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/. Embryo!, exp. Morph. Vol. 50, pp. 99-109, 1979
Printed in Great Britain © Company of Biologists Limited 1979
Inhibition of tooth germ differentiation in vitro
1
By K I R S T I H U R M E R I N T A , I R M A T H E S L E F F
AND LAURI S A X E N
1
1
From the Department
of Pathology,
University
of Helsinki,
Finland
SUMMARY
Molar tooth germs from mouse embryos were studied in a Trowell-type organ culture.
After 5 days of culture the odontoblasts had secreted predentine and the ameloblasts had
differentiated. When cultured in the presence of 10-50 /m. diazo-oxo-norleucine (DON),
which is a glutamine analogue, the differentiation of odontoblasts was inhibited, but the
teeth looked otherwise healthy. When DON was added after 2 days of culture in control
medium (at this tims the odontoblasts in the cuspal area were already differentiated), it did
not inhibit predentine secretion, ameloblast differentiation, nor enamel secretion. However,
this was seen only in the cuspal area and the boundary to the undifferentiated, more cervical
cells was distinct.
The results support the concept that the mechanism of the differentiation of odontoblasts
is different from that of the ameloblasts. We have shown earlier that a close association
between the basement msmbrane and the mesenchymal cells is required for odontoblast
differentiation. Because DON inteiferes with glycosaminoglycan and glycoprotein synthesis
we suggest that D O N inhibits odontoblast differentiation by affecting the mesenchymal cell
surface and/or the basement membrane.
INTRODUCTION
The tooth-forming cells, the odontoblasts and the ameloblasts, differentiate
in the bell-shaped tooth germ. The epithelial enamel organ surrounds the dental
papilla, which is a condensation of mesenchymal cells derived from the neural
crest. The epithelial and mesenchymal cells are separated from each other by a
basal lamina, which is continuous during the early stages of differentiation
(Reith, 1967; Silva & Kailis, 1972; Kallenbach, 1976; Meyer, Farbe, Staubli &
Ruch, 1977). Differentiation of the cells occurs in an exact sequence. First the
mesenchymal cells become polarized and start to secrete predentine. Thereafter
the cells of the enamel epithelium differentiate into ameloblasts and secrete the
organic matrix of enamel.
Transfilter studies have indicated that the first step requires a close association
between the interacting mesenchymal and epithelial cells (Thesleff, Lehtonen,
Wartiovaara & Saxen, 1977). This association was localized to the interface
1
Authors' address: Department of Pathology, University of Helsinki, Haartmaninkatu 3,
00290 Helsinki 29, Finland.
100
K. HURMERINTA, I. THESLEFF AND L. SAXEN
between the mesenchymal cells and the basement membrane under the enamel
epithelium (Thesleff, Lehtonen & Saxen, 1978).
As differentiation of the epithelial cells into the ameloblasts is preceded by
predentine secretion by odontoblasts, this has been suggested as inducing
ameloblasf differentiation (Ruch, Farbe, Karcher-Djuricic & Staubli, 1974).
However, also at this stage the basal lamina is degraded and epithelial microvilli project through its discontinuities so making contact with the odontoblast
cell processes (Kallenbach, 1971; Slavkin & Bringas, 1976; Meyer et al. 1977).
These cell-to-cell contacts have been suggested as playing a role in the determination of ameloblasts (Slavkin & Bringas, 1976).
The aim of this study was to examine the effects of the glutamine analogue
diazo-oxo-norleucine (DON) on the differentiation of odontoblasts and ameloblasts. By blocking the utilization of glutamine in the transamidination reactions
DON interferes with at least two important metabolic pathways: the synthesis
of glycosaminoglycans (GAG) and glycoproteins (Ghosh, Blumenthai, Davidson
& Roseman, 1960; Telser, Robinson & Dorfman, 1965) and the synthesis of
purines (Buchanan, 1973). DON inhibits palatal epithelial cell adhesions in
vitro apparently by blocking the synthesis and secretion of the surface-associated
glycoproteins (Greene & Pratt, 1977). Furthermore, DON inhibits kidney
tubule induction in the metanephric mesenchyme probably by blocking the
synthesis of GAG and glycoproteins (Ekblom et al. 1979). Since these molecules
are important constituents of the basement membrane and the cell surface,
inhibition of their synthesis could also be expected to interfere with the differentiation of odontoblasts and ameloblasts.
MATERIAL AND METHODS
Tooth buds were dissected from 16-, 17- and 18-day-old hybrid mouse
embryos, BALBc/CBA. The day of vaginal plug was designated as day 0. The
lower jaw was removed and the first mandibular molars were dissected free
from surrounding tissue in phosphate buffered saline. For recombination
cultures the enamel organ was separated from the dental papilla by treatment
with cold 2-25 % trypsin - 0-75 % pancreatin solution for 10 min and after
15min of incubation in culture medium at room temperature the two components were mechanically separated and recombined on Millipore filter.
A Trowell-type organ culture was used, in which the explants were supported
by a piece of TAWP-Millipore filter (thickness 25 /im and nominal pore size
0-8 jLtm) on a metal grid. The medium consisted of BGJb medium (Difco)
supplemented with 20 % horse serum (Flow laboratories), 10 % chick embryo
extract and 0-9 mM ascorbic acid (Thesleff, 1976). According to the Difco
formula BGJb contains 1-37 mM glutamine but because our medium had been
stored in a liquid form for approximately 1 year, most of the glutamine should
have decomposed. According to Tritsch & Moore (1962) a 30 % reduction
takes place in 30 days at 4 °C.
Inhibition of tooth differentiation by DON
101
The culture dishes were kept in a humidified incubator at 37 °C in an atmosphere of 5 % CO2 in air. The explants were cultured for 5-14 days with daily
changes of the medium. The explants were fixed in Zenker's solution, embedded
in Tissue prep® (Fisher Scientific Company, Fair Lawn, New Jersey) and
serially sectioned at 7 /tm. They were stained with Mallory's phosphotungstic
acid-haematoxylin, which stains the predentine red and the enamel matrix dark
bluish-black.
6-Diazo-5-oxo-L-norleucine (DON) and amino imidazole carboxamide (A1C)
were gifts from Dr Robert M. Pratt, National Institute of Dental Research,
N1H, Bethesda, Maryland. DON was used in concentrations of 10-50 JLLM and
AIC at a concentration of 10 mM. Glutamine and glucosamine were obtained
from Sigma Chemical Company, St Louis, Missouri, and they were used at a
concentration of 10 mM.
RESULTS
(a) Differentiation of odontoblasts
The mandibular first molar of a 16-day-old mouse embryo is in the early bell
stage of development. The epithelial and mesenchymal cells are histologically
undifferentiated. After 6 days of culture the mesenchymal cells had differentiated
into odontoblasts, which secreted predentine and the onset of ameloblast
differentiation was also seen frequently (Fig. 1A, Table 1). DON (10-50 jm)
inhibited odontoblast differentiation. Predentine secretion was not seen in any
of the 20 tooth germs grown in the presence of DON (Fig. IB). Even the
explants grown in 50 JLIM DON appeared viable by light microscope examination
(see Fig. 2D).
Even in 17-day-old tooth germs no histological sign of differentiation is seen.
After 5 days of culture in control medium predentine had regularly been secreted
in a continuous layer extending from one cusp to another. At this time polarization of ameloblasts was frequently seen and sometimes enamel matrix had been
secreted by the ameloblasts (Fig. 1C). In 27 of the 46 tooth germs grown in the
presence of DON (10-50 /LM) no predentine secretion was seen (Fig. ID). In 19
tooth germs a small amount of predentine had been secreted at the cuspal tips.
In these explants a distinct boundary was noted between the predentine secreting
odontoblasts and the undifferentiated more cervical mesenchymal cells.
In 18-day-old tooth germs the mesenchymal cells in the cuspal area are
aligned and this we consider the first sign of differentiation. DON (10-50 JLIM)
did not inhibit predentine secretion by the odontoblasts in the cuspal area.
However, the differentiated area was restricted to the cuspal tips so that there
was a distinct boundary between the tips and the undifferentiated more cervical
area (Table 1). In the control explants predentine had always been secreted as a
continuous layer extending from one cusp to another.
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K. H U R M E R I N T A , I. THESLEFF AND L. SAXEN
Fig. 1. Photomicrographs illustrating the effect of DON on mouse molar tooth
germs in vitro. Mallory's phosphotungstic acid-haematoxylin stain. (A) A 16-dayold tooth germ cultured for 6 days in control medium. Predentine (PD) has been
secreted by differentiated odontoblasts. Ameloblasts have started to polarize.
(B) A 16-day-old tooth germ cultured for 6 days in the presence of 10 /m DON.
Mesenchymal cells are undifferentiated. (C) A 17-day-old tooth germ cultured for
5 days in control medium. The differentiated odontoblasts have secreted predentine and ameloblasts have polarized and started the secretion of enamel matrix.
(D) A 17-day-old tooth germ cultured for 5 days in the presence of 30 /.CM DON.
No sign of differentiation of mesenchymal cells (M) into odontoblasts is seen. M,
mesenchymal cells; O, odontoblasts; PD, predentine; A, ameloblasts; EM, enamel
matrix.
Inhibition of tooth differentiation by DON
103
Table 1. Effect of 30 JM DON on odontoblast differentiation in 16-, 17- and
18-day-old tooth germs after 5 or 6 days of culture
Number of explants showing predentine (PD) secretion, used as a criterion for differentiation
Age
16-day
PD
NoPD
17-day
PD
NoPD
18-day
PD
No PD
Control
7
0
21
0
3
0
DON30/tM
0
7
7*
13
4*
0
* The borderline between the differentiated cuspal area and the undifferentiated more
cervical area was distinct.
(b) Differentiation of ameloblasts
The effect of DON on ameloblast differentiation and the secretion of enamel
matrix was studied in experiments in which 17-day-old tooth germs were precultivated for 2 days in control medium and then for 7 days in the presence of
DON (10-30 fiM). After the first 2 days the mesenchymal cells in the cuspal area
had already aligned (Fig. 2A) and occasionally secreted predentine. In the
control teeth, which were cultured for the whole 9-day period without DON, a
thick layer of predentine was formed and the epithelial cells had differentiated
into ameloblasts and frequently secreted enamel matrix (Fig. 2B). In the cuspal
area of DON-treated tooth germs an equal amount of predentine had been
secreted. Ameloblasts had differentiated in all 16 explants and enamel matrix
had been secreted in 13 of them (Fig. 2C). However, a distinct demarcation line
was observed between the odontoblasts secreting predentine and the undifferentiated mesenchymal cells (Fig. 2D).
(c) Culture of recombined epithelium and mesenchyme
In explants in which the epithelium and mesenchyme of 17-day-old tooth
germs were separated and subsequently recombined, predentine had always
been formed after 5 days of culture (Fig. 3 A). DON (10-30 /m) inhibited the
secretion of predentine, used as a criterion for odontoblast differentiation, in
all 20 explants (Fig. 3B).
(d) Reversibility and prevention of the DON effect
When 16-day-old tooth germs were transferred to control medium after
7 days of culture in the presence of DON (10 JUM), recovery was regular. After
5 days of subculture, predentine and enamel matrix had been secreted in all
three explants (Fig. 4 A).
The inhibitory effect of DON on odontoblast differentiation was fully over-
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K. H U R M E R I N T A , I. THESLEFF AND L. SAXEN
c
Fig. 2. Photomicrographs illustrating the effect of DON on the 17-day-old tooth
germs precultured in control medium for 2 days. Mallory's phosphotungstic acidhaematoxylin stain. (A) The tooth germ after 2 days of preculture in control medium.
Mesenchymal cells have aligned under the epithelium at the cuspal tips. (B) A tooth
germ cultured for 9 days in control medium. Odontoblasts have secreted predentine
and ameloblasts have formed enamel matrix. (C) A tooth germ precultured for
2 days in control medium and 7 days in the presence of 30 /xu DON. Predentine
has been secreted by the odontoblasts and enamel matrix by the ameloblasts.
(D) The epithelial-mesenchymal interface in the intercuspal area of a similar explant
as in C. DON concentration is 50 /*M. A distinct boundary (arrow) is seen between
the differentiated and undifferentiated area. M, mesenchymal cells; O, odontoblasts;
PD, predentine; A, ameloblasts; EM, enamel matrix.
Inhibition of tooth differentiation by DON
105
Fig. 3. Recombined explants of the epithelial and mesenchymal components of
17-day-old tooth germs cultured for 4 days. Mallory's phosphotungstic acidhaematoxylin stain. (A) In control medium the mesenchymal cells have differentiated
into odontoblasts and secreted predentine. (B) In the presence of 10 /*M DON the
mesenchymal cells have not differentiated. M, mesenchymal cells; O, odontoblasts;
PD, predentine.
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K. HURMERINTA, I. THESLEFF AND L. SAXEN
*
Fig. 4. Photomicrographs illustrating the reversibility and prevention of the DON
effect on the 16-day-old molar tooth germs in vitro. Mallory's phosphotungstic
acid-haematoxylin stain. (A) A tooth germ precultured in the presence of 10 fiu
DON for 7 days and subsequently in control medium for 5 days. Predentine has been
secreted by the odontoblasts and enamel matrix by the ameloblasts. (B) A tooth germ
cultured for 7 days in the presence of 10 [M. DON and 10 mM glutamine. The
odontoblasts have secreted predentine and the ameloblasts are polarized. O,
odontoblasts; PD, predentine; A, ameloblasts; EM, enamel matrix.
ridden by the excess of glutamine. In all four 16-day-old tooth germs cultured
in the presence of DON (10/AM) and glutamine (10 mM) for 7 days predentine
had been secreted and ameloblasts were polarized (Fig. 4B).
Neither glucosamine nor the purine analogue, amino imidazol carboxamidine
(AIC) overrode the effect of DON on odontoblast differentiation. Glucosamine
(10 mM) alone did not inhibit normal development in the four 16-day-old tooth
germs examined, but when both glucosamine (10/AM) and DON (10/*M) were
present the survival of most of the eight explants was poor. When the 16-day-old
tooth germs were cultured in the presence of AIC (10 mM) normal differentiation
was prevented in all the four explants. When both AIC (10 mM) and DON
(10 /.IM) were present odontoblast differentiation was not observed in the three
explants examined.
DISCUSSION
Our results show that in the developing tooth diazo-oxo-norleucine (DON)
inhibits the differentiation of odontoblasts but not that of the ameloblasts.
Furthermore, no effect on the secretion of predentine and enamel matrix could
be observed in light microscopy. In tooth germs of 16-day-old mouse embryos
Inhibition of tooth differentiation by DON
107
DON completely inhibited odontoblast differentiation and subsequent secretion
of predentine. In 17-day-old tooth germs odontoblast differentiation was inhibited in half of the explants whereas in the rest predentine had been secreted
in the cuspal area. We suggest that the cuspal cells had already been determined
in vivo prior to the explantation although histological differentiation could not
be observed. This suggestion is supported by the results of culture of more
advanced tooth germs. Predentine was seen in the cuspal area of all 10 DON
treated 18-day-old tooth germs. Furthermore, in explants in which the cuspal
odontoblasts had been allowed to differentiate over 2 days of culture prior to
DON addition, differentiation proceeded only in the cuspal area, and the
boundary with the undifferentiated more cervical area was clear-cut. In recombination cultures of 17-day-old tooth germs DON inhibited odontoblast
differentiation in all explants. This was probable due to the loss of basement
membrane during enzymic separation of the epithelium and the mesenchyme
(Thesleff et ah 1978), resulting in loss of the possible existing alignment of
mesenchymal cells at the cuspal tips. Thus it seems that DON had an effect
only on undifferentiated mesenchymal cells and that it did not affect predentine
secretion by already determined odontoblasts.
That DON did not affect secretion of predentine could have been expected
since 99 % of predentine is collagen (Eastoe, 1967), and it has been reported
that DON does not inhibit collagen synthesis (Bhatnager & Rapaka, 1971).
Agents interfering with collagen synthesis have been shown to inhibit tooth
morphogenesis (Galbraith & Kollar, 1974; Ruch et ah 1974). The actual effect
of these substances on odontoblast differentiation is, however, difficult to
evaluate since they have obviously prevented the secretion of predentine. Thus
it has not been possible to distinguish the effect of these agents on the epitheliomesenchymal interaction leading to odontoblast differentiation from their
obvious effect on the expression of the differentiated state of these cells, i.e. on
secretion of predentine. Collagen may well play an important role in these
interactive events (Hetem, Kollar, Cutler & Yaeger, 1975) but it is improbable
that the collagen molecule itself would induce differentiation. Attempts to
induce odontoblast differentiation by various collagen substrates have failed
(Koch, 1975; Thesleff, 1978).
DON interferes with the synthesis of GAG and glycoproteins by inhibiting
the glutamine dependent conversion of fructose-6-phosphate to glucosaminophosphate (Ghosh et ah 1960; Telser et ah 1965). DON also combines irreversibly with the enzyme necessary for purine nucleoside synthesis (Buchanan, 1973).
An excess of glutamine overrode the effect of DON on odontoblast differentiation. The purine analogue AIC did not override the effect of DON. This
result is in accordance with the reports of Greene & Pratt (1977) and Ekblom
et a]. (1979). Glucosamine, which would be expected to overcome the block on
GAG and glucoproteins synthesis (Telser et ah, 1965) did not normalize
development. This may depend on a toxic effect of glucosamine on embryonic
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K. HURMERINTA, I. THESLEFF AND L. SAXEN
tissue (Kim & Conrad, 1974). Similar results have been reported in other studies
as well (Ekblom et al. 1979). Therefore, these experiments are not contradictory
to the conclusion that odontoblast differentiation may depend on GAG and
glycoprotein synthesis.
The mechanism of the DON-resistant induction of ameloblasts seems to be
different from that of the odontoblasts, because the same DON concentrations
which inhibited the differentiation of odontoblast had no effect on that of
ameloblasts. For ameloblast determination actual contacts between, the epithelial and the mesenchymal cells have been considered necessary (Kallenbach,
1971; Slavkin & Bringas, 1976) as in the induction of metanephric kidney
tubules (Saxen et al. 1976). The latter has, however, been shown to be DONsusceptible (Ekblom et al. 1979). This suggests that either the two contactmediated interactions operate via different mechanisms, or, that ameloblast
differentiation is determined by compounds of the extracellular matrix. Since
our results seem to exclude the significance of extracellular GAGs and glycoproteins, collagen might still be considered as a possible mediator of the
inductive signal for ameloblast differentiation (Ruch et al. 1974).
Results of our transfilter studies have suggested that odontoblast differentiation is preceded by a close contact between mesenchymal cell processes and
the basement membrane on the epithelial side (Thesleff et al. 1978). We suggest
that the inhibitory effect of DON on odontoblast differentiation was due to its
effect on the structure and constitution of the basement membrane and/or of
the mesenchymal cell periphery. The changed structure may not allow the
transfer of inductive signals which are normally mediated via contacts between
cells and extracellular matrix.
We wish to thank Dr Robert M. Pratt, National Institute of Dental Research, NIH
Bethesda, Maryland, for providing DON and AIC. This investigation was supported by the
Emil Aaltonen Foundation and by the Finnish Dental Society.
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EHB
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