in a buffer (100 mM KC1;
30 mM HEPES, pH 7.2; 5 mM MgC1,; and 2 mM EGTA)
containing 0.2% saponin without chemical fixation for
20-60 min. After detergent extraction, tissues were frozen by the Freon or metal contact method. Freezesubstitution and embedding were the same as described
above.
Conventional Processing
For a control, tissues were first fmed for 2 hrs in 1%
tannic acid, 2% paraformaldehyde (PA), and 2.5% GA
buffered with 0.1 M cacodylate (pH 7.2) and then postfured in buffered 2% OsO,. After dehydration through a
graded series of acetones, the samples were embedded.
Ultrathin sections were doubly stained with uranyl
acetate and lead citrate or left unstained. They were
examined with a Hitachi 500 electron microscope at an
accelerating voltage of 100 kV.
RESULTS
General Features
The degree of ultrastructural preservation of rapidly
frozen osteoclasts was dependent on the size of ice crystals that caused the deformation of various structures.
The essential ultrastructural features observed after
quick freezing were similar to those seen after conventional furation (Fig. 1).However, variable improvements for morphological preservation could be achieved
by using quick freezing (QF) and freeze-substituion(FS).
The nuclear membrane displayed an apparently
smoother contour after quick freezing than after conventional processing. Various spherical vacuoles close
to the ruffled border, which were often coalesced with
each other or the ruffled border, seemed t o be true vac-
uoles. All the membrane components were well preserved, showing a trilaminar substructure without signs
of rupture (Fig. 2). Calcified matrix was also retained
well in situ (Figs. 1, 2). Mineral crystals from the dissolved bone were often observed in the vacuoles and in
the extracellular channels of the ruffled border (Fig. 2).
At low magnification, the cytoplasmic matrix of the osteoclast was uniformly well preserved (Fig. 1).
Regarding the reproducibility of the method utilizing
direct quick freezing without cryoprotectant, this method
imposed restrictions. On the other hand, chemical furation before freezing remarkably decreased the ice crystal damage in the sample. However, several differences
between the morphology of fresh-frozen and fmed-frozen
materials became evident. At low magnification, the
cytoplasmic matrix of chemically fixed specimens exhibited a granular appearance (Fig. 3). The contents of
vacuoles seemed to have a condensed or aggregated
form. Flocculent materials adhering to the ruffled border membrane, in the quick frozen sample (Figs. 2, see
also Fig. 5), were absent from the ruffled border after
chemical fixation and freeze-substitution (see Fig. 8).
Following saponin extraction, a finely disorganized
meshwork consisting of microfilaments was observed
beneath the ruffled border, whereas a loose network of
thicker filaments showing 10-12 nm in diameter was
visible in the deeper cytoplasm (Fig. 4).
Ruffled Border
The methods employed in the present study gave different ultrastructural pictures of the ruffled border (Figs.
5-7). In directly fresh-frozen and freeze-substituted
samples, a uniform appearance caused by a densely
interconnected, disorganized microfilament meshwork
was revealed (Fig. 5). In some places, protuberances of
electron-dense material projected from the inside of the
plasma membrane into the cytoplasm. Fuzzy material
appeared to coat the outside of the ruffled border membrane. In well-preserved cytoplasm it was difficult to
identify the cytoskeletal structure. In the sample treated
by saponin before quick freezing, a clearer picture of
the cytoskeletal organization and undercoat structure
was obtained (Fig. 6). Complex, disorganized, interconnected microfilaments (5-7 nm in diameter) were still
preserved. Neither microtubules nor intermediate filaments could be detected.
In the conventionally processed samples, cytoplasmic
structure in the ruffled border fingers was poorly preserved (Fig. 7). A partial membrane rupturing was frequently encountered. Extracellular fuzzy material was
also undetectable.
Freeze-substitution after aqueous chemical fixation
(CF) also provided better ultrastructural preservation
than did conventional processing (Fig. 8).The interfibrillar substance seen in directly frozen, freeze-substituted samples (Fig. 5) was not observed in samples
subjected to an initial aqueous chemical fixation. Distortion of the filamentous network was also recognizable. Along the inner side of the ruffled border membrane,
small protuberances and undercoat material were observed (Fig. 8).Casual contacts among the outer leaflets
of the ruffled fingers were also often encountered. After
freeze-substitution, the asymmetry of the ruffled border
membrane became apparent, since the inner leaflet was
more obvious than the outer one (Figs. 5, 8).
FREEZE-SUBSTITUTED OSTEOCLAST
Within the cytoplasm adjacent to the ruffled border,
the contents and limiting membrane in various vesicles
and vacuoles seemed to be well preserved. The fusion
of the d e d border and vesicular membranes was rarely
observed. Most vesicles and vacuoles seemed not to make
contact with the extracellur space (Fig. 1).
Clear Zone
The boundary between the clear zone and the ruffled
border was obscure in some cases. The clear zone was
characterized by an area that was organelle free except
for a few vesicular structures. After quick freezing, the
typical clear zone exhibited a dense, interconnected
meshwork similar to that in the ruffled border. In addition, electron-dense amorphous material and a few
parallel arrays of intermediate filaments (10-12 nm in
diameter) were recognizable (Figs. 9-13). The typical
clear zone in longitudinal sections exhibited bundles of
filamentous structures associated with electron-dense
dark bands (Fig. 9). On the contrary, in cross sections
through the clear zone, the amorphous material was
observed to have a uniform cloudy appearance (Fig. 10).
At high magnification, intermediate filaments were seen
interwoven among the disorganized microfilments with
amorphous, electron-dense material (Fig. 11). The contour of the clear zone membrane followed that of the
bone surface. In most cases. the clear zone showed a
smooth contour (Fig. 12). Even after quick freezing, a
constant narrow extracellular space in the contact area
where the clear zone was closest to the bone surface
was observed. Within the space, structures connecting
the cell membrane to the bone surface were revealed
only after quick freezing (Fig. 12). Along the inner side
of the clear zone membrane, an intimate relationship
was suggested between the microfilaments and the
membrane via the undercoat material. Saponin treatment clarified the cytoskeletal organization of the clear
zone: Microfilaments formed a network, while intermediate filaments tended to make a few ordered arrays
(Fig. 13).
DISCUSSION
Although a large amount of information on osteoclast
fine structure has been accumulated using conventional
processing (Gothlin and Ericsson, 1976; Holtrop and
King, 1977), recent methods utilizing quick freezing followed by freeze-substitution have been demonstrated
to be superior to the conventional method for preser-
Fig. 2. The ruffled fingers aRer QF and FS show a homogenous appearance, which reflects filamentous and interfibrillar materials, while
the clear zone contains a few thicker filaments. Arrowheads indicate
bone crystals in the channels of the ruffled border. Fuzzy material (asterisks) adhem to the rutned border membrane.Bar = 0.5 )~m.x 38,000.
Fig. 3. This section was treated with chemical furation (CF) before
freezing followed by freeze-substitution.A granularity of cytoplasmic
matrix becomes evident in comparison with Figure 2. The fuzzy coat
has almost disappeared from the ruffled border (RB).The content of
vacuoles (V) appears to be condensed. CZ, clear zone. Bar = 0.5 pm.
x 33,600.
Fig. 4. The sample was extracted with saponin before CF, freezing,
and freeze-substitution. Cytoskeletal components become more clearly
visualized. The ruffled border (RB)contains a fine filamentous network,
while the remaining cytoplasm exhibits a loose network of thicker fdaments. Bar = 0.5 pm. ~ 3 2 , 0 0 0 .
327
vation of the ultrastructure of calcifyingtissue (Akisaka
and Shigenaga, 1983; Goldberg and Escaig, 1984; Akisaka et al., 1987). It can be concluded from these results that quick freezing can minimize the disadvantage
of chemical fixation.
In this study, a great improvement on the preservation of osteoclast ultrastructure was achieved by quick
freezing and freeze-substitution. The combination of detergent extraction with freeze-substitution also allowed
a good visualization of cytoskeletal structures. The application of the metal contact freezing technique to mineralized tissue seems to be an extremely difficult
approach because 1) the hardness of the calcified matrix
prevents firm contact between the sample and the cooled
copper block, which could lead to uneven heat transfer;
and 2) the calcified matrix may have areas of different
thermal conductivity. In fact, during this study, only a
few osteoclasts were successfully preserved at the electron microscopic level because of severe ice crystal damage. On the other hand, chemical fixation with aqueous
aldehydes before freezing provided more reproducible
results, but made it difficult to retain some labile materials and delicate cytoskeletal structures (Bridgman
and Reese, 1984). However, it is apparent that freezesubstitution even after an initial aqueous chemical fixation exhibited better morphological preservation than
did the conventional processing alone.
The finding of the microfilament meshwork in the
ruffled border of the osteoclast was previously undescribed. These filaments may be actin, as judged from
their diameter of 5-7 nm. It is impossible, however,
with further study using the antibody localization technique, to determine the exact nature of the filaments.
Apparently, the d e d border of the osteolcast is filled
with a disorganized,interconnected microfilament meshwork and shows extremely variable forms. It is most
likely that these differences in cytoskeletal organization may reflect the different functional roles. Probably,
the shape of the ruffled border reflects its degree of
involvement in bone resorption. It is well known that
calcitonin induces the detachment of the cell body of
the osteoclast from the bone surface and the shortening
of the ruMed fingers (Kallio et al., 1972), while parathyroid hormone may function to develop the ruffled
border (Holtrop et al., 1974). Consequently, one may
speculate that the disorganized meshwork structure
would be a more adaptable or flexible form to allow
changes in the ruffled fingers than would the highly
ordered arrays of filaments.
Marchisio e t al. (1984) showed multiple actincontaining dots in cultured osteoclasts that seemed ultrastructurally to correspond to the clear zone of in vivo
osteoclasts. In the clear zone of osteoclasts, the microfilaments were decorated by heavy meromyosin, which
identified actin filaments (King and Holtrop, 1975). The
present study demonstrated that the clear zone was
composed of a disorganized microfilament meshwork,
parallel arrays of intermediate filaments, and electrondense amorphous material. This amorphous material
showed parallel dark bands in the longitudinal section,
while the clear zone had a uniform cloudy appearance
in cross section. Morphological differences in the clear
zone may be dependent on the environment in vivo or
in vitro (Malkani et al., 1973; Marchisio et al., 1984).
The adhesive properties of isolated osteoclasts may
be ascribed to the focal contact between the cell and the
Fig,5.The ruffled fingers aRer QF and FS. Small protuberances are
visible on the inner leaflet (arrowheads).The inner leaflet is thicker than
the outer leaflet. The fuzzy coat (asterisks) is well preserved. Bar = 0.5
pm. ~85,000.
Fig. 6.Saponin treatment before QF followed by FS. The filamentous
structures and undercoat material (arrowheads)become evident in the
ruffled hgers. Bar = 0.5 pm. x 55,100.
Fig. 7.Conventional processing (CP). Filamentous structures in the
ruffled border are indistinguishable.Bar = 0.5 pm. x 59,000.
Fig. 8. A cross sectioon of the d e d fingers after chemical furation
(CF) and FS. Small protuberances are visible along the inner side of the
plasma membrane (arrowheads), but other fragile cytoskeletal structures appear to be deformed because of the aqueous chemical furation.
A casual contact between the ruffled fmgers shows a gap junction-like
appearance (arrows). Bar = 0.1 pm. x 135,200.
Fig. 9. A osteoclast containing a typical clear zone (CZ). Parallel dark
bands (arrows) with the bundle of filaments are visible in the longitudinal section of the clear zone. QF and FS. Bar = 1pm. x 12,600. Inset:
A higher magnification of the parallel dark bands. Two types of filaments
were identificable: microfilament (mf) and intermediate filament (if).Bar
= 0.5 pm. ~46,200.
331
FREEZE-SUBSTITUTEDOSTEOCLAST
substratum (Marchisio et al., 1984). Similar focal contact between cells and substratum has been reported
for various types of motile cells (Izzard and Lochner,
1976; Wehland et al., 1979; Chen and Singer, 1982). A
similar observation was also made in the clear zone of
the in vivo osteoclast at sites of attachment. At the
contact site, the dense filamentous network can stabilize the clear zone membrane, which may strengthen
the adhesion between the cell and the bone surface. The
present study showed the presence of cementing material within the narrow extracellular space between
the clear zone membrane and bone. However, the chemical nature of the cementing material detected by freezesubstitution in the osteoclast is completely unknown.
Cytochemical differences between the ruMed border
and clear zone membrane have been suggested (Doty
and Schofield, 1976; Akisaka and Gay, 1986). They differ morphologically as well. Kallio et al. (1971) showed
fine bristle-like structures along the inside of the ruffled
border membrane. The present freeze-substitution work
exhibited a difference in undercoat structure: small protuberances associated with electron-densematerial were
seen in the undercoat of the ruffled border membrane
but not in that of the clear zone membrane. Different
preparative procedures have led to various interpretations of the ultrastructure of undercoat structures.
Undercoat structures may be composed of various enzymes or cytoskeletal components.
The superiority of the quick freezing technique for
the preservation of osteoclast ultrastructure was also
demonstrated in the fuzzy coat along the extracellular
aspect of the ruffled border. Within the enclosed microenvironment of the ruffled border, its membrane is
exposed to the extracellular fluid which contains various hydrolytic enzymes and organic acids (Vaes, 1968;
Doty and Schofield, 1972). Such an environment of the
ruffled border region is destructive for the integrity of
osteclasts. It is, therefore, likely that the fuzzy coat may
play a protective role against the extracellular resorbing fluid.
In conclusion, the present method-quick freezing followed by freeze-substitution-seems to provide a more
accurate picture of the ultrastructural details of in vivo
osteoclasts, especially of the ruffled border and clear
zone. Quick freezing can stabilize fragile structures,
while freeze-substitution allows anhydrous treatment
of the tissue. By use of these techniques several preFig. 10.A cross-sectional view of the QF and FS clear zone. The amorphous material appears to be a continous form within the network of
filaments. Bar = 1 Fm. X 14,400.
Fig. 11. A high-power of cross-sectioned clear zone after QF and FS.
Within the amorphous material, a filamentous network appears to be
composed of microfilaments (mfl and intermediate filaments (If).Bar =
0.5 pm. x 57,200.
Fig. 12.A longitudinal section of the clear zone adjacent to the calcified
matrix. The filamentous network appears to be in direct wntact with
the clear zone membrane. Within a narrow extracellular space, bridging
structures connect the cell membrane with the surface of calcified matrix.
QF and FS. Bar = 0.1 pm. x 132,000.
Fig. 13. A few microprojections from the clear zone into the bone.
Microfilaments and intermediate filaments seem to be interwoven. This
CF-fixed sample was treated with saponin followed by freeze-substitution. Bar = 0.5 pm. ~ 4 0 , 0 0 0 .
viously unreported observations have been made regarding osteoclast ultrastructure.
ACKNOWLEDGMENTS
The authors are indebted to Mrs. Yoshi Shirana for
typing the manuscript. This investigation was supported in part by Grant-in-Aid for Scientific Research
No. 62570808 from the Japanese Ministry of Education,
Science, and Culture to T.A.
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