J. Embryol. exp. Morph. Vol. 23, J, pp. 185-211, 1970
185
Printed in Great Britain
The inhibitory effect of tetracycline on
osteogenesis in organ culture
By I L K K A K A I T I L A , 1 J O R M A W A R T I O V A A R A , 1
OSSI L A I T I N E N 2 AND L A U R I SAXÉN 1
From the Third Department of Pathology and Department of
Medical Chemistry, University of Helsinki
Two characteristics of the tetracycline-group antibiotics have recently focused
the special attention of teratologists: these drugs are known to pass readily
across the placental'barrier' (Charles, 1954; Frost & Villanueva, 1960; Gibbons
& Reichelderfer, 1960; Hughes, Lee & Flood, 1965; Simpson, Burnette &
Bawden, 1967), and they are incorporated into the foetal skeleton, where they
may remain for long periods and can be detected owing to their brilliant
fluorescence (Rail, Loo, Lane & Kelly, 1957; Milch, Rail & Tobie, 1957, 1958;
Bevelander, Rolle & Cohlan, 1961; Urist & Ibsen, 1963; Michelson, 1964).
The question of whether they exert a harmful effect on bone formation and
mineralization in mammals, however, is still a matter of controversy. There is no
doubt that tetracycline interferes with mineralization of the skeletal tissue in
certain lower vertebrates (Bevelander, Goldberg & Nakahara, 1960; Bevelander
& Cohlan, 1962), and prevents calcification of mammalian bones in organ culture (Saxen, 1966«, b). Reports of in vivo studies in mammalian species have
yielded more confusing results.
In 1957, Filippi & Mela reported minor skeletal malformations in mouse
foetuses after maternal tetracycline administration, and later Cohlan, Bevelander
& Tiamsic (1963) observed depression of the growth rate of the long bones of
premature children receiving high doses of tetracycline. Correspondingly, a
reduction of the foetal weight has been reported to result from administration of
tetracycline to the mother (Bevelander & Cohlan, 1962; Cohlan et al. 1963).
On the other hand, neither experiments on rats nor observations on children
have confirmed all these reports, as Oxytetracycline appears to have no adverse
effects on mineralization (Chu, O'Hara & Keitel, 1963; Likins & Pakis, 1965). In
addition to differences in the dose and mode of administration, these conflicting
results may also be due to differences between the various members of the
1
Authors' address: Third Department of Pathology, Laboratory of Experimental Embryology, University of Helsinki, Helsinki 25, Finland.
2
Authors' address: Department of Medical Chemistry, University of Helsinki, Helsinki 10,
Finland.
186
I. KAITILA AND OTHERS
tetracycline group, as suggested by comparative studies both in vivo and in vitro
(Owen, 1963; Hughes et al 1965; Saxén, 19666).
Consequently, the exact site of action of tetracycline and the mechanism by
which it inhibits osteogenesis are not known. High concentrations of the drug
in the culture medium of embryonic bones seem to interfere with the uptake of
labelled thymidine and proline, suggesting that the synthesis of DNA and protein are affected (Bennet, Proffit & Norton, 1967), but the conclusion is based on
experiments employing concentrations much higher than those needed to prevent cumulative calcium uptake and the elongation of the mineralized zone
(Saxén, 19666). In a recent study, the results suggested that tetracycline injected
into pregnant rats inhibits the biosynthesis of collagen in the foetal bone and
skin (Halme & Aer, 1968). The antibiotic action of tetracyclines has been
attributed to several possible mechanisms. Interference with protein synthesis,
inhibition of phosphorylation and of oxidation of glutamate, or inhibition of
bacterial oxygen consumption have been suggested (see Goodman & Gilman,
1965). As a potent chelate, tetracycline may act directly by binding divalent
cations and thus either interfering with the formation of bone mineral crystals
or removing cations participating in enzymic processes (Ibsen & Urist, 1962;
Finerman & Milch, 1963; Urist & Ibsen, 1963; Sternberg, 1966). At the moment,
it has not been established which of these many alternatives are operative in the
inhibitory action of mineralization and whether we are dealing with a multitude
of different effects on the chain reaction, starting from the matrix synthesis and
ending in the formation of bone minerals.
The mechanism of bone calcification process has been, until now, only partially
elucidated. It includes, however, the formation of the osteoid matrix, where the
Proteinpolysaccharides and collagen are the most important components. The
sulphated mucopolysaccharides and collagen are both synthesized by the same
cells in the embryonic cartilage (Godman & Porter, 1960; Prockop, Pettengill &
Holtzer, 1964). The evidence indicates that gradual depolymerization of
chondroitic mucopolysaccharides precedes mineralization (Gersh, 1952;
Dziewiatkowski, 1966) and that there is a close parallel relation between collagen biosynthesis and mineralization in osteogenesis (cf. Glimcher, 1959;
Laitinen, 1967; Bauer & Shtacher, 1968) and between collagen and mineral
turnover in bone (Firschein, 1967). Collagen, which comprises about 95 % of
the organic bone matrix, contains about 14% of the hydroxyproline found
almost exclusively in collagen and formed from proline by hydroxylation only
(cf. Prockop & Kivirikko, 1967).
As previous experiments performed in organ cultures of embryonic bones
have suggested the usefulness of this method, some of the unsolved problems
related to the inhibitory action of tetracycline on osteogenesis were re-analysed
in these conditions. It was hoped especially that these highly standardized
conditions would allow comparative studies with different concentrations of the
drug, and thus enable a distinction to be made between its different toxic and/or
Tetracycline and osteogenesis
187
more specific actions. To give a more detailed impression of the effects of organ
culture on osteogenesis, a brief comparison of growth in vitro and in vivo will
also be presented.
MATERIAL AND METHODS
Organ culture
Random-bred Swiss mice were used throughout the work. The day 0 of
pregnancy was determined by the presence of a vaginal plug. Bone rudiments
from 16- to 17-day embryos were isolated under a dissecting microscope without
enzyme treatment. The rudiments were carefully freed from as much of the
surrounding tissue as possible without damage to the periosteum. The bones
were cultivated in a chemically defined medium, BGJb (Biggers, Gwatkin &
Heyner, 1961) supplemented with ascorbic acid, which has been shown to
maintain good viability and growth of the skeletal tissues and to allow
mineralization to take place (Reynolds, 1966; Saxen, 1966Ö, b). The culture
dishes, each with ten rudiments and 10 ml of medium, were incubated in a
humidified 5 % C0 2 atmosphere at 37 °C.
Tetracycline
The purified tetracycline hydrochloride was acquired through the courtesy of
the Pfizer Corporation. A stock solution was prepared by diluting dry powder in
sterile distilled water. ThepH remained constant in the buffered culture medium.
Measurements of length and weight
The length of the mineralized zone was measured from drawings of individual
bone rudiments made with a camera lucida, with which the dense calcified zone
was clearly distinguishable from the translucent, non-mineralized epiphyseal
parts of the bone. The actual lengths were calculated from these measurements.
The dry weights of the rudiments were determined with a 'Cahn' electric microbalance by a method described by Biggers (1960).
Histology
The undecalcified rudiments were fixed in 10 % formalin or in Zenker solution. Histological preparations stained with H.E., toluidine blue, PAS and von
Kossa stain were made by conventional methods.
Electron microscopy
After cultivation, the bones were rinsed with phosphate-buffered saline,
fixed for 30 min at 4 °C with 4 % glutaraldehyde in phosphate buffer at pH 7-2
and postfixed for 30 min with 1 % Os0 4 at the same temperature and in the same
buffer (Millonig, 1962; Sabatini, Bensch & Barrnett, 1963). The fixed bones were
188
I. KAITILA AND OTHERS
dehydrated in graded alcohol and embedded in Epon 812. Thin sections were cut
with glass knives on a Porter-Plum MT-2 microtome, stained with lead citrate
(Reynolds, 1963), and examined and photographed in a Philips 200 electron
microscope.
Radioactive labelling
Sodium [ S]sulphate (spec. act. 35-0 mCi/mM), [45Ca]calcium chloride
(spec. act. 8-25 and ll-8mCi/mM), [A/e-3H]thymidine (spec. act. 20600
mCi/mivi) and [3H]tetracycline hydrochloride (spec. act. 80-0 mCi/mM) were
obtained from the New England Nuclear Corporation, Boston, Mass., and
[l-3H]proline (spec. act. 100 mCi/mM) from the Radiochemical Centre, Amersham, England. The label was either added to the original culture medium or in
pulse experiments the unlabelled medium was replaced with fresh medium containing the label. The isotope concentrations used are indicated in the legends
to the figures.
After labelling, the bone rudiments were harvested, washed for 15 min in cold
distilled water and placed for 1 h in the counting vials to dry in an incubator
at 60 °C. Hyamine hydroxide (Nuclear Enterprises, Sighthill, Edinburgh) was
added for 10 min. Scintillation liquid (Nuclear Enterprises, Sighthill, Edinburgh)
or PPO, 5 g/1, and dimethyl-POPOP, 0-3 g/1, in toluene was used as scintillation
fluid. The radioactivity was determined by an 'I.D.L., Tritomat 6020' or
'Packard Tricarb 3375' liquid scintillation counter. For the determination of
45
Ca- and 3H-activities in the double-labelling experiment, the bones were
carefully washed after culturing with distilled water and hydrolysed in 6 N-HC1
at 138 °C for 8 h. The hydrolysates were then evaporated to dryness and dissolved in water. 45Ca content was then measured from the aliquots with a gasflow counter (Tracerlab) after previous precipitation of calcium with ammonium
oxalate. The supernatants obtained were further evaporated to dryness at
110 °C for 5 h and dissolved again in water. Samples of the aliquots were taken
for assay of total 3H content in Bray's solution and of the specific activity of
hydroxyproline by the method of Peterkofsky & Prockop (1962) after previous
separation of [3H]proline from hydroxy[3H]proline with a Dowex 50-X8
column (hydrogen form).
35
FIGURE 1
Comparison of mouse embryonic bones in vivo and /'/; vitro and the effect of tetracycline on the morphology of bone. PAS stain, x 100.
(A) 16-day ulna.
(B) 17-day ulna.
(C) 16-day ulna cultivated for 12 days in vitro.
(D) 16-day ulna cultivated for 12 days in the presence of 1 /tg/ml of tetracycline
in vitro.
(E) As D, but tetracycline, 100/tg/ml.
189
Tetracycline and osteogenesis
Mfc
C A Ä ^ ^ i » ^™
a
*
190
I. KAITILA AND OTHERS
A utoradiography
Autoradiographs were prepared from tissue sections, using the stripping film
technique (Kodak AR-10). After 4 days exposure, the autoradiographs were
developed and the tissue sections stained with haematoxylin.
Measurement of total calcium
The total calcium of the bones was determined by dry combustion in a maximal temperature of 600 °C. The ash was dissolved in concentrated HCl subsequently diluted with distilled water. The calcium was measured in a Beekman
DU flame photometer at a wavelength of 422-7 mjn. The determinations were
performed from pooled samples of ten bones.
RESULTS
Comparison of development in vivo and in vitro
It was of interest to compare the extent of depression of development resulting
from the culture conditions. In histological preparations made after different
20
1-5
10
in vitro
0-5
10
12
Days in vitro
I
L
17
19
Days In vivo
Fig. 2. Comparison of the elongation of the mineralized zone of embryonic ulnae
in vivo and in vitro. Cultivation was begun on the 16th day. (Each dot is based on
measurements of J0-20 bones, mean ± S.D.)
Tetracycline and osteogenesis
191
periods of cultivation (Fig. 1 A, B, C) it was clearly seen that the mitotic activity
of the chondroblasts at the epiphyseal plate decreased during cultivation; the
proliferating zone, which is 20-30 cell layers thick in vivo, diminished continually,
until after 14-16 days in vitro only a few layers remained. The hypertrophy of the
chondroblasts, which normally occurs towards the metaphysis, was also retarded; beyond the proliferating zone there existed a resting zone 10-30 cell
layers thick, which was not found in vivo and where the cells were morphologically uniform. In the next zone the cells were flattened in the longitudinal plane,
another phenomenon that could not be found in vivo. After this zone, the
chondroblasts hypertrophied in a few layers as in vivo and the intercellular
matrix lost the metachromatic stainability typical of chondroitic tissue. The
periosteal cells and the matrix, as well as the osteoid tissue in the diaphysis,
were like in vivo rudiments. In the cultivated bones no traces of haematopoietic
tissue could be found. The length of the mineralized zone increased steadily
during cultivation, although at a greatly depressed rate (Fig. 2). The length
achieved in vivo during R days was reached after 12 days of cultivation. During
a 12-day cultivation period the total calcium content of a tibia was about
doubled, whereas in vivo it increased eightfold.
Localization of tetracycline in the bone
Tetracycline was localized in the bones by two methods: either by making
use of the typical bright yellow fluorescence of the drug or by employing
labelled tetracycline in autoradiography. When examined in ultraviolet light,
the tetracycline fluorescence was found to be localized exclusively in the
mineral of the diaphysis of bones grown in the presence of the drug. When
subsequent counterstaining of the bones was performed by von Kossa's method
for calcium, a very good correlation was found between the tetracycline fluorescence and the stained calcium (Fig. 3 A, B, B1). Bones were also cultured in
the presence of radioactive tetracycline for different periods of time. Autoradiographs prepared from them indicated that the drug is not incorporated into the
epiphyseal cartilage. The first grains were noted in the zone of hypertrophied
chondroblasts, where the first calcium incorporation likewise takes place, as
judged by the distribution of the von Kossa stain. The bulk of the radioactive
tetracycline was found in the mineralized diaphysis on the mineral crystals
(Fig. 3C, D). The comparison of the U.V.-fluorescence and autoradiographic
45
Ca grains in the same section showed an exact correspondence; the drug was
localized on the mineralizing trabeculae (Fig. 4A, B).
Inhibition of calcification by tetracycline
The localization of the tetracycline fluorescence and the radioactivity to the
calcifying areas of the bone matrix is accompanied by inhibition of mineralization, as has previously been reported (Saxén, 1965, 1966a, b). The inhibitory
effect of tetracycline was further confirmed here by the use of randomized pairs
92
I. KAITILA AND OTHERS
Tetracycline and osteogenesis
193
of bones, consisting of the right and left long bones of the same embryos, one of
each pair being chosen, according to random numbers, to be treated with
tetracycline, while the other served as the control. With these pairs, the effect of
the drug was assessed by the elongation of the mineralized zone and by the
uptake of 45Ca. Fig. 5 shows the effect of different concentrations of tetracycline
on the mineralization of 16-day mouse embryo ulnae cultivated for 11 days in
the presence of 0-1 /^Ci/ml 45Ca in the medium. The results indicate that in all
bone pairs tetracycline causes inhibition in the elongation of the calcified zone
and a decrease in the uptake of radiocalcium. A dose response is also presented,
in which as little as 1 /^g/ml tetracycline decreases the incorporation of 45Ca by
40 % while an effect of 70-75 % inhibition is reached with 10 ^g/ml tetracycline. The depressive effect on the elongation of the calcified zone is even
more pronounced (Fig. 6).
The effect of tetracycline on the synthesis of DNA
The effect of tetracycline on the synthesis of DNA was studied to establish
the growth-depressing concentration of the drug. It can be seen (Fig. 7) that
tetracycline up to 10/*g/ml had no effect on the uptake of labelled thymidine,
whereas 100 /tg/ml greatly depressed the synthesis of DNA. Recently, it has
been found in a similar organ culture system, but with a different medium, that
the same kind of reduction in the synthesis of DNA occurs in concentrations of
tetracycline exceeding 50 yttg/ml (Bennet et al. 1967).
The effect of tetracycline on the dry weight of the bone
To obtain further knowledge of the effect of tetracycline on the overall growth
of bone rudiments in vitro, ulnar bones were cultivated in numbers of 5 to 10 for
different periods of time in the presence of 10-0 /^g/rnl of tetracycline (Fig. 8).
The results are expressed as the dry weight for an individual bone. The amount
FIGURE 3
Localization and effect of tetracycline on bone.
(A) 16-day embryonic ulna grown for 5 days in culture, stained by von Kossa's
method for calcium, x 100.
(B) Ultraviolet photograph of a similar bone to that in A, but grown in a
medium containing 5 /ig/ml tetracycline.
(B1) The same section as in B after staining for calcium by von Kossa's method.
The localization of the U.V.-fluorescence in the calcified area is clear.
(C) Autoradiograph of the epiphyseo-diaphyseal transition zone where calcium can
be seen in B 1 .16-day embryonic ulna grown for 6 days in the presence of 5/*g/ml
of [3H]tetracycline. The drug is seen to be localized in the calcifying matrix between
the hypertrophied chondroblasts. x 1500.
(D) Autoradiograph of a periosteal section. The [3H]tetracycline grains are localized
on the foci of calcifying matrix.
Insert shows source of material for C and D.
194
I. KAITILA AND OTHERS
if K * '
JWfih
+
W> AC"
Tetracycline and osteogenesis
195
of tissue increased about 40 % in the controls during the first 4 days, after which
the rate of increase slowed down. The dry weight of the bones cultivated in
tetracycline-containing medium increased only 10 % during the first 4 days.
Thereafter, a slow degradation of the already formed tissue seemed to take place
and after 11 days of cultivation the weight was about the same as at the beginning.
On the 11th day there was a difference of about 45 % between the dry weights of
the control and the experimental bones.
The effect of tetracycline on the morphology of the bone
The morphology of the cells in the various zones of ulnar bone rudiments was
relatively little changed by different concentrations of tetracycline in the
medium (Fig. 1C, D, E). The two additional zones of chondrocytes that were
not seen in vivo and which were described above in the control cultures could
still be found at the epiphyseal plate after 12 days cultivation of 16-day embryonic bones in a medium containing 1-0 or 10-0/tg/ml of tetracycline. No intensely
stained matrix was found between the hypertrophied chondrocytes after PAS
staining, as reported by Rolle (1967). On the other hand, the von Kossa stain
revealed slight amounts of calcium in these regions, as was also noticed by her.
A poorly formed diaphyseal matrix of reduced density and a short mineralized
zone were clearly seen in bones treated with 10-0^g/ml of tetracycline. In
sections stained with toluidine blue a gradual loss of metachromasia at the
metaphysis with advancing age could be seen in the control and in the tetracycline-treated bones.
Electron microscopy of calcification under the influence of tetracycline
For the electron microscopic studies of the inhibition of calcification by
tetracycline, ulnae of 16-day mouse embryos were cultured in the presence of
5 /tg/ml tetracycline or as controls for varying lengths of time up to 13 days
in vitro. Special attention was paid to the calcified parts where tetracycline was
localized by its fluorescence and by autoradiographic methods as described
above.
Tetracycline treatment did not seem to have any apparent effect on the ultrastructure of the osteoblasts in the area of calcification (Fig. 9 A). The nuclei
FIGURE 4
Comparison of the distribution of tetracycline fluorescence and radiocalcium. A
16-day embryonic ulna was grown for 3 days in the presence of 10 /ig/ml tetracycline
and 002/tCi/ml 45Ca. x 1500.
(A) Ultraviolet photograph of the bone, showing the localization of the typical
tetracycline fluorescence.
(B) Autoradiograph of the same section as in A, 45
showing the close correspondence of the localization of the newly deposited Ca and tetracycline on the
trabeculae of bone matrix.
13
EM 13 23
196
I. KAITILA AND OTHERS
Length of calcified zone
Incorporation of
45
Ca
u
3
M Start
O Control
ID Experiment
R Right L Left
u
0-5
10
1-5 mm
Inhibition of the
elongation
25
50
75Ci/100s/ A g
bone
Inhibition of the
incorporation of 4SCa
T C . 1 //g/cc
T C , 10/«g/cc
TC, 100//g/cc
25
50
75%
25
50
75%
Fig. 5. Inhibitory effect of different concentrations of tetracycline on the elongation
of the mineralized zone and on the uptake of 45Ca into bones cultured for 11 days.
The choice of the experimental bone from the same embryo was randomized. The
results are presented pair by pair. The percentage of inhibition is calculated from the
bone pairs and expressed as pooled samples.
Tetracycline and osteogenesis
197
1-2
Control
.
10
111
0-8
0-6
TC, 5//g/cc
i
I
i
I
I
i
i
4
6
8
10
12
16
Days of cultivation
Fig. 6. Effect of tetracycline on the elongation of the calcified zone. 16-day embryonic ulnae were cultivated for different periods of time. (Each dot is based on determinations from 10-20 bones, mean ± S.D.)
A
B
C
D
300
Control
T C , 1-0/<g/cc
T C , 100/zg/cc
T C , 1000//g/cc
rff
-h
200
U
100
C:
5
11
Days of cultivation
Fig. 7. Effect of tetracycline on [3H]thymidine uptake. 16-day embryonic ulnae were
cultivated for 5 and J1 days in the presence of 10, 100 and 1000/ig/ml of tetracycline. At the end of the cultivation a 2 h pulse with [3H]thymidine, 5 /tCi/ml,
was given. (Each column is based on determinations of 10 bones, mean" ± S.D.)
13-2
198
I. KAITILA AND OTHERS
were without signs of damage, and the cytoplasm of the cells was rich in mitochondria. An especially noticeable feature was the large amount of roughsurfaced endoplasmic reticulum which was frequently distended and filled with
homogeneous electron-dense material. Between the cells was a network of
unoriented fibres, mostly lacking clear periodicity (Fig. 9B). Numerous circular
sites of calcification of various sizes were in close relation to the matrix network.
At higher magnification a very elaborate network of thicker 350-500 Â fibres
Control
100
JO—°
'S
3 •
« 75 -
°
oo
'<D
Ó50
TC, 10Ag/cc
25
I
16
1
I
4
I
I
6
8
10
Days of cultivation
1
12
Fig. 8. Effect of tetracycline on dry weight of bone. 16-day embryonic ulnae grown
for different periods of time in the presence of 100/tg/ml of tetracycline. (Each dot
is based on a pooled sample of 10 bones.)
and thinner 200 Â fibrils was revealed, with dense particles or granules of 200500 Â in close association with the latter especially (Fig. 9B, Fig. 10 A, B). Some
granules contained radially arranged needles and appeared to be possible sites of
onset of calcification. The granules often seemed to contain a lighter core and a
more electron-dense ring in the periphery (Fig. 10B).
Electron micrographs of larger calcified sites showed a meshwork of partly
aligned needles of high electron density. The needles were 20-40 Â in diameter
FIGURE 9
(A) Electron micrograph of embryonic ulna grown in organ culture for 13 days in
the presence of 5 /*g/ml tetracycline. The osteoblasts show no sign of a toxic effect of
the drug. They have an elaborated distended endoplasmic reticulum. Abundant
fibres are present in the matrix. Smaller and larger electron-dense calcified areas
are seen adjacent to the fibres, x 7200.
(B) Higher magnification of an electron micrograph of the same specimen in which
a network of thick fibres is visible with an adjoining calcified area consisting of
small needle-type structures in a more heterogeneous substance, x 34000.
Tetracycline and osteogenesis
199
200
I. KAITILA AND OTHERS
Tetracycline
and osteogenesis
201
and embedded in a less electron-dense and more amorphous structure (Fig. 11 A).
Compared with the tetracycline-treated bones, the electron microscopy of the
calcified diaphysis in the control bones revealed a similar picture. The cells and
the matrix components did not indicate any differences. The areas of calcification
were more extensive and at higher magnification also had a more electrondense appearance (Fig. 11B).
The effect of tetracycline on the proteinpoly saccharides of the bone
The proteinpolysaccharides in the matrix of bone, which are mainly chondroitin sulphates, were labelled with radioactive sulphate. The 35 S0 4 autoradiography showed grains distributed all over the embryonic bone, but most abundantly in the epiphyseal portions of the long bone after 6-day cultivation and a
24 h pulse. The type of distribution was to be expected, because of the great
abundance of mucoproteins in the chondroitic intercellular matrix. No relation
to the localization of tetracycline was seen. In order to find out whether tetracycline in concentrations effectively inhibiting calcification would have any
influence on the chondroitin sulphate, which has been reported to undergo
degradation during the mineralization process (Dziewiatkowski, 1966), the
rudiments were cultured for 9 days in the presence of 35 S0 4 and 45Ca. The length
of the mineralized zone and the total calcium were determined from seven
rudiments at the beginning and end of cultivation as well as the uptake of
35
S0 4 and 45Ca (Fig. 12). Tetracycline, in concentrations of 1-0 and 10-0>g/ml,
had a markedly depressing effect on the growth of the mineralized zone, on the
total calcium and on the uptake of 45Ca of the bone. It did not seem to have a
significant influence on the uptake of 35 S0 4 . In an experiment elucidating the
cumulative incorporation of 35 S0 4 , the bone rudiments were cultured for 1, 3,
6,9, and 12 days with different concentrations of tetracycline (Fig. 13). The 35 S0 4
radioactivity of the rudiments increased continuously until the 9th day of cultivation, and tetracycline seemed to have no effect on the amounts of acid mucopolysaccharides in the rudiments.
FIGURE 10
(A) Electron micrograph of an ulna cultivated for 13 days in the presence of
5 /tg/ml tetracycline. Adjacent to the osteoblast an interlacing network of thin
fibrils can be seen. Seemingly attached to the fibrils are irregularly shaped granules,
which are also of varying size and electron density, x 34000.
(B) At a higher magnification a variety of different-sized particles are seen to be
attached to the thin fibrils. The arrow marks a radial arrangement of electron-dense
projections on one of the particles, x 130000.
202
I. KAITILA AND OTHERS
Tetracycline and
M
A
B
C
Start
Control
TC, 10/fg/cc
TC, 100//g/cc
203
osteogenesis
1000
A-
- 4000
0-9
m
E,
ö
0-8
X
800
- 3000
600
ra
U
2000
~n
i-*
O
r-
400
3 h
07 -
4l
- 1000
200
0-6 Length
Total Ca
45
Ca uptake
35
S uptake
9 days cultivation
Fig. 12. Effect of tetracycline on the uptake of 35S04 in concentrations inhibiting
calcification. The results of three parallel experiments are included. The length
of the mineralized zones of 7 ulnae were measured and the pooled sample then
used for determination of total calcium. The uptakes of 45Ca and 35S04 were
determined from 10 bones after a 24-h pulse of 002 /tCi/ml or 0-2/tCi/ml respectively in different cultures (mean ± S.D.).
The effect of tetracycline on the synthesis of collagen in the bone
In vitro experiments in our laboratory concerning the influence of tetracycline
on collagen biosynthesis suggested that the drug inhibits both the incorporation
of proline into protocollagen and the hydroxylation of proline into hydroxyproline (Halme, Kivirikko, Kaitila & Saxen, 1969). The results also suggested that
the tetracycline concentrations affecting collagen synthesis were higher than the
lowest concentrations still inhibiting mineralization, and that, therefore, the
final inhibition of osteogenesis can be obtained through two different pathways.
FIGURE .11
(A) Calcified site in an ulna grown for 8 days in the presence of 5 /tg/ml tetracycline.
Longitudinal and cross sections of needle-type structures are seen embedded in a
more heterogeneous material, x 335000.
(B) Extensive calcification in an ulna grown for 8 days in vitro. Thinner needles and
thicker fibrils extend from dense areas of calcification, x 130000.
204
I. K A I T I L A A N D
OTHERS
To analyse further this bimodal effect, a comparative study was made in which
both collagen biosynthesis and calcification were measured in the same bones.
Despite our recent finding, which showed the favourable effect of a daily change
of the medium on collagen biosynthesis, this experiment was performed under
conditions which were earlier tested and found suitable for mineralization,
i.e. without the daily replacement (Saxen, \966a). The bones were cultured for
4 and 8 days in the presence of different concentrations of tetracycline and
double-labelled during the last 24 h with radioactive calcium and proline. The
incorporation of 45Ca into bone rudiments was taken as a criterion for the
rate of mineralization and the incorporation of [3H]proline as hydroxy[3H]proline for the rate of collagen synthesis. The results showed (Table 1) that none
of the tetracycline concentrations used caused significant changes in the rate of
hydroxy[3H]proline synthesis or in the total uptake of 3 H. The incorporation of
45
Ca was slightly depressed in rudiments cultured in the medium containing
only 0 1 /tg/ml of tetracycline. Compared with the control rudiments, the
percentage incorporations of 45Ca on days 4 and 8 in dishes containing 1-0 jag/ml
of tetracycline were 72 and 77 % and in the ones containing 10-0/tg/ml 40 and
31 %, respectively. Slight retardation in the growth of the rudiments was also
observed in the dishes containing 10-0 /*g/ml of tetracycline.
Table 1. Effect of tetracycline on the dry weight, mineralization, and collagen
biosynthesis of bone after 4 and 8 days of culture
3
45
Dry we ight
Tetracycline
0«g/ml)
None
01
10
100
f
w
i
A
H total
Ca incorporation
(cpm/mg bone)
incorporation
(cpm x 102/mg)
A
i
r
4d
8d
4d
8d
4d
8d
87
90
78
66
92
84
82
60
2000
1880
1440
800
1720
1140
1320
540
404
387
412
399
266
310
291
358
•\
[3H]hypro. spec.
activ. (cpm/^g)
<
i
4d
8d
529
522
553
421
244
232
252
307
Cultures of 10 mouse embryonic ulnae were incubated under conditions described in the
text. 24 h before harvesting the bones were transferred to a medium containing 1 /id of
[3H]proline and 01 /tCi of 45Ca per ml. The dry weight is expressed per bone.
DISCUSSION
Comparison between the development of the bones in our culture conditions
and in vivo indicated a definite retardation, but suggested that developmental
events followed a normal course during the first 8-10 days of organ culture. The
experimental model-system thus appeared to be suitable for studies on the
effect of certain exogenous factors and their modes of action, and was used to
analyse the effect of tetracycline antibiotics. These have earlier been shown to
have a specific affinity for calcifying tissues (Milch et al. 1957; Bevelander et al.
1960), and our results, obtained in fluorescence microscopy and in autoradio-
Tetracycline and osteogenesis
205
3
graphs of bones treated with [ H]tetracycline, confirmed these observations.
It has previously been shown that tetracycline may interfere with the calcification
process both in vivo and in vitro (Introduction), and in the present study a significant inhibitory effect was observed with tetracycline concentrations as low as
1 /tg/ml.
A Control
14000
B TC, 0-1 //g/cc
C TC, 1 0 //g/cc
D TC, 10-0//g/cc
12000
10000
A
8000 -
u
6000
*
%h
4000
2000
CD
CD
3
6
Days of cultivation
CD
9
CD
12
Fig. 13. Effect of tetracycline on the cumulative uptake of 35S04.16-day embryonic
ulnae were cultivated for different periods of ti me in the presence of 0* .1, 1-0 and 10'O
/tg/ml tetracycline, and 1 /tCi/ml 35S04. (Each column is based on 5 bones,mean + S.D.).
The above observations in light and electron microscopy, as well as the determination of thymidine uptake, suggested that the effect on mineralization of
low tetracycline concentrations could not be attributed to an action affecting
cell viability or proliferation. Hence attention was focused on the formation
of the bone matrix, and the synthesis of its main organic components, proteinpolysaccharides and collagen fibres.
The role of proteinpolysaccharides in the calcification process is as yet obscure. It has been suggested that the ground substance polysaccharides might
206
I. KAITILA AND OTHERS
act as compatible polymer diluents or plasticizers for collagen structures (Milch,
1966). A remarkable loss of proteinpolysaccharides in the process of endochondral ossification has been noticed, probably due to a degrading proteaselike enzyme (Dziewiatkowski, 1966). If it is assumed that calcification is
preceded by this kind of degradation of acid mucopolysaccharides, it might be
proposed that tetracyclines would prevent the diminution of the proteinpolysaccharide content in bone rudiments when inhibiting calcification. As
chondroitin sulphate contains abundant sulphur, radiosulphate might serve as
a marker for the proteinpolysaccharides in the bone matrix. Under the conditions used, the concentration of tetracycline that was found to inhibit mineralization seemed to have no effect on the cumulative or short-term incorporation
of radiosulphate. The results have thus given no proof of a relation between the
inhibitory effect of tetracycline on osteogenesis and a specific influence on the
Proteinpolysaccharide degradation in the calcifying bone.
Our earlier quantitative evaluations on the effect of tetracycline on collagen
biosynthesis have demonstrated a clear inhibition at high concentrations (Halme
et al. 1969). Present results, on the other hand, indicate that inhibition of
mineralization can be obtained with lower drug concentrations. Our doublelabelling experiment recording the rate of mineralization and the synthesis
of collagen in the same bone rudiments shows a clear inhibition of mineralization in concentrations of 1-0 and 10-0/*g/ml, whereas the synthesis of collagen
seems to be unaffected.
Electron microscopy of cartilage calcification has been studied in detail
recently (Anderson, 1967; Bonucci, 1967). Two structural components of the
calcifying cartilage have attracted special interest: matrix fibres, presumably
collagen, in amorphous background material (Martin, 1954; Robinson &
Cameron, 1956; Scott & Pease, 1956; Godman & Porter, 1960), and dense
matrix granules (Robinson & Cameron, 1956; Godman & Porter, 1960;Takuma,
1960; Revel & Hay, 1963). The latter have been shown by Matukas, Panner &
Orbison (1967) preferentially to deposit colloidal iron, indicating that they
contain acid mucopolysaccharide. No differences in these structures between
tetracycline-treated and untreated bones were detected in this investigation. A
close relationship between proteinpolysaccharide and collagen has been detected
by chemical methods (Gross, Mathews & Dorfman, 1960; Campo & Dziewiatkowski, 1962), and in electron microscopy (Revel & Hay, 1963; Anderson,
1967; Matukas et al. 1967) fibrillar projections are seen to be attached to the
granules. The tetracycline treatment in the present study did not cause any
apparent changes in the relationship between the fibrils and granules.
The process of calcification has been reported to be associated with enlargement of the mucopolysaccharide granules in the zone of hypertrophic cells in
the cartilage (Matukas et al. 1967), and calcification starts with the appearance
of small needle-shaped apatite crystallites in these granules (Bonucci, 1967). The
localization of the crystallites to the granules could also be established in the
Tetracycline and osteogenesis
207
present case. Decalcification of ultrathin sections leaves an organic framework of
strands within the previously calcified granule, which has the appearance of a
'ghost', showing the sites of the apatite needles (Bonucci, 1967).
The tetracycline-treated bones and their controls in this study revealed an
apparently similar ultrastructure, with thicker (350-500 Â) fibres and thinner
(200 Â) fibrils, amorphous ground substance and homogeneous (200-500 Â)
granules, some of which contained electron-dense needlelike structures of 2040 Â diameter within them or were more heavily calcified. In essence, the
picture was like that in other cartilages or calcifying bones (Anderson, 1967;
Bonucci, 1967; Matukas et al. 1967). The inhibition of calcification by tetracycline in bone rudiments, demonstrated by the experiment with randomized
pairs of bones, was reflected in the ultrastructure only as a decrease in the
number of foci of calcification present.
It has been shown that tetracyclines are bound to the calcium rather than to
the organic matrix of bone (Finerman & Milch, 1963). Tetracycline has an
affinity for collagen in vitro but only in the presence of calcium and possibly only
by means of it (Jacobs, Harris, Katz & Glimcher, 1964). Constructed models of
unit cells of hydroxyapatite and tetracycline molecules have given strong support
for the view that tetracycline binds without steric strains to calcium atoms of
bone apatite (Perrin, 1965). The inhibition of calcification by tetracycline could
therefore well be a direct blocking effect of the further growth of a mineral
crystal, like that suggested for inorganic pyrophosphate in regulating the initiation of calcification (Fleisch et al. 1966).
Our present observations on the inhibitory effect of low tetracycline concentrations on mineralization in bones, where the synthesis of the organic
matrix was apparently unaffected, give support to the hypothesis of depressed
growth of mineral crystals. In high concentrations impairment of osteogenesis
seems to be due both to depressed matrix formation and to inhibited mineralization.
SUMMARY
1. The effect of tetracycline hydrochloride on osteogenesis was tested on
mouse embryonic long bones cultivated in vitro in a chemically defined medium.
In these conditions development was retarded, but morphological and electron
microscopic studies suggested a normal course of differentiative events.
2. Tetracycline at concentrations exceeding 1 /*g/ml had an inhibitory effect
on bone mineralization as judged from measurements on the elongation of
mineralized zone, the incorporation of radiocalcium and the increase in the
total amount of calcium in the rudiments.
3. No specific morphological changes were detected by light or electron
microscopy, apart from the decreased amount of histochemically stainable
calcium and of electron-dense bone mineral crystals.
4. Incorporation of labelled thymidine was not affected by tetracycline
208
I. KAITILA AND OTHERS
concentrations not exceeding 10/tg/ml, but was greatly inhibited at a concentration of 100/tg/ml.
5. Electron microscopy, histochemical stainings and determination of 35 S0 4
incorporation suggested bone proteinpolysaccharides were unaffected in the
presence of tetracycline at concentrations inhibiting calcification.
6. Negative results were also obtained as regards the inhibition of collagen
synthesis by drug concentrations of 10/*g/ml or less.
7. The results suggested that low concentrations of tetracycline antibiotics
prevent mineralization by exerting a direct action on the formation of bone
mineral crystals. An increased concentration may lead to a similar end-result
by affecting collagen biosynthesis, while still larger amounts of the drug interfere with DNA synthesis and cell proliferation as well as with general protein
synthesis.
RÉSUMÉ
Effet inhibiteur de la tetracycline sur VOsteogenese en culture d'organes
1. L'action du chlorhydrate de tetracycline sur l'ostéogenèse a été éprouvé
sur des os longs embryonnaires de souris cultivés in vitro dans un milieu chimiquement défini. Dans ces conditions, leur développement a été retardé, mais les
recherches morphologiques et au microscope électronique suggèrent que les
processus de différenciation se déroulent normalement.
2. A des concentrations supérieures à 1 /*g/ml, la tetracycline a eu un effet
inhibiteur sur la minéralisation de l'os, si on en juge d'après des mesures de
l'élongation de la zone minéralisée, l'incorporation de radiocalcium et l'accroissement de la teneur globale en calcium des ébauches.
3. On n'a pas décelé de modifications morphologiques spécifiques au microscope ordinaire ou électronique, en dehors de la diminution de la teneur en
calcium colorable histochimiquement et en cristaux minéraux osseux denses aux
électrons.
4. L'incorporation de thymidine marquée n'a pas été affectée par des concentrations de tetracycline n'excédant pas 10/^g/ml, mais a été fortement
inhibée à une concentration de 100^g/ml.
5. La microscopie électronique, les colorations histochimiques et la détermination de l'incorporation de 35 S0 4 permettent de penser que les polysaccharides protéiques de l'os ne sont pas affectés en présence de tetracycline à
des concentrations inhibant la calcification.
6. On a également obtenu des résultats négatifs en ce qui concerne l'inhibition
de la synthèse du collagène par des concentrations égales ou inférieures à
10/ig/ml.
7. Les résultats obtenus permettent de penser que de faibles concentrations de
tetracycline empêchent la minéralisation en exerçant une action directe sur la
formation des cristaux minéraux osseux. Une concentration accrue peut conduire à un résultat final similaire en affectant la biosynthèse du collagène, tandis
Tetracycline and osteogenesis
209
que des quantités encore plus élevées d'antibiotique interfèrent avec la synthèse
du DNA et la prolifération cellulaire ainsi qu'avec les synthèses protéiques
générales.
We greatly acknowledge the use of the facilities of the Electron Microscope Laboratory,
University of Helsinki, and the technical assistance of Mauri Nyholm, M.A. The work was
supported by grants from the Finnish National Research Council for Medical Sciences and
from the Finnish Medical Society 'Duodecim'.
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