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Immobilization and cartilage transformation into bone in the embryonic chick.

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Immobilization and Cartilage Transformation into Bone
in the Embryonic Chick
Department of Biology, Life Sciences Centre, Dalhousie University,
Hafifax, Nova Scotia, Canada
The fate of the secondary cartilage present on the membrane
bones of the embryonic chick has been studied after immobilization. Immobilization was achieved by the in vivo injection of paralysing drugs (tubocurare or
decamethonium), by grafting membrane bones onto the chorio-allantoic membrane, or by organ-culturing membrane bones in vitro. In all three situations the
cartilage was transformed into a bone-like tissue, the matrix losing its acid mucopolysaccharide, accumulating collagen and undergoing calcification. The chondrocytes shrank in size, came to resemble osteoblasts (osteocytes) and acquired
alkaline phosphatase activity.
In normal development this cartilage is not transformed into bone but is partly
replaced by bone and partly converted into a fibrocartilage which forms the definitive articular cartilage. Immobilization prevented this normal sequence.
Past studies on the transformation of cartilage to bone are reviewed and are
seen to be adaptations of a highly labile tissue to functional demands.
The secondary cartilage which forms on
the membrane bones of the skull and
mandible in the embryonic chick from ten
days of incubation onwards has been
shown to form only where the membrane
bones articulate with another part of the
musculoskeletal system and to be induced
by the intermittent pressure and tension
present at such articulations. This conclusion was based on anatomical studies of
the distribution of the cartilage (Murray,
’63; Hall, ’67a); failure of the cartilage to
form on membrane bones grafted to the
chorio-allantoic membrane or in paralyzed
embryos [Murray and Smiles, ’65; Murray
and Drachman, ’69); failure to form on
membrane bones cultured in vitro (Hall,
’67b; ’68a), and on the ability of the membrane bones to initiate chondrogenesis
when mechanical stimulation was artificially applied in vitro (Hall, ’67b; ’68a).
If the intermittent pressure and tension
was removed after chondrogenesis had
commenced (i.e., after 11 days of incubation) the cartilage already formed was lost
and no further chondrogenesis was initiated. The already-formed cartilage could
have suffered one of three fates: ( a ) it
might have been replaced by bone in the
ANAT. REC., 173: 391-404.
fashion of endochondral ossification; (b)
it might have been directly converted into
bone (“metaplasia”) or (c) the chondrocytes might have dedifferentiated to osteoprogenitor cells, redifferentiated as osteoblasts and deposited bone matrix.
These alternatives were studied on the
membrane bones of embryos immobilized
in one of three ways : (a)by the injection
of paralysing drugs; (b) by organ culture
in vitro; (c) by grafting to the chorioallantoic membrane.
Both organ culture and grafting removed
the membrane bones, not only from the
action of the muscular system, but also
from any vascular, hormonal or other systemic influences which might have acted
upon them. Paralysis was produced by injection of either decamethonium iodide or
D tubocurarine chloride onto the chorioallantoic membrane of intact embryos.
These agents have been shown to be effective paralysing agents of embryonic chicks
(Drachman and Coulombre, ’62; Drachman and Sokoloff, ’66; Murray and Smiles,
’65; Sullivan, ’66; ’67). Both act as neuromuscular blocking agents, decamethonium
Received March 6, ’72.Accepted May 24, ’72.
by depolaxizing the muscle membrane and
D tubocurarine by blocking the release
of acetylcholine from cholinergic nerve
The membrane bone chosen for study
was the quadratojugal, the posterior tip of
which consists of differentiating bone and
cartilage from 11 days of incubation onwards.
Eggs were incubated in a forced-draft
incubator at 37 I
.1” C and 57% relative
Injection of paralyzing drugs
Twenty-five embryos were injected with
decamethonium iodide (Koch-Light Labs.,
lot H4137) at ten days of incubation and
examined at 13 days of incubation. Doses
used were 0.625, 1.25, 2.50, 5.05 or 10.00
mg/embryo. A further 39 embryos were
injected with D tubocurarine chloride (Nutritional Biochemicals Corp., lot 3948) at
10 to 11 days of incubation and examined
at 13 days. Doses used were 1, 5, 10 or 20
mg/embryo. Sixteen embryos were untreated, examined at 13 days and served
as controls. A further six embryos were injected with 10 mg D tubocurarine chloride
at 13 days of incubation and examined at
19 days of incubation. Three embryos
served as controls.
The drugs were dissolved in sterile saline
(0.9% NaCl in distilled water) and injected in a volume of 0.5 cm”/embryo
using a pre-sterilized 2.5 cm3 Tuberculin
syringe. A pin-hole was made in the shell
and shell membrane, the drug injected
onto the chorio-allantoic membrane, the
hole sealed with Scotch tape and the egg
further incubated.
At recovery (13 or 19 days of incubation) the quadratojugals were removed
and processed for histological or histochemical analysis (see below).
Chorio-allantoic grafting
In order to further study the loss of
cartilage which follows removal of the
mechanical stimulus, quadratojugals from
12 day embryos (chondrogenesis in progress since day 11) were grafted onto the
chorio-allantoic membrane of eight day old
host embryos.
The quadratojugals were dissected from
donor 12 day embryos under sterile conditions. One quadratojugal was fixed for
histological analysis to assess the state of
chondrogenesis at the start of the experiment. The contralateral quadratojugal was
carefully cleaned of adherent connective
tissue, rinsed in sterile saline and grafted
to the chorio-allantoic membrane of the
host embryo. The hosts had been incubated
for eight days at the time of receipt of the
graft. A window was sawn in the shell of
the host over a bifurcation of the vitelline
blood vessels, a slit made in the shell membrane, the graft placed on the blood vessel,
the window replaced, sealed with scotch
tape and the host further incubated. Grafts
were normally recovered at or before the
hosts had reached 18 days of incubation,
the time at which the chorio-allantoic
membrane begins to break down, and by
which time the quadratojugal had been
grafted for ten days. For longer durations
as grafts the quadratojugals were removed
under sterile conditions from the 18 day
hosts and regrafted to a second eight day
host. In this way quadratojugals were
maintained as grafts from 0 to 21 days
(equivalent to 12 days embryonic age to
12 days post-hatching). At the time of recovery the grafts were fixed and processed
for histological and histochemical analysis
(see below).
Organ culture
The fate of the cartilage was also followed by organ culturing the quadratojugals from 12 day embryos. Again one
quadratojugal was fixed at the start to
serve as a control and the opposite quadratojugal cultured. The quadratojugals were
cultured on metal grids in a Liquid medium
in air. The medium used was BGJ (Difco
laboratories) supplemented with 40%
foetal calf serum. Fresh medium was
added every three days. A total of 20 quadratojugals were cultured for periods from
2 to 19 days and so paralled the time
course of the grafting experiment. At recovery the bones were processed for histological and histochemical analysis.
Histology and histochemistry
For histological analysis the bones were
fixed in Heidenhain’s susa for 24 hours,
washed in iodized alcohol, upgraded in
ethanol, cleared in xylene and embedded
in paraffin.
For histochemical analysis the bones
were fixed in 85% ethanol at 2" C for
four hours, rapidly upgraded, cleared and
embedded in low temp (52" C m.p.)
Alcian blue-ChlorantineFast red (ABCR,
ILison, '54) was used as the routine histological stain and to stain acid mucopolysaccharide. Non sulphated acid mucopolysaccharide was also visuaJized after alcian
blue at pH 2.75 ( 1 % aqueous alcian blue:
1% aqueous acetic acid ( 1:1) ), and sulphated acid mucopolysaccharide (predominantly chondroitin sulphate in embryonic chick cartilage) by ethanol-stable
metachromasia after 0.01% toluidine blue
(Ham and Harris, '50). The light green in
Masson's trichrome (Pantin, '60) was used
to visualize collagen; alizarin Red S to
visualize sites of calcification; the Gomori
method to visualize alkaline phosphatase;
and periodic acid SchifT (PAS) reagent to
visualize glycogen, mucoprotein and glycoprotein (Barka and Anderson, '65).
Calcium may give false localization in the
Gomori method. This was prevented by
decalcifying the sections in 5% EDTA and
reactivating the alkaline phosphatase with
1% sodium barbitone (Schajowicz and
Cabrini, '56).
Paralysis experiments
Paralysis was assessed as the absence of
movement of the embryo at the time of
examination (13 or 19 days of incubation).
D tubocurarine chloride at or above 10
mg/embryo and decamethonium iodide at
or above 1.25 mg/embryo completely paralysed the embryo.
All the quadratojugds from all the 13
day untreated embryos possessed prominent cartilage at their posterior hooked
tips (fig. 1). The chondrocytes were hypertrophic, there was little intercellular matrix
and the cartilage was differentiating from
a pool of undifferentiated cells common to
In those specimens given 10 or 20 mg
D tubocurarine chloride and examined at
13 days of incubation the presence or
absence of cartilage could be related to the
time of injection as follows:
10 mg at ten days - no cartilage present at 13 days; 10 mg at 11 days - none
or very little cartilage present at 13 days;
10 mg at 11-12 days (2 doses of 5 mg
each) - cartilage present at 13 days; 20
mg at 11-12 days ( 2 doses of 10 mg each)
- no cartilage present at 13 days.
These results confirmed earlier studies
(Murray, '63; Murray and Smiles, '65;
Hall, '68b, 70a) which indicated that
secondary cartilage began to differentiate
on the quadratojugal during the tenth day
of incubation. Paralysis beginning early
in the tenth day inhibited the initiation of
chondrogenesis, and as no cartilage was
present at the time of injection none was
found at 13 days. Paralysis at 11 to 12
days inhibited further chondrogenesis in
all specimens but some cartilage would
have been present at the time of injection
and in some specimens had not completely
disappeared by 13 days. However, with a
sufficiently high dose (20 mg) over the
eleventh to twelfth days this cartilage was
also lost.
The appearance of a typical specimen
given 10 mg D tubocurarine chloride at
ten days of incubation and examined at 13
days is shown in figure 2. The bone at the
tip of the quadratojugal (the site normally
occupied by cartilage) is very cellular, the
size of the cells more closely resembling
chondrocytes than osteocytes. Within this
material a few cells with capsules staining
blue after alcian blue but which were not
metachromatic after toluidine blue were
seen (fig. 3 ) . The matrix surrounding
these cells stained with neither stain,
whereas cartilage matrix stains with both.
These cells then had, on the basis of these
histochemical characteristics, ceased producing sulphated acid mucopolysaccharide,
had secreted a thin capsule of non-sulphated acid mucopolysaccharide but had
not elaborated this material further into
the matrix. Histologically they resembled
chondrocytes, but their matrix resembled
osseous matrix.
The same dose of D tubocurarine chloride given at 11 days of incubation, when
some cells would already have been committed to chondrogenesis and when some
cartilage would have already differentiated,
resulted in general inhibition of new chondrogenesis. Cartilage vestiges in the form
of isolated cells embedded in bone matrix
were all that remained of a n y cartilage
present at the time of treatment. Again
the bone at the tip of the quadratojugal
(the position normally occupied by cartilage) had a very cellular appearance.
Ten milligrams D tubocurarine chloride
given in two doses over the 11 to 12 day
embryonic period gave a similar picture
but with somewhat more cartilage vestiges
than if 10 mg were given at 11 days.
Evidently fewer chondrocytes were subject
to the paralysing effect of the drug and so
fewer were lost.
The quadratojugal from a control 19
day embryo is shown in figures 4 and 5.
Cartilage is being resorbed by, replaced by
or converted into bone, except at the articular surface where the chondrocytes are
closely packed and are forming the definitive articular cartilage (Hall, '67a, '68c).
These cells are still producing matrix as
shown by their metachromasia after toluidine blue (fig. 5). However, they grade
into bone and lose their metachromasia
away from the articular surface. This then
is the normal pattern of replacement of
this secondary cartilage.
A quadratojugal from an embryo given
10 mg D tubocurarine chloride at 13 days
and fixed at 19 days is shown in figure 6.
Chondrogenesis has been completely inhibited and their is no evidence of cartilage
present. The cartilage which is present in
the control (figs. 4, 5) has been completely
removed in the treated bone and its place
has been taken by bone. The articular surface has also lost its cartilage, indicating
the dependence of this cartilage on
Chondrogenesis was also completely inhibited in the quadratojugals examined
from 13 day embryos previously treated at
ten days with from 1.25 to 10 mg decamethonium iodide (figs. 7, 8). No cells with
alcian blue-staining capsules were seen
and no chondrocytes embedded in bone
were seen so that this treatment was rather
more effective in completely suppressing
chondrogenesis than was D tubocurarine
chloride at similar doses. The place normally occupied by cartilage was taken by
bone in all specimens.
Chorio-allantoic grafting,
organ culture
The question of the fate of the cartilage
on these membrane bones in the absence
of mechanical stimulation was further
analysed by grafting or organ culturing
the quadratojugals from 12 day embryos.
Typically the quadratojugal from a 12
day embryo consists of a prominent nodule
of cartilage actively differentiating from
the germinal cell pool at the tip (fig. 9).
All control specimens exhibited these
nodules. The germinal cells differentiate
as chondroblasts which undergo hypertrophy and deposit cartilage matrix. Both
osteogenesis and chondrogenesis are proceeding from the same pool of cells. The
border between the cartilage and the bone
is very sharp; there is little if any invasion
of bone fibres into the cartilage and no
evidence of resorption (fig. 9).
After two days in organ culture or on the
chorio-allantoic membrane as a graft the
cartilage matrix had lost its metachromasia
after toluidine blue and its staining after
alcian blue, indicating a loss of sulphated
and non-sulphated acid mucopolysaccharides (fig. 10). Biochemical data on
membrane bones from paralysed embryos
support this finding (Hall, unpublished
results). The boundary between the cartilage and the bone was still sharp (cf. figs.
9, 10) indicating minimal resorption of the
cartilage matrix.
After five days in vitro or on the chorioallantoic membrane areas of cartilage embedded in bone were found (fig. 11 and
compare with figs. 9, 10). The capsules
and intercellular matrices of these cells
stained very lightly after alcian blue but
quite strongly after the light green of Masson's trichrome (fig. 11). These histochemical reactions indicated decreased
amounts of acid mucopolysaccharide and
increased amounts of collagen within this
tissue, giving it an appearance intermediate between normal bone and cartilage.
This tissue was poorly calcified in comparison with bone (fig. 12), contained no
alkaline phosphatase in contrast to the intense activity within adjacent osteoblasts
(fig. 1 3 ) and was, along with the bone,
uniformly PAS positive. Such areas were
completely surrounded by bone and did
not abut onto a marrow cavity, i.e., they
were not undergoing resorption but were
apparently being transformed into osseous
tissue. The edges of these nodules graded
off into regular bone.
This tissue resembled bone even more
closely after it had been grafted to the
chorio-allantoic membrane for ten days.
The intercellular matrix stained red after
chlorantine fast red (cartilage does not
stain with this stain) but the cells have the
size and appearance of chondrocytes (fig.
14). In contrast to bone the matrix did not
!stain after toluidine blue (fig. 15).
After 14 or more days the cells within
this tissue had come to resemble osteocytes
in size and appearance (fig. 16), the
matrix was still toluidine blue negative
(fig. 17) but exhibited calcification and
the cells contained alkaline phosphatase.
Under polarised light this matrix and that
of the bone had the same characteristics,
i.e., trabeculae of woven bone.
Therefore, after nine days either in vitro
or on the chorioallantoic membrane little
if any sign of the previous extensive pad
of secondary cartilage was found. Instead
areas of cellular bone, in the position normally occupied by cartilage and which had
apparently arisen from transformation of
the cartilage matrix into bone matrix were
seen. The cells came to resemble osteocytes although the present study does not
establish to what degree the ongoing activity of these cells has changed.
Another possible mechanism for transforming cartilage into bone is for the cartilage to degenerate and undergo necrosis
and for osteoblasts to migrate into the
matrix and form osseous tissue. No evidence of cartilage necrosis was seen in the
cartilage studied here nor was evidence of
migration of cells into the cartilage found.
Bone may also replace cartilage if the
chondrocytes dedifferentiate and cease the
production of cartilage matrix products
and subsequently redifferentiate (perhaps
after undergoing division) into osteoblasts
and deposit bone matrix. No evidence of
dedifferentiation of the hypertrophic chondrocytes was found in this study and no
cell division was seen. Therefore the transformation observed appears to involve
transformation of one differentiated tissue
into another (as evidenced by the changing nature of the matrices produced) and
does not involve dedifferentiation (insofar
as this may be assessed with the criteria
The present study has shown that when
the secondary cartilage of the quadratojugal from the embryonic chick is removed
from the normal intermittent stresses acting upon it, by paralyzing embryos in vivo,
by grafting the membrane bones to the
chorio-allantoic membrane, or by organ
culturing the bones in vitro, the proliferating cells switch to forming bone and the
already-formed cartilage is transformed
into bone. The switch to osteogenesis by
cells which are bipotential for osteogenesis
and chondrogenesis, which were previously
producing cartilage and which are isolated
from mechanical stresses has been previously documented (Murray and Smiles,
'65; Hall, '67b; '68a; '68d). The transformation of the differentiated cartilage into
bone requires further comment.
The conversion of cartilage into bone or
the replacement of cartilage by bone has
been shown to occur in three ways; ( a ) by
resorption of the cartilage and its replacement by periosteal and endosteal bone;
( b ) by dedifferentiation of chondrocytes to
osteoprogenitor cells and their subsequent
redifferentiation as osteoblasts, or ( c ) by
the transformation of cartilage matrix to
bone matrix and/or of chondrocytes to
osteoblasts (osteocytes). In the present system this transformation proceeded by the
cartilage matrix losing acid mucopolysaccharide and acquiring collagen and the
staining properties of osteoid, the cells
acquiring alkaline phosphatase activity and
the matrix calcifying. This is the reverse
of the sequence observed when cartilage is
replaced by bone for then the matrix first
calcifies and is then broken down and replaced. The metabolic and functional levels
of activity of these cells could not be determined in detail from this study except
to show that they had detectible enzymatic
activity and that they evidently survived.
A combined ultrastructural and autoradiographical study of these cells is underway.
The resorption of calcified cartilage and
its replacement by bone, deposited by cells
other than those within the cartilage, is
the classically accepted mechanism of en-
dochondral ossification (Ham and Leeson,
'61; Bloom and Fawcett, '68). However,
recent experimental evidence has indicated
that during the normal process of endochondral ossification resorption of the
cartilage matrix does not necessarily mean
death of the chondrocyte (Holtrop, '71;
Melcher, '71a,b) and that the hypertrophic
chondrocytes may dedifferentiate to osteoprogenitor cells and redifferentiate as
osteoblasts which then deposit the bone
matrix (Crelin and Koch, '65, '67; Holtrop,
'66, '67; Melcher, '71a,b). If the mesenchymal primordium of the pubic bones of
foetal mice is exposed to tritiated thymidine and organ cultured (Crelin and Koch,
'65, '67) or if the rib from foetal mice is
similarly labelled and transplanted intramuscularly (Holtrop, '66, '67), the mesenchymal cells are found to be uniformly
heavily labeled. A cartilaginous mcdel then
differentiates in which only chondrocytes
show label. With the onset of osteogenesis
labeled osteoblasts and osteocytes are also
found and in the transplanted ribs labelled
osteoprogenitor cells are found. The conclusion drawn from these studies was that
during endochondral ossification chondrocytes dedifferentiate to osteoprogenitor
cells, redifferentiate as osteoblasts and deposit bone matrix. Such dedifferentiation
was not detected in the present study.
Considerable evidence has also accumulated in support of the thesis that chondrocytes may metaplase to osteoblasts without
the intermediate step of dedifferentiation. This has been shown in endochondral
ossification and on endochondral bones
(Retterer, '17, '20; Knese and Knoop, '61;
Enlow, '62; Friant, '59; Haines and
Mohuiddin, '68); in the repair of fractures
of the skull vault and long bones in mammals, reptiles, fish and amphibia (Pritchard
and Ruzicka, '50; Moss, '58; Young, '59;
Pritchard, Scott and Girgis, '56; Yamagishi
and Yoshimura, '55; Tallqvist, '62; Cabrini,
'61; Moss, '61; Bohatirchuk, '65, '69;
Robertson, '69); in antler formation in deer
(Goss, '70 for review); in normal development of the otic capsule, labyrinthine capsule of the inner ear, and incudomalleal
jcint of the middle ear in man (Gussen,
'68a,b, '72); in chondroidal ossification in
fish (Moss, '61; Weisel, '67); in transplanted fracture callus cartilage (Danis,
'57; Bridges and Pritchard, '58; Lacroix,
'61: Urist, Wallace and Adams, '65; Urist
and Adams, '68); in degenerative changes
(tracheopathia osteoplasia) in the trachea
and bronchus of man (Ashley, '70); in
cell cultures of endosteal bone of the chick
(Fell, '33; Roulet, '35); and in organ culture of the chick tibia (Glucksmann, '38;
Bassett and Herrmann, '61; Shaw and
Bassett, '64, '67).
This transformation has been described
as occuring by hypertrophic chondrocytes
shrinking to the size and shape of osteocytes, the matrix losing its basophilia and
reaction to stains for acid mucopolysaccharide and becoming more fibrous and
eosinophilic (Fell, '33; Roulet, '35; Glucksmann, '38; Pritchard and Ruzicka, '50;
Yamagishi and Yoshimura, '55; Pritchard
et al., '56; Danis, '57; Young, '59; Friant,
'59; Cabrini, '61; Enlow, '62; Haines and
Mohuiddin, '68; Robertson, '69; Gussen,
'72; Holtrop, '71). A similar transfonnation was found in the present study.
In osteogenesis in fish both cellular and
acellular bone may form from the transformation of chondroid into bone. In both
the chondroid matrix calcifies. In cellular
bone formation the chondrocytes transform into osteocytes whereas in acellular
bone formation the cells become pyknotic
and the cell space calcifies (Muss, '61;
Weisel, '67).
Shaw and Bassett ('64, '67) subjected
their cultures to oxygen levels of 5, 20, 35,
65 or 95% and found chondrocytes transforming to osteocytes and accumulating
collagen only with oxygen levels from 20
to 60%, indicating that this transformation was a response to a particular set of
environmental conditions. In the present
study the presence or absence of mechanical stress was evidently the important environmental parameter governing the fate
of the cartilage for in vivo the secondary
cartilage is not all transformed into bone
but is partly replaced by bone, partly converted into bone and partly converted into
(or at some sites replaced by) a fibrocartilage forming the definitive articular
cartilage of the adult joint (Hall, '67a,
'68c,d). The osseous replacement of the
cartilage is accompanied by chondrocyte
degeneration (Hall, '71a; Hall and Shorey,
'68) whereas immobilization results in
c!hondrocyte transformation and survival. with collagen (probably PPL-5) and to be
In normal development secondary carti- less tightly linked than that within the
lage whose chondrocytes are hypertrophic lacuna. Removal of this material would
is replaced at the articular surface by fibro- leave a matrix consisting of a collagenous
cartilage, whereas secondary cartilage framework as suggested by Pritchard and
whose chondrocytes do not hypertrophy is Ruzicka. It has further been shown (Hall,
transformed into fibrocartilage (Hall, '67a, '70a) that if the synthesis of chondroitin
'6Sd). In immobilization both cartilage sulphate is disrupted at the time when the
types are transformed into bone. The de- germinal cells have been determined for
pendence of the normal joint on mechani- chondrogenesis, chondroblasts may form
cal factors for its development has previ- but they fail to form chondrocytes, to
ously been shown (Drachman and Sokoloff, hypertrophy or to secrete matrix. Any vari'66; Murray and Drachman, '69). It ap- able which lowers the level of matrix acid
pears then that not only is mechanical mucopolysaccharide might well result in
stress required to initiate chondrogenesis loss of hypertrophy and leave the cells suson avian membrane bones but it is also ceptible to modulation to other skeletal cell
required for normal replacement of the types. The fact that some secondary carticartilage and for formation of normal tis- lage may be transformed not into bone but
!jues at the joint. Immobilization in the into an intermediate chondro-osseous tis(adultcould well lead to similar transforma- sue, characterised by high mucopolysacitions and be a factor in degenerative joint charide content (Gussen, '72) indicates a
strong association between the type of
(disease (Sokoloff, '69; Sood, '71).
It has been suggested, initially by Fell matrix and the stability of the tissue. In
( ' 3 3 ) and subsequently by Moss ('58) that this tissue the initial loss of acid mucopolythis cartilage is in a comparatively low saccharide is minimal and the cells restate of differentiation and therefore is able spond by producing an intermediate tissue.
to transform into bone. Evidence for the Fish also possess such an intermediate
low state of differentiation was the rela- tissue (Moss, '6 1 ) .
tively sparse amount of intercellular matrix
Evidently skeletal cells will differentiate
produced by the chondrocytes. Even if this as cartilage, bone or as chondroid bone, dedoes indicate low grade differentiation it pending on the environmental needs for a
does not explain how the matrix present, particular type of matrix. Fitton Jackson
which is normal cartilage matrix in all ('70) has shown that skeletal cells are able
respects is able to transform into bone to respond to changes in the macromolecumatrix. The suggestion made by Pritchard lar environment by altering their synthetic
and Ruzicka ('50), viz., that removal of the activity and it has been argued (Young,
acid mucopolysacchaxide from cartilage '64; Johnson, '64; Hall, '70b, '71b) that the
would leave a collagenous framework choice between producing cartilage or prowhich by impregnation with ossein could ducing bone is a matter of changing rates
be transformed to osteoid and subsequently of synthesis of existing products. It is percalcify, is borne out by the present histo- haps then only a semantic problem as to
chemical findings. Hypertrophic chondro- whether one considers this change from
cytes at such sites begin to secrete collagen cartilage to bone as a modulation or as a
(Shaw and Bassett, '67; Melcher, '71b).
metaplasia for both imply a reversible state
It has been shown that degradation of of differentiation. The transformation does
the protein-polysaccharide complex must not require the presence of specialized cells
occur before cartilage matrix can calcify such as chondroclasts or macrophages to
(Hisschman and Hirschman, '71 ). In the destroy the cartilage matrix before it bepresent system removal of the acid muco- comes osseous but requires that skeletal
polysaccharide from the matrix was the cells alter their activity in a relatively
first sign that chondrogenesis was ceasing. permanent way and so produce a matrix
The first acid mucopolysaccharide removed with new characteristics. Such transformawas that in the extralacunar area. This has tions are then a further indication of the
been shown by Eisenstein et al. ('71) to be highly responsive nature of skeletal tissue
the acid mucopolysaccharide associated to functional demand.
Supported by National Research Council of Canada grant A5056. The technical
assistance of Miss Joan Calder and of Mr.
Gary Akenhead is gratefully acknowledged.
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Figures 1 to 8 are from the paralysis experiments.
The quadratojugal from a n untreated 13 day embryo. Hypertrophic
cartilage is prominent and is differentiating as indicated by the
arrow. Alcian Blue-Chlorantine Red. x 165.
The quadratojugal from a 13 day embryo given 10 mg D tubocurarine
chloride at ten days. Bone has formed (arrow) where cartilage i s
normally found (cf. fig. 1). ABCR. x 165.
Specimen treated as was that in figure 2. Note islands of cartilaze
(open arrow) and developing bone (solid arrow). ABCR. X 165.
The quadratojugal from a n untreated 19 day embryo to show the
prominent cartilage and marrow cavity. ABCR. x 80.
The same specimen as in figure 4. Note the metachromatic capsules
around the chandrocytes (centre) and the loss of metachromasia as
they transform into bone (upper right). Toluidine blue. x 165.
I n a 19 day specimen treated with tubocurare at 13 days the cartilage is completely lost, bone having formed in its place. ABCR.
x 80.
The quadratojugals from 13 day embryos given 5 mg (fig. 7) or
10 mg (fig. 8 ) decamethonium iodide at ten days. Note complete
absence of cartilage and active ongoing osteogenesis. ABCR. X 165.
B. K. Hall
Figures 9 to 17 are from the chorio-allantoic grafting and organ culture
experiments. All are x 165.
The quadratojugal from a n untreated 12 day old embryo ( t h e control
for figs. 10 to 17). Note prominent cartilage ( c ) and bone ( b ) . ABCR.
10 The quadratojugal after two days in culture. Note reduced amount
of cartilage ( c ) and pale staining of matrix (cf. fig. 9). ABCR.
A quadratojugal after five days on the chorio-allantoic membrane.
The cartilage ( c ) stains uniformly with light green. Masson’s trichrome.
The same specimen as in figure 11 to show absence of calcification
i n the position of the cartilage ( p ) . Alizarin red S .
The same specimen as in figure 11 to show absence of alkaline phosphatase from cartilage cells ( p ) and presence in osteoblasts. Gomori
A quadratojugal after ten days as a graft. Note cellular appearance
of the bone i n the position normally occupied by cartilage (lower
right). ABCR.
15 The adjacent section to that shown in figure 14. Note absence of
staining of cellular bone as compared with dense staining of bone.
Toluidine blue.
A quadratojugal after 14 days as a graft. Note relatively uniform
appearance of bone and size of the cells (cf. fig. 1 4 ) . ABCR.
17 The adjacent section to that in figure 16. Note lack of staining of
newly transformed bone (cf. figs. 15, 16). Toluidine blue.
H. K. Hall
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transformation, immobilization, embryonic, chick, cartilage, bones
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