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In vitro transformation of chondroprogenitor cells into osteoblasts and the formation of new membrane bone.

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THE ANATOMICAL RECORD 206:373-383 (1983)
In Vitro Transformation of Chondroprogenitor Cells Into
Osteoblasts and the Formation of New
Membrane Bone
Division of Morphological Sciences, Faculty o f Medicine, Technion-Israel
Institute of Technology, H a i f q Israel (M.S., D. L., H. GJ and The MaxPlanck-Institute f i r Biochemie, Munich, Federal Republic of Germany
(M.A.L., K.u.d.M.)
Mandibular condyles of fetal mice 19 to 20 days in utero were
kept in a n organ culture system for up to 10 days. After 2 days in culture the
cartilage of the mandibular condyle appeared to have maintained all its inherent structural characteristics, including its various cell layers: chondroprogenitor, chondroblastic, and hypertrophic. After 5 days in culture no chondroblasts
could be seen and, instead, the entire cartilage was occupied by hypertrophic
chondrocytes. At the same time, the mesenchymal cells at the chondroprogenitor zone differentiated into osteoblasts which produced osteoid. Light microscopic examinations showed that the newly formed osteoid did not stain with
acidic toluidine blue or with alcian blue, but stained intensively with the van
Gieson stain and with Periodic acid-Schiff (PAS). The osteoid reacted with
antibodies against type I collagen but not with antibodies against type I1
collagen, Electron microscopic examinations showed that the mineralization
appeared to be associated with collagen fibers in bone rather than with matrix
vesicles in the cartilage. The process of bone formation progressed with time
and by the 10th day new bone replaced almost the entire cartilage, thus
forming an expanded layer of membrane bone. This in vitro system represents
a n experimental model whereby undifferentiated precursor cells transform
into osteoblasts with the subsequent formation of a typical membrane bone.
Recently, attempts were made to develop
in vitro systems which would provide appropriate models for studies on the mechanisms
involved in bone induction and bone formation. Simmons et al. (1982) described a combined in vitro-in vivo system for bone
formation, in which isolated osteoblasts were
placed in diffusion chambers and subsequently implanted in the peritoneal cavity of
mice. If, however, isolated bone cells are
grown in vitro in either primary cultures or
in established cell lines, they usually do not
form bone. The reason for that is as yet not
fully understood, and it has been postulated
that the currently used culture systems lack
some fundamental provisions essential for
bone morphogenesis (Wong, 1980).
The present study used a n organ culture
system of embryonic mandibular condylar
cartilage, a tissue that in vivo undergoes endochondral ossification (Silbermann and
Frommer, 1972, 1974). In vivo studies with
this tissue have shown that following trans@ 1983 ALAN R. LISS, INC
plantation the cartilage progenitor cells differentiate into osteoblasts (Meikle, 1973).
Charlier and Petrovic (1967) and Petrovic
(1972) were the first to culture isolated mandibular condyles, but they did not report on
bone formation in their explants. Hall (1981)
cultured quadratojugal cartilages of embryonic chicks and found chondroid bone formation if ascorbic acid was deleted from the
medium. Hence, the present study was undertaken to reevaluate the potential of condylar organ culture for studies related to in
vitro bone formation. We, hereby, report on a
reproducible system in which de novo membranous bone is produced within 7 days in
ICR mice which were pregnant 19-20 days
were anesthetized with ether. The uteri were
removed aseptically, placed in sterile Hank’s
Received November 30,1982; accepted April 25, 1983
solution, and the fetuses were delivered surgically. Using a surgical microscope, the
mandibular condyles were dissected away
from the mandibles, cleaned of all soft tissues
and of the underlying bone (parts of the mandibular ramus) and were then placed in a
fresh Hank’s solution a t 4°C. Explants consisting of clean cartilage (enveloped by a n
intact perichondrium) were transformed onto
Millipore filters (0.45 pm; Millipore Corp.,
Bedford, MA) cemented to stainless steel
grids (Falcon, Oxnard, CA) that were placed
in plastic disposable culture dishes (30 mm;
Falcon, CAI. The culture medium consisted
of Modified Eagle’s medium in Earle’s powder (Synthetic media, #410-1100, Gibco,
Grand Island, NY), supplemented with 10%
fetal calf serum (Gibco, Grand Island, NY)
and with Sigma’s (St. Louis, MO) ascorbic
acid (100 pg/ml), glycine (50 pg/ml), penicillin
(50 U/ml) and streptomycin (50 pg/ml). The
phosphate concentration in the medium was
3.0 mM and the pH 7.4. The medium was
pipetted into the culture dishes until it just
wetted the Millipore filters so that the explants were maintained at the medium-air
interface, in a humidified incubator at 37°C
in a n atmosphere of 5% COZ: 95% air. The
medium was changed every 48 hours and
specimens were obtained after 2, 5,7, and 10
days in culture.
antibodies were used. The latter were prepared against lathyritic rat skin collagen.
The immunization procedure was carried out
as described by Timpl et al. (1972).The purification of the antibodies was achieved via
immunoadsorption and their purity was determined by a n enzyme-linked immunosorbent assay (ELISA) as described by Gosslau
and Barrach (1979). For the localization of
type I1 collagen, guinea pig anti-chick type I1
collagen antibodies were used.’ Their preparation, purification, and specificity tests were
carried out as described by von der Mark et
al. (1976). We have also used rabbit antichick type I1 collagen antibodies, but they
did not cross react with the mouse type I1
Following incubation with the above antibodies (undiluted, 30 minutes, room temperature in a moist chamber) the sections were
rinsed three times with PBS and were subsequently reacted with fluorescein isothiocyanate (F1TC)-conjugated goat anti-rabbit
yG (1:20) or with rabbit anti-guinea pig yG
(1:20) for 30 minutes at room temperature.
The above antibodies were purchased from
Behringwerke (Marburg, Federal Republic of
Germany). Sections incubated with FITCconjugated rabbit anti-guinea pig yG alone
served as controls.
Electron Microscopy
Light Microscopy
Tissues were fixed in 5%glutaraldehyde in
Tissues that were designated for general 0.1 M cacodylate buffer, pH 7.4, at 4°C for
morphology and histochemical examinations l?h hours; postfixed in 1%Os04 in 0.1 M
were fixed in 80% ethanol for 30 minutes, cacodylate buffer, pH 7.4 for 1 hour; dehydehydrated in ethanols, and embedded in drated through graded ethanols and embedParaplast (Fisher Scientific Company, Pitts- ded flat in Epon. Thick sections (1pm) from
burgh, PA). Serial sections (6 pm in thick- the same blocks used for electron microscopy
ness) were stained with hematoxylin and were cut in the coronal plane and stained
eosin, toluidine blue (0.1%; pH 2.5), alcian with 1%toluidine blue or with Pearson’s silblue (0.5M; pH 5.8), van Gieson, von Kossa, ver gelatin stain. Adjacent thin sections were
and with the Periodic acid Schiff (PAS) stain. cut on LKB Ultratome I11 with a diamond
Explants that were designated for immuno- knife, mounted on No. 300 copper grids, and
fluorescence studies of collagen typing were stained for 10 minutes in saturated aqueous
not fixed but were immediately frozen in liq- uranyl acetate followed by staining for 10
uid nitrogen (-80”C), cut in a cryostat minutes in 0.2% lead citrate before exami(-25°C) and the sections (8 pm in thickness) nation with a Jeol 100 B electron microscope,
were mounted on microscope slides. A11 the operating at 60 kV.
frozen sections were first treated with 0.3 M
ethylenediaminetetraacetic acid (EDTA), pH
7.5, for 30 minutes a t room temperature folThe normal structure of mouse condylar
lowed by a 30-minute treatment with hy- cartilage, as seen in late fetal life, newborns,
alase (ovine testes hyaluronidase salt-free and neonatal animals, has been previously
analytical grade Serva #25118), 1 mg/ml in described (Silbermann and Frommer, 1973;
phosphate buffered saline (PBS), pH 7.2 at
room temperature. For the localization of
‘Kindly provided by Dr Waltraud Dessau, the Max Planck
type I collagen, rabbit anti-rat type I collagen Institut fur Biochemie, Munich, Federal Republic of Germany
Silbermann and Lewinson, 1978); thus,
rather than reiterating the material contained in these articles, we will refer to them
whenever appropriate.
At the age of 19-20 days embryonic life
(the time when the explants were removed
from the animals) the mandibular condyle is
elongated supero-inferiorly, while a large
proportion of the cartilage is comprised of
chondroblasts and hypertrophic chondrocytes. At 7 days postpartum the hypertrophic
zone is somewhat narrower, yet the progenitor and chondroblastic zones are still well
developed (Silbermann and Lewinson, 1978).
Bone or osteoid are never present within any
of the above cell layers; and replacement of
mineralized cartilage occurs only a t the condylar lower border along the ossification front
(Silbermann and Frommer, 1972).
Condyles that were cultured for 48 hours
showed a distinct morphological resemblance
to their age-matched tissues in vivo (1 to 2
days postpartum) (Fig. 1).The following cell
layers could be noted very distinctly: progenitor, chondroblastic, and hypertrophic. The
perichondrium was found to be very well organized and the cells along the chondroprogenitor zone formed a strip of closely packed
cells with many intercommunicating cell
processes. The intercellular matrix in the
above zone contained relatively few collagen
fibrils (Fig. 2). Farther away from the articular surface and below the chondroprogenitor zone the explants revealed a wide layer
of chondroblasts. The latter cells contained
the typical lakes of glycogen as well as a very
well-developed system of rough endoplasmic
reticulum with occasionally dilated cisternae. The Golgi apparatus appeared well developed and a small number of rounded
mitochondria1 profiles were randomly dispersed throughout the cytoplasm. The matrix surrounding the chondroblasts contained
fine fibrils of collagen and electron-dense
granules which represent matrical proteoglycans (Fig. 3). Early hypertrophic chondrocytes retained the above-mentioned lakes;
however, the glycogen was partly replaced
by electron-lucent vacuoles resembling lipid
droplets. These vacuoles varied in size and
appearance and they were never bounded by
a membrane (Fig. 4).In
there was
Fig. 1. Frontal section through condylar cartilage
a n increasing number of mitochondria which after 48 hours in culture. The tissue shows all the typical
were located mostlv along the DeriDheral
cell layers as are normally seen in a condyle of a new*
animal. A, articular surface; P, perichondrium; C,
parts of the cells. yiunghypertrophic chon- born
chondroprogenitor zone; B, chondroblastic zone; H, hy.
had a
drocytes alike chondroblasts
pertrophic zone. Stained with Pearson’s silver stain.
surface while multiple electron-dense gran- x 2407
Fig. 2. Electron micrograph of cells in the chondroprogenitor zone of a condyle that was cultured for 48 hours.
The cells reveal a high degree of resemblance to their
homologs in viva. The matrix contains fine collagen fibrils which lack any specific orientation. x 6,000.
ules appeared to be attached to their membrane. Hypertrophic chondrocytes were
surrounded by a lacuna and the intercellular
matrix farther away from the cells exhibited
vesicular structures. The latter were bounded
by a trilaminar membrane and were often
associated with precipitates of hydroxyapatite (Fig. 5 ) .Fine collagen fibrils were always
encountered in this portion of the matrix (Fig.
5). Immunofluorescent analysis indicated a
positive reaction with anti type I antibodies
along the perichondrium (Fig. 6) and a strong
positive reaction with antiserum against type
I1 collagen throughout the cartilaginous tissue (Fig. 7). Following 5 days in culture, the
cartilage became occupied by hypertrophic
chondrocytes which were surrounded by mineralized matrix. At this same time the zone
of chondroprogenitor cells as well as some
areas along the perichondrium revealed clear
signs of new bone formation (Fig. 8). Immunofluorescent examination indicated that the
matrix in the new site of bone formation
contained type I collagen (Fig. 9). Electron
micrographs from the above area showed that
the newly formed collagen was irregularly
oriented (Fig. 10)and that the fibers revealed
the typical cross-banding pattern as normally seen in bone collagen. Furthermore, in
the osteoid the hydroxyapatite crystals were
noted to lie in close association with collagen
fibers (Fig. 11)rather than with matrix vesicles, as was shown in cartilage (Fig. 5 ) . The
ossification process continued to progress and
by 7 days larger portions of the condylar
cartilage were replaced by the new osteoid
which was undergoing mineralization (Fig.
12). By this time the explants revealed the
appearance of multinucleated giant cells;
which were located close to the newly formed
bone (Fig. 13).When 7-day-old explants were
stained with either acidic toluidine blue or
with alcian blue the new bone did not stain
while the cartilage stained strongly. On the
other hand, when sections were stained with
either the van Gieson stain or with PAS the
Fig. 3. A section through a more mature chondroblast
and its adjacent matrix as seen in an explant cultured
for 48 hours. Note the prominent nucleolus, the welldeveloped rough endoplasmic reticulum, the Golgi apparatus (G), and the intracellular lakes of glycogen (GI)
so characteristic for these cells. The matrix contains few
collagen fibrils and proteoglycan granules. x 8,000.
Fig. 4. The appearance of an early hypertrophic chondrocyte in a condyle after 48 hours in culture. The cell’s
cytoplasm shows remnants of the glycogen lakes (G1)
which are largely replaced by lipidlike vacuoles (v). Multiple rounded mitochondria (m) are seen along with multiple strands of rough endoplasmic reticulum. A newly
formed lacuna (L) is seen pericellularly. X 8,000.
Fig. 5. A section through the intercellular matrix at
the level of the mineralization front in a condyle cultured for 48 hours. Matrix vesicles (rnv) which appear to
be bounded by a membrane are interspersed among the
collagen fibers. Most of these vesicles which serve as the
initial locus of matrix mineralization contain hydroxyapatite crystals. x 30,000.
Fig. 6. Frontal section through an explant that has
been cultured for 48 hours. The tissue was reacted with
antibodies against type I collagen. Note the positive
reaction along the perichondrium (PI, but not within the
cartilage (C). Frozen section. X 96.
Fig. 7. A similar section to that shown in Figure 6.
but reacted with antibodies against type I1 collagen. The
cartilage (C) responds strongly whereas the perichondrium (PI lacks any response. x 96.
Fig. 8. Coronal section through a n explant that has
been cultured for 5 days. It can be seen that almost the
entire cartilage is occupied by hypertrophic chondrocytes. The matrix surrounding many of the above cells
has undergone mineralization. Clear signs of new bone
formation are seen along the perichondrium (single arrows) and especially at the upper portion of the condyle
(double arrows). Epon embedded and stained with Pearson’s silver stain. X 192.
Fig. 9. A coronal section through the upper portion of
a condyle that has been cultured for 5 days. The tissue
was reacted with antibodies against type I collagen. A
strong reaction can be seen in the region of new bone
formation (B). x 384.
Fig. 10. Electron micrograph showing portions of two
osteoblasts (OBI and their adjacent matrix in the newly
formed osteoid. A large number of collagen fibers (COL)
occupy the matrix. x 10,000.
Fig. 11. A section through collagen fibers in the newly
formed osteoid. One of these fibers (arrow) is associated
with hydroxyapatite crystals, a characteristic feature for
osteoid mineralization. x 160,000.
Fig. 12. A coronal section of an explant that was
cultured for 7 days. In this specimen, the upper portion
of the condyle underwent an advanced stage of bone
formation (B). Note the alignment of giant cells along
the lower right margin of the condyle. x 192.
Fig. 13. A similar section to that shown in Figure 12,
showing a group of giant clasts (CL) around an isolated
area of mineralized matrix (M). Cartilage cells tC) at
various stages of maturation are seen nearby. x 768.
Fig. 14. The appearance of a portion of an explant
that has been cultured for 10 days. A widespread area of
the original explant underwent ossification (B). The
newly formed bone is surrounded by a well organized
periosteum (PI. Remnants of cartilage (C) are seen alongside the bony tissue. Pearson’s silver stain. x 192.
bone stained heavily while the cartilage did
not. The new bone also reacted strongly with
antibodies against type I collagen but not
with the anti type I1 antibodies. By the tenth
day most explants showed large areas of
newly formed membranous bone (Fig. 14).
Osteocytes could be easily identified within
this tissue as well as osteoblasts that were
lining the periphery of the mineralized osteoid. The latter cells were surrounded by
several layers of fibroblasts thus forming a
continuous periosteum. At this time interval
remnants of cartilage tissue were still present adjacent to the new bone.
In the present study we were able to show
that if fetal condylar cartilage is cultured in
a medium containing serum the tissue maintains its original structure and organization
for a t least 48 hours. Recent studies in our
laboratory have indicated that during this
time period the progenitor cells continue to
synthesize DNA and proliferate (Maor and
Silbermann, 19811, while the chondroblasts
and chondrocytes participate actively in the
mineralization of the matrix (Silbermann et
al., 1981).This study also revealed that after
5 days in culture almost all the chondrocytes
in the tissue became hypertrophic. This could
suggest that 1) many of the hypertrophic
chondrocytes present at the time of explant
could be maintained in culture system; and
2) that the young chondroblasts became hypertrophic during the period of the culture.
It is interesting to note that very similar
findings have been described by Ronning and
Koski (19691, who transplanted rat mandibular condyles into the brains of inbred littermates, and found that after 5 days the thickness of the proliferative zone had been
reduced and that hypertrophic cells were
closer to the upper surface of the cartilage.
The present findings also indicate that in
vitro the progeny of the chondroprogenitor
cells differentiate into osteoblasts instead of
into chondroblasts. This shift in differentiation and the subsequent formation of bone
are apparent in areas that in vivo never show
bone or osteoid tissue (Silbermann and Frommer, 1972, 1973, 1974; Silbermann and Lewinson, 1978; Lewinson and Silbermann,
1982).The results of the various methodological approaches used (histological, histochemical, immunofluorescent and ultrastructural) pointed toward a genuine formation of osseous tissue which eventually re-
placed most of the original cartilage. The
premise for the de novo elaboration of bone
is supported by the following findings: 1)the
newly formed tissue did not stain with alcian
blue nor did it respond with metachromasia
when stained with acidic toluidine blue; 2) it
stained strongly with PAS and with the van
Gieson stain; 3) it reacted positively with
antibodies against type I collagen but not
with antibodies against type I1 collagen; 4)
the collagen fibers were banded and wider
(two to five times) in comparison to those of
cartilage; and 5) the mineralization process
appeared to be associated with collagen fibers rather than with matrix vesicles as is
normally the pattern in cartilage. However,
in order to further substantiate the specificity of the newly formed tissue, one ought to
check the presence of bone specific proteins,
i.e., osteonectin via the application of the
appropriate antibodies.
The apparent change in the level of oxygen
tension (in vitro versus in vivo) could be one
of the factors responsible for the shift in cell
differentiation as was observed in the present system. It became also apparent that our
organ culture supported the mineralization
of the newly formed osteoid. Recent, and as
yet unpublished, data from our laboratory
indicated that 45Ca is taken up by the mineralizing osteoid as well as oxytetracycline.
Of particular interest was the finding that
related to the appearance of multinucleated
giant cells in the region of the newly formed
bone. The concurrent presence of osteoblasts
and osteoclasts in the cultured tissue could
possibly indicate the existence of a coordinate control mechanism in this system of
bone formation. The apparent “coupling” of
bone formation to bone resorption has been
attributed to a compensatory mechanism involved in bone homeostasis (Ivey and Baylink, 1981). A possible precursor of these
newly formed osteoclasts are uninucleated
cells that were transferred to the culture system in the small capillaries of the perichondrium. The cells could be mononuclear
phagocytes (monocytes) or derivatives from
the same progenitor cells which give rise to
mononuclear phagocytes (Malone et al.,
1982). In the present study osteoblasts appeared to have preceded the osteoclasts (although the exact sequence of events has not
been as yet carefully checked); however, once
mineralization started osteoclasts were always present. The present observations also
tend to indicate that there seems to exist a
spatial and temporal relationship between
hypertrophying chondrocytes and osteodifferentiation. Scott-Savage and Hall (1980)
have indeed argued that in order for progenitor cells to differentiate into osteoblasts they
require a suitable microenvironment which
could be provided by hypertrophic chondrocytes and their mineralized matrix.
In conclusion, this study presented a n in
vitro system which could serve as a useful
tool for studies on bone induction and bone
formation. The tissue utilized comprised embryonic cartilage which contains a population of committed but undifferentiated progenitor cells. In a n in vitro environment these
cells differentiate into bone-forming cells and
synthesize type I collagen and osteoid which
eventually mineralizes and ossifies.
The authors thank Mrs. A. Yahalomi and
M. Brenman for their excellent technical assistance. This study was supported in part by
the Israel National Council for Research and
Development, Grant No. 2099.
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transformation, chondroprogenitor, formation, osteoblastic, membranes, new, vitro, bones, cells
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