J. Embryo/, exp. Morph. Vol. 26, 2, pp. 157-167, 1971
Printed in Great Britain
\ 57
A three-dimensional collagen network
for cartilage reconstruction
BY S. MOSKALEWSKI, M. KAMINSKI
From the Department of Histology and Embryology, School of Medicine,
Chalubinskiego 5, Warsaw
AND A. DUKWICZ
the Department of Laboratory Diagnostics, Postgraduate Medical
School, Ceglowska 80, Warsaw, Poland
SUMMARY
To obtain a three-dimensional network which would support cells during culture, cold
collagen solution was mixed with cells and converted into a gel at 37 °C. Gelation of collagen
did not influence cell viability.
The development of chondrocyte cultures in collagen gels depended on the distance
between the cells. Single chondrocytes were surrounded by a mucopolysaccharide ring. The
collagen fibres in parts of the cultures with moderate cell density were strongly alcian-bluepositive. Chondrocytes in crowded areas of cultures formed a cartilage matrix. Collagen gel
with Ehrlich ascites tumour cells and liver cells remained unchanged after cultivation.
Cultures of kidney cells and some chondrocyte cultures shrivelled, owing to partial collagen
digestion.
The delicate primary collagen network in some cultures partially transformed to much
thicker, long fibres or to distinct capsules around chondrocytes.
INTRODUCTION
Reconstituted collagen gel spread on a glass surface makes a useful substrate
for supporting the growth and differentiation of cells (Ehrmann & Gey, 1956;
Konigsberg & Hauschka, 1965). Cold salt solutions of collagen become converted into a firm gel at 37 °C (Gross, 1958; Gross & Kirk, 1958). Such collagen
could be used as a three-dimensional network in studies on tissue reconstruction
or on cell interactions. Cells introduced into the cold collagen solution would
remain in the suspension owing to its viscosity for the time necessary for gel
formation at higher temperature, and in this manner a collagen matrix with
uniformly distributed cells could be obtained. The following experiments were
performed to test this supposition in practice.
MATERIALS AND METHODS
Collagen preparation
Collagen was extracted from the skin of guinea-pigs with weights ranging from
100 to 500 g. Purification of collagen was based on previously published methods
II
E M B 26
158
S. MOSKALEWSKI, M. KAMINSKI AND A. DUKWICZ
(Gross, 1958; Gross & Kirk, 1958). The skin, shaved and cleared of adhering
tissues, was cut into small pieces and stored for 24 h at — 20 °C. It was then
extracted on a gyro-rotatory shaker with tenfold amount of 0-45 M NaCl for
24 h at 4 °C. The supernatant was discarded and the skin was extracted under
the same conditions with 0-5 M acetic acid. The extract was cleared by centrifugation at 10000 g for 20 min and dialysed against phosphate buffer fi = 0-45,
pH 7-6, for 24-48 h at 0 °C. The collagen was precipitated by addition of
approximately 14% of 96% ethanol (Gross, 1958), centrifuged at 10000 g
and lyophilized. It was then extracted in cold absolute acetone, dissolved in
0-5 M acetic acid, cleared by centrifugation and lyophilized again. The hydroxyproline content of purified collagen hydrolysed in 6 N-HC1 (Ogle, Arlinghaus &
Logan, 1962), as determined by the method of Mitomae? ah (1959), was 10-11 %,
depending on the sample. Purified collagen was stored at 0 °C. For use in
culture, collagen was sterilized with 70% ethanol, dissolved in sterile 0-5 M
acetic acid to give a 0-25, 0-5 or 1 % solution, dialysed against phosphate buffer
fi = 0-45, pH 7-6, for 24-48 h and afterwards for 24 h against Hanks solution.
Both dialysing fluids contained 50 units of penicillin and 50 /tg of streptomycin
per millilitre. Dialysis was performed at 0 °C. When outside the refrigerator,
collagen was always kept in iced water.
Testing of distribution and viability of cells in collagen gel
Ehrlich ascites tumour cells (EATC) were harvested 6-8 days after inoculation into A Prah mice and distributed into test-tubes to obtain, after centrifugation, 2-5 x 106, 1-2 x 107 and 2-5 x 107 cells in the sediment. Such cells were
suspended in 250 /A of 0-25, 0-5 or 1 % collagen solution. After mixing, the
cell suspension was sucked into a glass tube (diameter 2 mm) and set in a vertical
position by pressing the tubes into 'Sira' adhesive wax (B.D.H.). Gelation of
collagen frequently occurred after a few minutes at room temperature, but all
tubes were kept for 30 min at 37 °C. Afterwards the gel formed with the cells was
blown out into the fixative. Other samples of gel were digested on the gyrorotatory shaker with a 0-25 % solution of bacterial collagenase (Biomed, Warsaw)
at 37 °C until the collagen dissolved. The viability of cells was estimated by the
trypan blue exclusion test (Pappenheimer, 1917) and compared with that of a
sample of cells kept under the same conditions in Hanks' solution.
Culture of chondrocytes in gel
Cartilage was dissected from the Anlagen of long bones of 20- to 25-day-old
rabbit embryos. Chondrocytes were isolated with trypsin and collagenase
according to the method described before (Kawiak, Moskalewski & Darzynkiewicz, 1965; Gaillard, Moskalewski, Verhoog & Wassenaar, 1969). Control
pieces of cartilage were fixed. Samples of chondrocytes, 0-5 x 107 or 1 x 107 in
number, were suspended in 100/A of 0-5 % collagen solution and prepared for
culture. In early experiments, 25 /A aliquots of collagen with chondrocytes were
placed on cover-glasses and left until gelation in a moist chamber at 37 °C. Then
Cartilage reconstruction
159
they were transferred on to the lens paper and cultured in metal grids in modified
embryological watch-glasses (Moskalewski, 1969). Since the collagen gel
proved to be very susceptible to handling and collapsed even after the slightest
touch, in further experiments collagen with cells was put into a small trough with
a 25 /d capacity, made of a Plexiglass ring with a Millipore filter bottom
(150 /an thick, pores 0-45 /on). Such a trough floated on the medium surface, so
the metal grid was unnecessary. Chondrocytes were cultured for 3, 6, 9 and 12
days. Some chondrocytes in gel were fixed without culturing to serve as a control.
The culture medium consisted of inactivated calf serum (20 %), Hanks' solution
with 0-5 % lactalbumin hydrolysate (15 %) and medium 199 (65 %). Vitamin C
(100/*g/ml), penicillin (50 units/ml) and streptomycin (50/tg/ml) were added.
The gaseous phase was 5 % CO2 in air.
Cultures of other tissues
For comparison, cultures of EATC or kidney and liver cells were started in a
similar manner, 5 x 106 cells/100 p\ of 0-5 % collagen. Suspensions of cells were
obtained from rabbit embryos by digestion for 30 min of minced tissues in
enzymic solution (Gaillard et al. 1969) used for chondrocyte isolation. All
cultures were fixed after 3 days. A total of 50 cartilage and 70 other tissues
cultures was studied.
Hydroxyproline determination in culture material
It was observed that some chondrocyte and all kidney-cell cultures shrivel.
Histological evidence suggested collagen digestion. To study the eventual
changes in collagen content, cultures of cells isolated from young mouse kidneys
and EATC were started. EATC cultures served as controls, since they never
shrivelled and the collagen gel seemed unchanged. Both EATC and kidney
fragments were kept for 30 min in the enzymic solution used for isolation and
then rinsed three times with culture medium to remove enzymes. Cells, 5 x 106
in number, were mixed in test-tubes with 100 ji\ of collagen solution and distributed into four troughs. Additionally, 100 /i\ of collagen was distributed into
four troughs without cells. After 3 days of culture, collagen from each group of
cultures was pooled, together with distilled water in which the troughs had been
rinsed. It was expected that digestion of collagen would be accompanied by
diffusion of free amino acids or peptides into the medium. Therefore both collagen
samples and 1 ml of pooled medium from the particular groups of cultures were
dried at 80 °C, hydrolysed in HC1 (Ogle, Arlinghaus & Logan, 1962) and hydroxyproline was determined (Mitoma et al. 1959). The hydroxyproline content of
samples of 5 x 106 kidney and EATC secured at the beginning of the experiments was also tested. Determinations were made into two independent
experiments.
160
S. MOSKALEWSKI, M. KAMINSKI AND A. DUKWICZ
Histological procedure
All material was fixed in Schaffer's fluid. Sections 6 /im thick were stained
with hematoxylin-eosin, by the Mallory-Azan method (Romeis, 1948), by the
Hansen modification of the van Gieson method for collagen fibres (Romeis,
1948) and with alcian blue at pH 0-4 to detect sulphate radicals (Mowry, 1963;
Quintarelli, Scott & Dellovo, 1964). Some sections, after alcian blue, were
counterstained with trioxyhematoxylin and picrofuchsin according to Hansen
to demonstrate simultaneously acid mucopolysaccharides and collagen fibres.
For comparison of cell densities, the number of cells per section area was
estimated with the use of a diaphragm inserted into the eyepiece with surface
area 2-5 x 103 /*m2. The mean number of chondrocytes was calculated from ten
measurements in various cultures. Areas for counting were chosen according to
the distribution of acid mucopolysaccharides.
RESULTS
Distribution and viability of EATC in collagen gel
The density of EATC in collagen gel was approximately the same at the top
and the bottom of the collagen cylinder formed in the glass tube. The viability
of cells was not impaired (Table 1) by the process of collagen gelation. The
collagen network in 0-25 and 0-5% collagen was very delicate. It was more
pronounced in 1 % collagen.
Table 1. Percentage of dead cells in EATC liberated from the
collagen gel*
Collagen
No. of cells per 100 /*1 of collagen
(%)
ixlO6
5xlO 6
IxlO 7
0-25
0-5
10
Control in Hanks
90
60
8-1
7-1
10-3
111
60
80
7-2
70
70
6-2
* Determined from 200 inspected cells. Values represent the arithemical mean from three
experiments.
Culture of chondrocytes in gel
After 3 days about 75 % of the cultures in troughs had shrivelled to %-j- of the
initial volume. Shrivelling occurred also in all cultures kept on lens paper. The
morphological structure of the cultures varied to a great extent, even if they
originated from the same chondrocyte suspension and were kept in vitro for
the same period. The majority of cells had ovoid or spherical nuclei, but some
fibroblast-like cells with elongated nuclei were also present. Such cells were
Cartilage reconstruction
161
occasionally located on the periphery of chondrified cultures, simulating
perichondrium. Mitoses were rarely observed. All cultures contained a variable
number of cells with disintegrating or pyknotic nuclei. The number of such cells
seemed to increase in older cultures. The distribution of cells as seen in collagen
gel fixed without cultivation was not quite uniform (Fig. 1); there were foci of
several or more cells in close contact and areas devoid of cells. In shrivelled
cultures the distribution of cells was even more variable. In many areas of
culture a network of collagen fibres similar to that in non-cultivated material
was present. There were also thicker fibres, distributed occasionally in a haphazard manner, but usually parallel to the surface of the culture. In the collagen
*
•
'
'
'
"
'
•
•
•
•
•
Fig. 1. Non-shrivelled collagen network with chondrocytes, after 3 days of culture.
H. and E., x 160.
network some cells lay singly or in groups of two or three (mean - 6 cells/
diaphragm surface area). They were usually surrounded by a narrow ring of
material strongly stained with alcian blue (Fig. 2). Such rings had a similar size
independently of the age of the culture. They were absent around fibroblast-like
cells. Further from cells, collagen fibres were unstained or stained only weakly
with alcian blue. In other areas where the cells were more densely packed
(mean - 19 cells/diaphragm surface area), a considerable amount of alcianblue-stainable material was distributed along the collagen fibres and between
them (Fig. 2). In such areas longitudinal, rather thick collagen fibres were
frequently observed. In areas with densely packed cells (mean - 30 cells/
diaphragm surface area), distinct foci of cartilage were present; or the whole
culture, or its part, particularly in the case of the shrivelled ones (Fig. 3), was
chondrified. In older cultures the density of cells in cartilage decreased owing
162
S. MOSKALEWSKI, M. KAMINSKI AND A. D U K W I C Z
Fig. 2. Matrix formation in collagen network within 3 days of culture. Single
chondrocytes surrounded by a layer of sulphated mucopolysaccharides. Among the
more densely packed chondrocytes greater accumulation of alcian-blue-stainable
material. Alcian blue at pH 0-4, x 400.
Fig. 3. Cartilage nodules separated by thick collagen fibres after 9 days of culture.
Trioxyhematoxylin and picrofuchsin, x 400.
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163
to their degeneration. Frequently cartilage was formed at the periphery of the
culture, while in its middle part only traces of alcian-blue-stainable material and
a small number of cells was present. After the alcian blue-picrofuchsin sequence,
in areas with less developed cartilage alcian blue staining was masked by
picrofuchsin. In better-developed matrix, after alcian blue staining no picrofuchsin stainable material could be demonstrated. Chondrocytes in cartilage
usually lay in small cavities surrounded in younger cultures by round or oval
collagen capsules uniformly stained withpicrofuchsin. The wall of these capsules
Fig. 4. Collagen network with EATC after 3 days of culture. Azan, x 400.
was much thicker than that of single fibres in the delicate collagen network at
the beginning of culture. In older cultures with a great amount of alcian-bluestainable material, collagen staining of capsules was weak or vanished. Some
chondrocytes formed distinct isogeneic groups. In cartilage with well-formed
matrix, chondrocytes were frequently hypertrophied. Pieces of rabbit cartilage
did not contain in their matrix any picrofuchsin-stainable material.
Culture of EATC and liver cells
Cultures of these cells (Fig. 4) were never shrivelled. The cells within the
collagen gel were distributed almost uniformly. They lay in close contact with
collagen fibres. Many cells were degenerated.
164
S. MOSKALEWSKI, M. KAMINSKI AND A. DUKWICZ
Culture of kidney cells
All cultures were shrivelled to \-^ of the initial volume. Cells in the peripheral
part of the cultures usually formed a compact arrangement; in the middle part
they were mostly degenerated. The original delicate collagen network was not
preserved. There were some rather thick long fibres on the periphery of some
cultures, but most of the collagen was present in the form of more or less
separate clumps (Fig. 5). The cells lay usually in the spaces between the collagen
clumps. Cultures of mouse kidney used for hydroxyproline determination were
shrivelled or partially liquefied.
>
.
*
*
•
Fig. 5. Disrupted and clumped collagen network with kidney cells after
3 days of culture. Azan, x 400.
Table 2. Hydroxyproline content in pooled cultures* and medial expressed
as percentage of that in corresponding controls (100%)J
Exp. I
Exp. II
Collagen
after
'culture'
Medium
(1 ml)
Collagen
+ EATC
Medium
(1 ml)
87-5
106-5
87-7
102-7
79-3
71-9
70-8
96-6
Collagen
+ kidney
cells
Medium
(1 ml)
40-8
35-6
* 100 fi\ of collagen solution with cells distributed into four troughs.
t 0-5 ml of medium per culture.
% 100/il of collagen prior to culture, or 1 ml of medium.
1190
135-9
Cartilage reconstruction
165
Hydroxyproline determination
The data presented in Table 2 indicate that the hydroxyproline content of
EATC cultures is lower than that of samples of collagen kept in troughs and
used as reference standard for comparison. Kidney-cell cultures lost a considerable amount of hydroxyproline, which was compensated by the increase of
hydroxyproline content in the medium. Samples of kidney and EATC
practically did not contain hydroxyproline.
DISCUSSION
Three-dimensional sponges are occasionally used in tissue culture (Leighton,
1951; Kalus & O'Neal, 1968; Leighton, Justh & Esper, 1967). The cells penetrate
into the sponge from the outside, and therefore such a system is based on a
different principle from the collagen network used in the present study. It
appears that the collagen network may offer a support for the embedded cells
provided that it remains undigested. The position of some cells (e.g. EATC,
liver cells) in the collagen network remains unchanged during culture and
they seem to be immobilized among the collagen fibres. Kidney cells evoked
collagen shrivelling and release of hydroxyproline into the medium.
The diminished amount of hydroxyproline in EATC cultures as compared
with that in collagen kept in culture without cells most probably reflects the
loss of collagen on the walls of the test-tube occurring during the mixing of
collagen solution with cells. Much lower values of hydroxyproline in kidneycell cultures accompanied by an increase of hydroxyproline in the medium seems
to be due to collagen digestion, and release of some material into the medium.
Thus, both morphological and chemical evidence is in favour of the explanation
that shrivelling of cultures is due to partial collagen digestion. Many mammalian
cells (Eisen, Jeffrey & Gross, 1968; Houck & Sharma, 1968; Lazarus, Brown,
Daniels & Fullmer, 1968) are known to produce collagenase, which could
explain the digestion of collagen in culture, but the influence of other proteases
cannot be excluded. If collagenase is the enzyme responsible, then the shrivelling
of cultures could probably be prevented by the use of its specific peptide
inhibitor (Heyns & Legler, 1960).
A certain degree of shrivelling occurred in most chondrocyte cultures.
Chondrocytes in epiphyseal cartilages are known to be highly heterogeneous
(Cooper & Lash, 1964). Therefore it is quite possible that only some chondrocytes - for example, the hypertrophied ones - produce collagenolytic enzymes
and the number of such chondrocytes may be influenced by the dissection or
isolation procedure or by the age of the embryos.
Apart from the digestion, collagen fibres in gel seem to undergo some other
changes, probably also due to the activity of cells. In kidney cultures, fragmented
collagen deposits were formed with much more compact arrangement of collagen
than in the original gel. In kidney and chondrocyte cultures longitudinal collagen
fibres were occasionally observed. Chondrocytes were surrounded by collagen
166
S. MOSKALEWSKI, M. KAMI&SKI AND A. DUKWICZ
capsules thicker and more regularly shaped than the fibres of primary gel. Such
rearrangement occurring in cultures could be due to several factors in which
collagen digestion and mechanical compression of collagen may play an
important role. It is not clear why partial digestion of collagen was accompanied
by shrivelling of the culture. However, little is known about the interactions
between cells and formation of intercellular substance: collagen gel with
embedded cells could serve as a model in such studies, even if it is not the exact
replica of the situation in vivo.
Owing to the fact that the viability of cells in collagen is not influenced by
the gelation of collagen, such a model can be used for estimation of the role of
the distance between cells in tissue reconstruction or its influence on cell
metabolism. Such influence is exemplified in experiments on cartilage reconstruction. Alcian-blue-positive material stained at low pH in cartilage represents
sulphated mucopolysaccharides (Mowry, 1963; Quintarelli et al. 1964). They
are deposited around single cells forming a ring. When the cell density increases,
the amount of mucopolysaccharides produced by them is sufficient to saturate
the adjacent collagen fibres, which nevertheless remain recognizable even in
12-day-old cultures. Only in areas with high cell density was a sufficient amount
of acid mucopolysaccharides accumulated to form cartilage matrix with the
typical appearance. It is unknown whether, in such matrix, original fibres
remain and are masked by mucopolysaccharides or whether they are digested
and are eventually replaced by the fibres produced by chondrocytes. At present,
the number of chondrocytes available is too low for more extensive trials, but
if a homogeneous population of chondrocytes actively producing intercellular
substance could be obtained (Coon, 1966), cartilage produced and modelled
in vitro in the collagen gel would probably find some application in plastic
surgery.
The authors are indebted to Mrs W. Zielinska for her skilful technical assistance.
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