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The development of the periodontium. A transplantation and autoradiographic study

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The Development of the Periodontiurn. A Transplantation
and Autoradiographic Study
A. R. TEN CATE, C . MILLS AND G . SOLOMON
Faculty of Dentistry, University of Toronto, Toronto 101, Canada
ABSTRACT
First molar tooth germs were dissected from one-day-old mice;
placed for one hour in McCoy’s medium containing 10 pc tritiated thymidine and
transplanted subcutaneously into young adult animals of the same strain. Seven,
14, 21 and 28 days after implantation the host animals were sacrificed and the
transplants harvested. The transplants were then serially sectioned and autoradiographs prepared. Control sections were prepared of first molar tooth germs
in situ, after dissection from the jaws and after labelling with tritiated thymidine.
Forty-nine of the 115 transplanted tooth germs continued development with the
formation of enamel, dentine, cement, periodontal ligament and bone. In some
instances the transplanted tooth germs “erupted through the skin with the establishment of an epithelial attachment. Examination of control sections showed
that the transplants consisted of dental organ, dental papilla and a layer of ectw
mesenchymal cells continuous with the dental papilla and investing the dental
organ. Examination of autoradiographs of the transplants showed labelling of
cementoblasts and periodontal ligament fibroblasts, thereby establishing their
origin from the ectomesenchymal cells investing the tooth germ.
The dental follicle is considered to con- and that only undifferentiated epithelial
sist of all the tissue between the develop- and ectomesenchymal cells remained scating tooth and the developing bone, and tered throughout the interstices of the
to be the formative organ of alveolar sponge. Significantly, when such late culbone, periodontal ligament and cement tures were transplanted subcutaneously
(Orban, ’62). Three layers are described into isologous newborn recipients, they dewithin this follicle, one associated with veloped into recognizable teeth with cement
bone, one associated with the tooth, and formation on the root dentine, indicating
an intermediate layer. Several investi- the ectomesenchymal derivation of this
gators have hinted at the distinctiveness of hard tissue. On the other hand, Hoffman
the inner layer (Fearnhead, ’61; Tonge, (’60) successfully transplanted developing
’63; Scott and Symons, ’67) on the basis molar teeth from the jaws of newborn
that it is continuous with the dental papilla hamsters into a subcutaneous site in adult
around the rim of the dental enamel organ animals. In this alien site, development
and therefore of ectomesenchymal deriva- continued with the formation of roots,
tion. Ten Cate (’69) has suggested on the cement, periodontal ligament and “alvebasis of histological, histochemical, em- olar” bone, and it was argued that the epibryological, and transplantation studies, thelial component of the tooth germ was
that the term “dental follicle” should be re- capable of organizing “foreign” connective
served for the layer immediately in contact tissue to differentiate into alveolar bone,
with the dental organ and continuous with periodontal ligament and cement. Howthe dental papilla, since cement, a true ever, it was pointed out that “additional
dental tissue, probably stems from it. Evi- evidence should be obtained to establish
dence supporting this ar,aument comes conclusively that no transplanted cells were
from the studies of Main (’66), who the precursors to the periodontal tissues
showed that when mouse tooth germs were formed. Without doubt, a certain few cells
adhered to the outer enamel epithelium
cultured on gelatin sponges for 37 days they lost their characteristic morphology
Received Nnv. 6, ’70. Accepted Dec. 8, ’70.
ANAT. REC., 170: 365-380.
365
366
A. R. TEN CATE, C. MILLS AND G. SOLOMON
and were transplanted. It seems improbable that these cells could have been responsible for the extensive formation of
periodontium routinely seen around transplanted teeth after 28 days in the host subcutaneous tissues.”
The contribution of these few cells adhering to the outer dental epithelium to
the formation of the periodontium, which
we regard as the innermost layer of the
classical dental follicle, is reported in this
communication.
MATERIALS AND METHODS
First molar tooth germs were dissected
from the jaws of one day old Connaught
strain mice. After decapitation the jaws
were separated by making a backward incision through the ramus of the developing mandible on each side with a razor
blade. With the aid of a dissecting microscope, the sites of first molar tooth germs
could be readily seen as rounded swellings
in both the mandible and maxilla. Using
sterile cataract needles the overlying oral
mucosa was gently teased away from over
the selected tooth germ and, with a spoon
shaped corneal knife, the tooth germ lifted
from its developing crypt.
Tooth germs removed in this way were
then treated in one of the following ways.
Some tooth germs were placed into Bouin’s
fixative, processed by routine histological
methods, and stained with either haematoxylin and eosin, or silver impregnated to
demonstrate reticulin to serve as controls
for the dissection technique. Other tooth
germs were placed into 1 ml of McCoy’s ’
culture medium at 37.5”C. When five or
six tooth germs had been placed into the
culture medium, 10 pc of tritiated thymi.
dine was added to the solution. In some
instances this amount of tritiated thymidine was either doubled or tripled. After
one hour in the culture medium plus tritiated thymidine, the tooth germs were removed and washed thoroughly in three
changes of McCoy’s medium.
At this point some of the tooth germs
were fixed in Bouin’s, processed, and autoradiographs of one week exposure prepared. Such autoradiographs served as
controls to check the uptake of tritiated
thymidine by the explanted tooth germs.
All the remaining tooth germs were trans-
planted subcutaneously into young adult
host animals of the same strain.
Transplantation was performed as follows. The host was anaesthetized with
nembutal (3.5 mg/100 mg body weight)
and the fur of the back shaved. Three incisions, each approximately 0.5 cm long,
were made parallel to and 1 cm away from
the line of the spinal column. Cold (nontritiated) thymidine at 37.5”C was dripped
into each incision and, after this rinse of
cold thymidine, two labelled tooth germs
were pipetted into a subcutaneous position
through each incision. The incisions were
then closed with transparent steri-tape and
the host animal returned to its cage.
Host animals were sacrificed 7, 21 and
28 days after introduction of the transplanted tooth germs. After sacrifice the
skin of the back was incised along the
mid-line and the skin reflected. The skin
was pinned down at its edges and the
transplanted tooth germs identified with
the aid of a dissecting microscope. Bouin’s
fixative was dripped onto each transplant
and then the transplant with surrounding
skin dissected out and pinned to a thin
cork board. The cork board and transplant
were then placed into Bouin’s fixative. After
fixation the transplants were demineralized
in a 1:1 solution of 20% sodium citrate
and 45% formic acid and then embedded
in paraffin wax following routine histological procedures. Sections were cut serially
at 8 and mounted on glass slides. Every
fifth section of a series was stained with
haematoxylin and eosin. The remaining
sections were dewaxed in xylol, rinsed in
absolute alcohol and air dried. These were
dipped in Kodak NTB-2 emulsion in the
dark and exposed for three weeks. After
exposure, the slides were developed, counterstained with haematoxylin and permanent preparations made.
A total of 30 tooth germs were prepared
as controls for the dissection procedure,
20 as controls to check the uptake of tritiated thymidine in the culture medium
and 115 transplanted subcutaneously. In
addition, three one-day-old mice were used
to prepare sections of first molar tooth
germs in situ. These sections were stained
with either haematoxylin and eosin or
1 Supplied by the Grand Island Biological Company,
Grand Island, New York.
DEVELOPMENT OF THE PERIODONTIUM
367
found some distance away from the epithelial root sheath cells (figs. 7, 8).
Three weeks after transplantation the
RESULTS
tooth germs showed continued development
Control tooth germs in situ
of both the crown and root and also forand after dissection
mation of cement, periodontal ligament
Examination of first molar tooth germs and alveolar bone (figs. 9, 10).
Examination of the autoradiographs of
in situ in the jaws of one-day-old mice
showed the presence of an inner investing three week transplants showed labelling
layer, continuous with the dental papilla, of cementoblasts and fibroblasts of the
and surrounding the dental organs (figs. 1, ligament in the region of the cement2). Examination of first molar tooth germs enamel junction (fig. 11). Whether cells
after dissection from the jaws of one-day- associated with the formed bone were
old mice showed that in all instances the labelled was equivocal. Whilst labelled
dissected tooth germ consisted of the den- cells were found (fig. 12), the grain count
tal or enamel organ, the dental papilla, was so low (3-4 grains per cell) that the
and the innermost layer of the dental fol- possibility of this being background radialicle (fig. 3). In no instances did we find tion could not be discounted. Four week
additional tissue of the classical follicle transplants (and in 2 instances, 3 week
(indeed, in several of the specimens ex- transplants) showed further dental develamined, parts of the investing layer and opment and “eruption” of teeth through
outer enamel epithelium were missing due the skin of the back (figs. 13, 14). Whether
or not transplanted tooth germs erupted
to careless handling).
seemed to be dependent upon orientation
Control tooth germs after dissection and of the transplant. Only if the crown of the
labelling with tritiated thymidine
transplant faced the skin surface did
Examination of this material revealed eruption occur (fig. 15). Erupted teeth alconsiderable uptake of tritiated thymidine ways showed the development of periodonby the cells of the dental papilla, the in- tal tissues and organization of the periternal enamel epithelium, the stratum in- odontal fibroblasts along the lines of the
termedium and the inner layer of the dental classical fibre bundles of the normal perifollicle (fig. 4). The uptake of tritiated odontal ligament. Thus (fig. 14) the fibrothymidine was judged as intense from the blasts showed oblique orientation between
fact that only a one week exposure of the bone and cement, a transverse orientation
autoradiograph was required to produce a in the region of the cement enamel juncgrain count so dense that no count was tion, and an oblique orientation corresponding to the free gingival fibres supporting
possible.
the “epithelial attachment.” AutoradioTransplanted tooth germs
graphs of erupted transplants showed, in
Of the 115 tooth germs transplanted additon to labelled cementoblasts and fibrosubcutaneously, 49 continued development blasts of the periodontal ligament, labelled
successfully and were harvested. After dental epithelial cells forming a component
scven days, tooth development had ad- of the developing attachment (fig. 16).
vanced considerably with the formation of The other component consisted of undentine and enamel (fig. 5). Most im- labelled cells of the host skin epidermis.
portant from the standpoint of the problem One transplant was at the stage where the
under investigation was the fmding that tooth was about to break through the skin
Hertwig’s root sheath had formed (fig. 6 ) epidermis. The epidermis overlying the
and initiated root formation. Autoradio- transplanted tooth showed evidence of prographs of seven-day implants showed the liferative activity and fusion with the denpresence of labelled cells on the outer as- tal epithelium (fig. 17); a configuration
pect of Hertwig’s root sheath in a location similar to that found in the oral cavity as
coinciding with that of the inner layer of the erupting tooth penetrates the oral epithe dental follicle of normal dental devel- thelium. Finally, in one three-week unopment. In addition, labelled cells were erupted transplant, there was some evisilver impregnated for the demonstration
of reticulin.
368
A. R. TEN CATE, C. MILLS AND G. SOLOMON
dence of lymphocyte accumulation in the
connective tissue located on the outer surface of the newly formed alveolar bone
(fig. 9).
DISCUSSION
The purpose of this investigation was
to identify those structures of the periodontium derived from the ectomesenchyma1 inner layer of the classical dental follicle, The dissection of tooth germs at the
late bell stage of development from the
jaws was found to be a relatively simple
procedure, made easy because the enamel
organ and dental papilla are encapsulated
by this inner investing layer. We are confident that all the tooth germs dissected
from the jaws and used for this investigation were encapsulated by this layer. Indeed, Hoffman’s (’60) original transplantation studies stressed the difficulty of obtaining enamel organ and papilla devoid
of this investing layer.
The introduction of tritiated thymidine
into the dissected tooth germs was satisfactorily achieved and its uptake was as
anticipated from previous labelling studies
of the tooth germ in situ (Starkey, ’63;
Scott, ’69 ). The experiment reported
here was designed to ensure as heavy a
labelling as possible with tritiated thymidine as it was anticipated that dilution of
the label with successive cell divisions
might hinder identification of cells at later
stages of development. The fact that development continued satisfactorily in the
presence of high labelling made us confident that the ionizing radiations did not
affect cellular differentiation and function
to any significant degree. Also, the successive rinses in McCoy’s medium after
labelling o€ the dissected tooth germs, and
the flooding of the implantation site with
cold thymidine, reduced the possibility of
the transfer of tritiated thymidine from the
implant to the host cells.
The continued development of tooth
germs in a subcutaneous site, with the formation of alveolar bone, periodontal ligament and cement, has been reported previously in the hamster (Hoffman, ’60). In
this study, significant additional features
of dental development have been observed;
namely the development of enamel, the
eruption of transplanted teeth and addi-
tional organization of the periodontal ligament. With respect to the development of
the periodontal tissues in this alien situation, Hoffman (’60) concluded that the
transplanted developing tooth formed periodontal ligament and alveolar bone in
f‘oreign connective tissue under the organizing influence of the epithelial cells of
Hertwig’s root sheath. Though he recognized the possibility that the cells adhering
to the outer enamel epithelium may have
proliferated he felt that it was improbable
that these few cells could have been responsible for “the extensive formation of
periodontium routinely seen around transplanted teeth.”
Our study indicates that these few cells,
the inner layer of the dental follicle, do
indeed contribute to the formation of the
periodontium. That the cementoblasts arise
from cells of the inner layer of the follicle
seems to us proven by the ready identification of labelled cementoblasts, indicating
their origin from transplanted tissue. The
ease of identification can be attributed to
their relatively heavy labelling. We would
anticipate that these cells would retain a
lot of label for the progenitor cells of the
cementoblasts at the late bell stage of tooth
development are unlikely to undergo too
many divisions before differentiation, so
dilution of the label would be minimal.
If this origin of cementoblasts from the
inner layer of the dental follicle is accepted, it substantiates the argument put
forward by Ten Cate (’69), who questioned
the suitability of terming thjs layer, which
is in immediate contact with the dental
organ and continuous with dental papilla,
the inner layer of the follicle. Instead it
was proposed that, since the dental organ
is the formative organ of enamel, and the
dental papilla the formative organ of the
dentine, the term dental follicle should be
reserved for this investing ectomesenchyma1 layer which forms cement, the remaining distinctive dental hard tissue. The term
perifollicular mesenchyme was suggested
for the remaining tissue of the classical
dental follicle.
This derivation of cement also has implications with respect to the evolutionary
development of the periodontium. Two
conflicting opinions exist (Noble, ’69). On
the one hand it is thought that the teeth
DEVELOPiMENT OF THE PERIODONTIUM
were originally attached to the jaw by an
underlying area of bone known as the bone
of attachment. The fibrous membrane
which developed between the tooth and
bone of attachment, as in the eel, is regarded as the earliest development of periodontal ligament, with the bone of attachment later developing into the alveolar
process and cement forming as a new tissue distinctive for socketed teeth. The alternative view is that the bone of attachment is homologous with cement (Scott
and Symons, ’67).
This study has shown that since the
origin of cement is from the dental follicle,
which is developmentally and morphologically derived from the same source as the
dental papilla and which is present in all
developing teeth, the latter view must be
supported.
The identification of labelled fibroblasts
in the periodontal ligament would also indicate that this structure is derived from
the inner layer of the classical dental follicle. However, it is not possible to be as
certain on this point. The fact that labelled
fibroblasts were found only in the region
of the cement-enamel junction can be explained on the basis that this region represents the original cervical loop area of the
transplant, and cell division here will have
bfen less than elsewhere to establish ligament fibroblasts. While our results indicate that some of the ligament fibroblasts
are derived from transplanted tissue. ultrastructural studies of the developing periodontium (Freeman and Ten Cate, ’71)
also indicate a perivascular source of ligament fibroblasts. As presumably no vessels
were transplanted, the possibility must be
recognized that periodontal fibroblasts
arise from both host and transplant tissue.
The question of the origin of “alveolar”
bone subcutaneously is much more difficult
to resolve, Though labelled cells associated
with the formed shell of bone were demonstrated, the labelling was minimal and the
possibility that this represents background
radiation cannot be excluded. The problem
here is that the bone develops some distance away from the tooth and therefore
if cells of the transplant do form bone,
they will have undergone several divisions
with corresponding dilution of the label.
It must be admitted that with respect to
369
the origin of “alveolar” bone our method is
inadequate. However, there is some indication that the bone may have been derived from transplanted tissue. This stems
from the finding, in one specimen, of
lymphocytes on the external aspect of the
formed bone. This could be indicative of
the initiation of a rejection mechanism.
If that were the case, i t is significant that
these cells occupy a situation external to
the bone and suggests that it is being rejected and therefore a derivation from
transplanted tissue.
Although this paper is not concerned
with enamel formation or tooth eruption
it is of interest that both occurred in this
study. Therefore, this model system could
be utilized for studies of amelogenesis and
tooth eruption. The fact that transplants
erupted only if orientated in the correct
position, and only when a periodontal ligament had formed, lends support to the
view that the force for tooth eruption resides in the ligament, as postulated by
Kostlan, Thorova and Skach (’60), Thomas
(‘65), and Berkovitz and Thomas (‘68). The
development of an epithelial attachment
between the erupted transplanted tooth and
skin is also a finding of interest. Histologically, the genesis of this attachment,
and also its structure, appear to resemble
the normal sequence of events, so the possibility exists of transplanted teeth being
used as a model system to study problems
associated with the genesis of the epithelial
attachment and the initiation of periodontal disease.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the
financial assistance of the Medical Research Council of Canada for support of
C. Mills and G. Solomon as summer students and for the grant in aid to A. R. Ten
Cate, grant MA-3693.
LITERATURE CITED
Berkovitz, B. K. B., and N. R. Thomas 1969
Unimpeded eruption i n the root-resected lower
incisor of the rat with a preliminary note on
root transection. Arch. oral Biol., 14: 771-780.
Fearnhead, R. W. 1961 The dental follicle. J.
Dent. Res., 40: 1278.
Freeman, E., and A. R. Ten Cate 1971 The
development of the periodontium. A n electron
inicroscopic study. J. Perio., in press.
Hoffman, R. L. 1960 Formation of periodontal
tissues around subcutaneously transplanted
hamster molars. J. Dent. Res., 39: 781-798.
370
A. R. TEN CATE, C. MILLS AND G . SOLOMON
Kostlan, J., J. Thorova and M. Skach 1960
Erupce hlodaveho zubo PO reselci jeho rustove
zony. Cas. Lek. ces., 60: 401-410.
Main, J. H. P. 1966 Retention of potential to
differentiate i n long term culture of tooth
germs. Science, 152: 778-780.
Noble, H. W. 1969 The evolution of the mammalian periodontiuni. In: The Biology of the
Periodontium. A. H. Melcher and W. H. Bowen,
eds. Academic Press, London, New York, pp.
1-26.
Orban, B. J. 1962 “Orban’s Oral Histology and
Embryology.” H. Sicher, ed. C. V. Mosby Company. St. Louis, U.S.A.
Scott, B. L. 1969 Tritiated thymidine study of
developing tooth germs and osteogenic tissue.
J. Dent. Res., 48: 753-760.
Scott, J. H., and N. B. B. Syinons 1967 “Introduction to Dental Anatomy.” E. and S. Livingstone Ltd., Edinburgh and London.
Starkey, W. E. 1963 Migration and renewel of
tritium labeled cells i n the developing enamel
organ of rabbits. Brit. Dent. J., 115: 143-153.
Ten Cate, A. R. 1969 The development of the
periodontium. In: The Biology of the Periodontium. A. H. Melcher and W. H. Bowen, eds.
Academic Press, London, New York, pp. 53-89.
Thomas, N. R. 1965 The process and mechanism of tooth eruption. Ph.D. Thesis. University of Rristol.
Tonge, C. H. 1963 The development and arrangement of the dental follicle. Tran5. European Orth. SOC.: 1-9.
PLATE 1
EXPLANATION OF FIGURES
1
First molar tooth germ in situ in a one day old mouse. Note the continuation of the dental papilla around the cervical loop (arrowed).
Haematoxylin and eosin. x 300.
Part of the first molar tooth germ of a one day old mouse showing the
layer of cells adjoining the external dental epithelium. Silver impregnation for reticulin. dp, dental papilla; ide, internal dental epithelium;
sr, stellate reticulum; e, external dental epithelium. x 300.
Part of the first molar tooth germ of a one day old mouse after dis.
section from the jaw. Notice the layer of cells (arrowed) continuous
with the dental papilla lying on the external surface of the dental
organ. Haematoxylin and eosin. dp, dental papilla; ide, internal dental
epithelium. x 300.
DEVELOPMENT OF THE PERIODONTIUM
A. R. Ten Cate, C. Mills and G . Solomon
PLATE 1
371
PLATE 2
EXPLANATION O F FIGURES
372
4
Autoradiograph of the first molar tooth germ of a one day old mouse.
The tooth germ illustrated was dissected from the jaw and labelled for
one hour with tritiated thymidine in McCoy’s medium. Notice the uptake of tritiated thyinidine by the cells of the dental papilla ( d p ) . the
internal dental epithelium and the cells (arrowed) on the surface of
the external dental epithelium. )< 1280.
5
Seven day subcutaneous implant of the first niolar tooth germ taken
from a one day old mouse. Notice continued development with the
formation of both dentine and enamel. No periodontal tissue has
formed at this stage. Haematoxylin and eosin. >( 120.
6
Seven day subcutaneous implant of the first molar tooth germ taken
from a one day old mouse. Root formation has begun with the development of Hertwig’s epithelial root sheath ( I s ) . d, dentine; dp,
dental papilla. Haeniatoxylin and eosin. A 120.
DEVELOPMENT OF THE PERIODONTIUM
A. R. Ten Cate, C. Mills and G. Solomon
PLATE 2
373
PLATE 3
EXPLANATION O F FIGURES
374
7
Autoradiograph of a seven day subcutaneous implant of the first molar
tooth germ taken from a one day old mouse. Labelled cells (circled)
can be seen on the outer aspect of Hertwig’s root sheath ( I s ) . dp,
dental papilla. x 640.
8
Autoradiograph of a seven day subcutaneous implant of the first molar
tooth germ taken from a one day old mouse. Labelled cells (circled)
can be seen on the outer aspect of the forming dentine ( d ) of the
root. dp, dental papilla. x 640.
9
Three week subcutaneous implant of the first molar tooth germ taken
from a one day old mouse. Development has continued with further
formation of dentine ( d ) , enamel ( e s ) and the formation of cement,
periodontal ligament and bone (b). The enamel has been lost during
demineralization and only a few remnants of enamel matrix occupy
the enamel space. l h e arrows indicate a n accumulation of lymphocytes. Haematoxylin and eosin. x 120.
10
Three week subcutaneous implant of the first molar tooth germ taken
from a one day old mouse. The periodontal ligament ( p l ) shows
oblique orientation of the fibroblasts. b, bone; c, cement and d, dentine. Haematoxylin and eosin. x 480.
DEVELOPMENT OF THE PERIODONTIUM
A. R. Ten Cate, C. Mills and G. Solomon
PLATE 3
375
PLATE 4
EXPLANATION OF FIGURES
11 Autoradiograph of a thrce week subcutaneous implant of the first
molar tooth germ taken from a one day old mouse. Three labelled
cells (circled) are shown. One such cell is a cementoblast; and
the remaining two fibroblasts of the ligament. d, dentine; c, cement;
es, enamel space; b, bone. x 480.
376
12
Autoradiograph of a three week subcutaneous implant of the first
molar tooth germ taken from a one dav old mouse. A cell (circled)
associated with the bone ( b ) has three silver grains associated with
it. x 480.
13
Four week subcutaneous implant of the first molar tooth germ taken
from a one day old mouse. This tooth h a s “erupted” through the skin.
Haematoxylin and eosin. x 48.
14
Four week subcutaneous implant of the first molar tooth germ taken
from a one day old mouse. This specimen has also “erupted” through
the skin ( s ) and shows the formation of a n epithelial attachment.
Note also the orientation of the periodontal ligament fibroblasts. d,
dentine; es, enamel space; b, bone. Haematoxylin and eosin. x 120.
DEVELOPMENT OF THE PERIODONTIUM
A. R. Ten Cate, C. Mills and G . Solomon
PLATE 4
377
PLATE 5
EXPLANATION O F FIGURES
15
Three week subcutaneous implant of the first molar tooth germ taken
from a one day old mouse. This specimen has continued development
but h a s not erupted as its orientation was away from the skin surface. Haematoxylin and eosin. x 64.
16 Autoradiograph of a three week subcutaneous implant of the first
molar tooth germ taken from a one day old mouse. The tooth has
“erupted’ and a n epithelial attachment has formed. The dental epithelial cells ( d e ) are labelled whereas the cells of the epidermis are
unlabelled. x 480.
17 Three week subcutaneous implant of the first molar tooth germ taken
from a one day old mouse. The tooth is about to break through the
epidermis of the skin (s). Note the proliferation of the epidermal cells
and fusion with the cells of the dental epithelium (de). Haematoxylin
and eosin. x 160.
378
DEVELOPMENT OF THE PERIODONTIUM
A. R. Ten Cate, C. Mills and G . Solomon
PLATE 5
379
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