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Morphology of osteoclasts in resorbing fetal rat bone explantsEffects of PTH and AIF in vitro.

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Morphology of Osteoclasts in Resorbing Fetal Rat Bone
Explants: Effects of PTH and AIF In Vitro
' Department of Pathology, Michael Reese Hospital and Medical Center, Chicago,
Illinois 6061 6; Department of Orthopedic Surgery, Rush Medical College,
Chicago, Illinois 60612; and Department of Periodontology, Haruard
School of Dental Medicine, Boston, Massachusetts 021 15
Osteoclastic bone resorption was studied using 45Ca-labeled
fetal rat bones cultured in the presence of parathyroid hormone (PTH) and an
anti-invasion factor (AIF) derived from bovine hyaline cartilage which is enriched in a collagenase inhibitor. The specific morphological expressions of osteoclasts cultured in PTH and AIF were observed in both light and electron microscopy and analyzed cytometrically. Stimulation of bone resorption with PTH
revealed significant increases in the numbers and activity of osteoclasts,
whereas bones cultured in the presence of AIF showed significant decreases in
numbers of osteoclasts and altered cell features including the loss of osteoclast
contact with bone surfaces. These structural modifications were evaluated with
45Carelease data derived from matched-pair explants of fetal rat bones, revealing the existence of a relationship between resorptive states of the cultured
bones and morphological expressions of osteoclastic activity.
Bone resorption is a sequence of cell-mediated events which result in the controlled removal of both mineral and organic matrix
components. One cell, the multinucleated osteoclast, resorbs bone through direct contact
with bone surfaces. This singular role of the
osteoclast was first recognized morphologically nearly a century ago (Kolliker, 1873). Only
recently, however, has the probable sequence
of events involved in osteoclast-mediated bone
resorption been elucidated (Burstone et al.,
'67; Vaes, '68; Lucht, '73b; Lucht and Maunsbach, '73). Osteoclastic bone resorption can
usually be assessed by the number of osteoclasts on bone surfaces and by the subcellular
morphological expressions of resorptive activity. These multinucleated cells may arise
through fusion, differentiation, or modulation
from mononuclear precursors (Hall, '75;
Jotereau and LeDouarin, '78). Their physiology can be influenced and directed by known
regulators such a s parathyroid hormone
(PTH), calcitonin (CT), osteoclast activating
factor (OAF), prostaglandin E (PGE), steroids, vitamin D metabolites, heparin, anti-invasion factor (AIF) and others (Goldhaber,
'65; Horton e t al., '72; Raisz et al., '72; Kallio,
'72; Tashjian e t al., '77; Wong e t al., '77;
ANAT. REC. (1979) 194: 311-324.
Miller, '78; Horton e t al., '78a). Yet, the exact
mechanism of osteoclastic bone resorption remains unclear. However, a concensus is now
being approached concerning certain cellular
events which accompany osteoclastic resorption (Hancox and Boothroyd, '64; Lucht, '74;
Holtrop and King, '77; Gothlin and Ericsson,
'76). The assumption that membrane alterations such as ruffling and the enlargement of
clear zones are singular indices of a cell's
ability to resorb bone must be assessed in the
light of recent reports that some osteoclasts as
well as other normal single-nucleated cell
types of the mononuclear phagocytic system
not giving evidence of such morphology are
also capable of resorbing bone and actively
phagocytosing mineral and matrix (Galasko,
'76; Mundy et al., '77; Kahn et al., '78).
However significant the osteoclast may be
in the process of bone resorption, loss of mineral from bony matrices may also result from
the action of invasive malignant mononucleated cells such as VX-2 carcinoma cells
(Galasko, '761, mammary carcinoma cells, and
osteosarcoma cells (Kuettner and Pauli, '78).
These various examples of non-osteoclastic inReceived Oct. 3, '78. Accepted Jan. 29. '79.
vasive cells which appear capable of directly
resorbing bone tissue suggest a n intriguing relationship between such cells and the invasive
capability of osteoclasts. Such observations
further the possibility that a variety of cells
and cellular mechanisms exist to promote
localized bone loss.
Recent information indicates that the regulation of osteoclastic activity may occur in
part due to the effects of local activators and
mediators (Horton et al., '72; Horton et al.,
'74; Trummel e t al., '75; Horton et al., '78a).
Rationale for such regulation of cell activity
exists in the observation that lymphoid elements elaborate a number of defined biologically-active effector molecules which are
known to mediate connective tissue cell activi t y (Pick and Turk, '72). Examples for selective stimulation of specific cells are evidenced by increased metabolic states in diseased tissues (Dayer et al., '77). While it is
still speculative t h a t many previously identified effector molecules participate in the
pathogenesis of bone loss, their participation
in tissue metabolism is clearly indicated
through in vitro studies (Pick and Turk, '72).
The generation of the lymphokine OAF (osteoclast-activating factor) is dependent upon a
macrophage-lymphocyte interaction (Horton
et al., '74). OAF activity in vitro is expressed
as an increase in the number and activity of
osteoclasts, and resembles the effect of parathyroid hormone (PTH) by promoting bone resorption and inducing the release of mineral
and matrix components from bone (Horton et
al., '72).
Collagen, the major organic component of
the bone matrix, is degraded by collagenase, a
proteolytic enzyme assumed to participate in
the process of bone resorption (Sakamoto et
al., '75; Vaes, '71). Recently collagenase activity derived from skin and tumor cell lines has
been shown to be effectively inhibited by a low
molecular weight cartilage-derived protease
inhibitor (Kuettner et al., '76; Kuettner and
Pauli, '78). Subsequently, we reported that a
specific fraction of a cartilage extract, which
we termed anti-invasion factor (AIF) and
which is enriched in protease inhibitors including an inhibitor of collagenase, is capable
of blocking both PTH and OAF-induced osteoclastic bone resorption in cultured fetal rat
bone (Kuettner et al., '76; Horton et al., '78a).
Furthermore, the effect of AIF was shown to
be reversible. This current report further
documents the cytometric expression of the
effects of AIF on PTH-stimulated osteoclasts.
The organ culture technique which measures the resorption of fetal long bone explants has been fully described (Raisz and
Niemann, '69). Briefly, paired shafts of both
the radii and ulnae from 19-day-oldrat fetuses
were radiolabeled by injection of the mother
with 45Ca.These shafts were cultured in BGJb
medium (Gibco) supplemented with 1 mg
bovine serum albumin (Pentex) per milliliter
with or without the purified specific cartilage
fraction, AIF, or in such medium containing
additions of 2.8 I.U. of PTH (Inolex) every 48
hours. The cultures were maintained for up to
144 hours and the medium, with or without
fresh additives, was changed every other day.
The degree of bone resorption was quantitated
by liquid scintillation spectrometry from the
radioactivity in counts per minute of 45Ca
present in the culture medium a t each 48-hour
interval and the 45Caradioactivity remaining
in the cultured bone shafts after 144 hours of
The cartilage-derived inhibitor of bone resorption was prepared from 1M NaCl extracts
from slices of fresh bovine nasal septa1 cartilage (Kuettner e t al., '76). After decantation, the NaCl concentration was raised to 3 M
by addition of solid NaC1. The extract was
then ultrafiltrated on an Amicon XM-50 membrane and afterwards concentrated and dialyzed into physiological saline with an Amicon
UM-2 membrane to yield a protein concentration of 5 mg/ml. This cartilage-derived AIF
with a molecular weight between 50,000 and
2,000 Daltons was essentially free of proteoglycan and collagen, as analyzed for uronic
acid and hydroxyproline, and enriched in protease inhibitors including the collagenase inhibitor. After Millipore filtration for sterilization, aliquots were stored frozen at - 70" until
tested. For bone cultures AIF was diluted with
medium to yield a concentration of 300 p g protein/ml.
Morphological and quantitative cytometric
analyses were performed on the cultured
bones following each 48-hour incubation period. Bones from culture were placed in 2.5%
glutaraldehyde in 0.1 M cacodylate buffer, pH
7.4. Following several days of fixation, the
undecalcified specimens were rinsed in cacodylate buffer containing 3% sucrose (to adjust the osmotic pressure of the buffer) and
post-fixed for two hours in 2% OsO, in 0.2 M
collidine buffer. Specimens were then dehydrated in graded ethanol and embedded in
Epon 812 for longitudinal sectioning. Thin
(0.5 pm) sections were stained with boratetoluidine blue and observed under light microscopy. Ultrathin (0.08 /*m) sections were
placed on Parlodion-coated 150 mesh copper
grids and stained with 5%uranyl acetate and
2%lead citrate. A Phillips 300 electron microscope was used to examine the tissues which,
due to the extended in vitro conditions and the
metabolic challenges associated with the presence of PTH and AIF, resulted in certain
shortcomings with regard to ultimate resolution in the electron microscope. Bones from
analogous experiments were routinely processed, embedded in paraffin or plastic (Cole
and Sykes, ‘741, and stained with hematoxylin
and eosin for observation by light microscopy.
Cell and nuclear counts of osteoclasts were determined from the total number of cells in
complete midsagittal serial sections from experimental and control bones. The data are expressed as the average from six midsagittal
sections from each bone plus or minus the
standard error of the mean (Rowe and Hausmann, ’76). The percent of 45Careleased from
each bone into the medium was used to quantitate bone resorption; this data was compared to the ultrastructural features of osteoclasts in the cultured bones.
Observed osteoclasts in sections derived
from cultured control bones exhibited characteristic structural features consistent with
previous reports (Lucht, ’73b,c; Lucht, ’74;
Gothlin and Ericsson, ’76; Holtrop and King,
’77). Such cells were apposed to bone surfaces
undergoing resorption and displayed moderate
development of the ruffled border and clear
zone areas (fig. 1). Numerous small vacuoles,
sparse development of endoplasmic reticulum,
and many mitochondria were evident in all osteoclasts examined. Free ribosomes were present in abundance and appeared primarily in
the form of single and polyribosomal structures. Thin filamentous structures were frequently noted in the cytoplasm. A few cells appeared to have been participating in highly
active endocytosis of mineralized collagen
fibrils and bony fragments.
Osteoclasts in bone sections derived from
cultures stimulated with additions of PTH appeared increased in both size and number and
displayed alterations in structural features
(fig. 2). An increased development of organellar components was indicated by the appearance of enlarged ruffled borders and their sybjacent channel expansions, enlarged clear
zones, greater numbers of nuclear profiles, expanded perinuclear Golgi fields, and enlarged
mitochondria. Additionally, vacuolar bodies
subjacent to the channel expansions of the
ruffled border region were expanded and elongated. Mineralized material was observed to
be located within the ruffled border region
and in its subjacent vacuoles. Quantitative
data were obtained by examining six total
midsagittal sections from each bone shaft
without knowledge of slide identification
after the method of Rowe and Hausmann
(‘76).Cell and nuclear counts were determined
from the total number of osteoclasts in adjacent midsagittal sections from both experimental and control bones. Counts were averaged and expressed as the mean plus or minus
the standard error. Statistical significance of
the data was determined using the students’
“T” test. Cytometric analyses of sections
revealed that a t 48 hours the average number
of PTH-stimulated osteoclasts per section was
12.5 f 0.27 compared to a value of 8.5 f 0.16
(P = < 0.01) in sections from unstimulated
cultures. Additionally, differences in the number of osteoclast nuclear profiles was observed. Forty-eight hour control values of 4.3
f 0.12 nuclear profiles per osteoclast were
compared to values of 5.9 f 0.22 (P = < 0.01)
nuclear profiles per cell when bones were cultured in the presence of PTH.
In contrast, distinct changes in the presence
and structural features of osteoclasts were observed in unstimulated bones cultured solely
in the presence of the cartilage derived antiinvasion factor, AIF (figs. 3A,B). First, the
number of osteoclasts per section and the
number of nuclear profiles per osteoclast was
significantly decreased when compared with
unstimulated cultured bones without AIF.
Following 48 hours of culture in the presence
of AIF, only 5.3 f 0.03 osteoclasts per section
could be enumerated. Furthermore, the number of nuclear profiles in these cells were significantly reduced to 2.4 f 0.05 per cell (P =
< 0.01) when compared with sections of control bones. When bones were co-cultured for 48
hours in the presence of both PTH and AIF
(fig. 4) however, the number of osteoclasts and
osteoclast nuclear profiles were significantly
reduced as compared with those from PTHstimulated bones, wherein the values deter-
3 14
Effect of additions and removal of AIF on PTH-stimulated bone resorption in vitro
present (hr)
Mean % ? S.E. of '%a-release each 48-hr
interval from cultured fetal rat bones
0-48 hr
49-96 hr
97-144 hr
49-144 hr
23.4942.01 I
5.462 0.54
14.86?1.51 '
372823.27 I
' Significantly different from respective control culture, p < 0.01.
mined were 5.5 k 0.09 (P = < 0.01)osteoclasts per section and 3.1 f 0.11 (P = < 0.01)
osteoclast nuclear profiles per cell. Secondly,
the average size of the osteoclasts appeared to
be reduced, and this was reflected in the subjective observation of a higher nuclear to cytoplasmic ratio. Third, the most apparent alteration noted was that osteoclasts from bones
cultured in the presence of AIF appeared to
have physically separated or withdrawn from
bone surfaces. Accompanying structural
changes noted in these non-apposed osteoclasts were the appearance of fewer vacuoles
within their cytoplasm and a loss of ruffled
borders and clear zones. Interval sectioning of
occasional specimens revealed that AIF-inhibited osteoclasts did not show evidence of
ruffled borders in other levels of sectioning
and that the random sectioning approach was
valid for assumptions concerning the three-dimensional arrangement of such osteoclasts.
Further, a reduction in endocytosis was evidenced by minimal amounts of bone debris
within cell vacuoles. Only occasionally were
osteoclasts in these sections observed to be
somewhat apposed to bone surfaces. However,
the extent of the ruffled borders and clear
zones of these cells appeared greatly reduced
or absent indicating a lesser degree of activity
which conformed to the minimal amount of
45Ca released by bones cultured in the presence of AIF. Morphological evidence for the
apparent inactivation of osteoclasts includes
separation of the cell from the bone surface as
evidenced by the effect of AIF in this investigation. In those cultures to which AIF was
added, osteoclasts revealed early separation
and development of intermediate borders
(Lucht, '73b, '74).Such cell membranes appeared more flattened and had only a few cytoplasmic projections oriented toward the
bone surfaces which were a few microns away
(fig. 3B).
Structural characteristics of active osteoclasts appeared restored in cultured bones
from which AIF was removed (fig. 5 ) . Osteoclasts reestablished a relationship with bone
surfaces and evidenced resumption of active
resorption by development of the ruffled
border area and clear zone attachments to
bone. With increased time in culture (to 96
hours) after removal of AIF, osteoclast organelles such as vacuoles, ribosomal structures,
and perinuclear Golgi regions appeared developed, and dense fragments appeared contained within cytoplasmic vacuoles subjacent
to the expanded channels of the ruffled border
Measurements of the 45Ca-release data,
which reflected a quantitative indication of
the degree of bone resorption, supported the
morphological observations. Unstimulated
control cultures indicated an egress of 45Ca
from bone into the culture medium when measured a t the 48 hour time point (table l).
These values declined minimally during the
culture period of 49 to 96 hours and remained
steady to the conclusion of the experiment.
Bones cultured in the presence of PTH alone
revealed an accelerated loss of 45Cainto the
medium which continued from 49 to 96 hours
and with a slight decline by the conclusion of
the culture period. In those cultures to which
AIF was added during the first 48 hours, the
45Ca egress from bone was significantly reduced. The total amount of 45Careleased into
the medium at 144 hours of culture was below
all control values. When PTH was added to
cultures containing AIF for forty-eight hours,
45Carelease into the medium was comparable
to those bones cultured in the presence of AIF
alone. Upon removal of AIF and further cul-
ture in the presence of PTH alone from 49 to
144 hours, an increased release of 45Cafrom
the bones into the culture medium occurred
indicating resumption of resorption. In cultures to which AIF was added a t 49 to 144
hours of culture, PTH-stimulated resorption
was completely blocked.
by few cell membrane modifications and a
smoother surface of the underlying bone. Apparent cessation of resorptive activity is further typified by separation from the bone surface and conversion to a more intermediate
morphology of the cell membrane at the bone
In our studies, subjective observations reDISCUSSION
vealed that approximately seventy-five perMorphologic features of osteoclasts reflect cent of all observed osteoclasts in control cultheir functional states and may be observed tured bones were intimately associated with
with either light or electron microscopy. It has the bone surface. Under PTH stimulation, the
been clearly demonstrated that the number of increased number and activity of the osteoosteoclasts, the degree of development of cer- clasts was accompanied by a greater percenttain organelles, as well as the presence of the age of cells revealing an intimate tissue conruffled border region of the cell membrane and tact. On the other hand, in the presence of
its adjacent clear zone area are measurable AIF, approximately half of the observed osteostructural features which reflect the resorp- clasts were physically separated from the
tive degree of cellular activity. Such observa- bone surfaces. Upon removal of the inhibitor
tions have been correlated with in vitro 45Ca the majority of all observed osteoclasts were
release data and in vivo serum calcium level found to again have intimate contact. It is of
alterations in numerous investigations (King interest to note that in disease conditions,
et al., '75; Thompson et al., '75; Rowe and such as human osteopetrosis fetalis, the perHausmann, '76, '77; Horton et al., '78a,b; cent of osteoclasts lying free from bone surMiller, '78; Wezeman e t al., '78).
faces is not significantly altered although reOsteoclasts appear as multinucleated cells sorption of bone is markedly diminished even
in intimate contact with a mineralized bone when the osteoclast number is increased
surface. Their distinctive cytoplasmic organ- (Shapiro et al., '78). Thus, reduction in the
elles differ greatly from those in other bone rate or extent of bone resorption mediated by
cells. Multiple nuclei are associated with osteoclasts does not uniformly include separanumerous centrioles (Matthews et al., '67; tion of such cells, implying that many mechaLucht, '73a) and the perinuclear Golgi zones nisms exist to control osteoclast activity and
are well developed (Cameron, '68). Active os- which result in varying morphological exteoclasts also reveal distinctive adaptive fea- pressions.
tures whereby membrane infoldings a t reStructural evidence of cell attachment to
gions of the cell's interface with the bone sur- bone through clear zones and adjacent ruffled
face are accompanied by deep and irregular borders and also increased lysosomal activity
channels associated with a well developed sub- are representations of osteoclastic activity.
jacent vacuolar apparatus. The clear zone at- Whereas minimally active osteoclasts show a
tachments to the bone surface are devoid of or- reduction in their complement of lysosomal
ganelles but this zone has been demonstrated bodies, highly active or stimulated osteoclasts
to contain actin-like filaments which are reveal enlarged perinuclear Golgi fields and
implicated as being important for osteoclast an accumulation of lysosomal vacuoles. Such
attachment to the bone surface (King and observations have provided investigators with
Holtrop, '75). The cell-bone surface interface suggestive evidence for a functional relationthus characteristically reflects the resorptive ship between Golgi bodies and the lysosomal
activity of the osteoclast. Whereas the pres- system in synthesis and transport of hydrolytence of an irregular zone of altered miner- ic enzymes during bone resorption (Cameron,
alized bone consisting of isolated fragments of '68). In our experiments such anticipated
mineralized collagen and mineralized masses organellar changes accompanied PTH-stimuin the region of the ruffled border indicates a lated resorptive states and were reversed
state of progressive resorption, other features when AIF was present in the culture media.
characterize more modest resorptive states. The principal structural alteration was, howAs described by Lucht ('72, '73b) as well as by ever, the AIF-induced separation of a majority
Dudley and Spiro ('611, a second type of osteo- of osteoclasts from the bone surface. Such an
clast-bone surface interface is characterized observation is not new to the literature con-
cerning inactive states of functional osteoclasts. Gaillard (’59), Dudley and Spiro (‘61)
and Lucht (’72, ’73b) have documented this
inactive and separated state of osteoclasts as
being indicative of an osteoclast incapable of
resorption. Indeed, Goldhaber’s (‘61) studies
demonstrated the osteoclast-bone surface relationship to coincide with the degree of cell
activity by varying oxygen tensions within
the tissue culture system.
Our present observations reinforce and
extend published observations concerning
modulation of osteoclast activity since the
presence of the cartilage-derived anti-invasion factor alters the cell’s resorptive state,
causes a reduction in those cellular characteristics which typify the highly active resorptive state, and results in a physical separation
of osteoclasts from bone surfaces. These morphological observations were supported by a
quantitative reduction in 45Ca released from
the bone explants when AIF was present. The
parallel between the movements of 45Cainto
the culture medium and the morphological
features of osteoclasts indicate that our system is an effective means to study expressions
and modulations of osteoclastic function.
Our cartilage derived anti-invasion factor
contains a spectrum of low molecular weight
proteins including protease inhibitors expressing the capacity to inhibit proteolytic enzymes such as trypsin as well as collagenolytic
activity. Thus, AIF is biologically capable of
blocking a n osteoclast-derived collagenase
assumed to be operative in the process of bone
resorption. An explanation for the separation
of osteoclasts from the bone surface in the
presence of AIF is presently unknown. Rose
and Robertson (‘77) studied the collagenolytic
activity of fibroblasts and suggested that the
fibroblastic collagenolytic agent is cell membrane-bound and must be in contact with collagen fibrils in order to effect lysis. This concept’ can be extrapolated to the osteoclast
suggesting that a biologically active tissue
fraction such as AIF would restrict the activity of an osteoclastic cell membrane collagenase to thereby result in the separation of
an osteoclast from the bone surface. Such observations reflect the ability of AIF to inhibit
bone resorption by modifying the behavior of
osteoclasts. Whether or not the separation of
the cell from the bone surface is a result of
intracellular or extracellular events is unknown.
Many agents such a s PTH, calcitonin, diphenylhantoin, vitamin D 3 metabolites, pros-
taglandins, osteoclast-activating factor, as
well as other naturally derived biologically active molecules have been reported to involve
distinctive alterations in osteoclast morphology. We have reported the reversible inhibition of osteoclastic bone resorption with the
naturally-occurring AIF which is derived
from cartilage (Horton e t al., ’78a,b). Such observations indicate the striking resemblance
of AIF inhibition to that of calcitonin inhibition (Lucht, ’73b) although the mechanism of
action may be quite different.
We have thus been able to superimpose the
effects of PTH-stimulation and inhibition of
proteases including collagenase by AIF upon
the normal range of expression of osteoclastic
activity, and further have demonstrated the
ability of AIF to reversibly modulate osteoclast function. Such morphologic expressions
of a reduced level of resorptive activity reflect
the response of a single bone cell population to
the presence of a naturally occurring factor
derived from cartilage. Our observations extend insight into possible mechanisms involved in normal and pathological processes
resulting in both generalized and localized
bone loss.
The authors wish to thank Ms. Manette
McReynolds and Ms. Mary Nelson for their
technical and secretarial assistance. This investigation was supported in part by Grants
GRS-5476, Arthritis Foundation (Illinois
Chapter), AM-09132, CA 21566, and YO1DE60025.
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2 Electronphotomicrograph of a portion of an osteoclast from a fetal r a t bone cultured in the presence of
PTH. The stimulated cell gives evidence of a well developed ruffled border (asterisks) and adjacent clear
zone (arrow) which are apposed to the bone surface. Subjacent channel expansions and vacuoles are present along with polyribosome clusters. Uranyl acetate and lead citrate. x 37,568.
1 Electronphotomicrograph of a portion of an osteoclast from a control cultured fetal rat bone. Portions of
the cytoplasm firmly apposed to the bone spicule show redundant membrane infoldings (asterisks) characteristic of types of ruffled borders. Numerous vacuoles, free ribosomes and mitochondria are present.
Uranyl acetate and lead citrate. x 18,616.
F. H. Wezernan. K. E. Kuettner and J. E. Horton
3B Electronphotomicrograph of a portion of a n osteoclast from a fetal r a t bone cultured in the presence of
AIF. Such osteoclasts gave evidence of an intermediate morphology of the cytoplasmic and membranous
portions of the cell toward bone. Separation from the bone surface was characterized by cell membrane
undulations and projections of cytoplasmic processes (asterisks) toward t h e bone surface. Uranyl acetate
and lead citrate. x 28,842.
3A Osteoclasts of fetal r a t bones cultured in t h e presence of AIF revealed significant separation from the
bone surfaces and a loss of cytoplasmic features such as ruffled borders and clear zones. Uranyl acetate
and lead citrate. x 16,240.
F. H. Wezeman, K. E. Kuettner and J. E. Horton
Osteoclasts of fetal r a t bones cultured in t h e presence of both PTH and AIF revealed continued separation
from t h e bone surfaces. Note the apparently higher nuclear: cytoplasmic ratio and occasional vacuoles
(asterisks) which are randomly distributed and not associated with a ruffled border. Uranyl acetate and
lead citrate. x 7,200.
5 Electronphotomicrograph of a portion of an osteoclast from a fetal rat bone cultured in the presence of
PTH after removal of AIF. Where such osteoclasts were found to be adjacent to bone evidence of a resumption of resorptive activity was noted. Endocytosis of mineralized fragments (arrows) was accompanied by what appeared to be a developing ruffled border (asterisks). Uranyl acetate and lead citrate.
X 28.842.
F. H. Wezeman, K. E. Kuettner and J. E. Horton
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explantseffects, aif, morphology, resorbing, pth, rat, vitro, bones, fetal, osteoclast
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