/. Embryo/, exp. Morph. Vol. 21, 1, pp. 131-48, February 1969
] 31
Printed in Great Britain
The influence of
the dental papilla on the development of tooth
shape in embryonic mouse tooth germs
By EDWARD J. KOLLAR 1 & GRACE R. BAIRD 2
From the Department of Anatomy and Zoller Dental Clinic,
The University of Chicago
Studies of epithelio-mesenchymal interactions during embryonic organogenesis have led to a number of conclusions regarding the nature of cellular and
tissue differentiation (McLoughlin, 1963; Grobstein, 1967). For example, the
importance of both the epithelium and the mesenchyme and the dependence of
some systems on a limited number of specific mesenchymal tissues have been
pointed out (Hilfer, 1968).
Intimately connected with the analysis of the factors that elicit differentiation
during such interactions is the question of structural specificity of the differentiated structure. Is the directive for the final form of the structure resident in the
epithelium, in the mesoderm, or in both? Can a seemingly stable epithelium
undergo transformation to a more labile state and respond to a new interaction
with the result that a new epithelial structure is formed (Billingham & Silvers,
1963, 1968)?
A number of examples indicate that the inductive stimulus as well as the
specificity for the type of structure formed from the epithelium resides in the
mesoderm (Cairns & Saunders, 1954; Sengel, 1958, 1964; Gomot, 1959). In
addition, Rawles (1963), in an elegant series of experiments, showed that the
age and competence of the two tissues are of fundamental importance during
the differentiation of chick skin. Her data clearly show that the developmental
age and sources of the tissues must be thoroughly examined if an understanding
of skin differentiation is to be achieved.
A similar question of the role of the epithelium vs. the mesoderm has
intrigued students of tooth germ development. The usual appraisal of tooth
development suggests that the dental epithelium is the controlling factor
directing whether a tooth germ will be incisiform or molariform. In view of
center advances in our understanding of epithelio-mesenchymal interactions, it
1
Author's address: Department of Anatomy, The University of Chicago, Chicago,
Illinois 60637, U.S.A.
2
Author's address: Zoller Dental Clinic, The University of Chicago, Chicago, Illinois
60637, U.S.A.
9-2
132
E. J. KOLLAR & G. R. BAIRD
is of interest to examine the tissue interactions operative in the developing tooth
germ.
The work reported here utilizes experimental combinations of dental
epithelium and dental papilla from 13- to 16-day-old mouse embryos. The
incisor and molar tooth germs were subjected to trypsinization and then
separated into their epithelial and mesodermal components. The isolated
components were combined as homologous (control) and heterologous (experimental) recombinants. In addition, the influence of the developmental age of
the components was investigated. The data indicate that the shape of the tooth
germ is a function of the source of the dental papilla.
MATERIALS AND METHODS
Molar and incisor tooth germs from the mandibles of 13- to 16-day C57/10
mouse embryos were used. The ages of the embryos were determined by the
appearance of a vaginal plug and confirmed by the staging criteria of Griineberg
(1943). Dissected tooth germs were pooled and stored in a solution of 20%
foetal bovine serum in Tyrode's solution.
Trypsinization in the cold (Szabo, 1955; Rawles, 1963; Kollar, 1966) was used
to facilitate separation of the epithelial and mesodermal components of the tooth
germs. The dissected tooth germs were washed in Tyrode's solution and then
placed in a 1 % trypsin solution (Bacto-Difco, 1:250) in Tyrode's solution. The
tooth germs were incubated at 4 °C for 1-2 h, depending on the age of the tooth
germs; tooth germs from 15- and 16-day embryos required 2 h of incubation
before the components of the germs would separate with ease. Although the
two components of younger germs floated free of each other, it was necessary
to separate the components of older germs gently with a sharp iris knife.
Following trypsinization, the isolated epithelial and mesodermal components
were stored in a solution of foetal bovine serum and Tyrode's (1:1, v/v).
Control and experimental recombinants were constructed by transferring the
appropriate components to Falcon organ culture dishes containing 1 ml of
medium. Care was taken to arrange the two components so that the enamel
organ approximated the dental papilla.
The culture medium has been described elsewhere (Kollar, 1968; Kollar &
Baird, 1968). One ml of Eagle's basal medium containing 10% foetal bovine
serum, 1 % glutamine, 0-4 % agar and 50 units/ml each of penicillin and
streptomycin was used in each dish. The control and experimental recombinants
were aspirated free of any fluid used to transfer the epithelium and mesoderm.
The explants were incubated at 37 °C in a humidified 5 % CO2-95 % air gas
mixture. The explants were transferred to fresh medium every 2 days and
harvested after 6 days in culture. The explants were fixed in Zenker's acetic acid
and stained with hematoxylin and eosin.
Dental papilla and tooth shape
133
RESULTS
Normal development
The development of rodent tooth germs in vivo and in vitro has been described
in detail (Glasstone, 1938; Lefkowitz, Bodecker & Mardfin, 1953; Cohn, 1957;
Hay, 1961; Glasstone, 1967; Kollar & Baird, 1968). However, the main stages
of incisor and molar development will be mentioned here in order to orient the
reader. The incisor begins as an epithelial downgrowth in association with a
mass of mesodermal cells, the future dental papilla, on the twelfth day of
gestation. Continued epithelial invasion and the concomitant differentiation of
ameloblasts and odontoblasts results in a typically chisel-shaped tooth germ. By
the sixteenth day of gestation, the inner enamel organ and the odontoblasts have
differentiated into a pattern typical of the rodent incisor; differentiation is well
advanced on the labial side and absent on the lingual side.
The early stages of molar tooth germ development in the mouse embryo are
similar in that an epithelial thickening invades the mandibular mesenchyme in
association with the future dental papilla. By the fourteenth day of gestation, the
molar can be recognized by the trough-like shape of the germ. In section, the
molar germ is characterized by a broader association of the enamel organ and
the dental papilla. In addition, the cusp pattern of the molar is foreshadowed;
cytodifferentiation is not limited to one side of the tooth germ but is present
along the entire interface of the inner enamel organ and the preodontoblastic
layer of the mesoderm.
Thus, two features of the incisor and molar tooth germs aid in the identification of the developing structures in control and experimental recombinants—
the generalized shape of the germ and the extent of the cytodifferentiation along
the length of the enamel organ. In this study, these criteria have been applied to
the scoring of experimental explants as either incisiform or molariform.
Development of the isolated components
Trypsinization apparently separated the dental components at the level of the
epithelio-mesenchymal interface. Separation of the two components from young
animals may occur while the tissue is incubating (Plate 1, figs. A, B). On the
other hand, the complex association of epithelial downgrowth and mesenchyme
from the mandibles of older embryos required that separation be completed by
carefully manipulating the two components (Plate 1, figs. C, D). Careful
inspection of the isolated components, examination of histological preparations
of trypsinized tissues and the absence of tooth development in the fragments
when they were cultured in isolation confirmed the effectiveness of the
separation.
Isolated epithelium grown in the absence of mesodermal tissue became
disorganized and died. After 2 days in culture there was virtually nothing left of
these explants of isolated enamel organs; some keratinizing epithelial cells could
134
E. J. KOLLAR & G. R. BAIRD
be found. On the other hand, the mesodermal component cultured in isolation
remained viable. Indeed, when mandibular mesenchyme was included in the
explant, mitotic activity of the cells was seen and bone formation continued.
However, tooth structures did not develop in the mesenchyme in the absence of
the epithelium.
Development of the recombinants
Sixty-four categories of combinations of incisor and molar epithelia
(IE13.16 and ME13.16) and of incisor and molar papillae (IPi3.16 and MP13.16)
were prepared from the tooth germs of 13- to 16-day embryos: (1) eight homologous and isochronal recombinants that are equivalent to control reconstructions following trypsinization, e.g. IE13IP13, ME14MP14, etc.; (2) twenty-four
recombinants whose components are homologous with respect to the source of
the tooth germs but heterochronal with respect to the ages of the components,
e.g. IE13IP14, ME15MP14, etc.; (3) eight heterologous and isochronal recombinants that test the response of the epithelial components when confronted
with a foreign dental papilla from a similarly aged embryo, e.g. IE13MP13,
ME15TP15, etc.; and (4) twenty-four heterologous and heterochronal recombinants, e.g. IE13MP15, IE13MP16, etc.
In the following sections, no attempt is made to document each of the sixtyfour kinds of control or experimental recombinants constructed from the
isolated components of incisor and molar tooth germs. The results are
consistent throughout all categories. Therefore, selected examples of each
category have been documented.
(1) Homologous and isochronal {control) recombinants
Control recombinants were reconstructed immediately following separation
of the component tissue sheets; epithelium and mesoderm from homologous
EXPLANATION OF PLATES
Scale line represents 40/* throughout. All recombinants were cultured for 6 days.
PLATE 1
Fig. A. Section of a 13-day incisor rudiment. The epithelial downgrowth (£) and dental
mesenchyme (DM) have been separated at the epithelio-mesenchymal border, x 203.
Fig. B. Section of the isolated enamel organ of a 14-day molar tooth germ, x 230.
Fig. C. Section of a 15-day incisor tooth germ fixed immediately after trypsin treatment.
Separation of the two components can be seen along the labial edge (L) of the tooth germ.
x90.
Fig. D. Section of a 16-day molar tooth germ. Note the stability of the dental papilla (DP)
despite 2 h incubation in trypsin. x 150.
Fig. E. Section of a homologous and isochronal recombinant of 13-day incisor components,
IE13IP13. Note the differential cytodifferentiation along the epithelio-mesenchymal border.
x275.
Fig. F. Section of a homologous and isochronal recombinant of 15-day molar components,
ME15MP15. x203.
Dental papilla and tooth shape
135
E. J. KOLLAR & G. R. BAIRD
Dental papilla and tooth shape
137
tooth germs of the same developmental stage were combined to test the effect of
trypsinization on the subsequent developmental performance of such explants.
In all cases, these homologous and isochronal recombinants continued their
development and produced characteristic tooth structures in vitro. Incisor
epithelium from J3-day-old embryos (IE13) was combined with its homologous
and isochronal mesoderm (IP13) and cultured for 6 days. Such an explant
resulted in a tooth germ with characteristic chisel shape and demonstrated the
differential cytodifferentiation along one side, of the explant that is characteristic
of the rodent incisor (Plate 1, fig. E). Similarly, molar recombinations of this
category from an older developmental age, e.g. ME15MP15, developed typical
molar structures (Plate 1, fig. F). Note that, in contrast to the incisor combinations, the cusp pattern could be seen and that there was a similar state of
cytodifferentiation along the entire interface of the pre-ameloblast and preodontoblast surfaces. These data clearly indicated that the separation, reconstitution, and subsequent period of culture did not seriously impair the development of these combinations.
(2) Homologous and hetewchronal recombinants
It was of interest to know whether an age disparity between homologous
epithelial and mesodermal components of the tooth germs could influence the
final structure of the recombinants. To this end, tooth-germ epithelium of one
developmental age (I E15) was combined with the homologous but heterochronal
mesodermal component of a germ from a different developmental stage (IP14).
As we noted in the previous section, recombinants of homologous components
of molar and incisor tooth germs developed characteristically shaped tooth
structures. In the cases reported here, heterochronal combinations similarly
produced structures that could be scored with confidence as incisiform or
PLATE 2
Fig. A. Section of a homologous and heterochronal recombinant of incisor components from
embryonic tooth germs of differing developmental ages, IE15IP14. Advanced cytodifferentiation is limited to one side of the explant. x 235.
Fig. B. Section of a typical molar developing in a recombinant of molar components from
embryos of differing ages, ME14MP15. x 230.
Fig. C. Section of a recombinant of molar components of dissimilar embryonic ages,
ME14MPir, x275.
Fig. D. Section of a heterologous and isochronal recombinant consisting of a molar epithelium and an incisor papilla from 14-day embryonic tooth germs, ME14IP14. Note the
differential cytodifferentiation characteristic of the rodent incisor, x 275.
Fig. E. Section of a recombinant of molar epithelium and an incisor papilla from 15-day
embryonic tooth germs, ME151P15. Note the advanced cytodifferentiation along one side of
the explant characteristic of the rodent incisor, x 230.
Fig. F. Section of a heterologous recombinant from 13-day molar and incisor rudiments,
ME18IP13. x345.
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E. J. KOLLAR & G. R. BAIRD
molariform. Recombinants such as IE15IP14 (Plate 2, fig. A) were clearly
incisiform; molariform germs were obtained with ME14MP15 (Plate 2, fig. B)
and ME14MP16 (Plate 2, fig. C).
(3) Heterologous and isochronal recombinants
Recombinants of heterologous components from similarly staged embryos
developed typical tooth structures. The striking result was that the shape of the
developing tooth germ was consistently related to the source of the mesodermal
component. Thus, ME14IP14 recombinants (Plate 2, fig. D) resulted in advanced
tooth germs that clearly showed the differential cytodifferentiation characteristic
of the incisor germ. Similarly, ME151P15 (Plate 2, fig. E) recombinants were
incisiform. When similar combinations of 13-day tooth-germ components were
examined (Plate 2, fig. F), sites of tissue interaction were noted, but it was not
always possible to score these developing germs with confidence. Certain
suggestive features such as the differentially advanced development of one
portion of the explant or the generalized shape indicated the influence of the
dental papilla in these confrontations of components from the earliest tooth
germs used. This blurring of the structure was noted again in reciprocal
exchanges between epithelium and papilla of 16-day tooth germs (Plate 3, figs.
A, B). Again, the scoring was difficult but the influence of the mesoderm was
unmistakable.
(4) Heterologous and heterochronal recombinants
This category of recombinants represented the widest deviation from the
normal constitution of developing mouse tooth germs. Both the sources of the
dental components and the ages of these components varied. These recombinants
developed structures compatible with the notion that the structural specificity
of the developing tooth germ resides in the papilla. In most cases, the structures
PLATE 3
Fig. A. Section of an ME16IP16 recombinant. Note the smooth interface between the enamel
organ and the dental papilla. Note also the generalized chisel-shape of this tooth germ.
x225.
Fig. B. Section of a heterologous and isochronal recombinant, IE16MP16, that is the reciprocal
of fig. A. x275.
Fig. C. Section of a heterologous and heterochronal recombinant, ME13TP14. Note the chisel
shape and the differentially advanced cytodifTerentiation. x 235.
Fig. D. Section of an incisiform recombinant, ME14IP13, that is the temporal reciprocal of
fig. C. x 230.
Fig. E. Section of another recombination of the tooth germ components used in figs. C and
D. This recombinant, IE14MP13, is readily scored as molariform. x 195.
Fig. F. Section of a recombinant, ME13IP15, that confronts molar epithelium from a 13-day
embryonic tooth germ with a 15-day embryonic incisor papilla. By the double scoring
criteria, this is clearly an incisiform tooth germ, x 205.
Dental papilla and tooth shape
139
140
E. J. KOLLAR & G. R. BAIRD
were clearly incisiform or molariform. In some instances, however, distortion
of typical molar or incisor patterns were noticed. None the less, evidence of
mesodermal influence was obvious in these cases as well.
Recombinants of molar epithelium from 13-day embryos and incisor mesoderm from 14-day tooth germs (ME131P14) resulted in a structure approximating
D
PLATE 4
Fig. A. Section of a recombinant, IE13MP15, that is the reciprocal of Plate 3, fig. F. This germ
is incisiform. x 230.
Fig. B. Section of an incisiform recombinant, ME13IP16, consisting of molar epithelium from a
13-day tooth germ and a 16-day incisor dental papilla, x 165.
Fig. C. Section of a molariform recombinant, IE13MP16, that is the temporal and spatial
reciprocal of the recombinant seen in fig. B. x 195.
Fig. D. Section of a recombinant containing a molariform tooth germ that is the product of a
14-day incisor epithelium associated with a 16-day molar dental papilla, JE14MP16. x 275.
Dental papilla and tooth shape
141
the chisel shape of the incisor and showed differential cytodifferentiation along
one side of the explant (Plate 3, fig. C). On the other hand, some ME141P13
recombinants appeared more molar-like (Plate 3, fig. D); however, the advanced
differentiation of the pre-ameloblasts along one side of the explant indicated a
directive influence from the incisor papilla. Recombinants of ]E14MP13 resulted
in a clearer modification of the epithelium (Plate 3, fig. E); the general shape of
this experimental tooth germ was molariform. Apparently, when a greater age
disparity existed between the tissue better results were obtained. For example,
ME131P15 (Plate 3, fig. F) was scored as incisiform; however, the reciprocal
1E13MP15 (Plate 4, fig. A) was clearly a molar germ. The results from recombinants encompassing the greatest age disparity used in these studies (ME13IP16,
Plate 4, fig. B; IE13MP16, Plate 4, fig. C; and 1E14MP16, Plate 4, fig. D) further
substantiate the conclusion that the mesodermal components influenced the
final shape of the developing mouse tooth germ.
DISCUSSION
Epithelio-mesenchymal interactions in developing tooth germs
The mechanisms involved in tooth development remain obscure. Gaunt &
Miles (1967) in a thoughtful review have emphasized the possibility that the
dental papilla may be the inductive component in the relationship between the
ectodermally derived enamel organ and the mesoderm-like dental papilla. These
authors, in addition, examine the role of the neural crest ('ectomesenchyme') as
the source of the dental papilla. Unfortunately, at the present time, there are no
data to further clarify the possible inductive interactions or the roles of the two
components in the early ontogenetic history of developing tooth germs in the
mammal.
Undoubtedly, a crucial interaction does take place between the epithelial and
mesodermal components of older tooth germs. The classic experiments of
Huggins, McCarroll & Dahlberg (1934) demonstrated the absolute requirement
for both components in heterotopically transplanted tooth components. Only
when both components were transplanted to the abdominal wall of dogs did any
tooth structures develop. Similarly, Koch (1967) has shown that in embryonic
mouse tooth germs the isolated components fail to undergo cytodifferentiation
when grown in isolation from each other. However, these components deposit
extracellular matrices when grown on opposite sides of a Millipore filter in the
familar trans-filter culture system (Grobstein, 1956). The data described here
confirm these previous observations and are consistent with data from many
other studies of epithelio-mesenchymal interactions (McLoughlin, 1961; Kollar
1966; Grobstein, 1967). In all such systems, the mesodermal component fails to
differentiate and the epithelium, unless subjected to very specialized culture
conditions, keratinizes and dies.
The data from studies of developing tooth germs, as well as the extensive data
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E. J. KOLLAR & G. R. BAIRD
from other epithelio-mesenchymal systems, advance the view that important
tissue interactions probably operate in embryonic mouse tooth germs. However,
the essential observations of the inductive properties of the dental papilla are
lacking. In this regard, we have undertaken a series of experiments to test the
developmental properties of the dental papilla by confronting the isolated
papilla with epithelia from various sites in the embryonic mouse. Snout skin,
esophageal, and plantar epithelia have been combined with isolated incisor and
molar papilla. These preliminary experiments have produced suggestive,
interesting, but none the less negative, data. Caution must be exercised in
interpreting these negative data in the context of an epithelio-mesenchymal
inductive interaction in the murine tooth germ, since a limited range of tissue
sources and ages have been tested so far. The elegant experiments of Rawles
(1963) on chick skin development have demonstrated that the inductive properties of the mesenchyme and the competence of the epithelium depends on
spatial and temporal factors that must be examined through a wide range of
tissues and embryonic ages before definite conclusions can be reached.
The development of the tooth may be unique among the many specializations
of the skin in that both the epithelium and the mesodermal components undergo
marked cytodifferentiation and specialization during the course of development.
Whereas other epithelial appendages are characterized by a highly specialized
epithelial component and a seemingly unstructured mesodermal component, the
tooth germ is the product of a more complex, and possibly reciprocal, interaction. In the tooth germ, both the epithelial and mesodermal components
participate in the formation of structural elements. An analogous situation
exists in the metanephros; both the epithelium and mesoderm form complex
tubular structures. In the metanephros, the development of the mesodermal
component can be induced by a variety of agents; but the ureteric epithelium
has a specific dependence on the metanephrogenic mesoderm (Grobstein, 1955).
Thus, some epithelio-mesenchymal interactions are characterized by specific
and limiting tissue requirements (Hilfer, 1968). Until the dental papilla can be
tested at earlier ages and with a wide variety of epithelia, the questions of
induction and the nature of the interactions between the enamel organ and the
dental papilla must remain unanswered.
The emergence and control of tooth shape
Despite the lack of precise information concerning the inductive properties of
this interacting system and the processes involved in the formation of complex
three dimensional structure following the initial events, certain facets of these
interactions are coming to light. The data presented here indicate that dental
papillae confer specificity for final shape on developing tooth germs. These data
are substantiated by a number of studies; it is clear that in epithelio-mesenchymal
interactions structural specificity resides in the mesoderm (Cairns & Saunders,
1954; Zwilling, 1961; Sengel, 1964).
Dental papilla and tooth shape
143
The data presented here are consistent throughout the range of ages tested.
The conclusion is clear that from the 13th day of gestation the dental papilla
influences the shape of the tooth germ. This conclusion is based on the interpretation of sections of early embryonic tooth germs. While the experimental germs
compare well with sections of control histological preparations of comparable
ages, we are aware that advanced stages of tooth development would provide a
more stringent demonstration of the role of the mesoderm in the emergence of
tooth shape. Since this work was completed we have repeated these experiments
and have transplanted the recombinants to the anterior chambers of eyes of
homologous hosts. Advanced differentiation is achieved and enamel and dentine
are present in explants grown in this nutritionally favourable site. The data from
these new experiments confirm the conclusions presented here and will be
described elsewhere.
While this paper was being prepared, two preliminary reports appeared that
contradict the conclusions presented here. Dryburgh (1967), working with
younger stages than those used in this study, states that incisor germs are
produced in association with incisor epithelium in transposed exchanges of
epithelium and mesenchyme of early mandibles. Similarly Miller (1968), using
incisor and molar rudiments from 11- to 13-day embryonic mice, reports that
following separation, recombination and grafting to the chorioallantois of
embryonated chick eggs there is no evidence of mesodermal influence in the
structures formed. It is not possible to critically discuss these data taken from
preliminary reports.
The possibility that the epithelium of the presumptive incisor and molar
areas of the mandible from very young stages interacts with the mesoderm and
initiates the formation of stable incisor and molar dental papillae is being tested
in this laboratory. However, the present data suggest that whether, as Dryburgh
and Miller suggest, or not the initial events are indeed controlled by the epithelium, the subsequent stability and developmental performance of the papilla
are consistent and directive through the stages of early cytodifferentiation in the
mouse tooth germ.
The means by which the dental papilla exerts this influence is the central issue.
Gaunt & Miles (1967) have reviewed this question. The physical stability of both
the enamel organ and the dental papilla are striking. Immediately following
trypsinization and separation, these components retain their structural integrity
(Plate 1,figs.A-D). The dental papilla is more stable than the enamel organ and,
in older tooth germs, it could easily be recognized within the surrounding
mandibular mesenchyme as a morphological entity. However, during
recombination, the enamel organ collapses and folds. Soon after reconstruction,
the explants collapse and spread on the surface of the agar. It is doubtful
whether the dental papilla survives this flattening as a structural entity. Examination of control explants of isolated mesoderm confirms the loss of papillar
structure in denuded mesodermal fragments. Therefore, we find it difficult to
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E. J. KOLLAR & G. R. BAIRD
accept the simplest explanation of the influence of the dental papilla acting to
provide a template on to which the enamel organ displays itself during subsequent culturing.
The view that the stellate reticulum plays an important and perhaps a
directive role in the formation of tooth shape by virtue of its turgor pressure
has become untenable. In this study, the experimental manipulations and culture
conditions place the enamel organs of older tooth germs containing a welldeveloped stellate reticulum in an environment that frees the enamel organ
from the confines of the mandible and confronts it with new osmotic conditions.
Observations during the experiments indicate that the enamel organs of older
tooth germs were completely collapsed and, indeed, were folded when placed
on homologous or heterologous mesodermal fragments. Although these observations suggest that the original stellate reticulum does not participate in the
reconstructions of the tooth germs, it is clear from favorable sections that a
stellate reticulum is found in association with the experimental tooth germs. It is
not possible to know whether the experimental germs examined in this study
were the product of an enamel organ that arose de novo under the direction of
the dental papilla. Certainly, if the enamel organ and the stellate reticulum, in
particular, participate in moulding the shape of the tooth germ, they do so under
the influence of some cue in the mesoderm.
Butler (1956, 1967) emphasizes the importance of the dental papilla and
suggests that the rigid stellate reticulum prevents distortion by the expanding
dental papilla. He further proposes that differential growth rates in the enamel
organ and the dental papilla are responsible for the complex topological
patterns of the mammalian tooth.
The view that differential mitotic activity may be responsible for the shape of
the tooth is of added interest in view of the recent work on epithelio-mesenchymal interactions. The maintenance of a mitotically active basal cell population
in the skin (that may be equivalent to the inner enamel epithelium) is dependent
on the presence of the mesoderm (Wessells, 1962). In addition, the suggestion
has been made that the local epithelial mitotic rate may be controlled by the
dermis of that site (Van Scott, 1964). These properties of the mesoderm seem
to be likely candidates for further examination of the influence of the papilla on
the emergence of tooth shape.
Finally, those cases in which cytodifferentiation was sufficiently advanced to
be used as a scoring criterion suggest that the incisor and molar pattern of
matrix deposition is resident in the papilla. Furthermore, the execution of these
experiments introduces another facet of importance to the question of the
development of tooth shape. The delicate collapsed enamel organ could not be
oriented with certainty on the dental papilla. The random placement of the
epithelial component and subsequent reconstruction of a recognizable tooth
germ suggest that the epithelium retains a plasticity that can be influenced by
the dental papilla. Glasstone (1952) has shown that when a molar germ is
Dental papilla and tooth shape
145
bisected the two halves retain sufficient plasticity to regulate and form two
harmonious molar germs. A number of questions are raised by these observations. Can the entire enamel organ of an older and presumably stable tooth
germ reorganize into a new structural form? What portions of the oral epithelium or the enamel organ retain this plasticity? Are the tooth germs described
in this study the product of a reorganization of existing portions of the enamel
organ that have been marshalled into a new pattern? Does the dental papilla
from 13- to 16-day embryonic tooth germs induce the complete reorganization
of the enamel organ or of some unspecialized cell population in this structure?
These questions and the mechanism of such reorganizations in tooth germs are
being investigated further.
SUMMARY
1. This report describes the influence of the dental papilla on the emergence
of tooth shape in developing mouse tooth germs in vitro. Incisor and molar
tooth germs from 13- to 16-day embryonic mice were separated into their
epithelial and mesodermal components following treatment with trypsin. The
isolated components were then recombined as homologous (control) and
heterologous (experimental) recombinants and cultured on a complex medium
solidified with agar. In addition, the effect of the age of these tooth-germ
elements was studied by constructing homologous and heterologous recombinants in which the developmental age of the components varied. The generalized
shape of the germs as well as the typical patterns of cytodifferentiation were used
to score these explants as incisiform or molariform.
2. The isolated enamel organ and dental papilla do not develop tooth structures or undergo any recognizable cytodifferentiation into ameloblasts or
odontoblasts when grown in isolation. This observation suggests that, as in
other epithelio-mesenchymal associations in avian and mammalian structures,
an interaction between these two tissues is necessary if development is to
proceed.
3. Homologous and isochronal recombinants composed of epithelium and
mesoderm of the same origin and developmental stage produce type-specific
tooth germs. Incisor components produce incisors and molar components
reconstruct recognizable molar tooth germs.
4. In all other combinations of the tooth germ elements in which the site of
origin, the age of the components, or both parameters varied, the data consistently indicated a strong influence of the dental papilla on the emerging shape of
the tooth germ. Thus, when a molar enamel organ was confronted with an
incisor papilla, incisor-like tooth germs were formed.
5. These data indicate that the structural specificities for tooth shape reside
in the dental papilla. The manner by which these developmental cues provided
by the mesoderm may be translated to the tooth germs as a whole are discussed
in the context of other epithelio-mesenchymal interactions.
IO
1EEM2I
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E. J. KOLLAR & G. R. BAIRD
RESUME
VInfluence des papilles dentaires sur la forme des ebauches
dentaires de Vembryon de Sour is
1. Cet article decrit l'influence des papilles dentaires sur l'apparition des
formes des ebauches dentaires de l'embryon de souris cultivees in vitro. Les
composants epitheliaux et mesodermiques des ebauches d'incisives et de
molaires de l'embryon de souris de 13 a 16 jours ont ete separes apres traitement
a la trypsine. Ces composants ainsi isoles sont ensuite reassocies, en associations
homologues (temoins) ou heterologues (experimentales) et cultives sur un
milieu complexe solidifie par l'agar. De plus le role de chacun de ces elements a
ete etudie par des associations homologues et heterologues dans les quels les
constituants etaient d'ages differents. La forme generate des germes ainsi que les
structures speciflques de differenciation cellulaire ont ete prises en consideration
pour definir ces explants comme incisiformes ou molariformes.
2. L'organe adamantin et les papilles dentaires isoles ne donnent pas de
structures dentaires et ne subissent aucune differenciation visible en adamantoblastes ou odontoblastes. Cette observation suggere que, comme dans d'autres
associations epithelio-mesenchymateuses d'oiseau ou de mammifere, une
interaction entre ces deux tissus est necessaire pour obtenir un developpement.
3. Des associations formees d'epithelium et de mesoderme de meme origine et
de meme stade de developpement donnent des ebauches dentaires typiques. Les
composants d'incisives donnent des incisives et les composants de molaires
forment des ebauches molaires caracteristiques.
4. Dans toutes les associations d'elements d'ebauches centaires d'origine
differente ou d'age different, ou d'origine et d'age differents, les resultats
indiquent une forte influence des papilles dentaires sur la forme de l'ebauche
dentaire naissante. Ainsi, quand un organe adamantin de molaire est associe a
une papille d'incisive, l'ebauche edifie des structures incisiformes.
5. Ces resultats indiquent que les specificites structurales des formes dentaires
sont determinees par les papilles dentaires. Le probleme de savoir comment sont
transmises les informations du mesoderme en ce qui concerne le developpement
de l'ebauche dentaire dans son ensemble est discute dans le cadre d'autres
interactions epithelio-mesenchymateuses.
The authors are grateful to Dr Benson E. Ginsburg, who generously made his animal
colony and animal-care facilities available, and to Mrs W. Garner for her technical help.
This research was supported by USPHS General Research Support Grant FR-5367 and the
Inland Steel-Ryerson Foundation Faculty Fellowship.
Dental papilla and tooth shape
147
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