/. Embryol. exp. Morph. Vol. 34, 2, pp. 327-337, 1975
Printed in Great Britain
327
Electron microscopic studies on chick limb
cartilage differentiated in tissue culture
By SURESH C. GOEL 1 AND A. JURAND 2
From the Institute of Animal Genetics, Edinburgh
SUMMARY
The hind limb-bud mesenchyme of chick embryos 4-4£ days old was cultured in Eagle's
Minimum Essential Medium supplemented with both horse serum and fresh chick embryo
extract. Whereas no differences are seen at the light-microscope level, at the electron-microscope level the chondroblasts differentiated in tissue culture are noticeably different from those
differentiated in vivo, particularly in the possession of some cytoplasmic fibrils and vacuoles.
It is proposed that the secretion of the extracellular matrix alone is not sufficient to account
for the pattern of cellular arrangement in a cartilaginous condensation.
INTRODUCTION
Studies on cartilage in tissue culture started with the work of Strangeways &
Fell (1926) and Fell (1928). A number of workers have since been using cartilage
or presumptive cartilage tissue in culture (a) to evaluate the potency of mesodermal cells to form cartilage independent of the influences operating in vivo
(Zwilling, 1966; Hamburgh, 1971; Searls, 1973), (b) to test the effect of various
chemicals on physiology, morphology and differentiation of chondroblasts
(Medoff, 1967; Reynolds, 1967; Fell & Dingle, 1969; Glauert, Fell & Dingle,
1969; Levitt & Dorfman, 1974), and (c) to study the effects of genetic behaviour
of cells (Ede & Agerbak, 1968; Ede & Flint, 1972).
The cartilage differentiated in tissue culture is hardly distinguishable from
that differentiated in vivo at the light microscope level (Ede & Agerbak, 1968;
Goel, 1969). It is, however, known that tissues developed in tissue culture are
usually under certain stress from the environmental conditions (Levitt & Dorfman, 1974). The present communication reports the ultrastructural differences
between the cartilage cells differentiated in tissue culture and in vivo in the
developing hind limb-buds of chick embryos (Goel, 1970).
MATERIALS AND METHODS
The hind-limb buds from 4- to 4^-day-old chick embryos of Brown Leghorn
variety were used. The excised buds were washed twice in Hanks' balanced salt
1
2
21
Author's address: Department of Zoology, Poona University, Poona, India.
Author's address: Institute of Animal Genetics, Edinburgh, EH9 3JN, U.K.
E M B 34
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SURESH C. GOEL AND A. JURAND
solution (Paul, 1970) and twice in calcium- and magnesium-free balanced salt
solution (CMF; Moscona, 1961) and incubated in CMF for 15 min at 38 °C.
This was followed by further incubation for 7-10 min in CMF containing 1 %
trypsin (crystallized and lyopholized, from Worthington Biochemical Co.:
minimum activity 150 units/mg.*LU.B. system). After washing in Hanks'
balanced salt solution the buds were transferred to standard culture medium,
which consisted of Minimal Essential Medium of Eagle (with Earle's salts)
supplemented with 10 % horse serum (Flow Laboratories, Irvine, Scotland)
and 10-50% fresh chick embryo extract (from 8- to 10-day-old embryos in
50:50 Tyrode's solution; Paul, 1970). The ectodermal jacket was removed with
the help of tungsten needles and chondrogenic mesoderm was cut into small
fragments, approximately 0-75 mm3, with cataract knives or dispersed into single
cells by passing through a micropipette. Suspension cultures in plastic Petri
dishes or hanging drop cultures using depression slides were set up and culture
medium changed every 48 h. The pH of the medium was maintained at 6-8-7-0.
The cartilage nodules developed in about 40-48 h (see also Jackson, 1965;
Ede & Flint, 1972). The cultures were incubated up to 6 days at 37-5 ±0-5 °C.
The material was fixed in 1 or 2% osmium tetroxide in veronal buffer at
0-4 °C for 1 h. It was routinely processed and embedded in Araldite epoxyresin. For electron microscopy 70-90 nm thick sections were stained with
saturated solution of uranyl acetate or 20% uranyl nitrate (15 min) followed by
lead citrate (15 min), and viewed under an AEI EM 6 electron microscope. For
light microscopy Araldite sections 1 /im thick were stained with 0-5 % toluidine
blue (in 1 % solution of sodium tetraborate) at 38 °C for 15 mins (see Goel &
Jurand, 1972, for further details).
The light microscope autoradiography experiments using [3H]proline
(15/*Ci/ml of culture medium; specific activity 720mCi/mM; from Radio-
FIGURES
1-5
All the figures are from cartilage developed in culture for two or three days.
Fig. 1. Cartilage allowed to differentiate in tissue culture for two days and incubated
with [3H]proline (15/*Ci/ml of culture medium; specific activity 720 mCi/mvi) for
2 h. In the autoradiograph the grains are not present on the extracellular phase or
on a non-cartilaginous cell, x 1050.
Fig. 2. Cartilage differentiated in tissue culture, and incubated with [3H]proline (as in
Fig. 1) for 24 h. In the autoradiograph the grains are present on the extracellular
phase as well as on the cartilage cells, x 1600.
Fig. 3. Cartilage differentiated in tissue culture. The whorl-like arrangement of the
cells, as characteristic of limb cartilage (see Fig. 5), is seen, x 250.
Fig. 4. Cartilage differentiated in tissue culture. In this cartilaginous nodule the
amount of extracellular phase towards the centre is high, x 375.
Fig. 5. Epiphyseal cartilage from the third toe of hind limb-bud of stage-31 embryo.
The characteristic arrangement and appearance of cells in 'condensation' is evident.
Compare with the tissue differentiated in culture (Figs. 3 and 4). x 270.
Chick limb cartilage in tissue culture
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SURESH C. GOEL AND A. JURAND
chemical Centre, Amersham) indicate that the cartilage cells differentiated in
the tissue culture were healthy (Goel, 1969). Such cells actively synthesize and
export proteins into the extracellular phase (Figs. 1, 2).
RESULTS
The cartilage nodules are usually spheroidal structures. The general arrangement of the cells in the nodule is comparable to that in the cartilage condensation in the limbs in vivo (Figs. 3-5). The cells in the centre, like typical chondroblasts, have a scalloped outline, but may sometimes be rounded. Towards the
periphery of the nodule, in the sections, the cells are usually thin, elongated,
closely packed and concentrically arranged. These cells could be designated
as the perichondrial cells. The extracellular phase is sometimes extensive, particularly in the centre of the nodule, and stains metachromatically with toluidine
blue. The chondroblasts in the process of mitotic division are also noticeable
in some cases.
The nucleus of the chondroblasts is eccentrically placed and is enclosed in a
nuclear envelope 25-70 nm thick which is frequently studded with the ribosomes
on its outer surface (Figs. 6, 8). The nuclear pores, around 70 nm in diameter,
are common. The nuclear matrix is largely homogeneous and consists of
granules and fibrils. Some chromatin material is also present and a part of it
forms a very thin layer adjacent to the inner nuclear membrane. Usually there is
only one nucleolus with morphology similar to the nucleolus of the chondroblasts differentiated in vivo; the more electron-dense fibrillar areas of it are
completely enclosed in less electron-dense particulate areas (Fig. 7).
In the cytoplasm the ribosomes are distributed either singly or as polysomes.
The endoplasmic reticulum is granular and consists mainly of elongated cisternal
profiles around 120 nm in diameter. The dilated saccular cisternae are infrequent
FIGURES
6-10
Fig. 6. Cartilage differentiated in tissue culture. The matrix in the extracellular phase
is on the average not as extensive as in the in vivo cartilage, x 4400.
Fig. 7. Cartilage differentiated in tissue culture. The cell shows saccular cisterna of
endoplasmic reticulum (er), some perinuclear cytoplasmic fibrils and a characteristic
appearance of nucleus with nucleolus. x 20700.
Fig. 8. Cartilage differentiated in tissue culture. The Golgi apparatus lamellae (C),
cilium (c/) and centriole (c) are seen. The small vesicles below the plasmalemma are
possibly pinocytotic in origin, x 20700.
Fig. 9. Cartilage differentiated in tissue culture. Note the three vacuoles of differing sizes (arrows) and heterogeneous contents in one of them (v). x 25700.
Fig. 10. Cartilage differentiated in tissue culture. The Golgi apparatus and various
types of vacuoles are seen. The boundary membrane of one vacuole (v) is incomplete;
the small vesicles in the extracellular phase (arrow) are perhaps due to the opening
out of a vacuole. x 20700.
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SURESH C. GOEL AND A. JURAND
and measure up to l-lxO-9/tm. The vesicular profiles of the endoplasmic
reticulum are seldom seen (Fig. 8). The contents of the reticulum are homogeneous, amorphous and moderately electron-dense. The Golgi apparatus, like
that of the chondroblasts differentiated in vivo, consists of a very few lamellae,
some Golgi vacuoles and many vesicles (Fig. 10). The vacuoles may be as large
as 0-7 jiim in diameter and are usually electron-translucent but sometimes
contain a small quantity of materials which resemble the electron-dense granules
of the extracellular phase. It appears as if the larger Golgi vacuoles are formed
by the fusion of smaller ones. The area of the Golgi vacuoles and the saccular
cisternae of the endoplasmic reticulum can be easily distinguished from each
other, since, as in the chondroblasts differentiated in vivo, the area occupied
by the Golgi vacuoles is electron-translucent in appearance. Mitochondria are
frequent and vary in shape and size from oval structures, about 0-5 //m in diameter, to elongated ones about 3-0x0-7 jam in size. They have a moderately
electron-dense matrix with a few mitochondrial granules and many mitochondrial cristae. Certain vesicles located below the plasmalemma, probably of
pinocytotic origin, are of common occurrence (Figs. 8, 11). A centriole as well
as a cilium can be seen in Fig. 8, though the cilia are uncommon. Glycogen was
never observed in the chondroblasts.
Sometimes groups of fibrils, around 5-8 nm thick, can be seen in the perinuclear cytoplasm (Fig. 7). Moreover, in a few cases even bigger groups of
slightly thicker fibrils, around 12-16 nm thick, fill a considerable part of the
cytoplasm of the cells situated on the periphery of the condensation (Fig. 12).
A variety of vacuoles of varying sizes and contents are frequently present
(Figs. 9, 10). They are usually around 0-5-0-8/on in diameter although occasionally they may be as large as 1-6 x 2-5 /tm. A large number of them contain
small vesicles (50-70 nm in diameter); some, in addition, contain an amorphous
moderately electron-dense mass and various ill-formed membranous structures,
while a few are lipid droplet-like structures. Most of these structures are bound
by a single membrane, around 14 nm thick, but some are delimited by double
FIGURES
11-15
Fig. 11. Cartilage differentiated in tissue culture. The vesicle subjacent to plasmalemma is possibly due to pinocytosis. x 31000.
Fig. 12. Cartilage differentiated in tissue culture. A chondroblast showing a large
amount of cytoplasmic fibrils; such chondroblasts are found near the periphery
of the nodule. The arrows point to the plasmalemma. x 20700.
Fig. 13. Cartilage differentiated in tissue culture. The extracellular phase has
bundles of thin fibres of collagen, x 20700.
Fig. 14. Cartilage differentiated in tissue culture. Some of the fibres of extracellular phase are very close to the plasmalemma and appear to merge with it.
x 91000.
Fig. 15. Cartilage differentiated in tissue culture. The fibres of the extracellular
phase are straight and sometimes show an incipient banding (arrow); the electrondense granules (g) are few compared with the in vivo differentiated cartilage.
Chick limb cartilage in tissue culture
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SURESH C. GOEL AND A. JURAND
membranes. Vesicles around 5C-70 nm in diameter and similar to those in the
vacuoles are also present free in the cytoplasm, especially in the area of the
Golgi apparatus, suggesting the origin of vacuoles from this organelle. Moreover, the presence of vacuoles with incomplete boundary membranes in the
Golgi area also indicates their Golgi origin, though the variation in their structure suggests that they may be formed in more than one way. In one case it is
noted that the vacuole is opening to the extracellular phase (Fig. 10).
The process of 'ecdysis' or 'excortication' is more frequently found in these
cells as compared to those differentiated in vivo. The fibres of the extracellular
phase are often very near to or confluent with the plasmalemma (Fig. 14), and
sometimes enter in the cortical cytoplasm, and run a short distance subjacent
to the plasmalemma before merging into the cytoplasm. At such points of contact, however, the plasmalemma seems discontinuous or indistinct, possibly
because of tangential sectioning.
The extracellular phase contains numerous fibres embedded in a homogeneous amorphous mass (Fig. 13). The fibres, usually in bundles, are about
15 nm thick, short and straight (Fig. 15). The presence of a relatively large number of fibres towards the periphery, rather than in the centre, of the condensation
is noticeable. The electron-dense granules of the extracellular phase are neither
as frequent nor as well developed as in the cartilage differentiated in vivo; and
are usually completely missing in the cartilage from younger cultures.
DISCUSSION
The cytoplasmic fibrils, reported in the present study in the chondroblasts
differentiated in tissue culture, are not described in the chick hind-limb cartilage
differentiated in vivo (Goel, 1970). However, Searls, Hilfer and Mirow (1972)
report the occurrence of the perinuclear and cytoplasmic fibrils of similar diameter in the cartilage cells from chick wing-bud. Similar fibrils are also described
in the articular cartilage of rabbit (Palfrey & Davies, 1966), man (Meachim &
Roy, 1967), and mice (Silberberg, 1968), in amphibian limb cartilage (Revel &
Hay, 1963) and in other types of cells, especially those grown in tissue culture
(Spooner, Yamada & Wessells, 1971).
The nature and function of the cytoplasmic fibrils is uncertain but they have
been considered instrumental in cytokinesis (Schroeder, 1968), as part of cytoskeleton and important in cell locomotion and maintenance of cell shape
(Spooner et ah 1971; Searls et al. 1972), as signs of cellular degeneration and
ageing (Barnett, Cochrane & Palfrey, 1963), or as indicating metabolic disturbance (Silberberg, 1968). Our observations are in accord with the view of Meachim & Roy (1967) that the presence of fibrils in small quantities is not evidence
of chondrocyte degeneration but that the accumulation of large quantities is
indicative of degenerative changes. It is important to point out that none of
these authors interpret the fibrils to be precursors of collagen.
Chick limb cartilage in tissue culture
335
The chondroblasts differentiated in tissue culture, compared with those
differentiated in vivo (Goel, 1970), have a higher frequency of vacuoles (see also
Jackson, 1964; Eguchi & Okada, 1971). The vacuoles are reported in chondrocytes from a variety of animals (Silberberg, 1968; Serafini-Fracassini & Smith,
1974), but a very high frequency of vacuoles is a symptom of unhealthy state
of the cell (Glauert et al. 1969). It is therefore possible that the increased frequency of the vacuoles is the response of cells to the conditions prevailing in
tissue culture, and that some of the vacuoles are lysosomes (Norrevang, 1968;
Glauert et al. 1969) and may be autophagic in nature. It is plausible that the
presence of vacuoles escapes notice in the light-microscope observations unless
the vacuolar frequency is very high.
The glycogen has not been observed in the chondroblasts differentiated in
tissue culture (Eguchi & Okada, 1971), as also in the chick chondroblasts in
vivo (Goel & Jurand, 1972). On the other hand, Levitt & Dorfman (1974)
report the presence of numerous glycogen lakes in chick hind limb-bud cells
grown in tissue culture, and Searls et al. (1972) indicate the presence of glycogen
in undifferentiated mesenchyme cells of the chick wing-bud; but Searls et al.
(1972) do not comment on the presence of glycogen in chick limb cartilage.
Moreover, unfortunately, none of the authors presents electron micrographs
showing any definite glycogen granules in the cells. For this reason we retain
the view that in the chick limb epiphyseal cartilage glycogen is not present and
that the presence of glycogen is not essential for cartilage differentiation (Goel
& Jurand, 1972).
In the extracellular phase the 15 nm thick fibres are in all probability collagenous in nature. Similar fibres are reported in the chick hind limb-bud cartilage
(Goel, 1970), and wing-bud cartilage (Searls et al. 1972) differentiated in vivo.
Searls et al. (1972) consider the fibres to be [al(II)]3 form of collagen, whereas
Linsenmayer, Toole & Trelstad (1973) consider the chick cartilage collagen at
this stage to be largely [al] 3 ; however, there is evidence to the effect that the
[al] 3 collagen is very similar to, if not identical with, the [al(Il)] 3 collagen
(Linsenmayer, 1974). The thick collagen fibres with a periodicity of about 64 nm,
as reported by Searls et al. (1972) in the chick wing-bud cartilage, have not
been seen in the present study or earlier studies (Goel, 1970; Seegmiller, Fraser
& Sheldon, 1971) and it is generally believed that the acid mucopolysaccharides
of the cartilage matrix somehow interfere with the formation of collagen fibres
with a 64 nm periodicity (see Seegmiller et al. 1971; Serafini-Fracassini &
Smith, 1974).
The present observations on the arrangement and appearance of cells in the
cartilage differentiated in tissue culture shed light on the acquisition of pattern
of arrangement and appearance of the cartilage cells. Gould, Selwood, Day &
Wolpert (1974) suggest a simple model in which they consider the secretion of
the extracellular matrix as the main cause of orientation and flattening of the
cells. The present study as well as earlier in vivo results (Goel, 1970) indicate
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SURESH C. GOEL AND A. JURAND
that the secretion of the matrix is very likely responsible for the scalloped appearance of the cartilage cells, and also for their typical randomly spaced arrangement in the central region (Gould et al. 1974). On the other hand it seems
unlikely that the shape and arrangement of the more peripheral or perichondrial
cells is also due to the same factor. This suggestion is supported by our observations both in vivo and in tissue culture. In the tissue culture the cartilage acquires
a spherical shape, and concentric arrangement and flattened appearance of the
peripheral cells is produced (see also Ede & Flint, 1972) despite the fact that the
peripheral cells have practically no space restriction and can move outwards
rather than become flattened, if and when the synthesis of matrix exerts pressure
on them, as suggested by Gould et al. (1974). Moreover, the morphology and
organization of the cartilage nodule in tissue culture is a function of density
of inoculum and medium composition, including the presence of factors like
ascorbic acid (see Levitt & Dorfman, 1974). Even in vivo (Goel, 1969; Gould
et al. 1974) the concentric arrangement of the cells near the periphery of cartilage
condensation precedes the secretion of appreciable amounts of the matrix
which may be able to exert such a pressure. It is very likely that the arrangement
and possibly also the appearance of the cartilage cells is to a marked degree
influenced by the inherent pattern and shape of the cartilage concerned; for
example, the arrangement and the appearance of the cartilage and perichondrial
cells in a more or less spheroidal mesopodial element may not be the same as in
an elongated cylinder-shaped femur.
The authors wish to thank Dr D. A. Ede for reading the manuscript. Thanks are also due
to Mr F. M. Johnston for photographic assistance and to Mr E. D. Roberts for mounting the
micrographs.
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